U.S. patent application number 14/604530 was filed with the patent office on 2015-07-23 for fusion proteins and methods thereof.
The applicant listed for this patent is THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. Invention is credited to Antonio IAVARONE, Anna LASORELLA, Raul RABADAN.
Application Number | 20150203589 14/604530 |
Document ID | / |
Family ID | 53544211 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150203589 |
Kind Code |
A1 |
IAVARONE; Antonio ; et
al. |
July 23, 2015 |
FUSION PROTEINS AND METHODS THEREOF
Abstract
The invention discloses oncogenic fusion proteins. The invention
provides methods for treating gene-fusion based cancers.
Inventors: |
IAVARONE; Antonio; (New
York, NY) ; LASORELLA; Anna; (New York, NY) ;
RABADAN; Raul; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW
YORK |
New York |
NY |
US |
|
|
Family ID: |
53544211 |
Appl. No.: |
14/604530 |
Filed: |
January 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2013/051888 |
Jul 24, 2013 |
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14604530 |
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62096311 |
Dec 23, 2014 |
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61675006 |
Jul 24, 2012 |
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Current U.S.
Class: |
424/138.1 ;
435/15; 435/194; 435/6.11; 435/6.12; 435/7.23; 530/387.7; 536/23.2;
536/24.31; 536/24.33 |
Current CPC
Class: |
C12N 9/12 20130101; G01N
33/57492 20130101; C07K 14/82 20130101; G01N 2333/91205 20130101;
C07K 16/2863 20130101; C07K 2319/73 20130101; C12Q 1/6886 20130101;
C12Q 2600/158 20130101; C07K 16/18 20130101; C07K 14/47 20130101;
C07K 14/71 20130101; C07K 2319/00 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; G01N 33/574 20060101 G01N033/574; C12N 9/12 20060101
C12N009/12; C12Q 1/68 20060101 C12Q001/68; C07K 14/82 20060101
C07K014/82; C07K 14/435 20060101 C07K014/435 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Nos. R01CA101644, R01CA085628, R01CA131126, and R01CA178546 awarded
by the National Cancer Institute and R01NS061776 awarded by the
National Institute of Neurological Disorders and Stroke. The
Government has certain rights in the invention.
Claims
1. An antibody or antigen-binding fragment thereof, that
specifically binds to a purified fusion protein comprising a
tyrosine kinase domain of an FGFR protein fused to the TACC domain
of a transforming acidic coiled-coil-containing (TACC) protein.
2. The antibody or antigen-binding fragment of claim 1, wherein the
FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4.
3. The antibody or antigen-binding fragment of claim 1, wherein the
TACC protein is TACC1, TACC2, or TACC3.
4. The antibody or antigen-binding fragment of claim 1, wherein the
fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
5. The antibody or antigen-binding fragment of claim 4, wherein the
FGFR1-TACC1 fusion protein comprises the amino acid sequence of SEQ
ID NO: 150.
6. The antibody or antigen-binding fragment of claim 4, wherein the
FGFR3-TACC3 fusion protein comprises the amino acid sequence of SEQ
ID NO: 79, 158, 159, 160, or 161.
7. A composition for decreasing in a subject the expression level
or activity of a fusion protein comprising the tyrosine kinase
domain of an FGFR protein fused to the TACC domain of a TACC
protein, the composition in an admixture of a pharmaceutically
acceptable carrier comprising an inhibitor of the fusion
protein.
8. The composition of claim 7, wherein the TACC protein is TACC1,
TACC2, or TACC3.
9. The composition of claim 7, wherein the inhibitor comprises an
antibody that specifically binds to a FGFR-TACC fusion protein or a
fragment thereof; a small molecule that specifically binds to a
FGFR protein; a small molecule that specifically binds to a TACC
protein; an antisense RNA or antisense DNA that decreases
expression of a FGFR-TACC fusion polypeptide; a siRNA that
specifically targets a FGFR-TACC fusion gene; or a combination
thereof.
10. The composition of claim 7, wherein the FGFR protein is FGFR1,
FGFR2, FGFR3, or FGFR4.
11. The composition of claim 7, wherein the FGFR-TACC fusion
protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
12. The composition of claim 9, wherein the small molecule that
specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,
PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68,
AB1010, AP24534, E-7080, LY2874455, or a combination thereof.
13. A method for decreasing in a subject in need thereof the
expression level or activity of a fusion protein comprising the
tyrosine kinase domain of an FGFR protein fused to the TACC domain
of a TACC protein, the method comprising: (a) administering to the
subject a therapeutic amount of a composition of claim 7; and (b)
determining whether the fusion protein expression level or activity
is decreased compared to fusion protein expression level or
activity prior to administration of the composition, thereby
decreasing the expression level or activity of the fusion
protein.
14. A method of decreasing growth of a solid tumor in a subject in
need thereof, the method comprising administering to the subject an
effective amount of a FGFR fusion molecule inhibitor, wherein the
inhibitor decreases the size of the solid tumor and, wherein the
FGFR fusion comprises the tyrosine kinase domain of FGFR fused to
the TACC domain of TACC.
15. The method of claim 14, wherein the solid tumor comprises
glioblastoma multiforme, breast cancer, lung cancer, prostate
cancer, or colorectal carcinoma.
16. The method of claim 14, wherein the inhibitor comprises an
antibody that specifically binds to a FGFR-TACC fusion protein or a
fragment thereof; a small molecule that specifically binds to a
FGFR protein; a small molecule that specifically binds to a TACC
protein; an antisense RNA or antisense DNA that decreases
expression of a FGFR-TACC fusion polypeptide; a siRNA that
specifically targets a FGFR-TACC fusion gene; or a combination
thereof.
17. The method of claim 13 or 14, wherein the FGFR is FGFR1, FGFR2,
FGFR3, or FGFR4.
18. The method of claim 13 or 16, wherein the fusion protein is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
19. The method of claim 16, wherein the small molecule that
specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,
PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68,
AB1010, AP24534, E-7080, LY2874455, or a combination thereof.
20. A diagnostic kit for determining whether a sample from a
subject exhibits a presence of a FGFR fusion, the kit comprising at
least one oligonucleotide that specifically hybridizes to a FGFR
fusion, or a portion thereof, and wherein the FGFR fusion comprises
the tyrosine kinase domain of FGFR fused to the TACC domain of
TACC.
21. The kit of claim 20, wherein the oligonucleotides comprise a
set of nucleic acid primers or in situ hybridization probes.
22. The kit of claim 20, wherein the oligonucleotide comprises SEQ
ID NO: 162, 163, 164, 165, 166, 167, 168, 169, or a combination
thereof.
23. The kit of claim 21, wherein the primers prime a polymerase
reaction only when a FGFR fusion is present.
24. The kit of claim 20, wherein the determining comprises gene
sequencing, selective hybridization, selective amplification, gene
expression analysis, or a combination thereof.
25. A diagnostic kit for determining whether a sample from a
subject exhibits a presence of a FGFR fusion protein, the kit
comprising an antibody that specifically binds to a FGFR fusion
protein comprising SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158,
159, 160, or 161, wherein the antibody will recognize the protein
only when a FGFR fusion protein is present, and wherein the FGFR
fusion protein comprises a tyrosine kinase domain of an FGFR
protein fused to the TACC domain of a transforming acidic
coiled-coil-containing (TACC) protein.
26. The kit of claim 25, wherein the antibody is directed to an
FGFR fusion protein comprising SEQ ID NO: 79, 85, 86, 87, 88, 89,
150, 158, 159, 160, or 161.
27. The kit of claim 20 or 25, wherein the FGFR is FGFR1, FGFR2,
FGFR3, or FGFR4.
28. The kit of claim 20 or 25, wherein the FGFR fusion is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
29. A method for detecting the presence of a FGFR fusion in a human
subject, wherein the FGFR fusion comprises the tyrosine kinase
domain of FGFR fused to the TACC domain of TACC, the method
comprising: (a) obtaining a biological sample from the human
subject; and (b) detecting whether or not there is a FGFR fusion
present in the subject.
30. The method of claim 29, wherein the detecting comprises
measuring FGFR fusion protein levels by ELISA using an antibody
directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160,
or 161; western blot using an antibody directed to SEQ ID NO: 79,
85, 86, 87, 88, 89, 150, 158, 159, 160, or 161; mass spectroscopy,
isoelectric focusing, or a combination thereof.
31. The method of claim 29, wherein the detecting of step (b)
comprises detecting whether or not there is a nucleic acid sequence
encoding a FGFR fusion protein in the subject.
32. The method of claim 31, wherein the nucleic acid sequence
comprises any one of SEQ ID NOS: 1-77, 80-84, or 95-145.
33. The method of claim 31, wherein the detecting comprises using
hybridization, amplification, or sequencing techniques to detect a
FGFR fusion.
34. The method of claim 33, wherein the amplification uses primers
comprising SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, or
169.
35. The method of claim 29 or 31, wherein the FGFR is FGFR1, FGFR2,
FGFR3, or FGFR4.
36. The method of claim 29 or 31, wherein the FGFR fusion is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
37. A method of identifying a compound that decreases the oncogenic
activity of a FGFR-TACC fusion, the method comprising: a)
transducing a cell cultured in vitro with FGFR-TACC DNA; b)
contacting a cell with a ligand source for an effective period of
time; and c) determining whether the cells acquire the ability to
grow in anchorage-independent conditions, form multi-layered foci,
or a combination thereof, compared to cells cultured in the absence
of the test compound.
38. A purified fusion protein comprising the tyrosine kinase domain
of an FGFR protein fused to the TACC domain of a transforming
acidic coiled-coil-containing (TACC) protein.
39. The purified fusion protein of claim 38, wherein the FGFR
protein is FGFR1, FGFR2, FGFR3, or FGFR4.
40. The purified fusion protein of claim 38, wherein the TACC
protein is TACC1, TACC2, or TACC3.
41. The purified fusion protein of claim 38, wherein the fusion
protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
42. The purified fusion protein of claim 38, wherein the fusion
protein comprises SEQ ID NO: 79, SEQ ID NO: 158, SEQ ID NO: 159,
SEQ ID NO: 160, or SEQ ID NO: 161.
43. The purified fusion protein of claim 38, wherein the fusion
protein has a breakpoint comprising at least 3 consecutive amino
acids from amino acids 730-758 of SEQ ID NO: 90 and comprising at
least 3 consecutive amino acids from amino acids 549-838 of SEQ ID
NO: 92.
44. The purified fusion protein of claim 38, wherein the fusion
protein has a breakpoint comprising SEQ ID NO: 78, SEQ ID NO: 85,
SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO:89.
45. The purified fusion protein of claim 38, wherein the fusion
protein comprises SEQ ID NO: 150.
46. The purified fusion protein of claim 38, wherein the fusion
protein has a breakpoint comprising at least 3 consecutive amino
acids from amino acids 746-762 of SEQ ID NO: 146 and comprising at
least 3 consecutive amino acids from amino acids 572-590 of SEQ ID
NO: 148.
47. The purified fusion protein of claim 38, wherein the fusion
protein has a breakpoint comprising SEQ ID NO: 88.
48. A cDNA encoding a fusion protein comprising the tyrosine kinase
domain of FGFR fused to the TACC domain of TACC.
49. The cDNA of claim 48, wherein the FGFR is FGFR1, FGFR2, FGFR3,
or FGFR4.
50. The cDNA of claim 48, wherein the TACC is TACC1, TACC2, or
TACC3.
51. The cDNA of claim 48, wherein the fusion protein is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
52. The cDNA of claim 48, wherein the cDNA comprises SEQ ID NO:
94.
53. The cDNA of claim 48, where in the cDNA has a breakpoint
comprising at least 9 consecutive in-frame nucleotides from
nucleotides 2443-2530 of SEQ ID NO: 91 and comprising at least 9
consecutive in-frame nucleotides from nucleotides 1800-2847 of SEQ
ID NO: 93.
54. The cDNA of claim 48, where in the cDNA has a breakpoint
comprising any one of SEQ ID NOs: 1-77.
55. The cDNA of claim 48, wherein the cDNA comprises SEQ ID NO:
151.
56. The cDNA of claim 48, where in the cDNA has a breakpoint
comprising at least 9 consecutive in-frame nucleotides from
nucleotides 3178-3228 of SEQ ID NO: 147 and comprising at least 9
consecutive in-frame nucleotides from nucleotides 2092-2794 of SEQ
ID NO: 149.
57. The cDNA of claim 48, where in the cDNA has a breakpoint
comprising SEQ ID NO: 83.
58. The cDNA of claim 48, comprising a combination of exons 1-16 of
FGFR3 spliced 5' to a combination of exons 8-16 of TACC3, wherein a
breakpoint occurs in: a) any one of exons 1-16 of FGFR3 and any one
of exons 8-16 of TACC3; b) any one of introns 1-16 of FGFR3 and any
one of exons 8-16 of TACC3; c) any one of exons 1-16 of FGFR3 and
any one of introns 7-16 of TACC3; or d) any one of introns 1-16 of
FGFR3 and any one of introns 7-16 of TACC3.
59. The cDNA of claim 48, comprising a combination of exons 1-17 of
FGFR1 spliced 5' to a combination of exons 7-13 of TACC1, wherein a
breakpoint occurs in any one of exons 1-17 of FGFR3 and any one of
exons 7-13 of TACC3.
60. The cDNA of claim 48, comprising a combination of exons 1-18 of
FGFR2 spliced 5' to a combination of exons 1-23 of TACC2.
Description
[0001] This application is a continuation-in-part of International
Application No. PCT/US2013/051888, filed on Jul. 24, 2013, which
claims priority to U.S. Provisional Patent Application No.
61/675,006, filed on Jul. 24, 2012, the content of which is hereby
incorporated by reference in their entireties. This application
also claims priority to U.S. Provisional Patent Application No.
62/096,311, filed on Dec. 23, 2014, the content of which is hereby
incorporated by reference in its entirety.
[0003] All patents, patent applications and publications cited
herein are hereby incorporated by reference in their entirety. The
disclosures of these publications in their entireties are hereby
incorporated by reference into this application.
[0004] This patent disclosure contains material that is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure as it appears in the U.S. Patent and Trademark
Office patent file or records, but otherwise reserves any and all
copyright rights.
BACKGROUND OF THE INVENTION
[0005] Glioblastoma multiforme (GBM) is the most common form of
brain cancer and among the most incurable and lethal of all human
cancers. The current standard of care includes surgery,
chemotherapy, and radiation therapy. However, the prognosis of GBM
remains uniformly poor. There are few available targeted therapies
and none that specifically target GBM.
[0006] The target population of GBM patients who may carry
FGFR-TACC gene fusions and would benefit from targeted inhibition
of FGFR kinase activity is estimated to correspond to 6,000
patients per year world-wide.
SUMMARY OF THE INVENTION
[0007] The invention is based, at least in part, on the discovery
of a highly expressed class of gene fusions in GBM, which join the
tyrosine kinase domain of FGFR genes to the TACC domain of TACC1 or
TACC3. The invention is based, at least in part, on the finding
that FGFR-TACC fusions identify a subset of GBM patients who will
benefit from targeted inhibition of the tyrosine kinase activity of
FGFR. Identification of fusions of FGFR and TACC genes in
glioblastoma patients and other subjects afflicted with a
gene-fusion associated cancer (such as an epithelial cancer) are
useful therapeutic targets.
[0008] The invention is also based, at least in part, on the
discovery of gene fusions joining the tyrosine kinase domain of
FGFR genes to the TACC domain of TACC1 or TACC3 in grade II and III
glioma, The invention is based, at least in part, on the finding
that FGFR-TACC fusions identify a subset of grade II and III glioma
patients who will benefit from targeted inhibition of the tyrosine
kinase activity of FGFR. Identification of fusions of FGFR and TACC
genes in glioma patients are useful therapeutic targets.
[0009] An aspect of the invention provides for a purified fusion
protein comprising a tyrosine kinase domain of an FGFR protein
fused to a polypeptide that constitutively activates the tyrosine
kinase domain of the FGFR protein. In one embodiment, the FGFR
protein is FGFR1, FGFR2, FGFR3, or FGR4. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0010] An aspect of the invention provides for a purified fusion
protein comprising a transforming acidic coiled-coil (TACC) domain
fused to a polypeptide with a tyrosine kinase domain, wherein the
TACC domain constitutively activates the tyrosine kinase domain. In
one embodiment, the TACC protein is TACC1, TACC2, or TACC3. In
another embodiment, the purified fusion protein is essentially free
of other human proteins.
[0011] An aspect of the invention provides for a purified fusion
protein comprising the tyrosine kinase domain of an FGFR protein
fused 5' to the TACC domain of a transforming acidic
coiled-coil-containing (TACC) protein. In one embodiment, the FGFR
protein is FGFR1, FGFR2, FGFR3, or FGR4. In another embodiment, the
TACC protein is TACC1, TACC2, or TACC3. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0012] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR1-TACC1 nucleic acid, wherein FGFR1-TACC1
comprises a combination of exons 1-17 of FGFR1 located on human
chromosome 8p11 spliced 5' to a combination of exons 7-13 of TACC1
located on human chromosome 8p11, wherein a genomic breakpoint
occurs in any one of exons 1-17 of FGFR1 and any one of exons 7-13
of TACC1. In another embodiment, the purified fusion protein is
essentially free of other human proteins.
[0013] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR2-TACC2 nucleic acid, wherein FGFR2-TACC2
comprises a combination of any exons 1-18 of FGFR2 located on human
chromosome 10q26 spliced 5' to a combination of any exons 1-23 of
TACC2 located on human chromosome 10q26. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0014] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-16 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 8-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of exons 1-16 of FGFR3 and any one of exons 8-16
of TACC3. In another embodiment, the purified fusion protein is
essentially free of other human proteins.
[0015] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-18 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 4-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of exons 1-18 of FGFR3 and any one of exons 4-16
of TACC3. In another embodiment, the purified fusion protein is
essentially free of other human proteins.
[0016] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-16 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 8-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of introns 1-16 of FGFR3 and any one of exons
8-16 of TACC3. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0017] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-18 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 4-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of introns 1-18 of FGFR3 and any one of exons
4-16 of TACC3. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0018] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-16 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 8-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of exons 1-16 of FGFR3 and any one of introns
7-16 of TACC3. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0019] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-18 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 4-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of exons 1-18 of FGFR3 and any one of introns
3-16 of TACC3. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0020] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-16 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 8-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of introns 1-16 of FGFR3 and any one of introns
7-16 of TACC3. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0021] An aspect of the invention provides for a purified fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of exons 1-18 of FGFR3 located on human
chromosome 4p16 spliced 5' to a combination of exons 4-16 of TACC3
located on human chromosome 4p16, wherein a genomic breakpoint
occurs in any one of introns 1-18 of FGFR3 and any one of introns
3-16 of TACC3. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0022] An aspect of the invention provides for a synthetic nucleic
acid encoding the fusion proteins described above.
[0023] An aspect of the invention provides for a purified
FGFR3-TACC3 fusion protein comprising SEQ ID NO: 79, 158, 159, 160,
161, 539, 540, 541, 542, 543, 544, 545, 546, 547. In another
embodiment, the purified fusion protein is essentially free of
other human proteins.
[0024] An aspect of the invention provides for a purified
FGFR3-TACC3 fusion protein having a genomic breakpoint comprising
at least 3 consecutive amino acids from amino acids 730-758 of SEQ
ID NO: 90 and comprising at least 3 consecutive amino acids from
amino acids 549-838 of SEQ ID NO: 92. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0025] An aspect of the invention provides for a purified
FGFR3-TACC3 fusion protein having a genomic breakpoint comprising
at least 3 consecutive amino acids from amino acids 730-781 of SEQ
ID NO: 90 and comprising at least 3 consecutive amino acids from
amino acids 432-838 of SEQ ID NO: 92. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0026] An aspect of the invention provides for a purified
FGFR3-TACC3 fusion protein having a genomic breakpoint comprising
SEQ ID NO: 78. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0027] An aspect of the invention provides for a purified
FGFR3-TACC3 fusion protein having a genomic breakpoint comprising
any one of SEQ ID NOS: 85, 86, 87, 89, 516 or 518. In another
embodiment, the purified fusion protein is essentially free of
other human proteins.
[0028] An aspect of the invention provides for a purified
FGFR1-TACC1 fusion protein comprising SEQ ID NO: 150. In another
embodiment, the purified fusion protein is essentially free of
other human proteins.
[0029] An aspect of the invention provides for a purified
FGFR1-TACC1 fusion protein having a genomic breakpoint comprising
at least 3 consecutive amino acids from amino acids 746-762 of SEQ
ID NO: 146 and comprising at least 3 consecutive amino acids from
amino acids 572-590 of SEQ ID NO: 148. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0030] An aspect of the invention provides for a purified
FGFR1-TACC1 fusion protein having a genomic breakpoint comprising
at least 3 consecutive amino acids from amino acids 746-762 of SEQ
ID NO: 146 and comprising at least 3 consecutive amino acids from
amino acids 571-590 of SEQ ID NO: 148. In another embodiment, the
purified fusion protein is essentially free of other human
proteins.
[0031] An aspect of the invention provides for a purified
FGFR1-TACC1 fusion protein having a genomic breakpoint comprising
SEQ ID NO: 88. In another embodiment, the purified fusion protein
is essentially free of other human proteins.
[0032] An aspect of the invention provides for a purified DNA
encoding an FGFR3-TACC3 fusion protein comprising SEQ ID NO: 94,
530, 531, 532, 533, 534, 535, 536, 537, or 538. In another
embodiment, the purified fusion protein is essentially free of
other human proteins. An aspect of the invention provides for a
purified cDNA encoding an FGFR3-TACC3 fusion protein comprising SEQ
ID NO: 94, 530, 531, 532, 533, 534, 535, 536, 537, or 538.
[0033] An aspect of the invention provides for a synthetic nucleic
acid encoding an FGFR3-TACC3 fusion protein having a genomic
breakpoint comprising at least 9 consecutive in-frame nucleotides
from nucleotides 2443-2530 of SEQ ID NO: 91 and comprising at least
9 consecutive in-frame nucleotides from nucleotides 1800-2847 of
SEQ ID NO: 93.
[0034] An aspect of the invention provides for a synthetic nucleic
acid encoding an FGFR3-TACC3 fusion protein having a genomic
breakpoint comprising any one of SEQ ID NOS: 1-77, or 519-527.
[0035] An aspect of the invention provides for a synthetic nucleic
acid encoding an FGFR1-TACC1 fusion protein comprising SEQ ID NO:
151.
[0036] An aspect of the invention provides for a synthetic nucleic
acid encoding an FGFR1-TACC1 fusion protein having a genomic
breakpoint comprising at least 9 consecutive in-frame nucleotides
from nucleotides 3178-3228 of SEQ ID NO: 147 and comprising at
least 9 consecutive in-frame nucleotides from nucleotides 2092-2794
of SEQ ID NO: 149.
[0037] An aspect of the invention provides for a synthetic nucleic
acid encoding an FGFR1-TACC1 fusion protein having a genomic
breakpoint comprising SEQ ID NO: 83.
[0038] An aspect of the invention provides for an antibody or
antigen-binding fragment thereof, that specifically binds to a
purified fusion protein comprising a tyrosine kinase domain of an
FGFR protein fused to a polypeptide that constitutively activates
the tyrosine kinase domain of the FGFR protein. In one embodiment,
the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. In another
embodiment, the fusion protein is an FGFR-TACC fusion protein. In a
further embodiment, the FGFR-TACC fusion protein is FGFR1-TACC1,
FGFR2-TACC2, or FGFR3-TACC3. In some embodiments, the FGFR1-TACC1
fusion protein comprises the amino acid sequence of SEQ ID NO: 150.
In other embodiments, the FGFR3-TACC3 fusion protein comprises the
amino acid sequence of SEQ ID NO: 79, 158, 159, 160, 161, 539, 540,
541, 542, 543, 544, 545, 546, or 547.
[0039] An aspect of the invention provides for a composition for
decreasing in a subject the expression level or activity of a
fusion protein comprising the tyrosine kinase domain of an FGFR
protein fused to a polypeptide that constitutively activates the
tyrosine kinase domain of the FGFR protein, the composition in an
admixture of a pharmaceutically acceptable carrier comprising an
inhibitor of the fusion protein. In one embodiment, the fusion
protein is an FGFR-TACC fusion protein. In another embodiment, the
inhibitor comprises an antibody that specifically binds to a
FGFR-TACC fusion protein or a fragment thereof; a small molecule
that specifically binds to a FGFR protein; a small molecule that
specifically binds to a TACC protein; an antisense RNA or antisense
DNA that decreases expression of a FGFR-TACC fusion polypeptide; a
siRNA that specifically targets a FGFR-TACC fusion gene; or a
combination of the listed inhibitors. In a further embodiment, the
FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. In some embodiments,
the FGFR-TACC fusion protein is FGFR1-TACC1, FGFR2-TACC2, or
FGFR3-TACC3. In other embodiments, the small molecule that
specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,
PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68,
AB1010, AP24534, E-7080, LY2874455, or a combination of the listed
small molecules. In other embodiments, the small molecule that
specifically binds to a FGFR protein comprises an oral pan-FGFR
tyrosine kinase inhibitor. In other embodiments, the small molecule
that specifically binds to a FGFR protein comprises
JNJ-42756493.
[0040] An aspect of the invention provides for a method for
decreasing in a subject in need thereof the expression level or
activity of a fusion protein comprising the tyrosine kinase domain
of an FGFR protein fused to a polypeptide that constitutively
activates the tyrosine kinase domain of the FGFR protein. In one
embodiment, the method comprises administering to the subject a
therapeutic amount of a composition for decreasing the expression
level or activity in a subject of a fusion protein comprising the
tyrosine kinase domain of an FGFR protein fused to a polypeptide
that constitutively activates the tyrosine kinase domain of the
FGFR protein. In one embodiment, the method comprises obtaining a
sample from the subject to determine the level of expression of an
FGFR fusion molecule in the subject. In some embodiments, the
sample is incubated with an agent that binds to an FGFR fusion
molecule, such as an antibody, a probe, a nucleic acid primer, and
the like. In one embodiment, the detection or determining comprises
nucleic acid sequencing, selective hybridization, selective
amplification, gene expression analysis, or a combination thereof.
In another embodiment, the detection or determination comprises
protein expression analysis, for example by western blot analysis,
ELISA, immunostaining, or other antibody detection methods. In a
further embodiment, the method comprises determining whether the
fusion protein expression level or activity is decreased compared
to fusion protein expression level or activity prior to
administration of the composition, thereby decreasing the
expression level or activity of the fusion protein. In one
embodiment, the fusion protein is an FGFR-TACC fusion protein. In a
further embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or
FGFR4. In some embodiments, the FGFR-TACC fusion protein is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In one embodiment, the
composition for decreasing the expression level or activity of a
fusion protein comprises an antibody that specifically binds to a
FGFR-TACC fusion protein or a fragment thereof; a small molecule
that specifically binds to a FGFR protein; a small molecule that
specifically binds to a TACC protein; an antisense RNA or antisense
DNA that decreases expression of a FGFR-TACC fusion polypeptide; a
siRNA that specifically targets a FGFR-TACC fusion gene; or a
combination of the listed inhibitors. In a further embodiment, the
FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. In some embodiments,
the FGFR-TACC fusion protein is FGFR1-TACC1, FGFR2-TACC2, or
FGFR3-TACC3. In other embodiments, the small molecule that
specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,
PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68,
AB1010, AP24534, E-7080, LY2874455, or a combination of the small
molecules listed. In other embodiments, the small molecule that
specifically binds to a FGFR protein comprises an oral pan-FGFR
tyrosine kinase inhibitor. In other embodiments, the small molecule
that specifically binds to a FGFR protein comprises
JNJ-42756493.
[0041] An aspect of the invention provides for a method for
treating a gene-fusion associated cancer in a subject in need
thereof, the method comprising administering to the subject an
effective amount of a FGFR fusion molecule inhibitor. In one
embodiment, the gene-fusion associated cancer comprises an
epithelial cancer. In one embodiment, the gene-fusion associated
cancer comprises glioblastoma multiforme, breast cancer, lung
cancer, prostate cancer, or colorectal carcinoma. In one
embodiment, the gene-fusion associated cancer comprises bladder
carcinoma, squamous lung carcinoma and head and neck carcinoma. In
one embodiment, the gene-fusion associated cancer comprises glioma.
In one embodiment, the gene-fusion associated cancer comprises
grade II or III glioma. In one embodiment, the gene-fusion
associated cancer comprises IDH wild-type grade II or III glioma.
In one embodiment, the method comprises obtaining a sample from the
subject to determine the level of expression of an FGFR fusion
molecule in the subject. In some embodiments the sample from the
subject is a tissue sample. In some embodiments, the sample is a
paraffin embedded tissue section. In some embodiments, the tissue
sample from the subject is a tumor sample. In some embodiments, the
sample is incubated with an agent that binds to an FGFR fusion
molecule, such as an antibody, a probe, a nucleic acid primer, and
the like. In one embodiment, the detection or determining comprises
nucleic acid sequencing, selective hybridization, selective
amplification, gene expression analysis, or a combination thereof.
In another embodiment, the detection or determination comprises
protein expression analysis, for example by western blot analysis,
ELISA, immunostaining, or other antibody detection methods. In
another embodiment, the FGFR fusion protein comprises an FGFR
protein fused to a polypeptide that constitutively activates the
tyrosine kinase domain of the FGFR protein. In one embodiment, the
fusion protein is an FGFR-TACC fusion protein. In another
embodiment, the inhibitor comprises an antibody that specifically
binds to a FGFR-TACC fusion protein or a fragment thereof; a small
molecule that specifically binds to a FGFR protein; a small
molecule that specifically binds to a TACC protein; an antisense
RNA or antisense DNA that decreases expression of a FGFR-TACC
fusion polypeptide; a siRNA that specifically targets a FGFR-TACC
fusion gene; or a combination of the listed inhibitors. In a
further embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or
FGFR4. In some embodiments, the FGFR-TACC fusion protein is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In other embodiments, the
small molecule that specifically binds to a FGFR protein comprises
AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120,
BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or
a combination of the small molecules listed. In other embodiments,
the small molecule that specifically binds to a FGFR protein
comprises an oral pan-FGFR tyrosine kinase inhibitor. In other
embodiments, the small molecule that specifically binds to a FGFR
protein comprises JNJ-42756493.
[0042] An aspect of the invention provides for a method of
decreasing growth of a solid tumor in a subject in need thereof,
the method comprising administering to the subject an effective
amount of a FGFR fusion molecule inhibitor, wherein the inhibitor
decreases the size of the solid tumor. In one embodiment, the solid
tumor comprises glioblastoma multiforme, breast cancer, lung
cancer, prostate cancer, or colorectal carcinoma. In one
embodiment, the solid tumor comprises bladder carcinoma, squamous
lung carcinoma and head and neck carcinoma. In one embodiment, the
solid tumor comprises glioma. In one embodiment, the solid tumor
comprises grade II or III glioma. In one embodiment, the solid
tumor comprises IDH wild-type grade II or III glioma. In one
embodiment, the method comprises obtaining a sample from the
subject to determine the level of expression of an FGFR fusion
molecule in the subject. In some embodiments the sample from the
subject is a tissue sample. In some embodiments, the sample is a
paraffin embedded tissue section. In some embodiments, the tissue
sample from the subject is a tumor sample. In some embodiments, the
sample is incubated with an agent that binds to an FGFR fusion
molecule, such as an antibody, a probe, a nucleic acid primer, and
the like. In one embodiment, the detection or determining comprises
nucleic acid sequencing, selective hybridization, selective
amplification, gene expression analysis, or a combination thereof.
In another embodiment, the detection or determination comprises
protein expression analysis, for example by western blot analysis,
ELISA, immunostaining, or other antibody detection methods. In
another embodiment, the FGFR fusion protein comprises an FGFR
protein fused to a polypeptide that constitutively activates the
tyrosine kinase domain of the FGFR protein. In one embodiment, the
fusion protein is an FGFR-TACC fusion protein. In another
embodiment, the inhibitor comprises an antibody that specifically
binds to a FGFR-TACC fusion protein or a fragment thereof; a small
molecule that specifically binds to a FGFR protein; a small
molecule that specifically binds to a TACC protein; an antisense
RNA or antisense DNA that decreases expression of a FGFR-TACC
fusion polypeptide; a siRNA that specifically targets a FGFR-TACC
fusion gene; or a combination of the listed inhibitors. In a
further embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or
FGFR4. In some embodiments, the FGFR-TACC fusion protein is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In other embodiments, the
small molecule that specifically binds to a FGFR protein comprises
AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120,
BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or
a combination of the small molecules listed. In other embodiments,
the small molecule that specifically binds to a FGFR protein
comprises an oral pan-FGFR tyrosine kinase inhibitor. In other
embodiments, the small molecule that specifically binds to a FGFR
protein comprises JNJ-42756493.
[0043] An aspect of the invention provides for a diagnostic kit for
determining whether a sample from a subject exhibits a presence of
a FGFR fusion, the kit comprising at least one oligonucleotide that
specifically hybridizes to a FGFR fusion, or a portion thereof. In
one embodiment, the oligonucleotides comprise a set of nucleic acid
primers or in situ hybridization probes. In another embodiment, the
oligonucleotide comprises SEQ ID NO: 162, 163, 164, 165, 166, 167,
168, 169, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,
506, 507, 508, 509, 510 or a combination of the listed
oligonucleotides. In one embodiment, the primers prime a polymerase
reaction only when a FGFR fusion is present. In another embodiment,
the determining comprises gene sequencing, selective hybridization,
selective amplification, gene expression analysis, or a combination
thereof. In a further embodiment, the FGFR-fusion is an FGFR-TACC
fusion. In some embodiments, the FGFR is FGFR1, FGFR2, FGFR3, or
FGFR4. In other embodiments, the FGFR-TACC fusion is FGFR1-TACC1,
FGFR2-TACC2, or FGFR3-TACC3.
[0044] An aspect of the invention provides for a diagnostic kit for
determining whether a sample from a subject exhibits a presence of
a FGFR fusion protein, the kit comprising an antibody that
specifically binds to a FGFR fusion protein comprising SEQ ID NO:
79, 85, 86, 87, 88, 89, 150, 158, 159, 160, 161, 516, 518, 539,
540, 541, 542, 543, 544, 545, 546, or 547 wherein the antibody will
recognize the protein only when a FGFR fusion protein is present.
In one embodiment, the antibody directed to and FGFR fusion
comprising SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160,
161, 516, 518 539, 540, 541, 542, 543, 544, 545, 546, or 547. In a
further embodiment, the FGFR-fusion is an FGFR-TACC fusion. In some
embodiments, the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. In other
embodiments, the FGFR-TACC fusion is FGFR1-TACC1, FGFR2-TACC2, or
FGFR3-TACC3. In some embodiments the sample from the subject is a
tissue sample. In some embodiments, the sample is a paraffin
embedded tissue section. In some embodiments, the tissue sample
from the subject is a tumor sample.
[0045] An aspect of the invention provides for a method for
detecting the presence of a FGFR fusion in a human subject. In one
embodiment, the method comprises obtaining a biological sample from
the human subject. In some embodiments the sample from the subject
is a tissue sample. In some embodiments, the sample is a paraffin
embedded tissue section. In some embodiments, the tissue sample
from the subject is a tumor sample. In some embodiments, the sample
is incubated with an agent that binds to an FGFR fusion molecule,
such as an antibody. In another embodiment, the detection or
determination comprises protein expression analysis, for example by
western blot analysis, ELISA, immunostaining or other antibody
detection methods. In some embodiments, the method further
comprises assessing whether to administer a FGFR fusion molecule
inhibitor based on the expression pattern of the subject. In
further embodiments, the method comprises administering a FGFR
fusion molecule inhibitor to the subject. In other embodiments, the
FGFR fusion molecule inhibitor comprises an oral pan-FGFR tyrosine
kinase inhibitor. In other embodiments, the FGFR fusion molecule
inhibitor comprises JNJ-42756493. In another embodiment, the method
comprises detecting whether or not there is a FGFR fusion present
in the subject. In one embodiment, the detecting comprises
measuring FGFR fusion protein levels by ELISA using an antibody
directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160,
161, 516, 518 539, 540, 541, 542, 543, 544, 545, 546, or 547;
western blot using an antibody directed to SEQ ID NO: 79, 85, 86,
87, 88, 89, 150, 158, 159, 160, 161, 516, 518 539, 540, 541, 542,
543, 544, 545, 546, or 547; immunostaining using an antibody
directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160,
161, 516, 518, 539, 540, 541, 542, 543, 544, 545, 546, or 547; mass
spectroscopy, isoelectric focusing, or a combination of the listed
methods. In some embodiments, the FGFR-fusion is an FGFR-TACC
fusion. In other embodiments, the FGFR is FGFR1, FGFR2, FGFR3, or
FGFR4. In other embodiments, the FGFR-TACC fusion is FGFR1-TACC1,
FGFR2-TACC2, or FGFR3-TACC3.
[0046] An aspect of the invention provides for a method for
detecting the presence of a FGFR fusion in a human subject. In one
embodiment, the method comprises obtaining a biological sample from
a human subject. In some embodiments, the sample is incubated with
an agent that binds to an FGFR fusion molecule, such as a probe, a
nucleic acid primer, and the like. In other embodiments, the
detection or determination comprises nucleic acid sequencing,
selective hybridization, selective amplification, gene expression
analysis, or a combination thereof. In some embodiments, the method
further comprises assessing whether to administer a FGFR fusion
molecule inhibitor based on the expression pattern of the subject.
In further embodiments, the method comprises administering a FGFR
fusion molecule inhibitor to the subject. In another embodiment,
the method comprises detecting whether or not there is a nucleic
acid sequence encoding a FGFR fusion protein in the subject. In one
embodiment, the nucleic acid sequence comprises any one of SEQ ID
NOS: 1-77, 80-84, 95-145, 515, 517, 519-527, or 530-538. In another
embodiment, the detecting comprises using hybridization,
amplification, or sequencing techniques to detect a FGFR fusion. In
a further embodiment, the amplification uses primers comprising SEQ
ID NO: 162, 163, 164, 165, 166, 167, 168, 169, 495, 496, 497, 498,
499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509 or 510. In
some embodiments, the FGFR-fusion is an FGFR-TACC fusion. In other
embodiments, the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. In other
embodiments, the FGFR-TACC fusion is FGFR1-TACC1, FGFR2-TACC2, or
FGFR3-TACC3.
[0047] An aspect of the invention provides for a method of
initiating oncogenic transformation in vitro. The method comprises
(a) transducing cells cultured in vitro with FGFR-TACC fusion DNA;
and (b) determining whether the cells acquire the ability to grow
in anchorage-independent conditions, form multi-layered foci, or a
combination thereof.
[0048] An aspect of the invention provides for a method of
initiating oncogenic transformation in vivo. The method comprises
(a) transducing cells cultured in vitro with FGFR-TACC fusion DNA;
(b) injecting a mouse with the transduced cells; and (c)
determining whether a tumor grows in the mouse. In one embodiment,
the injecting is a subcutaneous or intracranial injection.
[0049] An aspect of the invention provides a method of identifying
a compound that decreases the oncogenic activity of a FGFR-TACC
fusion. The method comprises (a) transducing a cell cultured in
vitro with FGFR-TACC DNA; (b) contacting a cell with a ligand
source for an effective period of time; and (c) determining whether
the cells acquire the ability to grow in anchorage-independent
conditions, form multi-layered foci, or a combination thereof,
compared to cells cultured in the absence of the test compound.
[0050] In one embodiment, the method can comprise contacting a
sample from the subject with an antibody specific for a FGFR fusion
molecule, and determining the presence of an immune complex. In
another embodiment, the method can comprise contacting a sample
from the subject with an antibody specific for a FGFR molecule, or
a TACC molecule, and determining the presence of an immune complex.
In another embodiment, the antibody can recognize the FGFR3
C-terminal region, or the TACC3 N-terminal region, or a combination
thereof. In another embodiment, the antibody can recognize the
FGFR3 C-terminal region, or the TACC3 N-terminal region, or a
combination thereof. In another embodiment, the method can comprise
contacting a sample from the subject with an antibody specific for
a FGFR molecule, or a TACC molecule, or a FGFR fusion molecule, and
determining the amount of an immune complex formed compared to the
amount of immune complex formed in non-tumor cells or tissue,
wherein an increased amount of an immune complex indicates the
presence of an FGFR fusion.
[0051] In one embodiment, the method can comprise contacting a
sample from the subject with primers specific for a FGFR fusion
molecule, and determining the presence of an PCR product. In
another embodiment, the method can comprise contacting a sample
from the subject with primer specific for a FGFR molecule, or a
TACC molecule, and determining the presence of a PCR product. In
another embodiment, the primers can recognize the nucleic acids
encoding a FGFR3 C-terminal region, or nucleic acids encoding a
TACC3 N-terminal region, or a combination thereof. In another
embodiment, the method can comprise contacting a sample from the
subject with primers specific for a FGFR molecule, or a TACC
molecule, or a FGFR fusion molecule, and determining the amount of
PCR product formed compared to the amount of PCR product formed in
non-tumor cells or tissue, wherein an increased amount of PCR
product indicates the presence of an FGFR fusion.
[0052] An aspect of the invention provides for a purified fusion
protein comprising the tyrosine kinase domain of an FGFR protein
fused to the TACC domain of a transforming acidic
coiled-coil-containing (TACC) protein. In one embodiment, the FGFR
protein is FGFR1, FGFR2, FGFR3, or FGFR4. In one embodiment, the
TACC protein is TACC1, TACC2, or TACC3. In one embodiment, the
fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In one
embodiment, the fusion protein comprises SEQ ID NO: 79, SEQ ID NO:
158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO:
539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO:
543, SEQ ID NO: 545, SEQ ID NO: 546, or SEQ ID NO: 547. In one
embodiment, the fusion protein has a breakpoint comprising at least
3 consecutive amino acids from amino acids 730-758 of SEQ ID NO: 90
and comprising at least 3 consecutive amino acids from amino acids
549-838 of SEQ ID NO: 92. In one embodiment, the fusion protein has
a breakpoint comprising SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO:
86, SEQ ID NO: 87, SEQ ID NO:89, SEQ ID NO: 516, or SEQ ID NO:518.
In one embodiment, the fusion protein comprises SEQ ID NO: 150. In
one embodiment, the fusion protein has a breakpoint comprising at
least 3 consecutive amino acids from amino acids 746-762 of SEQ ID
NO: 146 and comprising at least 3 consecutive amino acids from
amino acids 572-590 of SEQ ID NO: 148. In one embodiment, the
fusion protein has a breakpoint comprising SEQ ID NO: 88.
[0053] An aspect of the invention provides for a cDNA encoding a
fusion protein comprising the tyrosine kinase domain of FGFR fused
to the TACC domain of TACC. In one embodiment the FGFR is FGFR1,
FGFR2, FGFR3, or FGFR4. In one embodiment, the TACC is TACC1,
TACC2, or TACC3. In one embodiment, the fusion protein is
FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In one embodiment, the
cDNA comprises SEQ ID NO: 94, SEQ ID NO: 530, SEQ ID NO: 531, SEQ
ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID
NO: 536, SEQ ID NO: 537 or SEQ ID NO: 538. In one embodiment, the
cDNA has a breakpoint comprising at least 9 consecutive in-frame
nucleotides from nucleotides 2443-2530 of SEQ ID NO: 91 and
comprising at least 9 consecutive in-frame nucleotides from
nucleotides 1800-2847 of SEQ ID NO: 93. In one embodiment, the cDNA
has a breakpoint comprising any one of SEQ ID NOs: 1-77, or SEQ ID
NOs: 519-527. In one embodiment, the cDNA comprises SEQ ID NO: 151.
In one embodiment, the cDNA has a breakpoint comprising at least 9
consecutive in-frame nucleotides from nucleotides 3178-3228 of SEQ
ID NO: 147 and comprising at least 9 consecutive in-frame
nucleotides from nucleotides 2092-2794 of SEQ ID NO: 149. In one
embodiment, the cDNA has a breakpoint comprising SEQ ID NO: 83. In
one embodiment, the cDNA comprises a combination of exons 1-16 of
FGFR3 spliced 5' to a combination of exons 8-16 of TACC3, wherein a
breakpoint occurs in: a) any one of exons 1-16 of FGFR3 and any one
of exons 8-16 of TACC3; b) any one of introns 1-16 of FGFR3 and any
one of exons 8-16 of TACC3; c) any one of exons 1-16 of FGFR3 and
any one of introns 7-16 of TACC3; or d) any one of introns 1-16 of
FGFR3 and any one of introns 7-16 of TACC3. In one embodiment, the
cDNA comprises a combination of exons 1-17 of FGFR1 spliced 5' to a
combination of exons 7-13 of TACC1, wherein a breakpoint occurs in
any one of exons 1-17 of FGFR3 and any one of exons 7-13 of TACC3.
In one embodiment, the cDNA comprises a combination of exons 1-18
of FGFR2 spliced 5' to a combination of exons 1-23 of TACC2.
BRIEF DESCRIPTION OF THE FIGURES
[0054] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
[0055] FIG. 1A is a graph that shows genes recurrently involved in
gene fusions in TCGA. Only genes involved in at least three gene
fusions across different samples are displayed.
[0056] FIGS. 1B-1, 1B-2, 1B-3 and 1B-4 show an FGFR3-TACC3 gene
fusion identified by whole transcriptome sequencing of GSCs. 76
split-reads (SEQ ID NOS: 2-77, respectively) are shown aligning on
the breakpoint. The predicted reading frame at the breakpoint is
shown at the top (FGFR3 nucleotide sequence (left) and TACC3
nucleotide sequence (right); SEQ ID NO: 1) with FGFR3 sequences
below the predicted reading frame (left) and TACC3 (right). The
putative amino acid sequence (SEQ ID NO: 78) corresponding to SEQ
ID NO: 1 is shown above the predicted reading frame.
[0057] FIG. 1C shows an FGFR3-TACC3 gene fusion identified by whole
transcriptome sequencing of GSCs. On the left, FGFR3-TACC3-specific
PCR from cDNA derived from GSCs and GBM is shown. On the right,
Sanger sequencing chromatogram shows the reading frame at the
breakpoint (SEQ ID NO: 80) and putative translation of the fusion
protein (SEQ ID NO: 85) in the positive samples.
[0058] FIG. 1D shows an FGFR3-TACC3 gene fusion identified by whole
transcriptome sequencing of GSCs. Amino acid sequence of the
FGFR3-TACC3 protein is shown (SEQ ID NO: 79). Residues
corresponding to FGFR3 or to TACC3 (underlined) are shown. The
fusion protein joins the tyrosine kinase domain of FGFR3 to the
TACC domain of TACC3.
[0059] FIG. 1E shows an FGFR3-TACC3 gene fusion identified by whole
transcriptome sequencing of GSCs. Genomic fusion of FGFR3 exon 17
with intron 7 of TACC3 is shown. In the fused mRNA, exon 16 of
FGFR3 is spliced 5' to exon 8 of TACC3. Filled arrows indicate the
position of the fusion-genome primers, which generate
fusion-specific PCR products in GSC-1123 and GBM-1123.
[0060] FIG. 2A shows recurrent gene fusions between FGFR and TACC
genes in GBM. Specifically, FGFR3-TACC3 gene fusions are shown that
were identified by exome sequencing analysis. Split-reads are shown
aligning the genomic breakpoints of FGFR3 and TACC3 genes in the
four TCGA GBM samples. For TCGA-27-1835, SEQ ID NO: 95 shows the
reading frame at the breakpoint (bold), while SEQ ID NOS: 96-107,
respectively, show alignments of the genomic breakpoints. For
TCGA-19-5958, SEQ ID NO: 108 shows the reading frame at the
breakpoint (bold), while SEQ ID NOS: 109-111, respectively, show
alignments of the genomic breakpoints. For TCGA-06-6390, SEQ ID NO:
112 shows the reading frame at the breakpoint (bold), while SEQ ID
NOS: 113-131, respectively, show alignments of the genomic
breakpoints. For TCGA-12-0826, SEQ ID NO: 132 shows the reading
frame at the breakpoint (bold), while SEQ ID NOS: 133-145,
respectively, show alignments of the genomic breakpoints.
[0061] FIG. 2B shows recurrent gene fusions between FGFR and TACC
genes in GBM. On the left, a gel of FGFR-TACC-specific PCR is shown
for FGFR3-TACC3 from a GBM cDNA sample. On the right, Sanger
sequencing chromatograms show the reading frame at the breakpoint
(SEQ ID NO: 81) and putative translation of the fusion protein (SEQ
ID NO: 86) in the positive samples.
[0062] FIG. 2C shows recurrent gene fusions between FGFR and TACC
genes in GBM. On the left, a gel of FGFR-TACC-specific PCR is shown
for FGFR3-TACC3 from a GBM cDNA sample. On the right, Sanger
sequencing chromatograms show the reading frame at the breakpoint
(SEQ ID NO: 82) and putative translation of the fusion protein (SEQ
ID NO: 87) in the positive samples.
[0063] FIG. 2D shows recurrent gene fusions between FGFR and TACC
genes in GBM. Co-outlier expression of FGFR3 and TACC3 in four GBM
tumors from Atlas-TCGA is shown in the plot.
[0064] FIG. 2E shows recurrent gene fusions between FGFR and TACC
genes in GBM. CNV analysis shows micro-amplifications of the
rearranged portions of the FGFR3 and TACC3 genes in the same four
Atlas-TCGA GBM samples.
[0065] FIG. 2F shows recurrent gene fusions between FGFR and TACC
genes in GBM. On the left, a gel of FGFR-TACC-specific PCR is shown
for FGFR1-TACC1 from a GBM cDNA sample. On the right, Sanger
sequencing chromatograms show the reading frame at the breakpoint
(SEQ ID NO: 83) and putative translation of the fusion protein (SEQ
ID NO: 88) in the positive samples.
[0066] FIG. 2G shows recurrent gene fusions between FGFR and TACC
genes in GBM. On the left, a gel of FGFR-TACC-specific PCR is shown
for FGFR3-TACC3 from a GBM cDNA sample. On the right, Sanger
sequencing chromatograms show the reading frame at the breakpoint
(SEQ ID NO: 84) and putative translation of the fusion protein (SEQ
ID NO: 89) in the positive samples.
[0067] FIG. 3A shows transforming activity of FGFR-TACC fusion
proteins. FGFR1-TACC1 and FGFR3-TACC3 induce anchorage-independent
growth in Rat1A fibroblasts. The number of soft agar colonies was
scored from triplicate samples 14 days after plating.
Representative microphotographs are shown.
[0068] FIG. 3B are photomicrographs showing of immunofluorescence
staining of tumors from mice injected with Ink4A;Arf-/- astrocytes
expressing FGFR3-TACC3 showing positivity for glioma-specific
(Nestin, Oig2 and GFAP) and proliferation markers (Ki67 and pHH3).
Sub-cutaneous tumors were generated by Ink4A;Arf-/- astrocytes
expressing FGFR-TACC fusions.
[0069] FIG. 3C shows Kaplan-Meier survival curves of mice injected
intracranially with pTomo-shp53 (n=8) or pTomo-EGFRvIII-shp53 (n=7)
(green line; "light grey" in black and white image) and
pTomo-FGFR3-TACC3-shp53 (n=8, red line; "dark grey" in black and
white image). Points on the curves indicate deaths (log-rank test,
p=0.00001, pTomo-shp53 vs. pTomo-FGFR3-TACC3-shp53).
[0070] FIG. 3D shows representative photomicrographs of Hematoxylin
and Eosin staining of advanced FGFR3-TACC3-shp53 generated tumors
showing histological features of high-grade glioma. Of note is the
high degree of infiltration of the normal brain by the tumor cells
Immunofluorescence staining shows that glioma and stem cell markers
(Nestin, Olig2 and GFAP), the proliferation markers (Ki67 and pHH3)
and the FGFR3-TACC3 protein are widely expressed in the
FGFR3-TACC3-shp53 brain tumors. F1-T1: FGFR1-TACC1; F3-T3:
FGFR3-TACC3; F3-T3-K508M: FGFR3-TACC3-K508M.
[0071] FIG. 4A shows that FGFR3-TACC3 localizes to spindle poles,
delays mitotic progression and induces chromosome segregation
defects and aneuploidy Constitutive auto-phosphorylation of
FGFR3-TACC3 fusion. Ink4A;Arf-/- astrocytes transduced with empty
lentivirus or a lentivirus expressing FGFR3-TACC3 or
FGFR3-TACC3-K508M were left untreated (0) or treated with 100 nM of
the FGFR inhibitor PD173074 for the indicated times.
Phospho-proteins and total proteins were analyzed by Western blot
using the indicated antibodies.
[0072] FIG. 4B shows that FGFR3-TACC3 localizes to spindle poles,
delays mitotic progression and induces chromosome segregation
defects. Photomicrographs are shown of confocal microscopy analysis
of FGFR3-TACC3 in Ink4A;Arf-/- astrocytes. Maximum intensity
projection of z-stacked images shows FGFR3-TACC3 (red; "dark grey"
in black and white image) coating the spindle poles of a
representative mitotic cell (upper panels). In telophase (lower
panels) FGFR3-TACC3 localizes to the mid-body. .alpha.-tubulin
(green; "grey" in black and white image), DNA (DAPI, blue; "light
grey" in black and white image).
[0073] FIG. 4C shows representative fluorescence video-microscopy
for cells transduced with vector or FGFR3-TACC3.
[0074] FIG. 4D shows a Box and Whisker plot representing the
analysis of the time from nuclear envelope breakdown (NEB) to
anaphase onset and from NEB to nuclear envelope reconstitution
(NER). The duration of mitosis was measured by following 50 mitoses
for each condition by time-lapse microscopy.
[0075] FIG. 4E shows that FGFR3-TACC3 localizes to spindle poles,
delays mitotic progression and induces chromosome segregation
defects. Representative images are shown of cells with chromosome
missegregation. Arrows point to chromosome misalignments, lagging
chromosomes, and chromosome bridges.
[0076] FIG. 4F shows quantitative analysis of segregation defects
in Rat1A expressing FGFR1-TACC1 and FGFR3-TACC3. F3-T3:
FGFR3-TACC3; F3-T3-K508M: FGFR3-TACC3-K508M.
[0077] FIG. 5A shows karyotype analysis of Rat1A cells transduced
with control, FGFR3, TACC3 or FGFR3-TACC3 expressing lentivirus.
Distribution of chromosome counts of cells arrested in mitosis and
analyzed for karyotypes using DAPI. Chromosomes were counted in 100
metaphase cells for each condition to determine the ploidy and the
diversity of chromosome counts within the cell population.
FGFR3-TACC3 fusion induces aneuploidy.
[0078] FIG. 5B shows representative karyotypes and FIG. 5C shows
distribution of chromosome counts of human astrocytes transduced
with control or FGFR3-TACC3 expressing lentivirus. Chromosomes were
counted in 100 metaphase cells for each condition to determine the
ploidy and the diversity of chromosome counts within the cell
population.
[0079] FIG. 5D shows quantitative analysis of chromosome number in
100 metaphase cells for each condition to determine the ploidy and
the diversity of chromosome counts within the cell population. (n=3
independent experiments).
[0080] FIG. 6A shows inhibition of FGFR-TK activity corrects the
aneuploidy initiated by FGFR3-TACC3. The upper panel is a karyotype
analysis of Rat1A cells transduced with control or FGFR3-TACC3
lentivirus and treated with vehicle (DMSO) or PD173470 (100 nM) for
five days. The lower panel shows the ploidy and the diversity of
chromosome counts within the cell population were determined by
quantitative analysis of chromosome number in 100 metaphase cells
for each condition.
[0081] FIG. 6B shows inhibition of FGFR-TK activity corrects the
aneuploidy initiated by FGFR3-TACC3. Correction of premature sister
chromatid separation (PMSCS) by PD173470 in cells expressing
FGFR3-TACC3. Panels show representative metaphase spreads. DNA was
stained by DAPI. FIG. 6C shows quantitative analysis of metaphases
with loss of sister chromatid cohesion (n=3; p=0.001, FGFR3-TACC3
treated with DMSO vs. FGFR3-TACC3 treated with PD173470).
[0082] FIG. 7A shows inhibition of FGFR-TK activity suppresses
tumor growth initiated by FGFR3-TACC3. Growth rate of Rat1A
transduced with the indicated lentiviruses and treated for three
days with increasing concentrations of PD173074. Cell growth was
determined by the MTT assay. Data are presented as the
means.+-.standard error (n=4).
[0083] FIG. 7B shows the growth rate of GSC-1123 treated with
PD173470 at the indicated concentrations for the indicated times.
Cell growth was determined by the MTT assay. Data are presented as
the means.+-.standard error (n=4).
[0084] FIG. 7C shows the growth inhibitory effect of silencing
FGFR3-TACC3 fusion. At the left, parallel cultures of GSC-1123
cells were transduced in triplicate. Rat1A cells expressing
FGFR3-TACC3 fusion were transduced with lentivirus expressing a
non-targeting shRNA (Ctr) or shRNA sequences targeting FGFR3 (sh2,
sh3, sh4). Five days after infection cells were plated at density
of 2.times.10.sup.4 cells/well in triplicate and the number of
trypan blue excluding cells was scored at the indicated times.
Infection with lentivirus expressing sh-3 and sh-4, the most
efficient FGFR3 silencing sequences reverted the growth rate of
FGFR3-TACC3 expressing cultures to levels comparable to those of
Rat1A transduced with empty vector. Values are the
means.+-.standard deviation (n=3). At the right sided figure,
GSC-1123 cells were transduced with lentivirus expressing a
non-targeting shRNA (sh-Ctr) or lentivirus expressing sh-3 and sh-4
sequences targeting FGFR3. Western Blot analysis was performed on
parallel cultures using the FGFR3 antibody to the detect
FGFT3-TACC3 fusion protein. .beta.-actin is shown as a control for
loading.
[0085] FIG. 7D shows that the FGFR inhibitor PD173074 suppresses
tumor growth of glioma sub-cutaneous xenografts generated by
Ink4A;Arf-/- astrocytes expressing FGFR3-TACC3. After tumor
establishment (200-300 mm.sup.3, arrow) mice were treated with
vehicle or PD173074 (50 mg/kg) for 14 days. Values are mean tumor
volumes.+-.standard error (n=7 mice per group).
[0086] FIG. 7E is a Kaplan-Meier analysis of glioma-bearing mice
following orthotopic implantation of Ink4A;Arf-/- astrocytes
transduced with FGFR3-TACC3. After tumor engraftment mice were
treated with vehicle (n=9) or AZD4547 (50 mg/kg) (n=7) for 20 days
(p=0.001).
[0087] FIG. 8 shows a schematic of the TX-Fuse pipeline for the
identification of fusion transcripts from RNA-Seq data generated
from nine GSC cultures. The continued figure shows a schematic of
the Exome-Fuse pipeline for the identification of gene fusion
rearrangements from DNA exome sequences of 84 GBM TCGA tumor
samples.
[0088] FIGS. 9A-D shows the validation of fusion transcripts
identified by RNA-seq of nine GSCs. Sanger sequencing chromatograms
show the reading frames at the breakpoint and putative translation
of the fusion proteins in the positive samples (right side). The
left side shows gels of RT-PCR conducted. (A) POLR2A-WRAP53. DNA
sequence disclosed as SEQ ID NO: 319 and protein sequence disclosed
as SEQ ID NO: 320. (B) CAPZB-UBR4. DNA sequence disclosed as SEQ ID
NO: 321 and protein sequence disclosed as SEQ ID NO: 322. (C)
ST8SIA4-PAM. DNA sequence disclosed as SEQ ID NO: 323 and protein
sequence disclosed as SEQ ID NO: 324. (D) PIGU-NCOA6. DNA sequence
disclosed as SEQ ID NO: 325 and protein sequence disclosed as SEQ
ID NO: 326.
[0089] FIGS. 9E-1, 9E-2, 9E-3, 9E-4, 9E-5, 9E-6, 9E-7, and 9E-8
show the fusion transcripts identified by whole transcriptome
sequencing of nine GSCs. 54 split-reads (SEQ ID NOS 329-382,
respectively, in order of appearance) are shown aligning on the
breakpoint of the POLR2A-WRAP53 fusion (SEQ ID NO: 327). The
predicted reading frame at the breakpoint is shown at the top with
POLR2A sequences in red (left) and WRAP53 in blue (right). Protein
sequence disclosed as SEQ ID NO: 328. On the continued page, 48
split-reads (SEQ ID NOS 385-432, respectively, in order of
appearance) are shown aligning on the breakpoint of the CAPZB-UBR4
fusion (SEQ ID NO: 383). The predicted reading frame at the
breakpoint is shown at the top with CAPZB sequences in red (left)
and UBR4 in blue (right). Protein sequence disclosed as SEQ ID NO:
384. On the continued page after, 29 split-reads (SEQ ID NOS
435-463, respectively, in order of appearance) are shown aligning
on the breakpoint of the ST8SIA4-PAM fusion (SEQ ID NO: 433). The
predicted reading frame at the breakpoint is shown at the top with
ST8SIA4 sequences in red (left) and PAM in blue (right). Protein
sequence disclosed as SEQ ID NO: 434. On the subsequent continued
page, 17 split-reads (SEQ ID NOS 466-482, respectively, in order of
appearance) are shown (top) aligning on the breakpoint of the
PIGU-NCOA6 fusion (SEQ ID NO: 464). The predicted reading frame at
the breakpoint is shown at the top with PIGU sequences in red
(left) and NCOA6 in blue (right). Protein sequence disclosed as SEQ
ID NO: 465. Also (below), 6 split-reads (SEQ ID NOS 485-490,
respectively, in order of appearance) are shown aligning on the
breakpoint of the IFNAR2-IL10RB fusion (SEQ ID NO: 483). The
predicted reading frame at the breakpoint is shown at the top with
IFNAR2 sequences in red (left) and IL10RB in blue (right). Protein
sequence disclosed as SEQ ID NO: 484.
[0090] FIG. 10A shows the analysis and validation of the expression
of fused transcripts in GSCs and GBM sample. Expression measured by
read depth from RNA-seq data. Light grey arcs indicate predicted
components of transcripts fused together. Overall read depth (blue;
"grey" in black and white image) and split insert depth (red; "dark
grey" in black and white image) are depicted in the graph, with a
50-read increment and a maximum range of 1800 reads. Note the very
high level of expression in the regions of the genes implicated in
the fusion events, particularly for FGFR3-TACC3.
[0091] FIG. 10B shows the analysis and validation of the expression
of fused transcripts in GSCs and GBM sample. Top panel, qRT-PCR
showing the very high expression of FGFR3 and TACC3 mRNA sequences
included in the FGFR3-TACC3 fusion transcript in GSC-1123. Bottom
panel, for comparison the expression of sequences of WRAP53 mRNA
included in the POLR2A-WRAP53 fusion in GSC-0114 is also shown.
[0092] FIG. 10C shows the expression of the FGFR3-TACC3 protein in
GSC-1123 and GBM-1123. Western blot analysis with a monoclonal
antibody, which recognizes the N-terminal region of human FGFR3
shows expression of a .about.150 kD protein in GSC-1123 but not in
the GSC cultures GSC-0331 and GSC-0114, which lack the FGFR3-TACC3
rearrangement.
[0093] FIG. 10D shows the analysis and validation of the expression
of fused transcripts in GSCs and GBM sample Immunostaining analysis
with the FGFR3 antibody of the tumor GBM-1123 (top panel) and a GBM
tumor lacking the FGFR3-TACC3 rearrangement. FGFR3 (red; "light
grey" in black and white image), DNA (DAPI, blue; "grey" in black
and white image). The pictures were taken at low (left) and high
(right) magnification.
[0094] FIGS. 10E-1, 10E-2, 10E-3, 10E-4, 10E-5, and 10E-6 shows
MS/MS analysis of the .about.150 kD fusion protein
immunoprecipitated by the monoclonal anti-FGFR3 antibody from
GSC-1123, identifying three unique peptides mapping to the FGFR3
(FGFR3 Peptide 1 (SEQ ID NO: 492), 2 (SEQ ID NO: 493), and 3 (SEQ
ID NO: 494)) and three peptides mapping to the C-terminal region of
TACC3 (TACC Peptide 1 (SEQ ID NO: 156), 2 (SEQ ID NO: 157), and 3
(SEQ ID NO: 491)).
[0095] FIGS. 11A-C shows Rat1A cells transduced with control
lentivirus or lentivirus expressing FGFR3, TACC3, FGFR3-TACC3 (FIG.
11A) that were analyzed by Western blot with an antibody
recognizing the N-terminus of FGFR3 (included in the FGFR3-TACC3
fusion protein) or the N-terminus of TACC3 (not included in the
FGFR3-TACC3 fusion protein). FIG. 11B shows quantitative Western
blot analysis of endogenous FGFR3-TACC3 in GSC-1123 compared with
lentivirally expressed FGFR3-TACC3 in Rat1A. FIG. 11C shows Western
blot analysis of FGFR3-TACC3 and FGFR3-TACC3-K508M in Rat1A.
.alpha.-tubulin is shown as a control for loading.
[0096] FIGS. 11D-F shows expression analyses of FGFR3-TACC3 fusion
construct (FIG. 11D) FGFR3 immunostaining of GBM-1123 (left, upper
panel), BTSC1123 (right, upper panel), mouse GBM induced by
FGFR3-TACC3 expressing lentivirus (left, lower panel), and
sub-cutaneous xenograft of mouse astrocytes transformed by
FGFR3-TACC3 fusion (right, lower panel); FGFR3-TACC3, red ("light
grey" in black and white image); DNA (DAPI), blue ("grey" in black
and white image). FIG. 11E shows quantification of FGFR3-TACC3
positive cells in the tumors and cultures of cells shown in FIG.
11D. FIG. 11F shows a quantitative Western blot analysis of ectopic
FGFR3-TACC3 fusion protein in mouse astrocytes and FGFR3-TACC3
induced mouse GBM (mGBM-15 and mGBM-17) compared with the
endogenous expression in GBM1123. .beta.-actin is shown as a
control for loading. F3-T3: FGFR3-TACC3. .alpha.-tubulin or
.beta.-actin is shown as a control for loading.
[0097] FIG. 12A shows a western blot. Ink4A;Arf-/- astrocytes
transduced with empty lentivirus or a lentivirus expressing
FGFR3-TACC3 were starved of mitogens and left untreated (time 0) or
treated with FGF-2 at concentration of 50 ng/ml for the indicated
times. Phospho-proteins and total proteins were analyzed by Western
blot using the indicated antibodies. .alpha.-tubulin is shown as a
control for loading.
[0098] FIG. 12B show western blots. Ink4A;Arf-/- astrocytes
transduced with empty lentivirus or a lentivirus expressing
FGFR3-TACC3 or FGFR3-TACC3-K508M were starved of mitogens and left
untreated (time 0) or treated for 10 min with FGF-1 at the
indicated concentrations. Phospho-proteins and total proteins were
analyzed by Western blot using the indicated antibodies.
.beta.-actin is shown as a control for loading.
[0099] FIG. 12C show western blots. Ink4A;Arf-/- astrocytes
transduced with empty lentivirus or a lentivirus expressing
FGFR3-TACC3 or FGFR3-TACC3-K508M were starved of mitogens and left
untreated (time 0) or treated for 10 min with FGF-8 at the
indicated concentrations. Phospho-proteins and total proteins were
analyzed by Western blot using the indicated antibodies.
.beta.-actin is shown as a control for loading.
[0100] FIGS. 12D-F shows mitotic localization of FGFR3-TACC3 fusion
protein. FIG. 12D shows maximum intensity projection confocal image
of a representative FGFR3-TACC3 expressing Ink4A;Arf-/- mouse
astrocyte at metaphase immunostained using the FGFR3 antibody (red;
"dark grey" in black and white image). FGFR3-TACC3 displays
asymmetric localization on top of one spindle pole. FIG. 12E shows
maximum intensity projection confocal image of a representative
TACC3 expressing Ink4A;Arf-/- mouse astrocyte at metaphase
immunostained with the TACC3 antibody (red; ("dark grey" in black
and white image). TACC3 staining coincides with the spindle
microtubules. FIG. 12F shows maximum intensity projection confocal
image of a representative FGFR3 expressing Ink4A;Arf-/- mouse
astrocyte at metaphase immunostained with the FGFR3 antibody (red;
("dark grey" in black and white image). FGFR3 does not show a
specific staining pattern in mitosis. Cells were co-immunostained
using .alpha.-tubulin (green; ("light grey" in black and white
image) to visualize the mitotic spindle. DNA was counterstained
with DAPI (blue; ("grey" in black and white image). Images were
acquired at 0.250 .mu.m intervals. Endogenous levels of FGFR3 or
TACC3 were undetectable under the applied experimental conditions.
F3-T3: FGFR3-TACC3.
[0101] FIG. 13A shows that the FGFR3-TACC3 protein induces
chromosomal missegregation, chromatid cohesion defects and
defective spindle checkpoint. Quantitative analysis of metaphase
spreads for chromosome segregation defects in Ink4A;ARF-/-
astrocytes expressing vector control or FGFR3-TACC3 (upper panel).
Microscope imaging analysis of chromosome segregation defects in
Ink4A;Arf-/- astrocytes expressing FGFR3-TACC3 or vector control.
Representative images of cells with chromosome missegregation.
Arrows point to chromosome misalignments, lagging chromosomes and
chromosome bridges.
[0102] FIGS. 13B-D shows representative images of premature sister
chromatid separation (PMSCS) in Ink4A;Arf-/- astrocytes (FIG. 13B)
and Rat1A cells (FIG. 13C) expressing FGFR3-TACC3. Left, panels
show representative metaphase spreads. Right, quantitative analysis
of metaphases with loss of sister chromatid cohesion. The number of
mitosis with PMSCS in Ink4A;Arf-/- astrocytes was scored in at
least 100 metaphases for each condition in three independent
experiments. The number of mitosis with PMSCS was scored in
triplicate samples of Rat1A cells. FIG. 13D is a graph showing
nocodazole was added for the indicated durations to Rat1A-H2B-GFP
cells transduced with the specified lentiviruses. The mitotic index
at each time point was determined by quantitating the
H2B-GFP-positive cells in mitosis at each time point. Data are
presented as average and standard deviation (n=3). F3-T3:
FGFR3-TACC3.
[0103] FIGS. 14A-B shows growth curves of human primary astrocytes
transduced with lentivirus expressing FGFR3-TACC3 fusion or the
empty vector. An analysis was conducted of FGFR3-TACC3 fusion
mediated growth alteration and specific effect of RTK inhibitors on
cells carrying FGFR-TACC fusions. FIG. 14A is a graph that shows
cell proliferation of human primary astrocytes transduced with
lentivirus expressing FGFR3-TACC3 fusion or the empty vector was
determined by the MTT assay 7 days after infection (passage 1).
Values are the means.+-.standard deviation (n=4). p-value: 0.0033.
FIG. 14B is a graph that shows cell proliferation of human primary
astrocytes transduced with lentivirus expressing FGFR3-TACC3 fusion
or the empty vector was determined by the MTT assay six weeks after
the infection (passage 10). Values are the means.+-.standard
deviation (n=4). p-value: 0.0018.
[0104] FIGS. 14C-D shows specific growth inhibitory effect by FGFR
inhibitors on FGFR-TACC fusion expressing cells. Cell growth was
determined by MTT assay. Rat1A cells transduced with the indicated
lentivirus were treated for three days with BGJ398 (FIG. 14C) or
AZD4547 (FIG. 14D) at the indicated concentration. Values are the
means.+-.standard error (n=4).
[0105] FIG. 14E shows the growth inhibitory effect of silencing
FGFR3-TACC3 fusion. (left) GSC-1123 cells were transduced in
triplicate with lentivirus expressing a non-targeting shRNA (Ctr)
or lentivirus expressing sh-3 and sh-4 sequences targeting FGFR3.
Five days after infection cells were plated at density of
2.times.10.sup.4 cells/well in triplicate and the number of trypan
blue excluding cells was scored at the indicated times. Values are
the means.+-.standard deviation (n=3). (right) Western Blot
analysis was performed on parallel cultures collected five days
after infection using the FGFR3 antibody to the detect FGFT3-TACC3
fusion protein. .beta.-actin is shown as a control for loading.
(**: p-value=<0.005; ***: p-value=<0.0001).
[0106] FIG. 15 shows a survival plot of cells treated with
PD173074, NVP-BGJ398, or AZD4547.
[0107] FIG. 16 shows an FGFR3-TACC3 gene fusion identified by whole
transcriptome sequencing of GSCs. The histogram describes the
absolute frequency of each forward and reverse sequence read
spanning the breakpoint.
[0108] FIG. 17 shows transforming activity of FGFR3-TACC3.
FGFR3-TACC3 induces anchorage-independent growth in Rat1A
fibroblasts (top panels) and a transformed phenotype in
Ink4A;Arf-/- primary astrocytes (bottom panels).
[0109] FIG. 18 shows transforming activity of FGFR3-TACC3.
Kaplan-Meier survival curves of mice injected intracranially with
pTomo-shp53 (n=8), pTomo-FGFR3-TACC3-shp53 (n=8) and
pTomo-EGFRvIII-shp53 (n=7) are shown. Points on the curves indicate
deaths (log-rank test, p=0.025, pTomo-shp53 vs.
pTomo-FGFR3-TACC3-shp53).
[0110] FIG. 19 shows that inhibition of FGFR-TK activity corrects
the aneuploidy and suppresses tumor growth initiated by
FGFR3-TACC3. Short-term growth inhibition assays are shown of Rat1A
transduced with the indicated lentivirus and treated with PD173470
at the indicated concentrations. Cells were treated for three days.
Cell viability was determined by the MTT assay. Error bars show
means.+-.standard error (n=4).
[0111] FIG. 20 is a growth inhibition assay of human astrocytes
transduced with the indicated lentivirus and treated for four days
with PD173470 at the indicated concentration. Cell viability was
determined by the MTT assay. Error bars show means.+-.standard
error (n=4).
[0112] FIG. 21 is a graph showing a growth inhibition assay of
human astrocytes transduced with the indicated lentivirus and
treated for four days with PD173470 at the indicated concentration.
Cell viability was determined by the MTT assay. Error bars show
means.+-.standard error (n=4).
[0113] FIG. 22 shows graphs of the survival of Rat1A cells in
short-term growth inhibition assays. (Top graph) Rat1A cells were
transduced with the indicated ptomo constructs and treated with
PD173074 at the indicated concentrations. Cells were treated for
three days. Cell viability was determined by the MTT assay. Error
bars show means.+-.standard error (n=4). In the bottom panel, a
western blot photograph is shown.
[0114] FIG. 23 shows that inhibition of FGFR-TK activity corrects
the aneuploidy and suppresses tumor growth initiated by
FGFR3-TACC3. A plot is shown of karyotype analysis of Rat1A cells
transduced with control or FGFR3-TACC3 lentivirus and treated with
vehicle (DMSO) or PD173470 (100 nM) for five days.
[0115] FIG. 24 shows Survival of glioma-bearing mice was tracked
following intracranial implantation of Ink4A;Arf-/- astrocytes
transduced with FGFR3-TACC3. After tumor engraftment mice were
treated with vehicle or AZD4547 (50 mg/kg) for 20 days (vehicle,
n=7; AZD4547, n=6; p=0.001).
[0116] FIG. 25 shows the position of the peptides from FIGS.
10E1-10E6 in the amino acid sequence of the FGFR3-TACC3 fusion
protein (SEQ ID NO: 79), which are highlighted in pink (FGFR3;
underlined) and blue (TACC3; dotted lines).
[0117] FIG. 26 shows Kaplan-Meier analysis of IDH mutant and
FGFR3-TACC3 positive human GBM. Log rank test p-value: 0.0169.
[0118] FIGS. 27A-B are pictures that shows tumor xenografts that
were induced following sub-cutaneous injection of Ink4A;Arf-/-
mouse astrocytes transduced with lentivirus expressing FGFR3-TACC3
(upper panel A, right flank) or FGFR1-TACC1 (lower panel B, right
flank) fusion, but not with the empty vector (upper panel, left
flank) or FGFR3-TACC3 carrying a K508M mutation in the kinase
domain (FGFR3-TACC3-K508M; lower panel, left flank).
[0119] FIG. 28 shows constitutive auto-phosphorylation of
FGFR3-TACC3 fusion. BTSC derived from FGFR3-TACC3 or RasV12 induced
mouse GBM were left untreated or treated with 500 nM PD173074 for
the indicated times. Phospho-proteins and total proteins were
analyzed by Western blot using the indicated antibodies.
.beta.-actin is shown as a control for loading.
[0120] FIG. 29 shows Z-stacked confocal images of the
representative FGFR3-TACC3 expressing Ink4A;Arf-/- mouse astrocyte
shown as a maximum intensity projection. Cells were immunostained
using FGFR3 (red; "dark grey" in black and white image) and
.alpha.-tubulin (green; ("light grey" in black and white image).
DNA was counterstained with DAPI (blue; ("grey" in black and white
image). Images were acquired at 0.250 .mu.m intervals. Coordinates
of the image series are indicated. F3-T3: FGFR3-TACC3.
[0121] FIG. 30 shows examples of SKY karyotype analysis painting
two different cells from the same culture of GSC-1123, illustrating
the ongoing CIN and aneuploidy. Details of the karyotype analysis
of 20 cells are reported in Table 6.
[0122] FIGS. 31-1, 31-2, and 31-3 are a graphical representation of
segmented CNVs data visualized using the Integrated Genomic Viewers
software. Three bladder Urothelial Carcinoma harbor FGFR3-TACC3
gene fusions (black box). Red indicates amplification (A), blue
indicates deletion (D).
[0123] FIGS. 32-1, 32-2, and 32-3 are a graphical representation of
segmented CNVs data visualized using the Integrated Genomic Viewers
software. One Breast Carcinoma harbors FGFR3-TACC3 gene fusions
(black box). Red indicates amplification (A), blue indicates
deletion (D).
[0124] FIGS. 33-1, 33-2, and 33-3 are a graphical representation of
segmented CNVs data visualized using the Integrated Genomic Viewers
software. One Colorectal Carcinoma harbors FGFR3-TACC3 gene fusions
(black box). Red indicates amplification (A), blue indicates
deletion (D).
[0125] FIGS. 34-1, 34-2, and 34-3 are a graphical representation of
segmented CNVs data visualized using the Integrated Genomic Viewers
software. One Lung Squamous Cell Carcinoma harbors FGFR3-TACC3 gene
fusions (black box). Red indicates amplification (A), blue
indicates deletion (D).
[0126] FIGS. 35-1, 35-2, and 35-3 are a graphical representation of
segmented CNVs data visualized using the Integrated Genomic Viewers
software. One Head and Neck Squamous Cell Carcinoma harbors
FGFR3-TACC3 gene fusions (black box). Red indicates amplification
(A), blue indicates deletion (D).
[0127] FIG. 36 shows the structure of FGFR-TACC gene fusions
identified by RT-PCR-Sanger sequencing (see also SEQ ID NOs:
530-547). Predicted FGFR-TACC fusion proteins encoded by the
transcripts identified by RT-PCR. Regions corresponding to FGFR3 or
TACC3 are shown in red or blue, respectively. FGFR1 and TACC1
corresponding regions are shown in yellow and green. On the left
are indicated the FGFR and TACC exons joined in the fused mRNA; the
presence of TACC3 introns is also reported when they are spliced in
the fusion cDNA. On the right, the number of patients harboring the
corresponding fusion variant is indicated. The novel transcripts
discovered in this study are highlighted in red. Black arrows
indicate the position of the primers used for the FGFR-TACC fusions
screening.
[0128] FIGS. 37A-H show the identification and immunostaining of
FGFR3-TACC3-positive tumors. Results from RT-PCR screening in
representative samples from the Pitie-Salp triere Hospital (A, C)
and the Besta (B, D) datasets. M, DNA ladder. Schematic
representation of the FGFR3-TACC3 fusion transcripts identified in
samples GBM-4620 (C) and GBM-021 (D). The junction sequences on the
mRNA (GBM-4620 (C) SEQ ID NO: 515; GBM-021 (D) SEQ ID NO: 517) and
the reading frame and translation (GBM-4620 (C) SEQ ID NO: 516;
GBM-021 (D) SEQ ID NO: 518) at the breakpoint are reported.
Representative microphotographs of H&E and FGFR3 immunostaining
in the FGFR3-TACC3 positive samples GBM-4620 (E) and GBM-021 (F)
and two FGFR3-TACC3 negative samples (panels G and H); a, H&E,
10.times. magnification; b, H&E, 40.times. magnification; c,
FGFR3, 10.times. magnification; d, FGFR3, 40.times.
magnification.
[0129] FIGS. 38A-D show pre-clinical evaluation of FGFR3-TACC3
inhibition by JNJ-42756493. (A) Mouse astrocytes expressing
FGFR3-TACC3 (F3T3), FGFR3-TACC3-KD (F3T3-KD) or the empty vector
(Vector) were treated with the indicated concentration of
JNJ-42756493. Cell viability was determined by the MTT assay. Error
bars show mean.+-.SEM (n=6). (B) Survival analysis of GIC28 1123
treated with JNJ-42756493. (C) The FGFR-TK inhibitor JNJ-42756493
suppresses tumor growth of subcutaneous tumors generated by
GIC-1123. After tumor establishment (arrow) mice were treated with
vehicle or JNJ-42756493 (12 mg/kg) for 14 days. Values are mean
tumor volumes.+-.SD, (n=9 mice per group). P-value of the slope
calculated from the treatment starting point (arrow) is 0.04. (D)
Photograph showing the tumors dissected from vehicle or
JNJ-42756493 treated mice after two weeks of treatment.
[0130] FIGS. 39A-G show baseline and post-treatment Magnetic
Resonance Imaging (MRI) of patients treated with JNJ-42756493.
Patient 1 (Panels A-D). (A) Post-gadolinium T1 weighted images show
the target lesion on the right parietal lobe. The interval (days)
from the beginning of follow-up is indicated above each MRI. (B)
Analysis of sum of product diameters (SPD) before and during the
anti-FGFR treatment (RANO criteria). (C) Analysis of tumor volume
(cm3) before and during the anti-FGFR treatment. During anti-FGFR
treatment a stabilization of the tumor was observed according to
RANO criteria and volumetry. (D) Perfusion images at baseline and
after 20 days of anti-FGFR treatment. rCBV (relative cerebral blood
volume). Post-gadolinium T1 weighted images with color overlay of
rCBV are shown. Patient 2 (Panels E-G). (E) Two different MRI slice
levels of superior and middle part of the lesion are presented. (F)
Analysis of sum of product diameters (SPD) before and during the
anti-FGFR treatment. During the anti-FGFR treatment a reduction of
22% of tumor size was observed. (G) Volumetric evaluation showed a
28% tumor reduction. Vertical red arrow indicates the start of
anti-FGFR treatment (baseline).
[0131] FIG. 40 shows the genomic PCR images and Sanger sequences of
FGFR3-TACC3 genomic breakpoints. Fusion specific PCR products and
Sanger sequencing chromatograms showing the FGFR3-TACC3 genomic
breakpoints (Sample #4451 SEQ ID NO:519; Sample #OPK-14 SEQ ID NO:
520; Sample #MB-22 SEQ ID NO: 521; Sample #3048 SEQ ID NO: 522;
Sample #4373 SEQ ID NO: 523; Sample #4867 SEQ ID NO: 524; Sample
#3808 SEQ ID NO: 525; Sample #27-1835 SEQ ID NO: 526; Sample
#06-6390 SEQ ID NO: 527). The genomic sequences corresponding to
FGFR3 and TACC3 are indicated in red or blue, respectively. M, DNA
ladder; C-, Negative Control.
[0132] FIG. 41 shows schematics of FGFR3-TACC3 genomic breakpoints.
Schematic representation of the genomic fusions between FGFR3 and
TACC3 compared to the corresponding mRNA. In red and blue are
reported the regions belonging to FGFR3 and TACC3, respectively.
The genomic breakpoint coordinates, according to the genome build
GRCh37/hg19, are indicated above each fusion gene.
[0133] FIGS. 42A-B show evaluation of the expression of FGFR3-TACC3
fusion elements. (A) Microphotographs of immunofluorescence
staining of a representative GBM harboring FGFR3-TACC3 fusion using
antibodies that recognize the N- and C-termini of FGFR3 (FGFR3-N,
FGFR3-C) and TACC3 (TACC3-N, TACC3-C), red. Nuclei are
counterstained with DAPI, blue. (B) Quantitative RT-PCR of four
representative GBM carrying FGFR3-TACC3 fusion and three negative
controls using primer pairs that amplify FGFR3 and TACC3 regions
included in or excluded from the fusion transcripts, as indicated
in the diagram. OAW28: ovarian cystoadenocarcinoma cell line
harboring wild type FGFR3 and TACC3 genes; GBM55 and GBM0822: GBM
harboring wild type FGFR3 and TACC3 genes; GBM3808; GBM1133;
GBM0826; GBM3048: GBM harboring FGFR3-TACC3 (F3-T3) fusion. Error
bars are SD of triplicate samples.
[0134] FIGS. 43A-C show the FGFR3-TACC3 fusion gene and protein are
retained in recurrent GBM. (A) FGFR3-TACC3 fusion specific RT-PCR
product from untreated and recurrent GBM from patient #3124. (B)
Sanger sequencing chromatogram showing the identical reading frame
at the breakpoint (SEQ ID NO: 517) and the putative translation of
the fusion protein (SEQ ID NO: 86) in the untreated and recurrent
tumor from the same patient. The fused exons at mRNA level are
shown. Regions corresponding to FGFR3 and TACC3 are indicated in
red and blue, respectively. T, threonine; S, serine; D, aspartic
acid; V, valine; K, lysine; A, alanine. (C) Representative
microphotographs of FGFR3 immunofluorescence (IF) staining in both
untreated and recurrent GBM. Blue staining, DAPI; Red staining,
FGFR3. 10.times. Magnification.
[0135] FIGS. 44A-B show PFS and OS of FGFR3-TACC3-positive glioma
patients. (A) Kaplan-Meier curves in IDH wild-type glioma patients
don't show significant differences in Progression Free Survival
(PFS) between FGFR3-TACC3 positive (N=12, median PFS=11.20 months)
and FGFR3-TACC3 negative (N=274, Median PFS=12.27 months) (P=0.85).
(B) Kaplan-Meier curves in IDH wild-type glioma patients don't show
significant differences in Overall Survival (OS) between
FGFR3-TACC3 positive (N=12, Median OS=32.80 months) and FGFR3-TACC3
negative (N=326, Median OS=18.60 months) (P=0.6). In red
FGFR3-TACC3 positive patients, in green FGFR3-TACC3 negative
patients. Open circles represent censored patients.
[0136] FIG. 45 shows analysis of SNP6.0 arrays of GBM harboring
CNVs of FGFR3 and TACC3 genomic loci. CNVs of the FGFR3/TACC3
genomic loci in "gain labeled" (LRR>0.2) TCGA samples. The CNA
magnitudes (expressed as log 2 ratio) were classified using simple
thresholds: deletion (x<-1), loss (-1<x.ltoreq.-0.2), gain
(0.2.ltoreq.x<1) or amplification (x>1). Gains are in
gradient of red, loss in gradient of blue. Samples with uniform
gains/amplification of FGFR3 and TACC3 lack FGFR3-TACC3 fusions.
Samples harboring FGFR3-TACC3 fusions (F3-T3) show
microamplifications involving the first FGFR3 exons, which are
spliced in the fusion gene.
DETAILED DESCRIPTION OF THE INVENTION
[0137] Glioblastoma multiformes (GBMs) are the most common form of
brain tumors in adults accounting for 12-15% of intracranial tumors
and 50-60% of primary brain tumors. GBM is among the most lethal
forms of human cancer. The history of successful targeted therapy
of cancer largely coincides with the inactivation of recurrent and
oncogenic gene fusions in hematological malignancies and recently
in some types of epithelial cancer. GBM is among the most lethal
and incurable forms of human cancer. Targeted therapies against
common genetic alterations in GBM have not changed the dismal
clinical outcome of the disease, most likely because they have
systematically failed to eradicate the truly addicting oncoprotein
activities of GBM. Recurrent chromosomal rearrangements resulting
in the creation of oncogenic gene fusions have not been found in
GBM.
[0138] GBM is among the most difficult forms of cancer to treat in
humans (1). So far, the therapeutic approaches that have been
tested against potentially important oncogenic targets in GBM have
met limited success (2-4). Recurrent chromosomal translocations
leading to production of oncogenic fusion proteins are viewed as
initiating and addicting events in the pathogenesis of human
cancer, thus providing the most desirable molecular targets for
cancer therapy (5, 6). Recurrent and oncogenic gene fusions have
not been found in GBM. Chromosomal rearrangements are hallmarks of
hematological malignancies but recently they have also been
uncovered in subsets of solid tumors (breast, prostate, lung and
colorectal carcinoma) (7, 8). Important and successful targeted
therapeutic interventions for patients whose tumors carry these
rearrangements have stemmed from the discovery of functional gene
fusions, especially when the translocations involve kinase-coding
genes (BCR-ABL, EML4-ALK) (9, 10).
[0139] A hallmark of GBM is rampant chromosomal instability (CIN),
which leads to aneuploidy (11). CIN and aneuploidy are early events
in the pathogenesis of cancer (12). It has been suggested that
genetic alterations targeting mitotic fidelity might be responsible
for missegregation of chromosomes during mitosis, resulting in
aneuploidy (13, 14).
[0140] Fibroblast growth factor receptors (FGFR) are transmembrane
receptors that bind to members of the fibroblast growth factor
family of proteins. The structure of the FGFRs consist of an
extracellular ligand binding domain comprised of three Ig-like
domains, a single transmembrane helix domain, and an intracellular
domain with tyrosine kinase activity (Johnson, D. E., Williams, E.
T. Structural and functional diversity in the FGF receptor
multigene family. (1993) Adv. Cancer Res, 60:1-41).
[0141] Transforming acidic coiled-coiled protein (TACC) stabilize
microtubules during mitosis by recruiting minispindles
(Msps)/XMAP215 proteins to centrosomes. TACCs have been implicated
in cancer.
[0142] From a medical perspective, the FGFR-TACC fusions provide
the first "bona-fide" oncogenically addictive gene fusions in GBM
whose identification has long been overdue in this disease.
[0143] Beside GBM, which features the highest grade of malignancy
among glioma (grade IV), lower grade glioma which include grade II
and grade III are a heterogeneous group of tumors in which specific
molecular features are associated with divergent clinical outcome.
The majority of grade II-III glioma (but only a small subgroup of
GBM) harbor mutations in IDH genes (IDH1 or IDH2), which confer a
more favorable clinical outcome. Conversely, the absence of IDH
mutations is associated with the worst prognosis (5).
[0144] Described herein is the identification of FGFR-TACC gene
fusions (mostly FGFR3-TACC3, and rarely FGFR1-TACC1) as the first
example of highly oncogenic and recurrent gene fusions in GBM. The
FGFR-TACC fusions that have been identified so far include the
Tyrosine Kinase (TK) domain of FGFR and the coiled-coil domain of
TACC proteins, both necessary for the oncogenic function of
FGFR-TACC fusions. FGFR3-TACC3 fusions have been identified in
pediatric and adult glioma, bladder carcinoma, squamous lung
carcinoma and head and neck carcinoma, thus establishing FGFR-TACC
fusions as one of the chromosomal translocation most frequently
found across multiple types of human cancers (6-15).
[0145] Here a screening method for FGFR-TACC fusions is reported
that includes a RT-PCR assay designed to identify the known and
novel FGFR3-TACC3 fusion transcripts, followed by confirmation of
the inframe breakpoint by Sanger sequencing. Using this assay, a
dataset of 584 GBM and 211 grade II and grade III gliomas has been
analyzed. It was determined that brain tumors harboring FGFR-TACC
fusions manifest strong and homogeneous intra-tumor expression of
the FGFR3 and TACC3 component invariably included in the fusion
protein, when analyzed by immunostaining. A significant clinical
benefit following treatment with a specific inhibitor of FGFR-TK is
reported in two GBM patients who harbored FGFR3-TACC3
rearrangement.
DNA and Amino Acid Manipulation Methods and Purification
Thereof
[0146] The practice of aspects of the present invention can employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Molecular Cloning A Laboratory Manual,
3.sup.rd Ed., ed. by Sambrook (2001), Fritsch and Maniatis (Cold
Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and
II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait
ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription and Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the series, Methods
In Enzymology (Academic Press, Inc., N.Y.), specifically, Methods
In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Immunochemical Methods In
Cell And Molecular Biology (Caner and Walker, eds., Academic Press,
London, 1987); Handbook Of Experimental Immunology, Volumes I-IV
(D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986). All patents, patent applications and
references cited herein are incorporated by reference in their
entireties.
[0147] One skilled in the art can obtain a protein in several ways,
which include, but are not limited to, isolating the protein via
biochemical means or expressing a nucleotide sequence encoding the
protein of interest by genetic engineering methods.
[0148] A protein is encoded by a nucleic acid (including, for
example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as
well as any form of corresponding RNA). For example, it can be
encoded by a recombinant nucleic acid of a gene. The proteins of
the invention can be obtained from various sources and can be
produced according to various techniques known in the art. For
example, a nucleic acid that encodes a protein can be obtained by
screening DNA libraries, or by amplification from a natural source.
A protein can be a fragment or portion thereof. The nucleic acids
encoding a protein can be produced via recombinant DNA technology
and such recombinant nucleic acids can be prepared by conventional
techniques, including chemical synthesis, genetic engineering,
enzymatic techniques, or a combination thereof. For example, a
fusion protein of the invention comprises a tyrosine kinase domain
of an FGFR protein fused to a polypeptide that constitutively
activates the tyrosine kinase domain of the FGFR protein. For
example, a fusion protein of the invention comprises a transforming
acidic coiled-coil (TACC) domain fused to a polypeptide with a
tyrosine kinase domain, wherein the TACC domain constitutively
activates the tyrosine kinase domain. An example of a FGFR1-TACC1
polypeptide has the amino acid sequence shown in SEQ ID NO: 150. An
example of a FGFR3-TACC3 protein is the polypeptide encoded by the
nucleic acid having the nucleotide sequence shown in SEQ ID NOs:
94, 530, 531, 532, 533, 534, 535, 536, 537, or 538. Examples of a
FGFR3-TACC3 polypeptide has the amino acid sequence shown in SEQ ID
NO: 79, 158, 159, 160, 161, 539, 540, 541, 542, 543, 544, 545, 546,
or 547.
[0149] The Genbank ID for the FGFR3 gene is 2261. Three isoforms
are listed for FGFGR3, e.g., having Genebank Accession Nos.
NP.sub.--000133 (corresponding nucleotide sequence
NM.sub.--000142); NP.sub.--001156685 (corresponding nucleotide
sequence NM.sub.--001163213); NP.sub.--075254 (corresponding
nucleotide sequence NM.sub.--022965).
[0150] SEQ ID NO: 90 is the FGFR3 Amino Acid Sequence, Transcript
Variant 1 (NP.sub.--000133; 806 aa). The location of exons are
marked by alternating underlining. Amino acids encoded by
nucleotides spanning exons are shaded in gray.
TABLE-US-00001 ##STR00001##
[0151] SEQ ID NO: 91 is the FGFR3 Nucleotide Sequence, Transcript
Variant 1 (NM.sub.--000142; 4304 bp).
TABLE-US-00002 1 gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt
cgcacggacg caccggcggg 61 ccgccggccg gagggacggg gcgggagctg
ggcccgcgga cagcgagccg gagcgggagc 121 cgcgcgtagc gagccgggct
ccggcgctcg ccagtctccc gagcggcgcc cgcctcccgc 181 cggtgcccgc
gccgggccgt ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 241
ggcccccgcc cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc
301 catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg
tggggcgagc 361 ggcagaagtc ccgggcccag agcccggcca gcaggagcag
ttggtcttcg gcagcgggga 421 tgctgtggag ctgagctgtc ccccgcccgg
gggtggtccc atggggccca ctgtctgggt 481 caaggatggc acagggctgg
tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 541 ggtgctgaat
gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca 601
gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga
661 agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt
actggacacg 721 gcccgagcgg atggacaaga agctgctggc cgtgccggcc
gccaacaccg tccgcttccg 781 ctgcccagcc gctggcaacc ccactccctc
catctcctgg ctgaagaacg gcagggagtt 841 ccgcggcgag caccgcattg
gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 901 ggaaagcgtg
gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 961
cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct
1021 gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg
agttccactg 1081 caaggtgtac agtgacgcac agccccacat ccagtggctc
aagcacgtgg aggtgaatgg 1141 cagcaaggtg ggcccggacg gcacacccta
cgttaccgtg ctcaagacgg cgggcgctaa 1201 caccaccgac aaggagctag
aggttctctc cttgcacaac gtcacctttg aggacgccgg 1261 ggagtacacc
tgcctggcgg gcaattctat tgggttttct catcactctg cgtggctggt 1321
ggtgctgcca gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg
1381 catcctcagc tacggggtgg gcttcttcct gttcatcctg gtggtggcgg
ctgtgacgct 1441 ctgccgcctg cgcagccccc ccaagaaagg cctgggctcc
cccaccgtgc acaagatctc 1501 ccgcttcccg ctcaagcgac aggtgtccct
ggagtccaac gcgtccatga gctccaacac 1561 accactggtg cgcatcgcaa
ggctgtcctc aggggagggc cccacgctgg ccaatgtctc 1621 cgagctcgag
ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg 1681
caagcccctt ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga
1741 caaggaccgg gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag
acgatgccac 1801 tgacaaggac ctgtcggacc tggtgtctga gatggagatg
atgaagatga tcgggaaaca 1861 caaaaacatc atcaacctgc tgggcgcctg
cacgcagggc gggcccctgt acgtgctggt 1921 ggagtacgcg gccaagggta
acctgcggga gtttctgcgg gcgcggcggc ccccgggcct 1981 ggactactcc
ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt 2041
gtcctgtgcc taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca
2101 cagggacctg gctgcccgca atgtgctggt gaccgaggac aacgtgatga
agatcgcaga 2161 cttcgggctg gcccgggacg tgcacaacct cgactactac
aagaagacaa ccaacggccg 2221 gctgcccgtg aagtggatgg cgcctgaggc
cttgtttgac cgagtctaca ctcaccagag 2281 tgacgtctgg tcctttgggg
tcctgctctg ggagatcttc acgctggggg gctccccgta 2341 ccccggcatc
cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa 2401
gcccgccaac tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc
2461 ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc
ttaccgtgac 2521 gtccaccgac gagtacctgg acctgtcggc gcctttcgag
cagtactccc cgggtggcca 2581 ggacaccccc agctccagct cctcagggga
cgactccgtg tttgcccacg acctgctgcc 2641 cccggcccca cccagcagtg
ggggctcgcg gacgtgaagg gccactggtc cccaacaatg 2701 tgaggggtcc
ctagcagccc accctgctgc tggtgcacag ccactccccg gcatgagact 2761
cagtgcagat ggagagacag ctacacagag ctttggtctg tgtgtgtgtg tgtgcgtgtg
2821 tgtgtgtgtg tgtgcacatc cgcgtgtgcc tgtgtgcgtg cgcatcttgc
ctccaggtgc 2881 agaggtaccc tgggtgtccc cgctgctgtg caacggtctc
ctgactggtg ctgcagcacc 2941 gaggggcctt tgttctgggg ggacccagtg
cagaatgtaa gtgggcccac ccggtgggac 3001 ccccgtgggg cagggagctg
ggcccgacat ggctccggcc tctgcctttg caccacggga 3061 catcacaggg
tgggcctcgg cccctcccac acccaaagct gagcctgcag ggaagcccca 3121
catgtccagc accttgtgcc tggggtgtta gtggcaccgc ctccccacct ccaggctttc
3181 ccacttccca ccctgcccct cagagactga aattacgggt acctgaagat
gggagccttt 3241 accttttatg caaaaggttt attccggaaa ctagtgtaca
tttctataaa tagatgctgt 3301 gtatatggta tatatacata tatatatata
acatatatgg aagaggaaaa ggctggtaca 3361 acggaggcct gcgaccctgg
gggcacagga ggcaggcatg gccctgggcg gggcgtgggg 3421 gggcgtggag
ggaggcccca gggggtctca cccatgcaag cagaggacca gggccttttc 3481
tggcaccgca gttttgtttt aaaactggac ctgtatattt gtaaagctat ttatgggccc
3541 ctggcactct tgttcccaca ccccaacact tccagcattt agctggccac
atggcggaga 3601 gttttaattt ttaacttatt gacaaccgag aaggtttatc
ccgccgatag agggacggcc 3661 aagaatgtac gtccagcctg ccccggagct
ggaggatccc ctccaagcct aaaaggttgt 3721 taatagttgg aggtgattcc
agtgaagata ttttatttcc tttgtccttt ttcaggagaa 3781 ttagatttct
ataggatttt tctttaggag atttattttt tggacttcaa agcaagctgg 3841
tattttcata caaattcttc taattgctgt gtgtcccagg cagggagacg gtttccaggg
3901 aggggccggc cctgtgtgca ggttccgatg ttattagatg ttacaagttt
atatatatct 3961 atatatataa tttattgagt ttttacaaga tgtatttgtt
gtagacttaa cacttcttac 4021 gcaatgcttc tagagtttta tagcctggac
tgctaccttt caaagcttgg agggaagccg 4081 tgaattcagt tggttcgttc
tgtactgtta ctgggccctg agtctgggca gctgtccctt 4141 gcttgcctgc
agggccatgg ctcagggtgg tctcttcttg gggcccagtg catggtggcc 4201
agaggtgtca cccaaaccgg caggtgcgat tttgttaacc cagcgacgaa ctttccgaaa
4261 aataaagaca cctggttgct aacctggaaa aaaaaaaaaa aaaa
[0152] SEQ ID NO: 528 is the FGFR3 wt cDNA Nucleotide Sequence
corresponding to the coding sequence of FGFR3 (2421 bp)
(NM.sub.--000142.4 NP.sub.--000133.1). The location of exons are
marked by alternating underlining.
TABLE-US-00003 ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCC
ATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGC
GTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAG
CAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGC
TGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTC
AAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGG
CCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGG
GCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCAC
TTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAA
GACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCC
CCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCC
GTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGC
AACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTC
CGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAAC
TACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACG
TACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTG
CAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGAC
GTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATC
CAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCG
GACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAAC
ACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACC
TTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATT
GGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAG
GAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGC
ATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTG
GCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGC
CTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACA
CCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACG
CTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGG
GAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAG
GGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGAC
AAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTG
AAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAG
ATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAAC
CTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTG
GAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGG
CGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCC
GAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAG
GTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCAC
AGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTC
GACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGG
ATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGT
GACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTG
GGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAG
CTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACA
CACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCC
TCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGT
GTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCG
CCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGCTCC
AGCTCCTCAGGGGACGACTCCGTGTTTGCCCACGACCTGCTGCCC
CCGGCCCCACCCAGCAGTGGGGGCTCGCGGACGTGA
[0153] The Genbank ID for the TACC3 gene is 10460. SEQ ID NO: 92 is
the TACC3 Amino Acid Sequence (NP.sub.--006333) (838 aa). The
location of exons are marked by alternating underlining. Amino
acids encoded by nucleotides spanning exons are shaded in gray.
Double underlining indicates the amino acid encoded by the
nucleotides shaded in grey in SEQ ID NO: 529.
TABLE-US-00004 ##STR00002##
[0154] SEQ ID NO: 93 is the TACC3 Nucleotide Sequence
(NM.sub.--006342) (2847 bp):
TABLE-US-00005 1 gcgtttgaaa ctccggcgcg ccggcggcca tcaagggcta
gaagcgcgac ggcggtagca 61 gctaggcttg gcccccggcg tggagcagac
gcggacccct ccttcctggc ggcggcggcg 121 cgggctcaga gcccggcaac
gggcgggcgg gcagaatgag tctgcaggtc ttaaacgaca 181 aaaatgtcag
caatgaaaaa aatacagaaa attgcgactt cctgttttcg ccaccagaag 241
ttaccggaag atcgtctgtt cttcgtgtgt cacagaaaga aaatgtgcca cccaagaacc
301 tggccaaagc tatgaaggtg acttttcaga cacctctgcg ggatccacag
acgcacagga 361 ttctaagtcc tagcatggcc agcaaacttg aggctccttt
cactcaggat gacacccttg 421 gactggaaaa ctcacacccg gtctggacac
agaaagagaa ccaacagctc atcaaggaag 481 tggatgccaa aactactcat
ggaattctac agaaaccagt ggaggctgac accgacctcc 541 tgggggatgc
aagcccagcc tttgggagtg gcagctccag cgagtctggc ccaggtgccc 601
tggctgacct ggactgctca agctcttccc agagcccagg aagttctgag aaccaaatgg
661 tgtctccagg aaaagtgtct ggcagccctg agcaagccgt ggaggaaaac
cttagttcct 721 attccttaga cagaagagtg acacccgcct ctgagaccct
agaagaccct tgcaggacag 781 agtcccagca caaagcggag actccgcacg
gagccgagga agaatgcaaa gcggagactc 841 cgcacggagc cgaggaggaa
tgccggcacg gtggggtctg tgctcccgca gcagtggcca 901 cttcgcctcc
tggtgcaatc cctaaggaag cctgcggagg agcacccctg cagggtctgc 961
ctggcgaagc cctgggctgc cctgcgggtg tgggcacccc cgtgccagca gatggcactc
1021 agacccttac ctgtgcacac acctctgctc ctgagagcac agccccaacc
aaccacctgg 1081 tggctggcag ggccatgacc ctgagtcctc aggaagaagt
ggctgcaggc caaatggcca 1141 gctcctcgag gagcggacct gtaaaactag
aatttgatgt atctgatggc gccaccagca 1201 aaagggcacc cccaccaagg
agactgggag agaggtccgg cctcaagcct cccttgagga 1261 aagcagcagt
gaggcagcaa aaggccccgc aggaggtgga ggaggacgac ggtaggagcg 1321
gagcaggaga ggaccccccc atgccagctt ctcggggctc ttaccacctc gactgggaca
1381 aaatggatga cccaaacttc atcccgttcg gaggtgacac caagtctggt
tgcagtgagg 1441 cccagccccc agaaagccct gagaccaggc tgggccagcc
agcggctgaa cagttgcatg 1501 ctgggcctgc cacggaggag ccaggtccct
gtctgagcca gcagctgcat tcagcctcag 1561 cggaggacac gcctgtggtg
cagttggcag ccgagacccc aacagcagag agcaaggaga 1621 gagccttgaa
ctctgccagc acctcgcttc ccacaagctg tccaggcagt gagccagtgc 1681
ccacccatca gcaggggcag cctgccttgg agctgaaaga ggagagcttc agagaccccg
1741 ctgaggttct aggcacgggc gcggaggtgg attacctgga gcagtttgga
acttcctcgt 1801 ttaaggagtc ggccttgagg aagcagtcct tatacctcaa
gttcgacccc ctcctgaggg 1861 acagtcctgg tagaccagtg cccgtggcca
ccgagaccag cagcatgcac ggtgcaaatg 1921 agactccctc aggacgtccg
cgggaagcca agcttgtgga gttcgatttc ttgggagcac 1981 tggacattcc
tgtgccaggc ccacccccag gtgttcccgc gcctgggggc ccacccctgt 2041
ccaccggacc tatagtggac ctgctccagt acagccagaa ggacctggat gcagtggtaa
2101 aggcgacaca ggaggagaac cgggagctga ggagcaggtg tgaggagctc
cacgggaaga 2161 acctggaact ggggaagatc atggacaggt tcgaagaggt
tgtgtaccag gccatggagg 2221 aagttcagaa gcagaaggaa ctttccaaag
ctgaaatcca gaaagttcta aaagaaaaag 2281 accaacttac cacagatctg
aactccatgg agaagtcctt ctccgacctc ttcaagcgtt 2341 ttgagaaaca
gaaagaggtg atcgagggct accgcaagaa cgaagagtca ctgaagaagt 2401
gcgtggagga ttacctggca aggatcaccc aggagggcca gaggtaccaa gccctgaagg
2461 cccacgcgga ggagaagctg cagctggcaa acgaggagat cgcccaggtc
cggagcaagg 2521 cccaggcgga agcgttggcc ctccaggcca gcctgaggaa
ggagcagatg cgcatccagt 2581 cgctggagaa gacagtggag cagaagacta
aagagaacga ggagctgacc aggatctgcg 2641 acgacctcat ctccaagatg
gagaagatct gacctccacg gagccgctgt ccccgccccc 2701 ctgctcccgt
ctgtctgtcc tgtctgattc tcttaggtgt catgttcttt tttctgtctt 2761
gtcttcaact tttttaaaaa ctagattgct ttgaaaacat gactcaataa aagtttcctt
2821 tcaatttaaa cactgaaaaa aaaaaaa
[0155] SEQ ID NO: 529 is the TACC3 wt cDNA Nucleotide Sequence
corresponding to the coding sequence of TACC3 (2517 bp)
(NM.sub.--006342.2, NP.sub.--006333.1). The location of exons are
marked by alternating underlining.
TABLE-US-00006
ATGAGTCTGCAGGTCTTAAACGACAAAAATGTCAGCAATGAAAAAAATACAGAAAATTGCGACTTCCTGT
TTTCGCCACCAGAAGTTACCGGAAGATCGTCTGTTCTTCGTGTGTCACAGAAAGAAAATGTGCCACCCAA
GAACCTGGCCAAAGCTATGAAGGTGACTTTTCAGACACCTCTGCGGGATCCACAGACGCACAGGATTCTA
AGTCCTAGCATGGCCAGCAAACTTGAGGCTCCTTTCACTCAGGATGACACCCTTGGACTGGAAAACTCAC
##STR00003##
TCTACAGAAACCAGTGGAGGCTGACACCGACCTCCTGGGGGATGCAAGCCCAGCCTTTGGGAGTGGCAGC
TCCAGCGAGTCTGGCCCAGGTGCCCTGGCTGACCTGGACTGCTCAAGCTCTTCCCAGAGCCCAGGAAGTT
CTGAGAACCAAATGGTGTCTCCAGGAAAAGTGTCTGGCAGCCCTGAGCAAGCCGTGGAGGAAAACCTTAG
TTCCTATTCCTTAGACAGAAGAGTGACACCCGCCTCTGAGACCCTAGAAGACCCTTGCAGGACAGAGTCC
CAGCACAAAGCGGAGACTCCGCACGGAGCCGAGGAAGAATGCAAAGCGGAGACTCCGCACGGAGCCGAGG
AGGAATGCCGGCACGGTGGGGTCTGTGCTCCCGCAGCAGTGGCCACTTCGCCTCCTGGTGCAATCCCTAA
GGAAGCCTGCGGAGGAGCACCCCTGCAGGGTCTGCCTGGCGAAGCCCTGGGCTGCCCTGCGGGTGTGGGC
ACCCCCGTGCCAGCAGATGGCACTCAGACCCTTACCTGTGCACACACCTCTGCTCCTGAGAGCACAGCCC
CAACCAACCACCTGGTGGCTGGCAGGGCCATGACCCTGAGTCCTCAGGAAGAAGTGGCTGCAGGCCAAAT
GGCCAGCTCCTCGAGGAGCGGACCTGTAAAACTAGAATTTGATGTATCTGATGGCGCCACCAGCAAAAGG
GCACCCCCACCAAGGAGACTGGGAGAGAGGTCCGGCCTCAAGCCTCCCTTGAGGAAAGCAGCAGTGAGGC
AGCAAAAGGCCCCGCAGGAGGTGGAGGAGGACGACGGTAGGAGCGGAGCAGGAGAGGACCCCCCCATGCC
AGCTTCTCGGGGCTCTTACCACCTCGACTGGGACAAAATGGATGACCCAAACTTCATCCCGTTCGGAGGT
GACACCAAGTCTGGTTGCAGTGAGGCCCAGCCCCCAGAAAGCCCTGAGACCAGGCTGGGCCAGCCAGCGG
CTGAACAGTTGCATGCTGGGCCTGCCACGGAGGAGCCAGGTCCCTGTCTGAGCCAGCAGCTGCATTCAGC
CTCAGCGGAGGACACGCCTGTGGTGCAGTTGGCAGCCGAGACCCCAACAGCAGAGAGCAAGGAGAGAGCC
TTGAACTCTGCCAGCACCTCGCTTCCCACAAGCTGTCCAGGCAGTGAGCCAGTGCCCACCCATCAGCAGG
GGCAGCCTGCCTTGGAGCTGAAAGAGGAGAGCTTCAGAGACCCCGCTGAGGTTCTAGGCACGGGCGCGGA
GGTGGATTACCTGGAGCAGTTTGGAACTTCCTCGTTTAAGGAGTCGGCCTTGAGGAAGCAGTCCTTATAC
CTCAAGTTCGACCCCCTCCTGAGGGACAGTCCTGGTAGACCAGTGCCCGTGGCCACCGAGACCAGCAGCA
TGCACGGTGCAAATGAGACTCCCTCAGGACGTCCGCGGGAAGCCAAGCTTGTGGAGTTCGATTTCTTGGG
AGCACTGGACATTCCTGTGCCAGGCCCACCCCCAGGTGTTCCCGCGCCTGGGGGCCCACCCCTGTCCACC
GGACCTATAGTGGACCTGCTCCAGTACAGCCAGAAGGACCTGGATGCAGTGGTAAAGGCGACACAGGAGG
AGAACCGGGAGCTGAGGAGCAGGTGTGAGGAGCTCCACGGGAAGAACCTGGAACTGGGGAAGATCATGGA
CAGGTTCGAAGAGGTTGTGTACCAGGCCATGGAGGAAGTTCAGAAGCAGAAGGAACTTTCCAAAGCTGAA
ATCCAGAAAGTTCTAAAAGAAAAAGACCAACTTACCACAGATCTGAACTCCATGGAGAAGTCCTTCTCCG
ACCTCTTCAAGCGTTTTGAGAAACAGAAAGAGGTGATCGAGGGCTACCGCAAGAACGAAGAGTCACTGAA
GAAGTGCGTGGAGGATTACCTGGCAAGGATCACCCAGGAGGGCCAGAGGTACCAAGCCCTGAAGGCCCAC
GCGGAGGAGAAGCTGCAGCTGGCAAACGAGGAGATCGCCCAGGTCCGGAGCAAGGCCCAGGCGGAAGCGT
TGGCCCTCCAGGCCAGCCTGAGGAAGGAGCAGATGCGCATCCAGTCGCTGGAGAAGACAGTGGAGCAGAA
GACTAAAGAGAACGAGGAGCTGACCAGGATCTGCGACGACCTCATCTCCAAGATGGAGAAGATCTGA
[0156] SEQ ID NO: 94 is the nucleotide sequence of FGFR3-TACC3.
TABLE-US-00007 1 gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt
cgcacggacg caccggcggg 61 ccgccggccg gagggacggg gcgggagctg
ggcccgcgga cagcgagccg gagcgggagc 121 cgcgcgtagc gagccgggct
ccggcgctcg ccagtctccc gagcggcgcc cgcctcccgc 181 cggtgcccgc
gccgggccgt ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 241
ggcccccgcc cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc
301 catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg
tggggcgagc 361 ggcagaagtc ccgggcccag agcccggcca gcaggagcag
ttggtcttcg gcagcgggga 421 tgctgtggag ctgagctgtc ccccgcccgg
gggtggtccc atggggccca ctgtctgggt 481 caaggatggc acagggctgg
tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 541 ggtgctgaat
gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca 601
gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga
661 agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt
actggacacg 721 gcccgagcgg atggacaaga agctgctggc cgtgccggcc
gccaacaccg tccgcttccg 781 ctgcccagcc gctggcaacc ccactccctc
catctcctgg ctgaagaacg gcagggagtt 841 ccgcggcgag caccgcattg
gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 901 ggaaagcgtg
gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 961
cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct
1021 gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg
agttccactg 1081 caaggtgtac agtgacgcac agccccacat ccagtggctc
aagcacgtgg aggtgaatgg 1141 cagcaaggtg ggcccggacg gcacacccta
cgttaccgtg ctcaagacgg cgggcgctaa 1201 caccaccgac aaggagctag
aggttctctc cttgcacaac gtcacctttg aggacgccgg 1261 ggagtacacc
tgcctggcgg gcaattctat tgggttttct catcactctg cgtggctggt 1321
ggtgctgcca gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg
1381 catcctcagc tacggggtgg gcttcttcct gttcatcctg gtggtggcgg
ctgtgacgct 1441 ctgccgcctg cgcagccccc ccaagaaagg cctgggctcc
cccaccgtgc acaagatctc 1501 ccgcttcccg ctcaagcgac aggtgtccct
ggagtccaac gcgtccatga gctccaacac 1561 accactggtg cgcatcgcaa
ggctgtcctc aggggagggc cccacgctgg ccaatgtctc 1621 cgagctcgag
ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg 1681
caagcccctt ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga
1741 caaggaccgg gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag
acgatgccac 1801 tgacaaggac ctgtcggacc tggtgtctga gatggagatg
atgaagatga tcgggaaaca 1861 caaaaacatc atcaacctgc tgggcgcctg
cacgcagggc gggcccctgt acgtgctggt 1921 ggagtacgcg gccaagggta
acctgcggga gtttctgcgg gcgcggcggc ccccgggcct 1981 ggactactcc
ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt 2041
gtcctgtgcc taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca
2101 cagggacctg gctgcccgca atgtgctggt gaccgaggac aacgtgatga
agatcgcaga 2161 cttcgggctg gcccgggacg tgcacaacct cgactactac
aagaagacaa ccaacggccg 2221 gctgcccgtg aagtggatgg cgcctgaggc
cttgtttgac cgagtctaca ctcaccagag 2281 tgacgtctgg tcctttgggg
tcctgctctg ggagatcttc acgctggggg gctccccgta 2341 ccccggcatc
cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa 2401
gcccgccaac tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc
2461 ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc
ttaccgtgac 2521 gtccaccgac tttaaggagt cggccttgag gaagcagtcc
ttatacctca agttcgaccc 2581 cctcctgagg gacagtcctg gtagaccagt
gcccgtggcc accgagacca gcagcatgca 2641 cggtgcaaat gagactccct
caggacgtcc gcgggaagcc aagcttgtgg agttcgattt 2701 cttgggagca
ctggacattc ctgtgccagg cccaccccca ggtgttcccg cgcctggggg 2761
cccacccctg tccaccggac ctatagtgga cctgctccag tacagccaga aggacctgga
2821 tgcagtggta aaggcgacac aggaggagaa ccgggagctg aggagcaggt
gtgaggagct 2881 ccacgggaag aacctggaac tggggaagat catggacagg
ttcgaagagg ttgtgtacca 2941 ggccatggag gaagttcaga agcagaagga
actttccaaa gctgaaatcc agaaagttct 3001 aaaagaaaaa gaccaactta
ccacagatct gaactccatg gagaagtcct tctccgacct 3061 cttcaagcgt
tttgagaaac agaaagaggt gatcgagggc taccgcaaga acgaagagtc 3121
actgaagaag tgcgtggagg attacctggc aaggatcacc caggagggcc agaggtacca
3181 agccctgaag gcccacgcgg aggagaagct gcagctggca aacgaggaga
tcgcccaggt 3241 ccggagcaag gcccaggcgg aagcgttggc cctccaggcc
agcctgagga aggagcagat 3301 gcgcatccag tcgctggaga agacagtgga
gcagaagact aaagagaacg aggagctgac 3361 caggatctgc gacgacctca
tctccaagat ggagaagatc tgacctccac ggagccgctg 3421 tccccgcccc
cctgctcccg tctgtctgtc ctgtctgatt ctcttaggtg tcatgttctt 3481
ttttctgtct tgtcttcaac ttttttaaaa actagattgc tttgaaaaca tgactcaata
3541 aaagtttcct ttcaatttaa acactgaaaa aaaaaaaa
[0157] SEQ ID NO: 530 is the nucleotide sequence (cDNA) of
FGFR3ex17-TACC3ex11. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00008
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
##STR00004##
[0158] SEQ ID NO: 531 is the nucleotide sequence (cDNA) of
FGFR3ex17-TACC3ex8. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00009
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
##STR00005##
[0159] SEQ ID NO: 532 is the nucleotide sequence (cDNA) of
FGFR3ex17-TACC3ex10. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00010
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
##STR00006##
[0160] SEQ ID NO: 533 is the nucleotide sequence (cDNA) of
FGFR3ex17-TACC3ex6. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00011
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022##
[0161] SEQ ID NO: 534 is the nucleotide sequence (cDNA) of
FGFR3ex18-TACC3ex13. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00012
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
CCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTAC
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029##
[0162] SEQ ID NO: 535 is the nucleotide sequence (cDNA) of
FGFR3ex18-TACC3ex9_INS66BP. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded. The
sequence corresponding the the 66 bp intronic insert is double
underlined:
TABLE-US-00013
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
CCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCAGGAGCAACGGCAG
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039##
##STR00040## ##STR00041##
[0163] SEQ ID NO: 536 is the nucleotide sequence (cDNA) of
FGFR3ex18-TACC3ex5. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00014
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058##
[0164] SEQ ID NO: 537 is the nucleotide sequence (cDNA) of
FGFR3ex18-TACC3ex5_INS33 bp. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded. The
sequence corresponding the the 33 bp intronic insert is double
underlined:
TABLE-US-00015
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
CCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTAC
TCCCCGGGTGGCCAGGACACCCCCAGCTCCAGCTCCTCAGGGGACGTGCGTGAGCCACCGCACCCGGCGT
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068##
##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074## ##STR00075##
[0165] SEQ ID NO: 538 is the nucleotide sequence (cDNA) of
FGFR3ex18-TACC3ex4. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded.
TABLE-US-00016
ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGT
CCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGA
GCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGG
CCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGC
TGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGT
ACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAG
GCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGC
TGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTC
CTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAG
TGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGT
TTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGC
GGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGAC
GCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC
CCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCA
CAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCAC
TCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATG
CAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCG
CCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAG
CGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGT
CCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCT
GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAG
GCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATG
CCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAA
CATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAG
GGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC
CGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTA
CTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTG
ATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACG
GCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGT
CTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTG
GAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGT
ACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA
CCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTAC
##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080##
##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085##
##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093##
[0166] The Genbank ID for the FGFR1 gene is 2260. Eight isoforms
are listed for FGFR1, e.g., having Genebank Accession Nos.
NP.sub.--001167534 (corresponding nucleotide sequence
NM.sub.--001174063); NP.sub.--001167535 (corresponding nucleotide
sequence NM.sub.--001174064); NP.sub.--001167536 (corresponding
nucleotide sequence NM.sub.--001174065); NP.sub.--001167537
(corresponding nucleotide sequence NM.sub.--001174066);
NP.sub.--001167538 (corresponding nucleotide sequence
NM.sub.--001174067); NP.sub.--056934 (corresponding nucleotide
sequence NM.sub.--015850); NP.sub.--075593 (corresponding
nucleotide sequence NM.sub.--023105); NP.sub.--075594
(corresponding nucleotide sequence NM.sub.--023106);
NP.sub.--075598 (corresponding nucleotide sequence
NM.sub.--023110).
[0167] SEQ ID NO: 146 is the FGFR1 Amino Acid Sequence for isoform
10, having Genebank Accession No. NP.sub.--001167534 (820 aa):
TABLE-US-00017 1 MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV
ESFLVHPGDL LQLRCRLRDD 61 VQSINWLRDG VQLAESNRTR ITGEEVEVQD
SVPADSGLYA CVTSSPSGSD TTYFSVNVSD 121 ALPSSEDDDD DDDSSSEEKE
TDNTKPNRMP VAPYWTSPEK MEKKLHAVPA AKTVKFKCPS 181 SGTPNPTLRW
LKNGKEFKPD HRIGGYKVRY ATWSIIMDSV VPSDKGNYTC IVENEYGSIN 241
HTYQLDVVER SPHRPILQAG LPANKTVALG SNVEFMCKVY SDPQPHIQWL KHIEVNGSKI
301 GPDNLPYVQI LKTAGVNTTD KEMEVLHLRN VSFEDAGEYT CLAGNSIGLS
HHSAWLTVLE 361 ALEERPAVMT SPLYLEIIIY CTGAFLISCM VGSVIVYKMK
SGTKKSDFHS QMAVHKLAKS 421 IPLRRQVSAD SSASMNSGVL LVRPSRLSSS
GTPMLAGVSE YELPEDPRWE LPRDRLVLGK 481 PLGEGCFGQV VLAEAIGLDK
DKPNRVTKVA VKMLKSDATE KDLSDLISEM EMMKMIGKHK 541 NIINLLGACT
QDGPLYVIVE YASKGNLREY LQARRPPGLE YCYNPSHNPE EQLSSKDLVS 601
CAYQVARGME YLASKKCIHR DLAARNVLVT EDNVMKIADF GLARDIHHID YYKKTTNGRL
661 PVKWMAPEAL FDRIYTHQSD VWSFGVLLWE IFTLGGSPYP GVPVEELFKL
LKEGHRMDKP 721 SNCTNELYMM MRDCWHAVPS QRPTFKQLVE DLDRIVALTS
NQEYLDLSMP LDQYSPSFPD 781 TRSSTCSSGE DSVFSHEPLP EEPCLPRHPA
QLANGGLKRR
[0168] SEQ ID NO: 147 is the FGFR1 Nucleotide Sequence for isoform
10, having Genebank Accession No. NM.sub.--001174063 (5895 bp):
TABLE-US-00018 1 agatgcaggg gcgcaaacgc caaaggagac caggctgtag
gaagagaagg gcagagcgcc 61 ggacagctcg gcccgctccc cgtcctttgg
ggccgcggct ggggaactac aaggcccagc 121 aggcagctgc agggggcgga
ggcggaggag ggaccagcgc gggtgggagt gagagagcga 181 gccctcgcgc
cccgccggcg catagcgctc ggagcgctct tgcggccaca ggcgcggcgt 241
cctcggcggc gggcggcagc tagcgggagc cgggacgccg gtgcagccgc agcgcgcgga
301 ggaacccggg tgtgccggga gctgggcggc cacgtccgga cgggaccgag
acccctcgta 361 gcgcattgcg gcgacctcgc cttccccggc cgcgagcgcg
ccgctgcttg aaaagccgcg 421 gaacccaagg acttttctcc ggtccgagct
cggggcgccc cgcagggcgc acggtacccg 481 tgctgcagtc gggcacgccg
cggcgccggg gcctccgcag ggcgatggag cccggtctgc 541 aaggaaagtg
aggcgccgcc gctgcgttct ggaggagggg ggcacaaggt ctggagaccc 601
cgggtggcgg acgggagccc tccccccgcc ccgcctccgg ggcaccagct ccggctccat
661 tgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg
agcgccggcc 721 gcggagctct tgcgaccccg ccaggacccg aacagagccc
gggggcggcg ggccggagcc 781 ggggacgcgg gcacacgccc gctcgcacaa
gccacggcgg actctcccga ggcggaacct 841 ccacgccgag cgagggtcag
tttgaaaagg aggatcgagc tcactgtgga gtatccatgg 901 agatgtggag
ccttgtcacc aacctctaac tgcagaactg ggatgtggag ctggaagtgc 961
ctcctcttct gggctgtgct ggtcacagcc acactctgca ccgctaggcc gtccccgacc
1021 ttgcctgaac aagcccagcc ctggggagcc cctgtggaag tggagtcctt
cctggtccac 1081 cccggtgacc tgctgcagct tcgctgtcgg ctgcgggacg
atgtgcagag catcaactgg 1141 ctgcgggacg gggtgcagct ggcggaaagc
aaccgcaccc gcatcacagg ggaggaggtg 1201 gaggtgcagg actccgtgcc
cgcagactcc ggcctctatg cttgcgtaac cagcagcccc 1261 tcgggcagtg
acaccaccta cttctccgtc aatgtttcag atgctctccc ctcctcggag 1321
gatgatgatg atgatgatga ctcctcttca gaggagaaag aaacagataa caccaaacca
1381 aaccgtatgc ccgtagctcc atattggaca tccccagaaa agatggaaaa
gaaattgcat 1441 gcagtgccgg ctgccaagac agtgaagttc aaatgccctt
ccagtgggac cccaaacccc 1501 acactgcgct ggttgaaaaa tggcaaagaa
ttcaaacctg accacagaat tggaggctac 1561 aaggtccgtt atgccacctg
gagcatcata atggactctg tggtgccctc tgacaagggc 1621 aactacacct
gcattgtgga gaatgagtac ggcagcatca accacacata ccagctggat 1681
gtcgtggagc ggtcccctca ccggcccatc ctgcaagcag ggttgcccgc caacaaaaca
1741 gtggccctgg gtagcaacgt ggagttcatg tgtaaggtgt acagtgaccc
gcagccgcac 1801 atccagtggc taaagcacat cgaggtgaat gggagcaaga
ttggcccaga caacctgcct 1861 tatgtccaga tcttgaagac tgctggagtt
aataccaccg acaaagagat ggaggtgctt 1921 cacttaagaa atgtctcctt
tgaggacgca ggggagtata cgtgcttggc gggtaactct 1981 atcggactct
cccatcactc tgcatggttg accgttctgg aagccctgga agagaggccg 2041
gcagtgatga cctcgcccct gtacctggag atcatcatct attgcacagg ggccttcctc
2101 atctcctgca tggtggggtc ggtcatcgtc tacaagatga agagtggtac
caagaagagt 2161 gacttccaca gccagatggc tgtgcacaag ctggccaaga
gcatccctct gcgcagacag 2221 gtgtctgctg actccagtgc atccatgaac
tctggggttc ttctggttcg gccatcacgg 2281 ctctcctcca gtgggactcc
catgctagca ggggtctctg agtatgagct tcccgaagac 2341 cctcgctggg
agctgcctcg ggacagactg gtcttaggca aacccctggg agagggctgc 2401
tttgggcagg tggtgttggc agaggctatc gggctggaca aggacaaacc caaccgtgtg
2461 accaaagtgg ctgtgaagat gttgaagtcg gacgcaacag agaaagactt
gtcagacctg 2521 atctcagaaa tggagatgat gaagatgatc gggaagcata
agaatatcat caacctgctg 2581 ggggcctgca cgcaggatgg tcccttgtat
gtcatcgtgg agtatgcctc caagggcaac 2641 ctgcgggagt acctgcaggc
ccggaggccc ccagggctgg aatactgcta caaccccagc 2701 cacaacccag
aggagcagct ctcctccaag gacctggtgt cctgcgccta ccaggtggcc 2761
cgaggcatgg agtatctggc ctccaagaag tgcatacacc gagacctggc agccaggaat
2821 gtcctggtga cagaggacaa tgtgatgaag atagcagact ttggcctcgc
acgggacatt 2881 caccacatcg actactataa aaagacaacc aacggccgac
tgcctgtgaa gtggatggca 2941 cccgaggcat tatttgaccg gatctacacc
caccagagtg atgtgtggtc tttcggggtg 3001 ctcctgtggg agatcttcac
tctgggcggc tccccatacc ccggtgtgcc tgtggaggaa 3061 cttttcaagc
tgctgaagga gggtcaccgc atggacaagc ccagtaactg caccaacgag 3121
ctgtacatga tgatgcggga ctgctggcat gcagtgccct cacagagacc caccttcaag
3181 cagctggtgg aagacctgga ccgcatcgtg gccttgacct ccaaccagga
gtacctggac 3241 ctgtccatgc ccctggacca gtactccccc agctttcccg
acacccggag ctctacgtgc 3301 tcctcagggg aggattccgt cttctctcat
gagccgctgc ccgaggagcc ctgcctgccc 3361 cgacacccag cccagcttgc
caatggcgga ctcaaacgcc gctgactgcc acccacacgc 3421 cctccccaga
ctccaccgtc agctgtaacc ctcacccaca gcccctgctg ggcccaccac 3481
ctgtccgtcc ctgtcccctt tcctgctggc aggagccggc tgcctaccag gggccttcct
3541 gtgtggcctg ccttcacccc actcagctca cctctccctc cacctcctct
ccacctgctg 3601 gtgagaggtg caaagaggca gatctttgct gccagccact
tcatcccctc ccagatgttg 3661 gaccaacacc cctccctgcc accaggcact
gcctggaggg cagggagtgg gagccaatga 3721 acaggcatgc aagtgagagc
ttcctgagct ttctcctgtc ggtttggtct gttttgcctt 3781 cacccataag
cccctcgcac tctggtggca ggtgccttgt cctcagggct acagcagtag 3841
ggaggtcagt gcttcgtgcc tcgattgaag gtgacctctg ccccagatag gtggtgccag
3901 tggcttatta attccgatac tagtttgctt tgctgaccaa atgcctggta
ccagaggatg 3961 gtgaggcgaa ggccaggttg ggggcagtgt tgtggccctg
gggcccagcc ccaaactggg 4021 ggctctgtat atagctatga agaaaacaca
aagtgtataa atctgagtat atatttacat 4081 gtctttttaa aagggtcgtt
accagagatt tacccatcgg gtaagatgct cctggtggct 4141 gggaggcatc
agttgctata tattaaaaac aaaaaagaaa aaaaaggaaa atgtttttaa 4201
aaaggtcata tattttttgc tacttttgct gttttatttt tttaaattat gttctaaacc
4261 tattttcagt ttaggtccct caataaaaat tgctgctgct tcatttatct
atgggctgta 4321 tgaaaagggt gggaatgtcc actggaaaga agggacaccc
acgggccctg gggctaggtc 4381 tgtcccgagg gcaccgcatg ctcccggcgc
aggttccttg taacctcttc ttcctaggtc 4441 ctgcacccag acctcacgac
gcacctcctg cctctccgct gcttttggaa agtcagaaaa 4501 agaagatgtc
tgcttcgagg gcaggaaccc catccatgca gtagaggcgc tgggcagaga 4561
gtcaaggccc agcagccatc gaccatggat ggtttcctcc aaggaaaccg gtggggttgg
4621 gctggggagg gggcacctac ctaggaatag ccacggggta gagctacagt
gattaagagg 4681 aaagcaaggg cgcggttgct cacgcctgta atcccagcac
tttgggacac cgaggtgggc 4741 agatcacttc aggtcaggag tttgagacca
gcctggccaa cttagtgaaa ccccatctct 4801 actaaaaatg caaaaattat
ccaggcatgg tggcacacgc ctgtaatccc agctccacag 4861 gaggctgagg
cagaatccct tgaagctggg aggcggaggt tgcagtgagc cgagattgcg 4921
ccattgcact ccagcctggg caacagagaa aacaaaaagg aaaacaaatg atgaaggtct
4981 gcagaaactg aaacccagac atgtgtctgc cccctctatg tgggcatggt
tttgccagtg 5041 cttctaagtg caggagaaca tgtcacctga ggctagtttt
gcattcaggt ccctggcttc 5101 gtttcttgtt ggtatgcctc cccagatcgt
ccttcctgta tccatgtgac cagactgtat 5161 ttgttgggac tgtcgcagat
cttggcttct tacagttctt cctgtccaaa ctccatcctg 5221 tccctcagga
acggggggaa aattctccga atgtttttgg ttttttggct gcttggaatt 5281
tacttctgcc acctgctggt catcactgtc ctcactaagt ggattctggc tcccccgtac
5341 ctcatggctc aaactaccac tcctcagtcg ctatattaaa gcttatattt
tgctggatta 5401 ctgctaaata caaaagaaag ttcaatatgt tttcatttct
gtagggaaaa tgggattgct 5461 gctttaaatt tctgagctag ggattttttg
gcagctgcag tgttggcgac tattgtaaaa 5521 ttctctttgt ttctctctgt
aaatagcacc tgctaacatt acaatttgta tttatgttta 5581 aagaaggcat
catttggtga acagaactag gaaatgaatt tttagctctt aaaagcattt 5641
gctttgagac cgcacaggag tgtctttcct tgtaaaacag tgatgataat ttctgccttg
5701 gccctacctt gaagcaatgt tgtgtgaagg gatgaagaat ctaaaagtct
tcataagtcc 5761 ttgggagagg tgctagaaaa atataaggca ctatcataat
tacagtgatg tccttgctgt 5821 tactactcaa atcacccaca aatttcccca
aagactgcgc tagctgtcaa ataaaagaca 5881 gtgaaattga cctga
[0169] SEQ ID NO: 185 is the FGFR1 Amino Acid Sequence for isoform
1, having Genebank Accession No. NP.sub.--075598 (822 aa):
TABLE-US-00019 1 MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV
ESFLVHPGDL LQLRCRLRDD 61 VQSINWLRDG VQLAESNRTR ITGEEVEVQD
SVPADSGLYA CVTSSPSGSD TTYFSVNVSD 121 ALPSSEDDDD DDDSSSEEKE
TDNTKPNRMP VAPYWTSPEK MEKKLHAVPA AKTVKFKCPS 181 SGTPNPTLRW
LKNGKEFKPD HRIGGYKVRY ATWSIIMDSV VPSDKGNYTC IVENEYGSIN 241
HTYQLDVVER SPHRPILQAG LPANKTVALG SNVEFMCKVY SDPQPHIQWL KHIEVNGSKI
301 GPDNLPYVQI LKTAGVNTTD KEMEVLHLRN VSFEDAGEYT CLAGNSIGLS
HHSAWLTVLE 361 ALEERPAVMT SPLYLEIIIY CTGAFLISCM VGSVIVYKMK
SGTKKSDFHS QMAVHKLAKS 421 IPLRRQVTVS ADSSASMNSG VLLVRPSRLS
SSGTPMLAGV SEYELPEDPR WELPRDRLVL 481 GKPLGEGCFG QVVLAEAIGL
DKDKPNRVTK VAVKMLKSDA TEKDLSDLIS EMEMMKMIGK 541 HKNIINLLGA
CTQDGPLYVI VEYASKGNLR EYLQARRPPG LEYCYNPSHN PEEQLSSKDL 601
VSCAYQVARG MEYLASKKCI HRDLAARNVL VTEDNVMKIA DFGLARDIHH IDYYKKTTNG
661 RLPVKWMAPE ALFDRIYTHQ SDVWSFGVLL WEIFTLGGSP YPGVPVEELF
KLLKEGHRMD 721 KPSNCTNELY MMMRDCWHAV PSQRPTFKQL VEDLDRIVAL
TSNQEYLDLS MPLDQYSPSF 781 PDTRSSTCSS GEDSVFSHEP LPEEPCLPRH
PAQLANGGLK RR
[0170] SEQ ID NO: 186 is the FGFR1 Nucleotide Sequence for isoform
1, having Genebank Accession No. NM.sub.--023110 (5917 bp):
TABLE-US-00020 1 agatgcaggg gcgcaaacgc caaaggagac caggctgtag
gaagagaagg gcagagcgcc 61 ggacagctcg gcccgctccc cgtcctttgg
ggccgcggct ggggaactac aaggcccagc 121 aggcagctgc agggggcgga
ggcggaggag ggaccagcgc gggtgggagt gagagagcga 181 gccctcgcgc
cccgccggcg catagcgctc ggagcgctct tgcggccaca ggcgcggcgt 241
cctcggcggc gggcggcagc tagcgggagc cgggacgccg gtgcagccgc agcgcgcgga
301 ggaacccggg tgtgccggga gctgggcggc cacgtccgga cgggaccgag
acccctcgta 361 gcgcattgcg gcgacctcgc cttccccggc cgcgagcgcg
ccgctgcttg aaaagccgcg 421 gaacccaagg acttttctcc ggtccgagct
cggggcgccc cgcagggcgc acggtacccg 481 tgctgcagtc gggcacgccg
cggcgccggg gcctccgcag ggcgatggag cccggtctgc 541 aaggaaagtg
aggcgccgcc gctgcgttct ggaggagggg ggcacaaggt ctggagaccc 601
cgggtggcgg acgggagccc tccccccgcc ccgcctccgg ggcaccagct ccggctccat
661 tgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg
agcgccggcc 721 gcggagctct tgcgaccccg ccaggacccg aacagagccc
gggggcggcg ggccggagcc 781 ggggacgcgg gcacacgccc gctcgcacaa
gccacggcgg actctcccga ggcggaacct 841 ccacgccgag cgagggtcag
tttgaaaagg aggatcgagc tcactgtgga gtatccatgg 901 agatgtggag
ccttgtcacc aacctctaac tgcagaactg ggatgtggag ctggaagtgc 961
ctcctcttct gggctgtgct ggtcacagcc acactctgca ccgctaggcc gtccccgacc
1021 ttgcctgaac aagcccagcc ctggggagcc cctgtggaag tggagtcctt
cctggtccac 1081 cccggtgacc tgctgcagct tcgctgtcgg ctgcgggacg
atgtgcagag catcaactgg 1141 ctgcgggacg gggtgcagct ggcggaaagc
aaccgcaccc gcatcacagg ggaggaggtg 1201 gaggtgcagg actccgtgcc
cgcagactcc ggcctctatg cttgcgtaac cagcagcccc 1261 tcgggcagtg
acaccaccta cttctccgtc aatgtttcag atgctctccc ctcctcggag 1321
gatgatgatg atgatgatga ctcctcttca gaggagaaag aaacagataa caccaaacca
1381 aaccgtatgc ccgtagctcc atattggaca tccccagaaa agatggaaaa
gaaattgcat 1441 gcagtgccgg ctgccaagac agtgaagttc aaatgccctt
ccagtgggac cccaaacccc 1501 acactgcgct ggttgaaaaa tggcaaagaa
ttcaaacctg accacagaat tggaggctac 1561 aaggtccgtt atgccacctg
gagcatcata atggactctg tggtgccctc tgacaagggc 1621 aactacacct
gcattgtgga gaatgagtac ggcagcatca accacacata ccagctggat 1681
gtcgtggagc ggtcccctca ccggcccatc ctgcaagcag ggttgcccgc caacaaaaca
1741 gtggccctgg gtagcaacgt ggagttcatg tgtaaggtgt acagtgaccc
gcagccgcac 1801 atccagtggc taaagcacat cgaggtgaat gggagcaaga
ttggcccaga caacctgcct 1861 tatgtccaga tcttgaagac tgctggagtt
aataccaccg acaaagagat ggaggtgctt 1921 cacttaagaa atgtctcctt
tgaggacgca ggggagtata cgtgcttggc gggtaactct 1981 atcggactct
cccatcactc tgcatggttg accgttctgg aagccctgga agagaggccg 2041
gcagtgatga cctcgcccct gtacctggag atcatcatct attgcacagg ggccttcctc
2101 atctcctgca tggtggggtc ggtcatcgtc tacaagatga agagtggtac
caagaagagt 2161 gacttccaca gccagatggc tgtgcacaag ctggccaaga
gcatccctct gcgcagacag 2221 gtaacagtgt ctgctgactc cagtgcatcc
atgaactctg gggttcttct ggttcggcca 2281 tcacggctct cctccagtgg
gactcccatg ctagcagggg tctctgagta tgagcttccc 2341 gaagaccctc
gctgggagct gcctcgggac agactggtct taggcaaacc cctgggagag 2401
ggctgctttg ggcaggtggt gttggcagag gctatcgggc tggacaagga caaacccaac
2461 cgtgtgacca aagtggctgt gaagatgttg aagtcggacg caacagagaa
agacttgtca 2521 gacctgatct cagaaatgga gatgatgaag atgatcggga
agcataagaa tatcatcaac 2581 ctgctggggg cctgcacgca ggatggtccc
ttgtatgtca tcgtggagta tgcctccaag 2641 ggcaacctgc gggagtacct
gcaggcccgg aggcccccag ggctggaata ctgctacaac 2701 cccagccaca
acccagagga gcagctctcc tccaaggacc tggtgtcctg cgcctaccag 2761
gtggcccgag gcatggagta tctggcctcc aagaagtgca tacaccgaga cctggcagcc
2821 aggaatgtcc tggtgacaga ggacaatgtg atgaagatag cagactttgg
cctcgcacgg 2881 gacattcacc acatcgacta ctataaaaag acaaccaacg
gccgactgcc tgtgaagtgg 2941 atggcacccg aggcattatt tgaccggatc
tacacccacc agagtgatgt gtggtctttc 3001 ggggtgctcc tgtgggagat
cttcactctg ggcggctccc cataccccgg tgtgcctgtg 3061 gaggaacttt
tcaagctgct gaaggagggt caccgcatgg acaagcccag taactgcacc 3121
aacgagctgt acatgatgat gcgggactgc tggcatgcag tgccctcaca gagacccacc
3181 ttcaagcagc tggtggaaga cctggaccgc atcgtggcct tgacctccaa
ccaggagtac 3241 ctggacctgt ccatgcccct ggaccagtac tcccccagct
ttcccgacac ccggagctct 3301 acgtgctcct caggggagga ttccgtcttc
tctcatgagc cgctgcccga ggagccctgc 3361 ctgccccgac acccagccca
gcttgccaat ggcggactca aacgccgctg actgccaccc 3421 acacgccctc
cccagactcc accgtcagct gtaaccctca cccacagccc ctgctgggcc 3481
caccacctgt ccgtccctgt cccctttcct gctggcagga gccggctgcc taccaggggc
3541 cttcctgtgt ggcctgcctt caccccactc agctcacctc tccctccacc
tcctctccac 3601 ctgctggtga gaggtgcaaa gaggcagatc tttgctgcca
gccacttcat cccctcccag 3661 atgttggacc aacacccctc cctgccacca
ggcactgcct ggagggcagg gagtgggagc 3721 caatgaacag gcatgcaagt
gagagcttcc tgagctttct cctgtcggtt tggtctgttt 3781 tgccttcacc
cataagcccc tcgcactctg gtggcaggtg ccttgtcctc agggctacag 3841
cagtagggag gtcagtgctt cgtgcctcga ttgaaggtga cctctgcccc agataggtgg
3901 tgccagtggc ttattaattc cgatactagt ttgctttgct gaccaaatgc
ctggtaccag 3961 aggatggtga ggcgaaggcc aggttggggg cagtgttgtg
gccctggggc ccagccccaa 4021 actgggggct ctgtatatag ctatgaagaa
aacacaaagt gtataaatct gagtatatat 4081 ttacatgtct ttttaaaagg
gtcgttacca gagatttacc catcgggtaa gatgctcctg 4141 gtggctggga
ggcatcagtt gctatatatt aaaaacaaaa aagaaaaaaa aggaaaatgt 4201
ttttaaaaag gtcatatatt ttttgctact tttgctgttt tattttttta aattatgttc
4261 taaacctatt ttcagtttag gtccctcaat aaaaattgct gctgcttcat
ttatctatgg 4321 gctgtatgaa aagggtggga atgtccactg gaaagaaggg
acacccacgg gccctggggc 4381 taggtctgtc ccgagggcac cgcatgctcc
cggcgcaggt tccttgtaac ctcttcttcc 4441 taggtcctgc acccagacct
cacgacgcac ctcctgcctc tccgctgctt ttggaaagtc 4501 agaaaaagaa
gatgtctgct tcgagggcag gaaccccatc catgcagtag aggcgctggg 4561
cagagagtca aggcccagca gccatcgacc atggatggtt tcctccaagg aaaccggtgg
4621 ggttgggctg gggagggggc acctacctag gaatagccac ggggtagagc
tacagtgatt 4681 aagaggaaag caagggcgcg gttgctcacg cctgtaatcc
cagcactttg ggacaccgag 4741 gtgggcagat cacttcaggt caggagtttg
agaccagcct ggccaactta gtgaaacccc 4801 atctctacta aaaatgcaaa
aattatccag gcatggtggc acacgcctgt aatcccagct 4861 ccacaggagg
ctgaggcaga atcccttgaa gctgggaggc ggaggttgca gtgagccgag 4921
attgcgccat tgcactccag cctgggcaac agagaaaaca aaaaggaaaa caaatgatga
4981 aggtctgcag aaactgaaac ccagacatgt gtctgccccc tctatgtggg
catggttttg 5041 ccagtgcttc taagtgcagg agaacatgtc acctgaggct
agttttgcat tcaggtccct 5101 ggcttcgttt cttgttggta tgcctcccca
gatcgtcctt cctgtatcca tgtgaccaga 5161 ctgtatttgt tgggactgtc
gcagatcttg gcttcttaca gttcttcctg tccaaactcc 5221 atcctgtccc
tcaggaacgg ggggaaaatt ctccgaatgt ttttggtttt ttggctgctt 5281
ggaatttact tctgccacct gctggtcatc actgtcctca ctaagtggat tctggctccc
5341 ccgtacctca tggctcaaac taccactcct cagtcgctat attaaagctt
atattttgct 5401 ggattactgc taaatacaaa agaaagttca atatgttttc
atttctgtag ggaaaatggg 5461 attgctgctt taaatttctg agctagggat
tttttggcag ctgcagtgtt ggcgactatt 5521 gtaaaattct ctttgtttct
ctctgtaaat agcacctgct aacattacaa tttgtattta 5581 tgtttaaaga
aggcatcatt tggtgaacag aactaggaaa tgaattttta gctcttaaaa 5641
gcatttgctt tgagaccgca caggagtgtc tttccttgta aaacagtgat gataatttct
5701 gccttggccc taccttgaag caatgttgtg tgaagggatg aagaatctaa
aagtcttcat 5761 aagtccttgg gagaggtgct agaaaaatat aaggcactat
cataattaca gtgatgtcct 5821 tgctgttact actcaaatca cccacaaatt
tccccaaaga ctgcgctagc tgtcaaataa 5881 aagacagtga aattgacctg
aaaaaaaaaa aaaaaaa
[0171] The Genbank ID for the TACC1 gene is 6867. Three isoforms
are listed for TACC1, e.g., having Genebank Accession Nos.
NP.sub.--006274 (corresponding nucleotide sequence
NM.sub.--001174063); NP.sub.--001167535 (corresponding nucleotide
sequence NM.sub.--001174064); NP.sub.--001167536 (corresponding
nucleotide sequence NM.sub.--001174065).
[0172] SEQ ID NO: 148 is the TACC1 Amino Acid Sequence for isoform
1, having Genebank Accession No. NP.sub.--006274 (805 aa):
TABLE-US-00021 1 MAFSPWQILS PVQWAKWTWS AVRGGAAGED EAGGPEGDPE
EEDSQAETKS LSFSSDSEGN 61 FETPEAETPI RSPFKESCDP SLGLAGPGAK
SQESQEADEQ LVAEVVEKCS SKTCSKPSEN 121 EVPQQAIDSH SVKNFREEPE
HDFSKISIVR PFSIETKDST DISAVLGTKA AHGCVTAVSG 181 KALPSSPPDA
LQDEAMTEGS MGVTLEASAE ADLKAGNSCP ELVPSRRSKL RKPKPVPLRK 241
KAIGGEFSDT NAAVEGTPLP KASYHFSPEE LDENTSPLLG DARFQKSPPD LKETPGTLSS
301 DTNDSGVELG EESRSSPLKL EFDFTEDTGN IEARKALPRK LGRKLGSTLT
PKIQKDGISK 361 SAGLEQPTDP VARDGPLSQT SSKPDPSQWE SPSFNPFGSH
SVLQNSPPLS SEGSYHFDPD 421 NFDESMDPFK PTTTLTSSDF CSPTGNHVNE
ILESPKKAKS RLITSGCKVK KHETQSLALD 481 ACSRDEGAVI SQISDISNRD
GHATDEEKLA STSCGQKSAG AEVKGEPEED LEYFECSNVP 541 VSTINHAFSS
SEAGIEKETC QKMEEDGSTV LGLLESSAEK APVSVSCGGE SPLDGICLSE 601
SDKTAVLTLI REEIITKEIE ANEWKKKYEE TRQEVLEMRK IVAEYEKTIA QMIEDEQRTS
661 MTSQKSFQQL TMEKEQALAD LNSVERSLSD LFRRYENLKG VLEGFKKNEE
ALKKCAQDYL 721 ARVKQEEQRY QALKIHAEEK LDKANEEIAQ VRTKAKAESA
ALHAGLRKEQ MKVESLERAL 781 QQKNQEIEEL TKICDELIAK LGKTD
[0173] SEQ ID NO: 149 is the TACC1 Nucleotide Sequence for isoform
1, having Genebank Accession No. NM.sub.--006283 (7802 bp):
TABLE-US-00022 1 agctgatgcg cgccccgccg gccgggaggc gggagtccgc
gagccgggag cgggagcagc 61 agaggtctag cagccgggcg ccgcgggccg
ggggcctgag gaggccacag gacgggcgtc 121 ttcccggcta gtggagcccg
gcgcggggcc cgctgcggcc gcaccgtgag gggaggaggc 181 cgaggaggac
gcagcgccgg ctgccggcgg gaggaagcgc tccaccaggg cccccgacgg 241
cactcgttta accacatccg cgcctctgct ggaaacgctt gctggcgcct gtcaccggtt
301 ccctccattt tgaaagggaa aaaggctctc cccacccatt cccctgcccc
taggagctgg 361 agccggagga gccgcgctca tggcgttcag cccgtggcag
atcctgtccc ccgtgcagtg 421 ggcgaaatgg acgtggtctg cggtacgcgg
cggggccgcc ggcgaggacg aggctggcgg 481 gcccgagggc gaccccgagg
aggaggattc gcaagccgag accaaatcct tgagtttcag 541 ctcggattct
gaaggtaatt ttgagactcc tgaagctgaa accccgatcc gatcaccttt 601
caaggagtcc tgtgatccat cactcggatt ggcaggacct ggggccaaaa gccaagaatc
661 acaagaagct gatgaacagc ttgtagcaga agtggttgaa aaatgttcat
ctaagacttg 721 ttctaaacct tcagaaaatg aagtgccaca gcaggccatt
gactctcact cagtcaagaa 781 tttcagagaa gaacctgaac atgattttag
caaaatttcc atcgtgaggc cattttcaat 841 agaaacgaag gattccacgg
atatctcggc agtcctcgga acaaaagcag ctcatggctg 901 tgtaactgca
gtctcaggca aggctctgcc ttccagcccg ccagacgccc tccaggacga 961
ggcgatgaca gaaggcagca tgggggtcac cctcgaggcc tccgcagaag ctgatctaaa
1021 agctggcaac tcctgtccag agcttgtgcc cagcagaaga agcaagctga
gaaagcccaa 1081 gcctgtcccc ctgaggaaga aagcaattgg aggagagttc
tcagacacca acgctgctgt 1141 ggagggcaca cctctcccca aggcatccta
tcacttcagt cctgaagagt tggatgagaa 1201 cacaagtcct ttgctaggag
atgccaggtt ccagaagtct ccccctgacc ttaaagaaac 1261 tcccggcact
ctcagtagtg acaccaacga ctcaggggtt gagctggggg aggagtcgag 1321
gagctcacct ctcaagcttg agtttgattt cacagaagat acaggaaaca tagaggccag
1381 gaaagccctt ccaaggaagc ttggcaggaa actgggtagc acactgactc
ccaagataca 1441 aaaagatggc atcagtaagt cagcaggttt agaacagcct
acagacccag tggcacgaga 1501 cgggcctctc tcccaaacat cttccaagcc
agatcctagt cagtgggaaa gccccagctt 1561 caaccccttt gggagccact
ctgttctgca gaactcccca cccctctctt ctgagggctc 1621 ctaccacttt
gacccagata actttgacga atccatggat ccctttaaac caactacgac 1681
cttaacaagc agtgactttt gttctcccac tggtaatcac gttaatgaaa tcttagaatc
1741 acccaagaag gcaaagtcgc gtttaataac gagtggctgt aaggtgaaga
agcatgaaac 1801 tcagtctctc gccctggatg catgttctcg ggatgaaggg
gcagtgatct cccagatttc 1861 agacatttct aatagggatg gccatgctac
tgatgaggag aaactggcat ccacgtcatg 1921 tggtcagaaa tcagctggtg
ccgaggtgaa aggtgagcca gaggaagacc tggagtactt 1981 tgaatgttcc
aatgttcctg tgtctaccat aaatcatgcg ttttcatcct cagaagcagg 2041
catagagaag gagacgtgcc agaagatgga agaagacggg tccactgtgc ttgggctgct
2101 ggagtcctct gcagagaagg cccctgtgtc ggtgtcctgt ggaggtgaga
gccccctgga 2161 tgggatctgc ctcagcgaat cagacaagac agccgtgctc
accttaataa gagaagagat 2221 aattactaaa gagattgaag caaatgaatg
gaagaagaaa tacgaagaga cccggcaaga 2281 agttttggag atgaggaaaa
ttgtagctga atatgaaaag actattgctc aaatgattga 2341 agatgaacaa
aggacaagta tgacctctca gaagagcttc cagcaactga ccatggagaa 2401
ggaacaggcc ctggctgacc ttaactctgt ggaaaggtcc ctttctgatc tcttcaggag
2461 atatgagaac ctgaaaggtg ttctggaagg gttcaagaag aatgaagaag
ccttgaagaa 2521 atgtgctcag gattacttag ccagagttaa acaagaggag
cagcgatacc aggccctgaa 2581 aatccacgca gaagagaaac tggacaaagc
caatgaagag attgctcagg ttcgaacaaa 2641 agcaaaggct gagagtgcag
ctctccatgc tggactccgc aaagagcaga tgaaggtgga 2701 gtccctggaa
agggccctgc agcagaagaa ccaagaaatt gaagaactga caaaaatctg 2761
tgatgagctg attgcaaagc tgggaaagac tgactgagac actccccctg ttagctcaac
2821 agatctgcat ttggctgctt ctcttgtgac cacaattatc ttgccttatc
caggaataat 2881 tgcccctttg cagagaaaaa aaaaaactta aaaaaagcac
atgcctactg ctgcctgtcc 2941 cgctttgctg ccaatgcaac agccctggaa
gaaaccctag agggttgcat agtctagaaa 3001 ggagtgtgac ctgacagtgc
tggagcctcc tagtttcccc ctatgaaggt tcccttaggc 3061 tgctgagttt
gggtttgtga tttatcttta gtttgtttta aagtcatctt tactttccca 3121
aatgtgttaa atttgtaact cctctttggg gtcttctcca ccacctgtct gatttttttg
3181 tgatctgttt aatcttttaa ttttttagta tcagtggttt tatttaagga
gacagtttgg 3241 cctattgtta cttccaattt ataatcaaga aggggctctg
gatccccttt taaattacac 3301 acactctcac acacatacat gtatgtttat
agatgctgct gctcttttcc ctgaagcata 3361 gtcaagtaag aactgctcta
cagaaggaca tatttccttg gatgtgagac cctattttga 3421 aatagagtcc
tgactcagaa caccaactta agaatttggg ggattaaaga tgtgaagacc 3481
acagtcttgg gttttcatat ctggagaaga ctatttgcca tgacgttttg ttgccctggt
3541 atttggacac tcctcagctt taatgggtgt ggccccttta gggttagtcc
tcagactaat 3601 gatagtgtct gctttctgca tgaacggcaa tatgggactc
cctccaagct agggtttggc 3661 aagtctgccc tagagtcatt tactctcctc
tgcctccatt tgttaataca gaatcaacat 3721 ttagtcttca ttatcttttt
tttttttttt gagacagagt ttcgatctat tttaagtatg 3781 tgaagaaaat
ctacttgtaa aaggctcaga tcttaattaa aaggtaattg tagcacatta 3841
ccaattataa ggtgaagaaa tgtttttttc ccaagtgtga tgcattgttc ttcagatgtt
3901 gaaaagaaag caaaaaatac cttctaactt aagacagaat ttttaacaaa
atgagcagta 3961 aaagtcacat gaaccactcc aaaaatcagt gcattttgca
tatttttaaa caaagacagc 4021 ttgttgaata ctgagaagag gagtgcaagg
agaaggtctg tactaacaaa gccaaattcc 4081 tcaagctctt actggactca
gttcagagtg gtgggccatt aaccccaaca tggaattttt 4141 ccatataaat
ctcaatgaat tccctttcat ttgaataggc aaacccaaat ccatgcaagt 4201
gttttaaagc actgtcctgt cttaatctta catgctgaaa gtcttcatgg tgatatgcac
4261 tatattcagt atacgtatgt tttcctactt ctcttgtaaa actgttgcat
gatccaactt 4321 cagcaatgaa ttgtgcctag tggagaacct ctatagatct
taaaaaatga attattcttt 4381 agcagtgtat tactcacatg ggtgcaatct
ttagccccag ggaggtcaat aatgtctttt 4441 aaagccagaa gtcacatttt
accaatatgc atttatcata attggtgctt aggctgtata 4501 ttcaagcctg
ttgtcttaac attttgtata aaaaagaaca acagaaatta tctgtcattt 4561
gagaagtggc ttgacaatca tttgagcttt gaaagcagtc actgtggtgt aatatgaatg
4621 ctgtcctagt ggtcatagta ccaagggcac gtgtctcccc ttggtataac
tgatttcctt 4681 tttagtcctc tactgctaaa taagttaatt ttgcattttg
cagaaagaaa cattgattgc 4741 taaatctttt tgctgctgtg ttttggtgtt
ttcatgttta cttgttttat attgatctgt 4801 tttaagtatg agaggcttat
agtgccctcc attgtaaatc catagtcatc tttttaagct 4861 tattgtgttt
aagaaagtag ctatgtgtta aacagaggtg atggcagccc ttccctagca 4921
cactggtgga agagacccct taagaacctg accccagtga atgaagctga tgcacaggga
4981 gcaccaaagg accttcgtta agtgataatt gtcctggcct ctcagccatg
accgttatga 5041 ggaaatatcc cccattcgaa cttaacagat gcctcctctc
caaagagaat taaaatcgta 5101 gcttgtacag atcaagagaa tatactgggc
agaatgaagt atgtttgttt atttttcttt 5161 aaaaataaag gattttggaa
ctctggagag taagaatata gtatagagtt tgcctcaaca 5221 catgtgaggg
ccaaataacc tgctagctag gcagtaataa actctgttac agaagagaaa 5281
aagggccggg cacagtggct tattcctgta atcccaacac tgtggaaggc cgaggcagga
5341 ggatcacttg agtccaggag tttgaaacct acctaggcaa catggtgaaa
ccttgtctct 5401 accaaaataa aaattagctg ggcatggtgg cacgtgcctg
tggtcccagc tacttgggag 5461 gctgaggtgg gagcctggga ggtcaaggct
gcagtgagcc atgatcatgc cactgcactc 5521 catcctgggt gacagcaaga
tcttgtctca aaaaaaaaaa aaaaaaaaaa aaaaccagga 5581 gtgaaaaagg
aaagtagaag gcagctgctg gcctagatgt tggtttggga atattaggtg 5641
atcctgttga gattctggat ccagagcaat ttctttagct tttgactttg ccaaagtgta
5701 gatagccttt atccagcagt attttaagtg gggaatgcaa cgtgaggcca
actgaacaat 5761 tccccccgtg gctgcccaga tagtcacagt caaggttgga
gagtctcctt ccagccagtg 5821 acctacccaa accttttgtt ctgtaaaact
gctctggaaa taccgggaag cccagttttc 5881 tcacgtggtt tctagcttct
tcagactcag cccaaattag gaagtgcaga agcacatgat 5941 ggtgaaaaac
ctaggatttg gcagccttcc agaatggtat ggaatctgag ggaagattta 6001
tgtttcgttt tggaggatag ctcaagttga attttctttc cagccagtta ccctttcaac
6061 ctacccatac tttgtacaac tcttacacaa atacttagat atttattaga
tagccctgaa 6121 ttcactctaa ttataaacag ggagtgtaaa ctgcccccag
atgttcctgg gctgggtaaa 6181 agcagctgga gtgaagcact cattttccat
aaaggtaaca aagggcagct cagtggttac 6241 tcaagctcaa aagggttttt
ttaagagcaa gcattggtta agtctgtgta tactgagttg 6301 gaagtgattt
cagcacattc ttttttagtg gagtgaaagt tctgaagccc ccttttaact 6361
tcctcttggt ttttcattat aattggtagc catctcatga actgtctctg actgttgtct
6421 ctttgtggtc atgtgattgt gagcttgctt tctgacttgc atttctgact
ttatcctgtt 6481 gttaggaaga tagaaactag gttttgaaag attacatgat
tcaagcgagg gattttaaag 6541 taaagatgta tttattctga agaatctaaa
agataacaga ttatttgctt atgaaagaac 6601 aatatagtct gggaatccca
gaatgtcaag ccaaaggtct aagaagtcat ctccttcaaa 6661 tactttaata
aagaagtatt tcgaggagat atctgtccaa aaaggtttga ctggcctcca 6721
gattccagtt atttttaaaa agcaacttac cactaaatcc ttgagtctcc atagagtaac
6781 agtaaagaaa ctgatgtaac agactctcct ctcaaaggat ctcctctgga
agagactatc 6841 agcggcagca ttctccaggg aagacccatc ccctagtgcc
agagcttgca tcctggagac 6901 taaagattgc acttttttgt agttttttgt
ccaaatgcaa tcccatttct gtgcctctta 6961 gcatgcagtt agatttggac
aaacaagatt cctaaggaat gactttatta actataatat 7021 ggttacagct
attatataaa tatatattct ggttatagtt ctaatatgga gatgttgtgt 7081
gcaatgctgg cctgtggtgg tctgtgtaat gctttaactt gtatggagga ggccaggctc
7141 agagctgaga tgtggcctga accttccctg tatcgatcct ttaatttaga
actgtcaaga 7201 tgtcactttc tccccctctg ccttttagtg gtatctgaca
tatactcaaa acagtaattt 7261 cctggtcaca tcattaactg ctaattctgt
atttataaag aattttcaga tggacatgta 7321 caaatttgaa ctcaaaccat
ccccagtcca gatacagggc agcgtgtagg tgaccacacc 7381 agagcctcag
cctcggtcct tctcagccgt cgggatagga tccaggcatt tcttttaaat 7441
ctcagaggta gcagtaaact tttcagtatt gctgttagca agtgtgtgtt
tgccaataga
7501 tacccattat actaatgtgc caagtaaatg ttcattgcac atctgcttcc
actgtgttcc 7561 cacgggtgcc atgaagtgtg tgaggagccc ctcatctgga
gggatgagtg ctgcgttgac 7621 tactgctatc aggattgtgt tgtgtggaat
attcatctac ataaatttta tatgcacagt 7681 aatttccctt tttatatgtc
aagtaactat ttgtaaaagt tatactcaca aattattata 7741 atgattacta
atatattttt tccatgtttc attgcctgaa taaaaactgt ttaccactgt 7801 ta
[0174] SEQ ID NO: 150 is the amino acid sequence of the FGFR1-TACC1
fusion protein.
TABLE-US-00023 MWSWKCLLFWAVLVTATLCTARPSPTLPEQDALPSSEDDDDDDDS
SSEEKETDNTKPNPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSS
GTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKG
NYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGS
NVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGV
NTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEA
LEERPAVMTSPLYLEIIIYCTGAFLISCMVGSVIVYKMKSGTKKS
DFHSQMAVHKLAKSIPLRRQVTVSADSSASMNSGVLLVRPSRLSS
SGTPMLAGVSEYELPEDPRWELPRDRLVLGKPLGEGCFGQVVLAE
AIGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKMIGKH
KNIINLLGACTQDGPLYVIVEYASKGNLREYLQARRPPGLEYCYN
PSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLV
TEDNVMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRI
YTHQSDVWSFGVLLWEIFTLGGSPYPGVPVEELFKLLKEGHRMDK
PSNCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRIVALTSNQGL
LESSAEKAPVSVSCGGESPLDGICLSESDKTAVLTLIREEIITKE
IEANEWKKKYEETRQEVLEMRKIVAEYEKTIAQMIEDEQRTSMTS
QKSFQQLTMEKEQALADLNSVERSLSDLFRRYENLKGVLEGFKKN
EEALKKCAQDYLARVKQEEQRYQALKIHAEEKLDKANEEIAQVRT
KAKAESAALHAGLRKEQMKVESLERALQQKNQEIEELTKICDELI AKLGKTD
[0175] SEQ ID NO: 151 is the nucleotide sequence that encodes the
FGFR1-TACC1 fusion protein.
TABLE-US-00024 atgtggagctggaagtgcctcctcttctgggctgtgctggtcaca
gccacactctgcaccgctaggccgtccccgaccttgcctgaacaa
gcccagccctggggagcccctgtggaagtggagtccttcctggtc
caccccggtgacctgctgcagcttcgctgtcggctgcgggacgat
gtgcagagcatcaactggctgcgggacggggtgcagctggcggaa
agcaaccgcacccgcatcacaggggaggaggtggaggtgcaggac
tccgtgcccgcagactccggcctctatgcttgcgtaaccagcagc
ccctcgggcagtgacaccacctacttctccgtcaatgtttcagat
gctctcccctcctcggaggatgatgatgatgatgatgactcctct
tcagaggagaaagaaacagataacaccaaaccaaaccgtatgccc
gtagctccatattggacatccccagaaaagatggaaaagaaattg
catgcagtgccggctgccaagacagtgaagttcaaatgcccttcc
agtgggaccccaaaccccacactgcgctggttgaaaaatggcaaa
gaattcaaacctgaccacagaattggaggctacaaggtccgttat
gccacctggagcatcataatggactctgtggtgccctctgacaag
ggcaactacacctgcattgtggagaatgagtacggcagcatcaac
cacacataccagctggatgtcgtggagcggtcccctcaccggccc
atcctgcaagcagggttgcccgccaacaaaacagtggccctgggt
agcaacgtggagttcatgtgtaaggtgtacagtgacccgcagccg
cacatccagtggctaaagcacatcgaggtgaatgggagcaagatt
ggcccagacaacctgccttatgtccagatcttgaagactgctgga
gttaataccaccgacaaagagatggaggtgcttcacttaagaaat
gtctcctttgaggacgcaggggagtatacgtgcttggcgggtaac
tctatcggactctcccatcactctgcatggttgaccgttctggaa
gccctggaagagaggccggcagtgatgacctcgcccctgtacctg
gagatcatcatctattgcacaggggccttcctcatctcctgcatg
gtggggtcggtcatcgtctacaagatgaagagtggtaccaagaag
agtgacttccacagccagatggctgtgcacaagctggccaagagc
atccctctgcgcagacaggtgtctgctgactccagtgcatccatg
aactctggggttcttctggttcggccatcacggctctcctccagt
gggactcccatgctagcaggggtctctgagtatgagcttcccgaa
gaccctcgctgggagctgcctcgggacagactggtcttaggcaaa
cccctgggagagggctgctttgggcaggtggtgttggcagaggct
atcgggctggacaaggacaaacccaaccgtgtgaccaaagtggct
gtgaagatgttgaagtcggacgcaacagagaaagacttgtcagac
ctgatctcagaaatggagatgatgaagatgatcgggaagcataag
aatatcatcaacctgctgggggcctgcacgcaggatggtcccttg
tatgtcatcgtggagtatgcctccaagggcaacctgcgggagtac
ctgcaggcccggaggcccccagggctggaatactgctacaacccc
agccacaacccagaggagcagctctcctccaaggacctggtgtcc
tgcgcctaccaggtggcccgaggcatggagtatctggcctccaag
aagtgcatacaccgagacctggcagccaggaatgtcctggtgaca
gaggacaatgtgatgaagatagcagactttggcctcgcacgggac
attcaccacatcgactactataaaaagacaaccaacggccgactg
cctgtgaagtggatggcacccgaggcattatttgaccggatctac
acccaccagagtgatgtgtggtctttcggggtgctcctgtgggag
atcttcactctgggcggctccccataccccggtgtgcctgtggag
gaacttttcaagctgctgaaggagggtcaccgcatggacaagccc
agtaactgcaccaacgagctgtacatgatgatgcgggactgctgg
catgcagtgccctcacagagacccaccttcaagcagctggtggaa
gacctggaccgcatcgtggccttgacctccaaccagtgggctgct
ggagtcctctgcagagaaggcccctgtgtcggtgtcctgtggagg
tgagagccccctggatgggatctgcctcagcgaatcagacaagac
agccgtgctcaccttaataagagaagagataattactaaagagat
tgaagcaaatgaatggaagaagaaatacgaagagacccggcaaga
agttttggagatgaggaaaattgtagctgaatatgaaaagactat
tgctcaaatgattgaagatgaacaaaggacaagtatgacctctca
gaagagcttccagcaactgaccatggagaaggaacaggccctggc
tgaccttaactctgtggaaaggtccctttctgatctcttcaggag
atatgagaacctgaaaggtgttctggaagggttcaagaagaatga
agaagccttgaagaaatgtgctcaggattacttagccagagttaa
acaagaggagcagcgataccaggccctgaaaatccacgcagaaga
gaaactggacaaagccaatgaagagattgctcaggttcgaacaaa
agcaaaggctgagagtgcagctctccatgctggactccgcaaaga
gcagatgaaggtggagtccctggaaagggccctgcagcagaagaa
ccaagaaattgaagaactgacaaaaatctgtgatgagctgattgc
aaagctgggaaagactgac
[0176] The Genbank ID for the FGFR2 gene is 2263. Eight isoforms
are listed for FGFR2, e.g., having Genebank Accession Nos.
NP.sub.--000132 (corresponding nucleotide sequence
NM.sub.--000141); NP.sub.--001138385 (corresponding nucleotide
sequence NM.sub.--001144913); NP.sub.--001138386 (corresponding
nucleotide sequence NM.sub.--001144914); NP.sub.--001138387
(corresponding nucleotide sequence NM.sub.--001144915);
NP.sub.--001138388 (corresponding nucleotide sequence
NM.sub.--001144916); NP.sub.--001138389 (corresponding nucleotide
sequence NM.sub.--001144917); NP.sub.--001138390 (corresponding
nucleotide sequence NM.sub.--001144918); NP.sub.--001138391
(corresponding nucleotide sequence NM.sub.--001144919);
NP.sub.--075259 (corresponding nucleotide sequence
NM.sub.--022970).
[0177] SEQ ID NO: 152 is the FGFR2 Amino Acid Sequence for isoform
1, having Genebank Accession No. NP.sub.--000132 (821 aa):
TABLE-US-00025 1 MVSWGRFICL VVVTMATLSL ARPSFSLVED TTLEPEEPPT
KYQISQPEVY VAAPGESLEV 61 RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE
YLQIKGATPR DSGLYACTAS RTVDSETWYF 121 MVNVTDAISS GDDEDDTDGA
EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP 181 AGGNPMPTMR
WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI 241
NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK
301 YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI
SFHSAWLTVL 361 PAPGREKEIT ASPDYLEIAI YCIGVFLIAC MVVTVILCRM
KNTTKKPDFS SQPAVHKLTK 421 RIPLRRQVTV SAESSSSMNS NTPLVRITTR
LSSTADTPML AGVSEYELPE DPKWEFPRDK 481 LTLGKPLGEG CFGQVVMAEA
VGIDKDKPKE AVTVAVKMLK DDATEKDLSD LVSEMEMMKM 541 IGKHKNIINL
LGACTQDGPL YVIVEYASKG NLREYLRARR PPGMEYSYDI NRVPEEQMTF 601
KDLVSCTYQL ARGMEYLASQ KCIHRDLAAR NVLVTENNVM KIADFGLARD INNIDYYKKT
661 TNGRLPVKWM APEALFDRVY THQSDVWSFG VLMWEIFTLG GSPYPGIPVE
ELFKLLKEGH 721 RMDKPANCTN ELYMMMRDCW HAVPSQRPTF KQLVEDLDRI
LTLTTNEEYL DLSQPLEQYS 781 PSYPDTRSSC SSGDDSVFSP DPMPYEPCLP
QYPHINGSVK T
[0178] SEQ ID NO: 153 is the FGFR2 Nucleotide Sequence for isoform
1, having Genebank Accession No. NM.sub.--000141 (4654 bp):
TABLE-US-00026 1 ggcggcggct ggaggagagc gcggtggaga gccgagcggg
cgggcggcgg gtgcggagcg 61 ggcgagggag cgcgcgcggc cgccacaaag
ctcgggcgcc gcggggctgc atgcggcgta 121 cctggcccgg cgcggcgact
gctctccggg ctggcggggg ccggccgcga gccccggggg 181 ccccgaggcc
gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 241
tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc
301 cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc
cgggaggctt 361 ggcgccggcg aagacccaag gaccactctt ctgcgtttgg
agttgctccc cgcaaccccg 421 ggctcgtcgc tttctccatc ccgacccacg
cggggcgcgg ggacaacaca ggtcgcggag 481 gagcgttgcc attcaagtga
ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 541 gcaggctgaa
ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 601
cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg
661 gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc
cggccctcct 721 tcagtttagt tgaggatacc acattagagc cagaagagcc
accaaccaaa taccaaatct 781 ctcaaccaga agtgtacgtg gctgcgccag
gggagtcgct agaggtgcgc tgcctgttga 841 aagatgccgc cgtgatcagt
tggactaagg atggggtgca cttggggccc aacaatagga 901 cagtgcttat
tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct 961
atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca
1021 cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa
gattttgtca 1081 gtgagaacag taacaacaag agagcaccat actggaccaa
cacagaaaag atggaaaagc 1141 ggctccatgc tgtgcctgcg gccaacactg
tcaagtttcg ctgcccagcc ggggggaacc 1201 caatgccaac catgcggtgg
ctgaaaaacg ggaaggagtt taagcaggag catcgcattg 1261 gaggctacaa
ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg 1321
acaagggaaa ttatacctgt gtagtggaga atgaatacgg gtccatcaat cacacgtacc
1381 acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga
ctgccggcaa 1441 atgcctccac agtggtcgga ggagacgtag agtttgtctg
caaggtttac agtgatgccc 1501 agccccacat ccagtggatc aagcacgtgg
aaaagaacgg cagtaaatac gggcccgacg 1561 ggctgcccta cctcaaggtt
ctcaaggccg ccggtgttaa caccacggac aaagagattg 1621 aggttctcta
tattcggaat gtaacttttg aggacgctgg ggaatatacg tgcttggcgg 1681
gtaattctat tgggatatcc tttcactctg catggttgac agttctgcca gcgcctggaa
1741 gagaaaagga gattacagct tccccagact acctggagat agccatttac
tgcatagggg 1801 tcttcttaat cgcctgtatg gtggtaacag tcatcctgtg
ccgaatgaag aacacgacca 1861 agaagccaga cttcagcagc cagccggctg
tgcacaagct gaccaaacgt atccccctgc 1921 ggagacaggt aacagtttcg
gctgagtcca gctcctccat gaactccaac accccgctgg 1981 tgaggataac
aacacgcctc tcttcaacgg cagacacccc catgctggca ggggtctccg 2041
agtatgaact tccagaggac ccaaaatggg agtttccaag agataagctg acactgggca
2101 agcccctggg agaaggttgc tttgggcaag tggtcatggc ggaagcagtg
ggaattgaca 2161 aagacaagcc caaggaggcg gtcaccgtgg ccgtgaagat
gttgaaagat gatgccacag 2221 agaaagacct ttctgatctg gtgtcagaga
tggagatgat gaagatgatt gggaaacaca 2281 agaatatcat aaatcttctt
ggagcctgca cacaggatgg gcctctctat gtcatagttg 2341 agtatgcctc
taaaggcaac ctccgagaat acctccgagc ccggaggcca cccgggatgg 2401
agtactccta tgacattaac cgtgttcctg aggagcagat gaccttcaag gacttggtgt
2461 catgcaccta ccagctggcc agaggcatgg agtacttggc ttcccaaaaa
tgtattcatc 2521 gagatttagc agccagaaat gttttggtaa cagaaaacaa
tgtgatgaaa atagcagact 2581 ttggactcgc cagagatatc aacaatatag
actattacaa aaagaccacc aatgggcggc 2641 ttccagtcaa gtggatggct
ccagaagccc tgtttgatag agtatacact catcagagtg 2701 atgtctggtc
cttcggggtg ttaatgtggg agatcttcac tttagggggc tcgccctacc 2761
cagggattcc cgtggaggaa ctttttaagc tgctgaagga aggacacaga atggataagc
2821 cagccaactg caccaacgaa ctgtacatga tgatgaggga ctgttggcat
gcagtgccct 2881 cccagagacc aacgttcaag cagttggtag aagacttgga
tcgaattctc actctcacaa 2941 ccaatgagga atacttggac ctcagccaac
ctctcgaaca gtattcacct agttaccctg 3001 acacaagaag ttcttgttct
tcaggagatg attctgtttt ttctccagac cccatgcctt 3061 acgaaccatg
ccttcctcag tatccacaca taaacggcag tgttaaaaca tgaatgactg 3121
tgtctgcctg tccccaaaca ggacagcact gggaacctag ctacactgag cagggagacc
3181 atgcctccca gagcttgttg tctccacttg tatatatgga tcagaggagt
aaataattgg 3241 aaaagtaatc agcatatgtg taaagattta tacagttgaa
aacttgtaat cttccccagg 3301 aggagaagaa ggtttctgga gcagtggact
gccacaagcc accatgtaac ccctctcacc 3361 tgccgtgcgt actggctgtg
gaccagtagg actcaaggtg gacgtgcgtt ctgccttcct 3421 tgttaatttt
gtaataattg gagaagattt atgtcagcac acacttacag agcacaaatg 3481
cagtatatag gtgctggatg tatgtaaata tattcaaatt atgtataaat atatattata
3541 tatttacaag gagttatttt ttgtattgat tttaaatgga tgtcccaatg
cacctagaaa 3601 attggtctct ctttttttaa tagctatttg ctaaatgctg
ttcttacaca taatttctta 3661 attttcaccg agcagaggtg gaaaaatact
tttgctttca gggaaaatgg tataacgtta 3721 atttattaat aaattggtaa
tatacaaaac aattaatcat ttatagtttt ttttgtaatt 3781 taagtggcat
ttctatgcag gcagcacagc agactagtta atctattgct tggacttaac 3841
tagttatcag atcctttgaa aagagaatat ttacaatata tgactaattt ggggaaaatg
3901 aagttttgat ttatttgtgt ttaaatgctg ctgtcagacg attgttctta
gacctcctaa 3961 atgccccata ttaaaagaac tcattcatag gaaggtgttt
cattttggtg tgcaaccctg 4021 tcattacgtc aacgcaacgt ctaactggac
ttcccaagat aaatggtacc agcgtcctct 4081 taaaagatgc cttaatccat
tccttgagga cagaccttag ttgaaatgat agcagaatgt 4141 gcttctctct
ggcagctggc cttctgcttc tgagttgcac attaatcaga ttagcctgta 4201
ttctcttcag tgaattttga taatggcttc cagactcttt ggcgttggag acgcctgtta
4261 ggatcttcaa gtcccatcat agaaaattga aacacagagt tgttctgctg
atagttttgg 4321 ggatacgtcc atctttttaa gggattgctt tcatctaatt
ctggcaggac ctcaccaaaa 4381 gatccagcct catacctaca tcagacaaaa
tatcgccgtt gttccttctg tactaaagta 4441 ttgtgttttg ctttggaaac
acccactcac tttgcaatag ccgtgcaaga tgaatgcaga 4501 ttacactgat
cttatgtgtt acaaaattgg agaaagtatt taataaaacc tgttaatttt 4561
tatactgaca ataaaaatgt ttctacagat attaatgtta acaagacaaa ataaatgtca
4621 cgcaacttat ttttttaata aaaaaaaaaa aaaa
[0179] The Genbank ID for the TACC2 gene is 10579. Four isoforms
are listed for TACC2, e.g., having Genebank Accession Nos.
NP.sub.--996744 (corresponding nucleotide sequence
NM.sub.--206862); NP.sub.--996743 (corresponding nucleotide
sequence NM.sub.--206861); NP.sub.--996742 (corresponding
nucleotide sequence NM.sub.--206860); NP.sub.--008928
(corresponding nucleotide sequence NM.sub.--006997).
[0180] SEQ ID NO: 154 is the TACC2 Amino Acid Sequence for isoform
a, having Genebank Accession No. NP.sub.--996744 (2948 aa):
TABLE-US-00027 1 MGNENSTSDN QRTLSAQTPR SAQPPGNSQN IKRKQQDTPG
SPDHRDASSI GSVGLGGFCT 61 ASESSASLDP CLVSPEVTEP RKDPQGARGP
EGSLLPSPPP SQEREHPSSS MPFAECPPEG 121 CLASPAAAPE DGPQTQSPRR
EPAPNAPGDI AAAFPAERDS STPYQEIAAV PSAGRERQPK 181 EEGQKSSFSF
SSGIDQSPGM SPVPLREPMK APLCGEGDQP GGFESQEKEA AGGFPPAESR 241
QGVASVQVTP EAPAAAQQGT ESSAVLEKSP LKPMAPIPQD PAPRASDRER GQGEAPPQYL
301 TDDLEFLRAC HLPRSNSGAA PEAEVNAASQ ESCQQPVGAY LPHAELPWGL
PSPALVPEAG 361 GSGKEALDTI DVQGHPQTGM RGTKPNQVVC VAAGGQPEGG
LPVSPEPSLL TPTEEAHPAS 421 SLASFPAAQI PIAVEEPGSS SRESVSKAGM
PVSADAAKEV VDAGLVGLER QVSDLGSKGE 481 HPEGDPGEVP APSPQERGEH
LNTEQSHEVQ PGVPPPPLPK EQSHEVQPGA PPPPLPKAPS 541 ESARGPPGPT
DGAKVHEDST SPAVAKEGSR SPGDSPGGKE EAPEPPDGGD PGNLQGEDSQ 601
AFSSKRDPEV GKDELSKPSS DAESRDHPSS HSAQPPRKGG AGHTDGPHSQ TAEADASGLP
661 HKLGEEDPVL PPVPDGAGEP TVPEGAIWEG SGLQPKCPDT LQSREGLGRM
ESFLTLESEK 721 SDFPPTPVAE VAPKAQEGES TLEIRKMGSC DGEGLLTSPD
QPRGPACDAS RQEFHAGVPH 781 PPQGENLAAD LGLTALILDQ DQQGIPSCPG
EGWIRGAASE WPLLSSEKHL QPSQAQPETS 841 IFDVLKEQAQ PPENGKETSP
SHPGFKDQGA DSSQIHVPVE PQEDNNLPTH GGQEQALGSE 901 LQSQLPKGTL
SDTPTSSPTD MVWESSLTEE SELSAPTRQK LPALGEKRPE GACGDGQSSR 961
VSPPAADVLK DFSLAGNFSR KETCCTGQGP NKSQQALADA LEEGSQHEEA CQRHPGASEA
1021 ADGCSPLWGL SKREMASGNT GEAPPCQPDS VALLDAVPCL PALAPASPGV
TPTQDAPETE 1081 ACDETQEGRQ QPVPAPQQKM ECWATSDAES PKLLASFPSA
GEQGGEAGAA ETGGSAGAGD 1141 PGKQQAPEKP GEATLSCGLL QTEHCLTSGE
EASTSALRES CQAEHPMASC QDALLPAREL 1201 GGIPRSTMDF STHQAVPDPK
ELLLSGPPEV AAPDTPYLHV DSAAQRGAED SGVKAVSSAD 1261 PRAPGESPCP
VGEPPLALEN AASLKLFAGS LAPLLQPGAA GGEIPAVQAS SGSPKARTTE 1321
GPVDSMPCLD RMPLLAKGKQ ATGEEKAATA PGAGAKASGE GMAGDAAGET EGSMERMGEP
1381 SQDPKQGTSG GVDTSSEQIA TLTGFPDFRE HIAKIFEKPV LGALATPGEK
AGAGRSAVGK 1441 DLTRPLGPEK LLDGPPGVDV TLLPAPPARL QVEKKQQLAG
EAEISHLALQ DPASDKLLGP 1501 AGLTWERNLP GAGVGKEMAG VPPTLREDER
PEGPGAAWPG LEGQAYSQLE RSRQELASGL 1561 PSPAATQELP VERAAAFQVA
PHSHGEEAVA QDRIPSGKQH QETSACDSPH GEDGPGDFAH 1621 TGVPGHVPRS
TCAPSPQREV LTVPEANSEP WTLDTLGGER RPGVTAGILE MRNALGNQST 1681
PAPPTGEVAD TPLEPGKVAG AAGEAEGDIT LSTAETQACA SGDLPEAGTT RTFSVVAGDL
1741 VLPGSCQDPA CSDKAPGMEG TAALHGDSPA RPQQAKEQPG PERPIPAGDG
KVCVSSPPEP 1801 DETHDPKLQH LAPEELHTDR ESPRPGPSML PSVPKKDAPR
VMDKVTSDET RGAEGTESSP 1861 VADDIIQPAA PADLESPTLA ASSYHGDVVG
QVSTDLIAQS ISPAAAHAGL PPSAAEHIVS 1921 PSAPAGDRVE ASTPSCPDPA
KDLSRSSDSE EAFETPESTT PVKAPPAPPP PPPEVIPEPE 1981 VSTQPPPEEP
GCGSETVPVP DGPRSDSVEG SPFRPPSHSF SAVFDEDKPI ASSGTYNLDF 2041
DNIELVDTFQ TLEPRASDAK NQEGKVNTRR KSTDSVPISK STLSRSLSLQ ASDFDGASSS
2101 GNPEAVALAP DAYSTGSSSA SSTLKRTKKP RPPSLKKKQT TKKPTETPPV
KETQQEPDEE 2161 SLVPSGENLA SETKTESAKT EGPSPALLEE TPLEPAVGPK
AACPLDSESA EGVVPPASGG 2221 GRVQNSPPVG RKTLPLTTAP EAGEVTPSDS
GGQEDSPAKG LSVRLEFDYS EDKSSWDNQQ 2281 ENPPPTKKIG KKPVAKMPLR
RPKMKKTPEK LDNTPASPPR SPAEPNDIPI AKGTYTFDID 2341 KWDDPNFNPF
SSTSKMQESP KLPQQSYNFD PDTCDESVDP FKTSSKTPSS PSKSPASFEI 2401
PASAMEANGV DGDGLNKPAK KKKTPLKTDT FRVKKSPKRS PLSDPPSQDP TPAATPETPP
2461 VISAVVHATD EEKLAVTNQK WTCMTVDLEA DKQDYPQPSD LSTFVNETKF
SSPTEELDYR 2521 NSYEIEYMEK IGSSLPQDDD APKKQALYLM FDTSQESPVK
SSPVRMSESP TPCSGSSFEE 2581 TEALVNTAAK NQHPVPRGLA PNQESHLQVP
EKSSQKELEA MGLGTPSEAI EITAPEGSFA 2641 SADALLSRLA HPVSLCGALD
YLEPDLAEKN PPLFAQKLQE ELEFAIMRIE ALKLARQIAL 2701 ASRSHQDAKR
EAAHPTDVSI SKTALYSRIG TAEVEKPAGL LFQQPDLDSA LQIARAEIIT 2761
KEREVSEWKD KYEESRREVM EMRKIVAEYE KTIAQMIEDE QREKSVSHQT VQQLVLEKEQ
2821 ALADLNSVEK SLADLFRRYE KMKEVLEGFR KNEEVLKRCA QEYLSRVKKE
EQRYQALKVH 2881 AEEKLDRANA EIAQVRGKAQ QEQAAHQASL RKEQLRVDAL
ERTLEQKNKE IEELTKICDE 2941 LIAKMGKS
[0181] SEQ ID NO: 155 is the TACC2 Nucleotide Sequence for isoform
a, having Genebank Accession No. NM.sub.--206862 (9706 bp)
TABLE-US-00028 1 gcctgctcca agggaaggat caggagagaa gaaacgcaaa
tcccagaacc gtgccaacat 61 ataaaacccc acattaaggg ttgtacagtg
cactgggatt tctcaagtca cccgcttggt 121 cctcttccaa gtatacttta
cttcctttca ttcctctcta aaactttttt aaaaactttc 181 actcctgctc
taaaagttat cttggtttct tactctacct tatgcccctt gggcgaattt 241
tttcctctga ggagggaaga atagagttgc tgctgcagac acatcagatt ccctactggt
301 aacagctgga gtgcgtcacc tctgacaaaa ttctggggac gctgggaaca
ctgaatcaac 361 atgggcaatg agaacagcac ctcggacaac cagaggactt
tatcagctca gactccaagg 421 tccgcgcagc cacccgggaa cagtcagaat
ataaaaagga agcagcagga cacgcccgga 481 agccctgacc acagagacgc
gtccagcatt ggcagcgttg ggcttggagg cttctgcacc 541 gcttctgaga
gttctgccag cctggatcca tgccttgtgt ccccagaggt gactgagcca 601
aggaaggacc cacagggagc cagggggcca gaaggttctt tgctgcccag cccaccaccg
661 tcccaggagc gagagcaccc ctcgtcctcc atgccctttg ccgagtgtcc
cccggaaggt 721 tgcttggcaa gtccagcagc ggcacctgaa gatggtcctc
agactcagtc tcccaggagg 781 gaacctgccc caaatgcccc aggagacatc
gcggcggcat ttcccgctga gagggacagc 841 tctactccat accaagagat
tgctgccgtc cccagtgctg gaagagagag acagccgaag 901 gaagaaggac
agaagtcctc cttctccttc tccagtggca tcgaccagtc acctggaatg 961
tcgccagtac ccctcagaga gccaatgaag gcaccgctgt gtggagaggg ggaccagcct
1021 ggtggttttg agtcccaaga gaaagaggct gcaggtggct ttccccctgc
agagtccagg 1081 cagggggtgg cttctgtgca agtgacccct gaggcccctg
ctgcagccca gcagggcaca 1141 gaaagctcag cggtcttgga gaagtccccc
ctaaaaccca tggccccgat cccacaagat 1201 ccagccccaa gagcctcaga
cagagaaaga ggccaagggg aggcgccgcc tcagtattta 1261 acagatgact
tggaattcct cagggcctgc catctcccta ggagcaattc aggggctgcc 1321
ccagaagcag aagtgaatgc cgcttcccag gagagctgcc agcagccagt gggagcatat
1381 ctgccgcacg cagagctgcc ctggggcttg ccaagtcctg ccctggtgcc
agaggctggg 1441 ggctctggga aggaggctct ggacaccatt gatgttcagg
gtcacccaca gacagggatg 1501 cgaggaacca agcccaatca agttgtctgt
gtggcagcag gcggccagcc cgaagggggt 1561 ttgcctgtga gccctgaacc
ttccctgctc actccgactg aggaagcaca tccagcttca 1621 agcctcgctt
cattcccagc tgctcagatt cctattgctg tagaagaacc tggatcatca 1681
tccagggaat cagtttccaa ggctgggatg ccagtttctg cagatgcagc caaagaggtg
1741 gtggatgcag ggttggtggg actggagagg caggtgtcag atcttggaag
caagggagag 1801 catccagaag gggaccctgg agaggttcct gccccatcac
cccaggagag gggagagcac 1861 ttgaacacgg agcaaagcca tgaggtccaa
ccaggagtac caccccctcc tcttcccaag 1921 gagcaaagcc atgaggtcca
accaggagca ccaccccctc ctcttcccaa ggcaccaagt 1981 gaaagtgcca
gagggccacc ggggccaacg gatggagcca aggtccatga agattccaca 2041
agcccagccg tggctaaaga aggaagcaga tcacctggtg acagccctgg aggaaaggag
2101 gaagccccag agccacctga tggtggagac ccagggaacc tgcaaggaga
ggactctcag 2161 gctttcagca gcaagcgtga tccagaagta ggcaaagatg
agctttcaaa gccaagcagt 2221 gatgcagaga gcagagacca tcccagctca
cactcagcac agccacccag aaaggggggt 2281 gctgggcaca cggacgggcc
ccactctcag acagcagagg ctgatgcatc tggcctacca 2341 cacaagctgg
gtgaggagga ccccgtcctg ccccctgtgc cagatggagc tggtgagccc 2401
actgttcccg aaggagccat ctgggagggg tcaggattgc agcccaaatg tcctgacacc
2461 cttcagagca gggaaggatt gggaagaatg gagtctttcc tgactttaga
atcagagaaa 2521 tcagattttc caccaactcc tgttgcagag gttgcaccca
aagcccagga aggtgagagc 2581 acattggaaa taaggaagat gggcagctgt
gatggggagg gcttgctgac gtccccagat 2641 caaccccgcg ggccggcgtg
tgatgcgtcg agacaggaat ttcatgctgg ggtgccacat 2701 cccccccagg
gggagaactt ggcagcagac ctggggctca cggcactcat cctggaccaa 2761
gatcagcagg gaatcccatc ctgcccaggg gaaggctgga taagaggagc tgcatccgag
2821 tggcccctac tatcttctga gaagcatctc cagccatccc aggcacaacc
agagacatcc 2881 atctttgacg tgctcaagga gcaggcccag ccacctgaaa
atgggaaaga gacttctcca 2941 agccatccag gttttaagga ccagggagca
gattcttccc aaatccatgt acctgtggaa 3001 cctcaggaag ataacaactt
gcccactcat ggaggacagg agcaggcttt gggatcagaa 3061 cttcaaagtc
agctccccaa aggcaccctg tctgatactc caacttcatc tcccactgac 3121
atggtttggg agagttctct gacagaagag tcagaattgt cagcaccaac gagacagaag
3181 ttgcctgcac taggggagaa gcggccagag ggagcatgcg gtgatggtca
gtcctcgagg 3241 gtctcgcctc cagcagcaga tgtcttaaaa gacttttctc
ttgcagggaa cttcagcaga 3301 aaggaaactt gctgcactgg gcaggggcca
aacaagtctc aacaggcatt ggctgatgcc 3361 ttggaagaag gcagccagca
tgaagaagca tgtcaaaggc atccaggagc ttctgaagca 3421 gctgatggtt
gttccccact ctggggcttg agtaagaggg agatggcaag tggaaacaca 3481
ggggaggccc caccttgtca gcctgactca gtagctctcc tggatgcagt tccctgcctg
3541 ccagccctgg cgcccgccag ccccggagtc acacccaccc aggatgcccc
agagacagag 3601 gcatgtgatg aaacccagga aggcaggcag caaccagtgc
cggccccgca gcagaaaatg 3661 gagtgctggg ccacttcgga tgcagagtcc
ccaaagcttc ttgcaagttt cccatcagct 3721 ggggagcaag gtggtgaagc
cggggctgct gagactggtg gcagcgctgg tgcaggagac 3781 ccaggaaagc
agcaggctcc ggagaaacct ggagaagcta ctttgagttg tggcctcctt 3841
cagactgagc actgccttac ctccggggag gaagcttcta cctctgccct acgtgagtcc
3901 tgccaagctg agcaccccat ggccagctgc caggatgcct tgctgccagc
cagagagctg 3961 ggtgggattc ccaggagcac catggatttt tctacacacc
aggctgtccc agacccaaag 4021 gagctcctgc tgtctgggcc accagaagtg
gctgctcctg acacccctta cctgcatgtc 4081 gacagtgctg cccagagagg
agcagaagac agtggagtga aagctgtttc ctctgcagac 4141 cccagagctc
ctggcgaaag cccctgtcct gtaggggagc ccccacttgc cttggaaaat 4201
gctgcctcct tgaagctgtt tgctggctcc ctcgcccccc tgttgcaacc aggagctgca
4261 ggtggggaaa tccctgcagt gcaagccagc agtggtagtc ccaaagccag
aaccactgag 4321 ggaccagtgg actccatgcc atgcctggac cggatgccac
ttctggccaa gggcaagcag 4381 gcaacagggg aagagaaagc agcaacagct
ccaggtgcag gtgccaaggc cagtggggag 4441 ggcatggcag gtgatgcagc
aggagagaca gagggcagca tggagaggat gggagagcct 4501 tcccaggacc
caaagcaggg cacatcaggt ggtgtggaca caagctctga gcaaatcgcc 4561
accctcactg gcttcccaga cttcagggag cacatcgcca agatcttcga gaagcctgtg
4621 ctcggagccc tggccacacc tggagaaaag gcaggagctg ggaggagtgc
agtgggtaaa 4681 gacctcacca ggccattggg cccagagaag cttctagatg
ggcctccagg agtggatgtc 4741 acccttctcc ctgcacctcc tgctcgactc
caggtggaga agaagcaaca gttggctgga 4801 gaggctgaga tttcccatct
ggctctgcaa gatccagctt cagacaagct tctgggtcca 4861 gcagggctga
cctgggagcg gaacttgcca ggtgccggtg tggggaagga gatggcaggt 4921
gtcccaccca cactgaggga agacgagagg ccagaggggc ctggggcagc ctggccaggc
4981 ctggaaggcc aggcttactc acagctggag aggagcaggc aggaattagc
ttcaggtctt 5041 ccttcaccag cagctactca ggagctccct gtggagagag
ctgctgcctt ccaggtggct 5101 ccccatagcc atggagaaga ggccgtggcc
caagacagaa ttccttctgg aaagcagcac 5161 caggaaacat ctgcctgcga
cagtccacat ggagaagatg gtcccgggga ctttgctcac 5221 acaggggttc
caggacatgt gccaaggtcc acgtgtgccc cttctcctca gagggaggtt 5281
ttgactgtgc ctgaggccaa cagtgagccc tggacccttg acacgcttgg gggtgaaagg
5341 agacccggag tcactgctgg catcttggaa atgcgaaatg ccctgggcaa
ccagagcacc 5401 cctgcaccac caactggaga agtggcagac actcccctgg
agcctggcaa ggtggcaggc 5461 gctgctgggg aagcagaggg tgacatcacc
ctgagcacag ctgagacaca ggcatgtgcg 5521 tccggtgatc tgcctgaagc
aggtactacg aggacattct ccgttgtggc aggtgacttg 5581 gtgctgccag
gaagctgtca ggacccagcc tgctctgaca aggctccggg gatggagggt 5641
acagctgccc ttcatgggga cagcccagcc aggccccagc aggctaagga gcagccaggg
5701 cctgagcgcc ccattccagc tggggatggg aaggtgtgcg tctcctcacc
tccagagcct 5761 gacgaaactc acgacccgaa gctgcaacat ttggctccag
aagagctcca cactgacaga 5821 gagagcccca ggcctggccc atccatgtta
ccttcggttc ctaagaagga tgctccaaga 5881 gtcatggata aagtcacttc
agatgagacc agaggtgcgg aaggaacaga aagttcacct 5941 gtggcagatg
atatcatcca gcccgctgcc cccgcagacc tggaaagccc aaccttagct 6001
gcctcttcct accacggtga tgttgttggc caggtctcta cggatctgat agcccagagc
6061 atctccccag ctgctgccca tgcgggtctt cctccctcgg ctgcagaaca
catagtttcg 6121 ccatctgccc cagctggtga cagagtagaa gcttccactc
cctcctgccc agatccggcc 6181 aaggacctca gcaggagttc cgattctgaa
gaggcatttg agaccccgga gtcaacgacc 6241 cctgtcaaag ctccgccagc
tccaccccca ccaccccccg aagtcatccc agaacccgag 6301 gtcagcacac
agccaccccc ggaagaacca ggatgtggtt ctgagacagt ccctgtccct 6361
gatggcccac ggagcgactc ggtggaagga agtcccttcc gtcccccgtc acactccttc
6421 tctgccgtct tcgatgaaga caagccgata gccagcagtg ggacttacaa
cttggacttt 6481 gacaacattg agcttgtgga tacctttcag accttggagc
ctcgtgcctc agacgctaag 6541 aatcaggagg gcaaagtgaa cacacggagg
aagtccacgg attccgtccc catctctaag 6601 tctacactgt cccggtcgct
cagcctgcaa gccagtgact ttgatggtgc ttcttcctca 6661 ggcaatcccg
aggccgtggc ccttgcccca gatgcatata gcacgggttc cagcagtgct 6721
tctagtaccc ttaagcgaac taaaaaaccg aggccgcctt ccttaaaaaa gaaacagacc
6781 accaagaaac ccacagagac ccccccagtg aaggagacgc aacaggagcc
agatgaagag 6841 agccttgtcc ccagtgggga gaatctagca tctgagacga
aaacggaatc tgccaagacg 6901 gaaggtccta gcccagcctt attggaggag
acgccccttg agcccgctgt ggggcccaaa 6961 gctgcctgcc ctctggactc
agagagtgca gaaggggttg tccccccggc ttctggaggt 7021 ggcagagtgc
agaactcacc ccctgtcggg aggaaaacgc tgcctcttac cacggccccg 7081
gaggcagggg aggtaacccc atcggatagc ggggggcaag aggactctcc agccaaaggg
7141 ctctccgtaa ggctggagtt tgactattct gaggacaaga gtagttggga
caaccagcag 7201 gaaaaccccc ctcctaccaa aaagataggc aaaaagccag
ttgccaaaat gcccctgagg 7261 aggccaaaga tgaaaaagac acccgagaaa
cttgacaaca ctcctgcctc acctcccaga 7321 tcccctgctg aacccaatga
catccccatt gctaaaggta cttacacctt tgatattgac 7381 aagtgggatg
accccaattt taaccctttt tcttccacct caaaaatgca ggagtctccc 7441
aaactgcccc aacaatcata caactttgac ccagacacct gtgatgagtc
cgttgacccc
7501 tttaagacat cctctaagac ccccagctca ccttctaaat ccccagcctc
ctttgagatc 7561 ccagccagtg ctatggaagc caatggagtg gacggggatg
ggctaaacaa gcccgccaag 7621 aagaagaaga cgcccctaaa gactgacaca
tttagggtga aaaagtcgcc aaaacggtct 7681 cctctctctg atccaccttc
ccaggacccc accccagctg ctacaccaga aacaccacca 7741 gtgatctctg
cggtggtcca cgccacagat gaggaaaagc tggcggtcac caaccagaag 7801
tggacgtgca tgacagtgga cctagaggct gacaaacagg actacccgca gccctcggac
7861 ctgtccacct ttgtaaacga gaccaaattc agttcaccca ctgaggagtt
ggattacaga 7921 aactcctatg aaattgaata tatggagaaa attggctcct
ccttacctca ggacgacgat 7981 gccccgaaga agcaggcctt gtaccttatg
tttgacactt ctcaggagag ccctgtcaag 8041 tcatctcccg tccgcatgtc
agagtccccg acgccgtgtt cagggtcaag ttttgaagag 8101 actgaagccc
ttgtgaacac tgctgcgaaa aaccagcatc ctgtcccacg aggactggcc 8161
cctaaccaag agtcacactt gcaggtgcca gagaaatcct cccagaagga gctggaggcc
8221 atgggcttgg gcaccccttc agaagcgatt gaaattacag ctcccgaggg
ctcctttgcc 8281 tctgctgacg ccctcctcag caggctagct caccccgtct
ctctctgtgg tgcacttgac 8341 tatctggagc ccgacttagc agaaaagaac
cccccactat tcgctcagaa actccaggag 8401 gagttagagt ttgccatcat
gcggatagaa gccctgaagc tggccaggca gattgctttg 8461 gcttcccgca
gccaccagga tgccaagaga gaggctgctc acccaacaga cgtctccatc 8521
tccaaaacag ccttgtactc ccgcatcggg accgctgagg tggagaaacc tgcaggcctt
8581 ctgttccagc agcccgacct ggactctgcc ctccagatcg ccagagcaga
gatcataacc 8641 aaggagagag aggtctcaga atggaaagat aaatatgaag
aaagcaggcg ggaagtgatg 8701 gaaatgagga aaatagtggc cgagtatgag
aagaccatcg ctcagatgat agaggacgaa 8761 cagagagaga agtcagtctc
ccaccagacg gtgcagcagc tggttctgga gaaggagcaa 8821 gccctggccg
acctgaactc cgtggagaag tctctggccg acctcttcag aagatatgag 8881
aagatgaagg aggtcctaga aggcttccgc aagaatgaag aggtgttgaa gagatgtgcg
8941 caggagtacc tgtcccgggt gaagaaggag gagcagaggt accaggccct
gaaggtgcac 9001 gcggaggaga aactggacag ggccaatgct gagattgctc
aggttcgagg caaggcccag 9061 caggagcaag ccgcccacca ggccagcctg
cggaaggagc agctgcgagt ggacgccctg 9121 gaaaggacgc tggagcagaa
gaataaagaa atagaagaac tcaccaagat ttgtgacgaa 9181 ctgattgcca
aaatggggaa aagctaactc tgaaccgaat gttttggact taactgttgc 9241
gtgcaatatg accgtcggca cactgctgtt cctccagttc catggacagg ttctgttttc
9301 actttttcgt atgcactact gtatttcctt tctaaataaa attgatttga
ttgtatgcag 9361 tactaaggag actatcagaa tttcttgcta ttggtttgca
ttttcctagt ataattcata 9421 gcaagttgac ctcagagttc ctgtatcagg
gagattgtct gattctctaa taaaagacac 9481 attgctgacc ttggccttgc
cctttgtaca caagttccca gggtgagcag cttttggatt 9541 taatatgaac
atgtacagcg tgcataggga ctcttgcctt aaggagtgta aacttgatct 9601
gcatttgctg atttgttttt aaaaaaacaa gaaatgcatg tttcaaataa aattctctat
9661 tgtaaataaa attttttctt tggatcttgg caaaaaaaaa aaaaaa
[0182] SEQ ID NO: 158 is the amino acid sequence of the
FGFR3-TACC3-1 fusion protein. The bolded text corresponds to the
FGFR3 protein:
TABLE-US-00029 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQ
EQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQ
RLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGE
DEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTP
SISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVV
ENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCK
VYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELE
VLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADE
AGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKI
SRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELP
ADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVA
VKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLY
VLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCA
YQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHN
LDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTL
GGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPS
QRPTFKQLVEDLDRVLTVTSTDFKESALRKQSLYLKFDPLLRDSPG
RPVPVATETSSMHGANETPSGRPREAKLVEFDFLGALDIPVPGPPP
GVPAPGGPPLSTGPIVDLLQYSQKDLDAVVKATQEENRELRSRCEE
LHGKNLELGKIMDRFEEVVYQAMEEVQKQKELSKAEIQKVLKEKDQ
LTTDLNSMEKSFSDLFKRFEKQKEVIEGYRKNEESLKKCVEDYLAR
ITQEGQRYQALKAHAEEKLQLANEEIAQVRSKAQAEALALQASLRK
EQMRIQSLEKTVEQKTKENEELTRICDDLISKMEKI
[0183] SEQ ID NO: 159 is the amino acid sequence of the
FGFR3-TACC3-2 fusion protein. The bolded text corresponds to the
FGFR3 protein:
TABLE-US-00030 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQ
EQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQ
RLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGE
DEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTP
SISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVV
ENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCK
VYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELE
VLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADE
AGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKI
SRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELP
ADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVA
VKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLY
VLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCA
YQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHN
LDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTL
GGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPS
QRPTFKQLVEDLDRVLTVTSTDVSAGSGLVPPAYAPPPAVPGHPSG
RPREAKLVEFDFLGALDIPVPGPPPGVPAPGGPPLSTGPIVDLLQY
SQKDLDAVVKATQEENRELRSRCEELHGKNLELGKIMDRFEEVVYQ
AMEEVQKQKELSKAEIQKVLKEKDQLTTDLNSMEKSFSDLFKRFEK
QKEVIEGYRKNEESLKKCVEDYLARITQEGQRYQALKAHAEEKLQL
ANEEIAQVRSKAQAEALALQASLRKEQMRIQSLEKTVEQKTKENEE LTRICDDLISKMEKI
[0184] SEQ ID NO: 160 is the amino acid sequence of the
FGFR3-TACC3-3 fusion protein. The bolded text corresponds to the
FGFR3 protein:
TABLE-US-00031 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQ
EQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQ
RLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGE
DEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTP
SISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVV
ENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCK
VYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELE
VLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADE
AGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKI
SRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELP
ADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVA
VKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLY
VLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCA
YQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHN
LDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTL
GGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPS
QRPTFKQLVEDLDRVLTVTSTDVPGPPPGVPAPGGPPLSTGPIVDL
LQYSQKDLDAVVKATQEENRELRSRCEELHGKNLELGKIMDRFEEV
VYQAMEEVQKQKELSKAEIQKVLKEKDQLTTDLNSMEKSFSDLFKR
FEKQKEVIEGYRKNEESLKKCVEDYLARITQEGQRYQALKAHAEEK
LQLANEEIAQVRSKAQAEALALQASLRKEQMRIQSLEKTVEQKTKE
NEELTRICDDLISKMEKI
[0185] SEQ ID NO: 161 is the amino acid sequence of the
FGFR3-TACC3-4 fusion protein. The bolded text corresponds to the
FGFR3 protein:
TABLE-US-00032 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQ
EQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQ
RLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGE
DEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTP
SISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVV
ENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCK
VYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELE
VLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADE
AGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKI
SRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELP
ADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVA
VKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLY
VLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCA
YQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHN
LDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTL
GGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPS
QRPTFKQLVEDLDRVLTVTSTDVKATQEENRELRSRCEELHGKNLE
LGKIMDRFEEVVYQAMEEVQKQKELSKAEIQKVLKEKDQLTTDLNS
MEKSFSDLFKRFEKQKEVIEGYRKNEESLKKCVEDYLARITQEGQR
YQALKAHAEEKLQLANEEIAQVRSKAQAEALALQASLRKEQMRIQS
LEKTVEQKTKENEELTRICDDLISKMEKI
[0186] SEQ ID NO: 539 is the amino acid sequence of
FGFR3ex17-TACC3ex11. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00033 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQ
LVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQV
LNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDT
GVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG
REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQT
YTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWL
KHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGE
YTCLAGNSIGESHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFF
LFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMS
SNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGE
GCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEM
MKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLD
YSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVL
VTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYT
HQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCT ##STR00094##
##STR00095## ##STR00096## ##STR00097##
[0187] SEQ ID NO: 540 is the amino acid sequence of
FGFR3ex17-TACC3ex8. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00034 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQ
LVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQV
LNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDT
GVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG
REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQT
YTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWL
KHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGE
YTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFF
LFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMS
SNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGE
GCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEM
MKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLD
YSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVL
VTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYT
HQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCT ##STR00098##
##STR00099## ##STR00100## ##STR00101## ##STR00102##
[0188] SEQ ID NO: 541 is the amino acid sequence of
FGFR3ex17-TACC3ex10. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00035 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVF
GSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHE
DSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYW
TRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGI
KLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPIL
QAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYV
TVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVL
PAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGS
PTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELEL
PADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKML
KDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKG
NLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQK
CIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAP
EALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMD
##STR00103##
[0189] SEQ ID NO: 542 is the amino acid sequence of
FGFR3ex17-TACC3ex6. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00036 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV
FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS
HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA
PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR
IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP
HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP
DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH
SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS
PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT
LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA
KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP
LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ
VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK
TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV
EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR ##STR00104##
[0190] SEQ ID NO: 543 is the amino acid sequence of
FGFR3ex18-TACC3ex13. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00037 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV
FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS
HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA
PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR
IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP
HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP
DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH
SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS
PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT
LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA
KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP
LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ
VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK
TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV
EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR ##STR00105##
[0191] SEQ ID NO: 544 is the amino acid sequence of
FGFR3ex18-TACC3ex9_INS66BP. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded. The
sequence corresponding the the 66 bp intronic insert is double
underlined:
TABLE-US-00038 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV
FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS
HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA
PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR
IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP
HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP
DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH
SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS
PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT
LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA
KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP
LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ
VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK
TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV
EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR ##STR00106##
[0192] SEQ ID NO: 545 is the amino acid sequence of
FGFR3ex18-TACC3ex5. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded:
TABLE-US-00039 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQL
VFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLN
ASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVD
TGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFR
GEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDV
LERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVN
GSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGN
SIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAA
VTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIA
RLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAE
AIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNII
NLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQ
LTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFG
LARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWE
IFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAP ##STR00107##
[0193] SEQ ID NO: 546 is the amino acid sequence of
FGFR3ex18-TACC3ex5_INS33 bp. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded. The
sequence corresponding the the 33 bp intronic insert is double
underlined:
TABLE-US-00040 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV
FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS
HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA
PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR
IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP
HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP
DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH
SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS
PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT
LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA
KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP
LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ
VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK
TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV
EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR ##STR00108##
[0194] SEQ ID NO: 547 is the amino acid sequence of
FGFR3ex18-TACC3ex4. The sequence corresponding to FGFR3 is
underlined. The sequence corresponding to TACC3 is shaded.
TABLE-US-00041 MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV
FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS
HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA
PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR
IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP
HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP
DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH
SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS
PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT
LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA
KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP
LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ
VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK
TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV
EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR ##STR00109##
[0195] The Genbank ID for the FGFR4 gene is 2264. Three isoforms
are listed for FGFR4, e.g., having Genebank Accession Nos.
NP.sub.--002002 (corresponding nucleotide sequence
NM.sub.--002011); NP.sub.--075252 (corresponding nucleotide
sequence NM.sub.--022963); NP.sub.--998812 (corresponding
nucleotide sequence NM.sub.--213647).
[0196] As used herein, a "FGFR fusion molecule" can be a nucleic
acid (e.g., synthetic, purified, and/or recombinant) which encodes
a polypeptide corresponding to a fusion protein comprising a
tyrosine kinase domain of an FGFR protein fused to a polypeptide
that constitutively activates the tyrosine kinase domain of the
FGFR protein, or a nucleic acid encoding a fusion protein
comprising a transforming acidic coiled-coil (TACC) domain fused to
a polypeptide with a tyrosine kinase domain, wherein the TACC
domain constitutively activates the tyrosine kinase domain. It can
also be a fusion protein comprising a tyrosine kinase domain of an
FGFR protein fused to a polypeptide that constitutively activates
the tyrosine kinase domain of the FGFR protein, or a fusion protein
comprising a transforming acidic coiled-coil (TACC) domain fused to
a polypeptide with a tyrosine kinase domain, wherein the TACC
domain constitutively activates the tyrosine kinase domain. For
example, a FGFR fusion molecule can include a FGFR1-TACC1 (e.g.,
comprising the amino acid sequence shown in SEQ ID NO: 150, or
comprising the nucleic acid sequence shown in SEQ ID NO: 88),
FGFR2-TACC2, FGFR3-TACC3 (e.g., comprising the amino acid sequence
shown in SEQ ID NOS: 79, 158-161, or 539-547 or comprising the
nucleic acid sequence shown in SEQ ID NOS: 80-82, 84, 94-145, 515,
517, 519-527, or 530-538), or other FGFR-TACC fusion proteins
(e.g., an N-terminal fragment of FGFR4 containing its tyrosine
kinase domain fused to a fragment of TACC1, TACC2, or TACC3). For
example, a FGFR fusion molecule can include a FGFR1-containing
fusion comprising the amino acid sequence corresponding to Genebank
Accession no. NP.sub.--001167534, NP.sub.--001167535,
NP.sub.--001167536, NP.sub.--001167537, NP.sub.--001167538,
NP.sub.--056934, NP.sub.--075593, NP.sub.--075594, or
NP.sub.--075598; or a FGFR1-containing fusion comprising the
nucleotide sequence corresponding to Genebank Accession no.
NM.sub.--001174063, NM.sub.--001174064, NM.sub.--001174065,
NM.sub.--001174066, NM.sub.--001174067, NM.sub.--015850,
NM.sub.--023105, NM.sub.--023106, or NM.sub.--023110. For example,
a FGFR fusion molecule can include a FGFR2-containing fusion
comprising the amino acid sequence corresponding to Genebank
Accession no. NP.sub.--000132, NP.sub.--001138385,
NP.sub.--001138386, NP.sub.--001138387, NP.sub.--001138388,
NP.sub.--001138389, NP.sub.--001138390, NP.sub.--001138391, or
NP.sub.--075259; or a FGFR2-containing fusion comprising the
nucleotide sequence corresponding to Genebank Accession no.
NM.sub.--000141, NM.sub.--001144913, NM.sub.--001144914,
NM.sub.--001144915, NM.sub.--001144916, NM.sub.--001144917,
NM.sub.--001144918, NM.sub.--001144919, or NM.sub.--022970. For
example, a FGFR fusion molecule can include a FGFR3-containing
fusion comprising the amino acid sequence corresponding to Genebank
Accession no. NP.sub.--000133, NP.sub.--001156685, or
NP.sub.--075254; or a FGFR3-containing fusion comprising the
nucleotide sequence corresponding to Genebank Accession no.
NM.sub.--000142, NM.sub.--001163213, or NM.sub.--022965. For
example, a FGFR fusion molecule can include a FGFR4-containing
fusion comprising the amino acid sequence corresponding to Genebank
Accession no. NP.sub.--002002, NP.sub.--075252, or NP.sub.--998812;
or a FGFR4-containing fusion comprising the nucleotide sequence
corresponding to Genebank Accession no. NM.sub.--002011,
NM.sub.--022963, or NM.sub.--213647. A FGFR fusion molecule can
also include a tyrosine kinase domain of an FGFR protein fused to a
protein encoded by any one of the genes listed in Table 7. A FGFR
fusion molecule can include a variant of the above described
examples, such as a fragment thereof. Table 7. Fusion Partners
TABLE-US-00042 gene gene gene gene ABCA13 C21orf29 CAMKK1 DNAJC6
ABCC1 CACNA1C CAMSAP1 DYRK3 ABCC12 CACNA1G CAMTA1 EIF2C2 ABCC6
CNTNAP4 CAP2 FAM184B ABL1 CUL3 CCDC147 FREM2 ADAM12 DMD CCDC158
GDPD2 ADCY10 DUSP27 CELF2 GLI3 ADCY2 ECE1 CILP IL1RN ADCY8 EYS
CMYA5 ISX AGBL4 FAM172A COL14A1 KIDINS220 AHNAK FAM184B CORO7 LRBA
ANXA7 FGFR4 CSMD2 LY75 AP4S1 ITGAV CUL3 MDH2 AQP2 LRP1 DDI2 MMP12
ARMC6 LY75 DEPDC5 N4BP2L2 ATP5B MAPKAP1 DEPDC7 NCF2 ATP6AP1L MYT1
DI10L NCOR1 ATP6V0D2 NCF2 DMD NCRNA00157 ATXN1 NCOR1 EDA NRXN3
BAHD1 NHSL2 EFHC1 PARP16 BBX NKAIN2 EFS PLA2G2F BCA10 NR3C1 EIF2C2
PLEK2 C15orf23 NUP188 ENTPD2 PRKCH C15orf33 OSBPL10 EYS PTPRS
C21orf29 PACSIN1 FAM160A1 ROBO1 C2CD3 PARP16 MUSK SASH3 C6orf170
PDZRN4 NEUROG1 SH3BP5 C7orf44 POLM NHSL2 SLC44A2 CACNA1C PPP1R3A
NR3C1 SLC5A4 CACNA1G PSEN1 ODZ1 SNX5 FAM168A PTPRD PCDH12 SORCS2
FAM172A PTPRS PLCL1 SRRM1 FAM192A RALYL PLEKHM3 SSX3 FAM19A2 RERE
PLOD3 STAG2 FBXL4 RIMBP2 PRKCH STK24 FH RNF216 PSEN1 SURF6 FREM2
SDAD1 SEPT5 SYNPO2 GAPVD1 SEC14L3 SLC44A2 TAF1 GLI3 SH3RF3 SNTA1
TMEM80 GPR182 SLC9A1 USP48 TNFRSF10B GSTA3 SMOC2 VSNL1 TTYH1 IGFBP3
SNX5 WDFY1 UNC93B1 ITGA9 TACC2 WISP2 VSNL1 ITGB2 SRGAP1 XRRA1 XRCC4
JOSD2 SSX3 LRRC4B ZNF410 KIDINS220 SUMF1 LRRK2 TRIOBP LAMA2 SYNPO2
MAPKAP1 TTYH1 LCLAT1 TNFRSF10B MST1R LRBA LIN9
[0197] For example, a FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 648-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can also include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 549-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 613-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 488-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-781 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 689-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-765 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 583-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-767 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 462-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-767 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 472-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-780 of FGFR3 (e.g. SEQ ID NO: 90) fused
to the amino acid sequence corresponding residues 432-838 of TACC3
(e.g. SEQ ID NO: 92). A FGFR fusion molecule can include a
FGFR1-containing fusion comprising the amino acid sequence
corresponding to residues 1-762 of FGFR1 (e.g. SEQ ID NOS: 146,
185) fused to the amino acid sequence corresponding residues
571-805 of TACC1 (e.g. SEQ ID NO: 148). For example, a FGFR fusion
molecule can include SEQ ID NOs: 539-543, 545, and 547.
[0198] The alteration in a chromosome region occupied by a FGFR
fusion molecule, e.g., a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or
other FGFR-TACC nucleic acid, can result in amino acid
substitutions, RNA splicing or processing, product instability, the
creation of stop codons, production of oncogenic fusion proteins,
frame-shift mutations, and/or truncated polypeptide production. A
FGFR fusion molecule can include FGFR and TACC exons joined in the
fused mRNA or cDNA. A FGFR fusion molecule can also include FGFR
and TACC exons joined in the fused mRNA or cDNA along with the
presence of FGFR or TACC introns that are spliced in the fusion
cDNA. FGFR or TACC introns can encode amino acids of the FGFR
fusion molecule. For example, a FGFR fusion molecule can include a
FGFR3-containing fusion comprising the amino acid sequence
corresponding to residues 1-765 of FGFR3 (e.g. SEQ ID NO: 90) fused
to a 22 amino acid sequence encoded by a TACC3 intron fused to the
amino acid sequence corresponding to residues 583-838 of TACC3
(e.g. SEQ ID NO: 92). For example, a FGFR fusion molecule can
include a FGFR3-containing fusion comprising the amino acid
sequence corresponding to residues 1-767 of FGFR3 (e.g. SEQ ID NO:
90) fused to a 11 amino acid sequence encoded by a TACC3 intron
fused to the amino acid sequence corresponding to residues 472-838
of TACC3 (e.g. SEQ ID NO: 92). For example, a FGFR fusion molecule
can include SEQ ID NOs: 544 and 546.
[0199] A FGFR fusion protein can also include a fusion protein
encoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3
comprises a combination of introns and exons 1-18 of FGFR3 located
on human chromosome 4p16 spliced 5' to a combination of introns and
exons 4-16 of TACC3 located on human chromosome 4p16, wherein a
genomic breakpoint occurs in any one of introns or exons 1-18 of
FGFR3 and any one of introns or exons 3-16 of TACC3. For example, a
FGFR fusion protein can also include a fusion protein encoded by an
FGFR3-TACC3 nucleic acid, wherein the nucleic acid comprises exons
1-17 of FGFR3 located on human chromosome 4p16 spliced 5' to exons
11-16 of TACC3 located on human chromosome 4p16. In one embodiment,
a genomic breakpoint occurs in intron 17 of FGFR3 and in intron 10
of TACC3. For example, a FGFR fusion protein can also include a
fusion protein encoded by an FGFR3-TACC3 nucleic acid, wherein the
nucleic acid comprises exons 1-17 of FGFR3 located on human
chromosome 4p16 spliced 5' to exons 8-16 of TACC3 located on human
chromosome 4p16. In one embodiment, a genomic breakpoint occurs in
intron 17 of FGFR3 and in intron 7 of TACC3. For example, a FGFR
fusion protein can also include a fusion protein encoded by an
FGFR3-TACC3 nucleic acid, wherein the nucleic acid comprises exons
1-17 of FGFR3 located on human chromosome 4p16 spliced 5' to exons
10-16 of TACC3 located on human chromosome 4p16. In one embodiment,
a genomic breakpoint occurs in exon 18 of FGFR3 and in intron 9 of
TACC3. For example, a FGFR fusion protein can also include a fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic
acid comprises exons 1-17 of FGFR3 located on human chromosome 4p16
spliced 5' to exons 6-16 of TACC3 located on human chromosome 4p16.
In one embodiment, a genomic breakpoint occurs in intron 17 of
FGFR3 and in intron 5 of TACC3. In one embodiment, a genomic
breakpoint occurs in intron 17 of FGFR3 and in exon 5 of TACC3. For
example, a FGFR fusion protein can also include a fusion protein
encoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic acid
comprises exons 1-18 of FGFR3 located on human chromosome 4p16
spliced 5' to exons 13-16 of TACC3 located on human chromosome
4p16. In one embodiment, a genomic breakpoint occurs in exon 18 of
FGFR3 and in intron 12 or exon 13 of TACC3. For example, a FGFR
fusion protein can also include a fusion protein encoded by an
FGFR3-TACC3 nucleic acid, wherein the nucleic acid comprises exons
1-18 of FGFR3 located on human chromosome 4p16 spliced 5' to a
portion of intron 8 of TACC3 and exons 9-16 of TACC3 located on
human chromosome 4p16. In one embodiment, a genomic breakpoint
occurs in exon 18 of FGFR3 and in intron 8 of TACC3. For example, a
FGFR fusion protein can also include a fusion protein encoded by an
FGFR3-TACC3 nucleic acid, wherein the nucleic acid comprises exons
1-18 of FGFR3 located on human chromosome 4p16 spliced 5' to exons
5-16 of TACC3 located on human chromosome 4p16. In one embodiment,
a genomic breakpoint occurs in exon 18 of FGFR3 and in intron 4 or
exon 5 of TACC3. For example, a FGFR fusion protein can also
include a fusion protein encoded by an FGFR3-TACC3 nucleic acid,
wherein the nucleic acid comprises exons 1-18 of FGFR3 located on
human chromosome 4p16 spliced 5' to a portion of intron 4 of TACC 3
and exons 5-16 of TACC3 located on human chromosome 4p16. In one
embodiment, a genomic breakpoint occurs in exon 18 of FGFR3 and in
intron 4 or exon 5 of TACC3. For example, a FGFR fusion protein can
also include a fusion protein encoded by an FGFR3-TACC3 nucleic
acid, wherein the nucleic acid comprises exons 1-18 of FGFR3
located on human chromosome 4p16 spliced 5' to exons 4-16 of TACC3
located on human chromosome 4p16. In one embodiment, a genomic
breakpoint occurs in exon 18 of FGFR3 and in intron 3 or exon 4 of
TACC3. For example, a FGFR fusion protein can also include a fusion
protein encoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic
acid comprises exons 1-17 of FGFR3 and a portion of intron 17 of
FGFR3 located on human chromosome 4p16 spliced 5' to exons 9-16 of
TACC3 located on human chromosome 4p16. In one embodiment, a
genomic breakpoint occurs in intron 17 of FGFR3 and in exon 9 of
TACC3. For example, a FGFR fusion protein can also include a fusion
protein encoded by an FGFR1-TACC1 nucleic acid, wherein the nucleic
acid comprises exons 1-17 of FGFR1 located on human chromosome 8p11
spliced 5' to exons 7-13 of TACC1 located on human chromosome 8p11.
In one embodiment, a genomic breakpoint occurs in intron 17 or exon
17 of FGFR1 and in intron 6 or exon 7 of TACC1.
[0200] In one embodiment, the coordinates comprising FGFR3
translocations comprise chr4:1,795,039-1,810,599. In a further
embodiment, the genomic breakpoint coordinate according to the
genome build GRCh37/hg19 for FGFR3 is chr4:1,808,808,
chr4:1,808,843, chr4:1,809,083, chr4:1,808,785, chr4:1,808,700,
chr4:1,808,864, chr4:1,808,678, chr4:1, 808,798, or chr4:1,808,723.
In a further embodiment, the coordinates comprising TACC3
translocations comprise chr4:1,723,217-1,746,905. In a further
embodiment, the genomic breakpoint coordinate according to the
genome build GRCh37/hg19 for TACC3 is chr4:1,732,648,
chr4:1,732,757, chr4:1,739,187, chr4:1,737,091, chr4:1,737,062,
chr4:1,737741, chr4:1,739,662, chr4:1,739,600, or
chr4:1,738,989.
[0201] The nucleic acid can be any type of nucleic acid, including
genomic DNA, complementary DNA (cDNA), recombinant DNA, synthetic
or semi-synthetic DNA, as well as any form of corresponding RNA. A
cDNA is a form of DNA artificially synthesized from a messenger RNA
template and is used to produce gene clones. A synthetic DNA is
free of modifications that can be found in cellular nucleic acids,
including, but not limited to, histones and methylation. For
example, a nucleic acid encoding a FGFR fusion molecule can
comprise a recombinant nucleic acid encoding such a protein. The
nucleic acid can be a non-naturally occurring nucleic acid created
artificially (such as by assembling, cutting, ligating or
amplifying sequences). It can be double-stranded or
single-stranded.
[0202] The invention further provides for nucleic acids that are
complementary to a FGFR fusion molecule. Complementary nucleic
acids can hybridize to the nucleic acid sequence described above
under stringent hybridization conditions. Non-limiting examples of
stringent hybridization conditions include temperatures above
30.degree. C., above 35.degree. C., in excess of 42.degree. C.,
and/or salinity of less than about 500 mM, or less than 200 mM.
Hybridization conditions can be adjusted by the skilled artisan via
modifying the temperature, salinity and/or the concentration of
other reagents such as SDS or SSC.
[0203] According to the invention, protein variants can include
amino acid sequence modifications. For example, amino acid sequence
modifications fall into one or more of three classes:
substitutional, insertional or deletional variants. Insertions can
include amino and/or carboxyl terminal fusions as well as
intrasequence insertions of single or multiple amino acid residues.
Insertions ordinarily will be smaller insertions than those of
amino or carboxyl terminal fusions, for example, on the order of
one to four residues. Deletions are characterized by the removal of
one or more amino acid residues from the protein sequence. These
variants ordinarily are prepared by site-specific mutagenesis of
nucleotides in the DNA encoding the protein, thereby producing DNA
encoding the variant, and thereafter expressing the DNA in
recombinant cell culture.
[0204] In one embodiment, a FGFR fusion molecule comprises a
protein or polypeptide encoded by a nucleic acid sequence encoding
a FGFR fusion molecule, such as the sequences shown in SEQ ID NOS:
80-82, 84, 94-145, 515, 517, 519-527, or 530-538. In some
embodiments, the nucleic acid sequence encoding a FGFR fusion
molecule is about 70%, about 75%, about 80%, about 85%, about 90%,
about 93%, about 95%, about 97%, about 98%, or about 99% identical
to SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or 530-538. In
another embodiment, the polypeptide can be modified, such as by
glycosylations and/or acetylations and/or chemical reaction or
coupling, and can contain one or several non-natural or synthetic
amino acids. An example of a FGFR fusion molecule is the
polypeptide having the amino acid sequence shown in SEQ ID NOS: 79,
88, 150, 158-161, or 539-547. In some embodiments, the FGFR fusion
molecule that is a polypeptide is about 70%, about 75%, about 80%,
about 85%, about 90%, about 93%, about 95%, about 97%, about 98%,
or about 99% identical to SEQ ID NOS: 79, 88, 150, 158-161, or
539-547. In another embodiment, a FGFR fusion molecule can be a
fragment of a FGFR fusion protein. For example, the FGFR fusion
molecule can encompass any portion of at least about 8 consecutive
amino acids of SEQ ID NOS: 79, 88, 150, 158-161, or 539-547. The
fragment can comprise at least about 10 amino acids, a least about
20 amino acids, at least about 30 amino acids, at least about 40
amino acids, a least about 50 amino acids, at least about 60 amino
acids, or at least about 75 amino acids of SEQ ID NOS: 79, 88, 150,
158-161, or 539-547. Fragments include all possible amino acid
lengths between about 8 and 100 about amino acids, for example,
lengths between about 10 and 100 amino acids, between about 15 and
100 amino acids, between about 20 and 100 amino acids, between
about 35 and 100 amino acids, between about 40 and 100 amino acids,
between about 50 and 100 amino acids, between about 70 and 100
amino acids, between about 75 and 100 amino acids, or between about
80 and 100 amino acids. Fragments include all possible amino acid
lengths between about 100 and 800 amino acids, for example, lengths
between about 125 and 800 amino acids, between about 150 and 800
amino acids, between about 175 and 800 amino acids, between about
200 and 800 amino acids, between about 225 and 800 amino acids,
between about 250 and 800 amino acids, between about 275 and 800
amino acids, between about 300 and 800 amino acids, between about
325 and 800 amino acids, between about 350 and 800 amino acids,
between about 375 and 800 amino acids, between about 400 and 800
amino acids, between about 425 and 800 amino acids, between about
450 and 800 amino acids, between about 475 and 800 amino acids,
between about 500 and 800 amino acids, between about 525 and 800
amino acids, between about 550 and 800 amino acids, between about
575 and 800 amino acids, between about 600 and 800 amino acids,
between about 625 and 800 amino acids, between about 650 and 800
amino acids, between about 675 and 800 amino acids, between about
700 and 800 amino acids, between about 725 and 800 amino acids,
between about 750 and 800 amino acids, or between about 775 and 800
amino acids.
[0205] Chemical Synthesis.
[0206] Nucleic acid sequences encoding a FGFR fusion molecule can
be synthesized, in whole or in part, using chemical methods known
in the art. Alternatively, a polypeptide can be produced using
chemical methods to synthesize its amino acid sequence, such as by
direct peptide synthesis using solid-phase techniques. Protein
synthesis can either be performed using manual techniques or by
automation. Automated synthesis can be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer).
[0207] Optionally, polypeptides fragments can be separately
synthesized and combined using chemical methods to produce a
full-length molecule. For example, these methods can be utilized to
synthesize a fusion protein of the invention. In one embodiment,
the fusion protein comprises a tyrosine kinase domain of an FGFR
protein fused to a polypeptide that constitutively activates the
tyrosine kinase domain of the FGFR protein. In another embodiment,
a fusion protein comprises a transforming acidic coiled-coil (TACC)
domain fused to a polypeptide with a tyrosine kinase domain,
wherein the TACC domain constitutively activates the tyrosine
kinase domain. An example of a fusion protein is the FGFR1-TACC1
polypeptide, which comprises the amino acid sequence shown in SEQ
ID NO: 150. An example of a fusion protein is the FGFR3-TACC3
polypeptide, which has the amino acid sequence comprising SEQ ID
NO: 79, 158, 159, 160, 161, 539, 540, 541, 542, 543, 544, 545, 546,
or 547.
[0208] Obtaining, Purifying and Detecting FGFR Fusion
Molecules.
[0209] A polypeptide encoded by a nucleic acid, such as a nucleic
acid encoding a FGFR fusion molecule, or a variant thereof, can be
obtained by purification from human cells expressing a protein or
polypeptide encoded by such a nucleic acid. Non-limiting
purification methods include size exclusion chromatography,
ammonium sulfate fractionation, ion exchange chromatography,
affinity chromatography, and preparative gel electrophoresis.
[0210] A synthetic polypeptide can be substantially purified via
high performance liquid chromatography (HPLC), such as ion exchange
chromatography (IEX-HPLC). The composition of a synthetic
polypeptide, such as a FGFR fusion molecule, can be confirmed by
amino acid analysis or sequencing.
[0211] Other constructions can also be used to join a nucleic acid
sequence encoding a polypeptide/protein of the claimed invention to
a nucleotide sequence encoding a polypeptide domain which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). Including cleavable linker sequences (i.e., those
specific for Factor Xa or enterokinase (Invitrogen, San Diego,
Calif.)) between the purification domain and a polypeptide encoded
by a nucleic acid of the invention also can be used to facilitate
purification. For example, the skilled artisan can use an
expression vector encoding 6 histidine residues that precede a
thioredoxin or an enterokinase cleavage site in conjunction with a
nucleic acid of interest. The histidine residues facilitate
purification by immobilized metal ion affinity chromatography,
while the enterokinase cleavage site provides a means for purifying
the polypeptide encoded by, for example, an FGFR1-TACC1,
FGFR2-TACC2, FGFR3-TACC3, other FGFR-TACC, FGFR-containing, or TACC
containing nucleic acid.
[0212] Host cells which contain a nucleic acid encoding a FGFR
fusion molecule, and which subsequently express the same, can be
identified by various procedures known to those of skill in the
art. These procedures include, but are not limited to, DNA-DNA or
DNA-RNA hybridizations and protein bioassay or immunoassay
techniques which include membrane, solution, or chip-based
technologies for the detection and/or quantification of nucleic
acid or protein. For example, the presence of a nucleic acid
encoding a FGFR fusion molecule can be detected by DNA-DNA or
DNA-RNA hybridization or amplification using probes or fragments of
nucleic acids encoding the same. In one embodiment, a nucleic acid
fragment of a FGFR fusion molecule can encompass any portion of at
least about 8 consecutive nucleotides of SEQ ID NOS: 80-82, 84,
94-145, 515, 517, 519-527, or 530-538. In another embodiment, the
fragment can comprise at least about 10 consecutive nucleotides, at
least about 15 consecutive nucleotides, at least about 20
consecutive nucleotides, or at least about 30 consecutive
nucleotides of SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527,
530-538. Fragments can include all possible nucleotide lengths
between about 8 and about 100 nucleotides, for example, lengths
between about 15 and about 100 nucleotides, or between about 20 and
about 100 nucleotides. Nucleic acid amplification-based assays
involve the use of oligonucleotides selected from sequences
encoding a FGFR fusion molecule nucleic acid, or FGFR fusion
molecule nucleic acid to detect transformants which contain a
nucleic acid encoding a protein or polypeptide of the same.
[0213] Protocols are known in the art for detecting and measuring
the expression of a polypeptide encoded by a nucleic acid, such as
a nucleic acid encoding a FGFR fusion molecule, using either
polyclonal or monoclonal antibodies specific for the polypeptide.
Non-limiting examples include enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), immunostaining, and fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay using monoclonal antibodies reactive to two
non-interfering epitopes on a polypeptide encoded by a nucleic
acid, such as a nucleic acid encoding a FGFR fusion molecule, can
be used, or a competitive binding assay can be employed.
[0214] Labeling and conjugation techniques are known by those
skilled in the art and can be used in various nucleic acid and
amino acid assays. Methods for producing labeled hybridization or
PCR probes for detecting sequences related to nucleic acid
sequences encoding a protein, such as FGFR fusion molecule,
include, but are not limited to, oligolabeling, nick translation,
end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, nucleic acid sequences, such as nucleic acids
encoding a FGFR fusion molecule, can be cloned into a vector for
the production of an mRNA probe. Such vectors are known in the art,
are commercially available, and can be used to synthesize RNA
probes in vitro by addition of labeled nucleotides and an
appropriate RNA polymerase such as T7, T3, or SP6. These procedures
can be conducted using a variety of commercially available kits
(Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable
reporter molecules or labels which can be used for ease of
detection include radionuclides, enzymes, and fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, and/or magnetic particles.
[0215] A fragment can be a fragment of a protein, such as a FGFR
fusion protein. For example, a fragment of a FGFR fusion molecule
can encompass any portion of at least about 8 consecutive amino
acids of SEQ ID NOS: 79, 88, 150, 158-161, or 539-547. The fragment
can comprise at least about 10 consecutive amino acids, at least
about 20 consecutive amino acids, at least about 30 consecutive
amino acids, at least about 40 consecutive amino acids, a least
about 50 consecutive amino acids, at least about 60 consecutive
amino acids, at least about 70 consecutive amino acids, at least
about 75 consecutive amino acids, at least about 80 consecutive
amino acids, at least about 85 consecutive amino acids, at least
about 90 consecutive amino acids, at least about 95 consecutive
amino acids, at least about 100 consecutive amino acids, at least
about 200 consecutive amino acids, at least about 300 consecutive
amino acids, at least about 400 consecutive amino acids, at least
about 500 consecutive amino acids, at least about 600 consecutive
amino acids, at least about 700 consecutive amino acids, or at
least about 800 consecutive amino acids of SEQ ID NOS: 79, 88, 150,
158-161, or 539-547. Fragments include all possible amino acid
lengths between about 8 and 100 about amino acids, for example,
lengths between about 10 and about 100 amino acids, between about
15 and about 100 amino acids, between about 20 and about 100 amino
acids, between about 35 and about 100 amino acids, between about 40
and about 100 amino acids, between about 50 and about 100 amino
acids, between about 70 and about 100 amino acids, between about 75
and about 100 amino acids, or between about 80 and about 100 amino
acids.
Cell Transfection
[0216] Host cells transformed with a nucleic acid sequence of
interest can be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
polypeptide produced by a transformed cell can be secreted or
contained intracellularly depending on the sequence and/or the
vector used. Expression vectors containing a nucleic acid sequence,
such as a nucleic acid encoding a FGFR fusion molecule, can be
designed to contain signal sequences which direct secretion of
soluble polypeptide molecules encoded by the nucleic acid. Cell
transfection and culturing methods are described in more detail
below.
[0217] A eukaryotic expression vector can be used to transfect
cells in order to produce proteins encoded by nucleotide sequences
of the vector, e.g. those encoding a FGFR fusion molecule.
Mammalian cells can contain an expression vector (for example, one
that contains a nucleic acid encoding a fusion protein comprising a
tyrosine kinase domain of an FGFR protein fused to a polypeptide
that constitutively activates the tyrosine kinase domain of the
FGFR protein, or a nucleic acid encoding a fusion protein comprises
a transforming acidic coiled-coil (TACC) domain fused to a
polypeptide with a tyrosine kinase domain, wherein the TACC domain
constitutively activates the tyrosine kinase domain) via
introducing the expression vector into an appropriate host cell via
methods known in the art.
[0218] A host cell strain can be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed polypeptide encoded by a nucleic acid, in the desired
fashion. Such modifications of the polypeptide include, but are not
limited to, acetylation, carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational
processing which cleaves a "prepro" form of the polypeptide also
can be used to facilitate correct insertion, folding and/or
function. Different host cells which have specific cellular
machinery and characteristic mechanisms for post-translational
activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available
from the American Type Culture Collection (ATCC; 10801 University
Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure
the correct modification and processing of the foreign protein.
[0219] An exogenous nucleic acid can be introduced into a cell via
a variety of techniques known in the art, such as lipofection,
microinjection, calcium phosphate or calcium chloride
precipitation, DEAE-dextran-mediated transfection, or
electroporation. Electroporation is carried out at approximate
voltage and capacitance to result in entry of the DNA construct(s)
into cells of interest (such as glioma cells (cell line SF188),
neuroblastoma cells (cell lines IMR-32, SK-N-SH, SH-F and SH-N),
astrocytes and the like). Other transfection methods also include
modified calcium phosphate precipitation, polybrene precipitation,
liposome fusion, and receptor-mediated gene delivery.
[0220] Cells that will be genetically engineered can be primary and
secondary cells obtained from various tissues, and include cell
types which can be maintained and propagated in culture.
Non-limiting examples of primary and secondary cells include
epithelial cells, neural cells, endothelial cells, glial cells,
fibroblasts, muscle cells (such as myoblasts) keratinocytes, formed
elements of the blood (e.g., lymphocytes, bone marrow cells), and
precursors of these somatic cell types.
[0221] Vertebrate tissue can be obtained by methods known to one
skilled in the art, such a punch biopsy or other surgical methods
of obtaining a tissue source of the primary cell type of interest.
In one embodiment, a punch biopsy or removal (e.g., by aspiration)
can be used to obtain a source of cancer cells (for example, glioma
cells, neuroblastoma cells, and the like). A mixture of primary
cells can be obtained from the tissue, using methods readily
practiced in the art, such as explanting or enzymatic digestion
(for examples using enzymes such as pronase, trypsin, collagenase,
elastase dispase, and chymotrypsin). Biopsy methods have also been
described in U.S. Pat. No. 7,419,661 and PCT application
publication WO 2001/32840, and each are hereby incorporated by
reference.
[0222] Primary cells can be acquired from the individual to whom
the genetically engineered primary or secondary cells are
administered. However, primary cells can also be obtained from a
donor, other than the recipient, of the same species. The cells can
also be obtained from another species (for example, rabbit, cat,
mouse, rat, sheep, goat, dog, horse, cow, bird, or pig). Primary
cells can also include cells from a purified vertebrate tissue
source grown attached to a tissue culture substrate (for example,
flask or dish) or grown in a suspension; cells present in an
explant derived from tissue; both of the aforementioned cell types
plated for the first time; and cell culture suspensions derived
from these plated cells. Secondary cells can be plated primary
cells that are removed from the culture substrate and replated, or
passaged, in addition to cells from the subsequent passages.
Secondary cells can be passaged one or more times. These primary or
secondary cells can contain expression vectors having a gene that
encodes a FGFR fusion molecule.
Cell Culturing
[0223] Various culturing parameters can be used with respect to the
host cell being cultured. Appropriate culture conditions for
mammalian cells are well known in the art (Cleveland W L, et al., J
Immunol Methods, 1983, 56(2): 221-234) or can be determined by the
skilled artisan (see, for example, Animal Cell Culture: A Practical
Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford
University Press: New York, 1992)). Cell culturing conditions can
vary according to the type of host cell selected. Commercially
available medium can be utilized. Non-limiting examples of medium
include, for example, Minimal Essential Medium (MEM, Sigma, St.
Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma); Ham's
F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan,
Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media,
which are formulated for various cell types, e.g., CD-CHO Medium
(Invitrogen, Carlsbad, Calif.).
[0224] The cell culture media can be supplemented as necessary with
supplementary components or ingredients, including optional
components, in appropriate concentrations or amounts, as necessary
or desired. Cell culture medium solutions provide at least one
component from one or more of the following categories: (1) an
energy source, usually in the form of a carbohydrate such as
glucose; (2) all essential amino acids, and usually the basic set
of twenty amino acids plus cysteine; (3) vitamins and/or other
organic compounds required at low concentrations; (4) free fatty
acids or lipids, for example linoleic acid; and (5) trace elements,
where trace elements are defined as inorganic compounds or
naturally occurring elements that can be required at very low
concentrations, usually in the micromolar range.
[0225] The medium also can be supplemented electively with one or
more components from any of the following categories: (1) salts,
for example, magnesium, calcium, and phosphate; (2) hormones and
other growth factors such as, serum, insulin, transferrin, and
epidermal growth factor; (3) protein and tissue hydrolysates, for
example peptone or peptone mixtures which can be obtained from
purified gelatin, plant material, or animal byproducts; (4)
nucleosides and bases such as, adenosine, thymidine, and
hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as
gentamycin or ampicillin; (7) cell protective agents, for example
pluronic polyol; and (8) galactose. In one embodiment, soluble
factors can be added to the culturing medium.
[0226] The mammalian cell culture that can be used with the present
invention is prepared in a medium suitable for the type of cell
being cultured. In one embodiment, the cell culture medium can be
any one of those previously discussed (for example, MEM) that is
supplemented with serum from a mammalian source (for example, fetal
bovine serum (FBS)). In another embodiment, the medium can be a
conditioned medium to sustain the growth of host cells.
[0227] Three-dimensional cultures can be formed from agar (such as
Gey's Agar), hydrogels (such as matrigel, agarose, and the like;
Lee et al., (2004) Biomaterials 25: 2461-2466) or polymers that are
cross-linked. These polymers can comprise natural polymers and
their derivatives, synthetic polymers and their derivatives, or a
combination thereof. Natural polymers can be anionic polymers,
cationic polymers, amphipathic polymers, or neutral polymers.
Non-limiting examples of anionic polymers can include hyaluronic
acid, alginic acid (alginate), carageenan, chondroitin sulfate,
dextran sulfate, and pectin. Some examples of cationic polymers,
include but are not limited to, chitosan or polylysine. (Peppas et
al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug
Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944:
62-73). Examples of amphipathic polymers can include, but are not
limited to collagen, gelatin, fibrin, and carboxymethyl chitin.
Non-limiting examples of neutral polymers can include dextran,
agarose, or pullulan. (Peppas et al., (2006) Adv Mater. 18:
1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12;
Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).
[0228] Cells to be cultured can harbor introduced expression
vectors, such as plasmids. The expression vector constructs can be
introduced via transformation, microinjection, transfection,
lipofection, electroporation, or infection. The expression vectors
can contain coding sequences, or portions thereof, encoding the
proteins for expression and production. Expression vectors
containing sequences encoding the produced proteins and
polypeptides, as well as the appropriate transcriptional and
translational control elements, can be generated using methods well
known to and practiced by those skilled in the art. These methods
include synthetic techniques, in vitro recombinant DNA techniques,
and in vivo genetic recombination which are described in J.
Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al.,
1989, Current Protocols in Molecular Biology, John Wiley &
Sons, New York, N.Y.
FGFR Fusion Molecule Inhibitors
[0229] The invention provides methods for use of compounds that
decrease the expression level or activity of a FGFR fusion molecule
in a subject. In addition, the invention provides methods for using
compounds for the treatment of a gene-fusion associated cancer. In
one embodiment, the gene-fusion associated cancer is an epithelial
cancer. In one embodiment, the gene-fusion associated cancer
comprises glioblastoma multiforme, breast cancer, lung cancer,
prostate cancer, or colorectal carcinoma.
[0230] As used herein, a "FGFR fusion molecule inhibitor" refers to
a compound that interacts with a FGFR fusion molecule of the
invention and modulates its activity and/or its expression. For
example, the compound can decrease the activity or expression of a
FGFR fusion molecule. The compound can be an antagonist of a FGFR
fusion molecule (e.g., a FGFR fusion molecule inhibitor). Some
non-limiting examples of FGFR fusion molecule inhibitors include
peptides (such as peptide fragments comprising a FGFR fusion
molecule, or antibodies or fragments thereof), small molecules, and
nucleic acids (such as siRNA or antisense RNA specific for a
nucleic acid comprising a FGFR fusion molecule). Antagonists of a
FGFR fusion molecule decrease the amount or the duration of the
activity of an FGFR fusion protein. In one embodiment, the fusion
protein comprises a tyrosine kinase domain of an FGFR protein fused
to a polypeptide that constitutively activates the tyrosine kinase
domain of the FGFR protein (e.g., FGFR1-TACC1, FGFR2-TACC2,
FGFR3-TACC3 or other FGFR-TACC), or a fusion protein comprises a
transforming acidic coiled-coil (TACC) domain fused to a
polypeptide with a tyrosine kinase domain, wherein the TACC domain
constitutively activates the tyrosine kinase domain. Antagonists
include proteins, nucleic acids, antibodies, small molecules, or
any other molecule which decrease the activity of a FGFR fusion
molecule.
[0231] The term "modulate," as it appears herein, refers to a
change in the activity or expression of a FGFR fusion molecule. For
example, modulation can cause a decrease in protein activity,
binding characteristics, or any other biological, functional, or
immunological properties of a FGFR fusion molecule, such as an FGFR
fusion protein.
[0232] In one embodiment, a FGFR fusion molecule inhibitor can be a
peptide fragment of a FGFR fusion protein that binds to the protein
itself.
[0233] For example, the FGFR fusion polypeptide can encompass any
portion of at least about 8 consecutive amino acids of SEQ ID NOS:
79, 88, 150, 158-161, or 539-547. The fragment can comprise at
least about 10 consecutive amino acids, at least about 20
consecutive amino acids, at least about 30 consecutive amino acids,
at least about 40 consecutive amino acids, a least about 50
consecutive amino acids, at least about 60 consecutive amino acids,
at least about 70 consecutive amino acids, at least about 75
consecutive amino acids, at least about 80 consecutive amino acids,
at least about 85 consecutive amino acids, at least about 90
consecutive amino acids, at least about 95 consecutive amino acids,
at least about 100 consecutive amino acids, at least about 200
consecutive amino acids, at least about 300 consecutive amino
acids, at least about 400 consecutive amino acids, at least about
500 consecutive amino acids, at least about 600 consecutive amino
acids, at least about 700 consecutive amino acids, or at least
about 800 consecutive amino acids of SEQ ID NOS: 79, 88, 150,
158-161, or 539-547. Fragments include all possible amino acid
lengths between about 8 and 100 about amino acids, for example,
lengths between about 10 and about 100 amino acids, between about
15 and about 100 amino acids, between about 20 and about 100 amino
acids, between about 35 and about 100 amino acids, between about 40
and about 100 amino acids, between about 50 and about 100 amino
acids, between about 70 and about 100 amino acids, between about 75
and about 100 amino acids, or between about 80 and about 100 amino
acids. These peptide fragments can be obtained commercially or
synthesized via liquid phase or solid phase synthesis methods
(Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical
Approach. IRL Press, Oxford, England). The FGFR fusion peptide
fragments can be isolated from a natural source, genetically
engineered, or chemically prepared. These methods are well known in
the art.
[0234] A FGFR fusion molecule inhibitor can be a protein, such as
an antibody (monoclonal, polyclonal, humanized, chimeric, or fully
human), or a binding fragment thereof, directed against a FGFR
fusion molecule of the invention. An antibody fragment can be a
form of an antibody other than the full-length form and includes
portions or components that exist within full-length antibodies, in
addition to antibody fragments that have been engineered. Antibody
fragments can include, but are not limited to, single chain Fv
(scFv), diabodies, Fv, and (Fab').sub.2, triabodies, Fc, Fab, CDR1,
CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies,
bifunctional hybrid antibodies, framework regions, constant
regions, and the like (see, Maynard et al., (2000) Ann. Rev.
Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol.
9:395-402). Antibodies can be obtained commercially, custom
generated, or synthesized against an antigen of interest according
to methods established in the art (see U.S. Pat. Nos. 6,914,128,
5,780,597, 5,811,523; Roland E. Kontermann and Stefan Dubel
(editors), Antibody Engineering, Vol. I & II, (2010) 2.sup.nd
ed., Springer; Antony S. Dimitrov (editor), Therapeutic Antibodies:
Methods and Protocols (Methods in Molecular Biology), (2009),
Humana Press; Benny Lo (editor) Antibody Engineering: Methods and
Protocols (Methods in Molecular Biology), (2004) Humana Press, each
of which are hereby incorporated by reference in their entireties).
For example, antibodies directed to a FGFR fusion molecule can be
obtained commercially from Abcam, Santa Cruz Biotechnology, Abgent,
R&D Systems, Novus Biologicals, etc. Human antibodies directed
to a FGFR fusion molecule (such as monoclonal, humanized, fully
human, or chimeric antibodies) can be useful antibody therapeutics
for use in humans. In one embodiment, an antibody or binding
fragment thereof is directed against SEQ ID NOS: 79, 88, 150,
158-161, or 539-547.
[0235] Inhibition of RNA encoding a FGFR fusion molecule can
effectively modulate the expression of a FGFR fusion molecule.
Inhibitors are selected from the group comprising: siRNA;
interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed
DNAs; ribozymes; and antisense nucleic acids, which can be RNA,
DNA, or an artificial nucleic acid.
[0236] Antisense oligonucleotides, including antisense DNA, RNA,
and DNA/RNA molecules, act to directly block the translation of
mRNA by binding to targeted mRNA and preventing protein
translation. For example, antisense oligonucleotides of at least
about 15 bases and complementary to unique regions of the DNA
sequence encoding a FGFR fusion molecule can be synthesized, e.g.,
by conventional phosphodiester techniques (Dallas et al., (2006)
Med. Sci. Monit. 12(4):RA67-74; Kalota et al., (2006) Handb. Exp.
Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp.
Pharmacol. 173:243-59). Antisense nucleotide sequences include, but
are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA,
RNA and the like.
[0237] siRNA comprises a double stranded structure containing from
about 15 to about 50 base pairs, for example from about 21 to about
25 base pairs, and having a nucleotide sequence identical or nearly
identical to an expressed target gene or RNA within the cell. The
siRNA comprise a sense RNA strand and a complementary antisense RNA
strand annealed together by standard Watson-Crick base-pairing
interactions. The sense strand comprises a nucleic acid sequence
which is substantially identical to a nucleic acid sequence
contained within the target miRNA molecule. "Substantially
identical" to a target sequence contained within the target mRNA
refers to a nucleic acid sequence that differs from the target
sequence by about 3% or less. The sense and antisense strands of
the siRNA can comprise two complementary, single-stranded RNA
molecules, or can comprise a single molecule in which two
complementary portions are base-paired and are covalently linked by
a single-stranded "hairpin" area. See also, McMnaus and Sharp
(2002) Nat Rev Genetics, 3:737-47, and Sen and Blau (2006) FASEB
J., 20:1293-99, the entire disclosures of which are herein
incorporated by reference.
[0238] The siRNA can be altered RNA that differs from
naturally-occurring RNA by the addition, deletion, substitution
and/or alteration of one or more nucleotides. Such alterations can
include addition of non-nucleotide material, such as to the end(s)
of the siRNA or to one or more internal nucleotides of the siRNA,
or modifications that make the siRNA resistant to nuclease
digestion, or the substitution of one or more nucleotides in the
siRNA with deoxyribo-nucleotides. One or both strands of the siRNA
can also comprise a 3' overhang. As used herein, a 3' overhang
refers to at least one unpaired nucleotide extending from the
3'-end of a duplexed RNA strand. For example, the siRNA can
comprise at least one 3' overhang of from 1 to about 6 nucleotides
(which includes ribonucleotides or deoxyribonucleotides) in length,
or from 1 to about 5 nucleotides in length, or from 1 to about 4
nucleotides in length, or from about 2 to about 4 nucleotides in
length. For example, each strand of the siRNA can comprise 3'
overhangs of dithymidylic acid ("TT") or diuridylic acid
("uu").
[0239] siRNA can be produced chemically or biologically, or can be
expressed from a recombinant plasmid or viral vector (for example,
see U.S. Pat. No. 7,294,504 and U.S. Pat. No. 7,422,896, the entire
disclosures of which are herein incorporated by reference).
Exemplary methods for producing and testing dsRNA or siRNA
molecules are described in U.S. Patent Application Publication No.
2002/0173478 to Gewirtz, U.S. Pat. No. 8,071,559 to Hannon et al.,
and in U.S. Pat. No. 7,148,342 to Tolentino et al., the entire
disclosures of which are herein incorporated by reference.
[0240] In one embodiment, an siRNA directed to a human nucleic acid
sequence comprising a FGFR fusion molecule can be generated against
any one of SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527 or
530-538. In another embodiment, an siRNA directed to a human
nucleic acid sequence comprising a breakpoint of an FGFR fusion
molecule can be generated against any one of SEQ ID NOS: 1-77,
80-82, 84-145, 515, 517, 519-527 or 530-538. In one embodiment, the
hairpin sequences targeting the FGFR3 gene comprise SEQ ID NOS:
182, 183, or 184.
[0241] RNA polymerase III transcribed DNAs contain promoters, such
as the U6 promoter. These DNAs can be transcribed to produce small
hairpin RNAs in the cell that can function as siRNA or linear RNAs,
which can function as antisense RNA. The FGFR fusion molecule
inhibitor can comprise ribonucleotides, deoxyribonucleotides,
synthetic nucleotides, or any suitable combination such that the
target RNA and/or gene is inhibited. In addition, these forms of
nucleic acid can be single, double, triple, or quadruple stranded.
(see for example Bass (2001) Nature, 411:428-429; Elbashir et al.,
(2001) Nature, 411:494 498; U.S. Pat. No. 6,509,154; U.S. Patent
Application Publication No. 2003/0027783; and PCT Publication Nos.
WO 00/044895, WO 99/032619, WO 00/01846, WO 01/029058, WO
00/044914).
[0242] FGFR fusion molecule inhibitor can be a small molecule that
binds to a FGFR fusion protein described herein and disrupts its
function. Small molecules are a diverse group of synthetic and
natural substances generally having low molecular weights. They can
be isolated from natural sources (for example, plants, fungi,
microbes and the like), are obtained commercially and/or available
as libraries or collections, or synthesized. Candidate small
molecules that inhibit a FGFR fusion protein can be identified via
in silico screening or high-through-put (HTP) screening of
combinatorial libraries according to methods established in the art
(e.g., see Potyrailo et al., (2011) ACS Comb Sci. 13(6):579-633;
Mensch et al., (2009) J Pharm Sci. 98(12):4429-68; Schnur (2008)
Curr Opin Drug Discov Devel. 11(3):375-80; and Jhoti (2007) Ernst
Schering Found Symp Proc. (3):169-85, each of which are hereby
incorporated by reference in their entireties.) Most conventional
pharmaceuticals, such as aspirin, penicillin, and many
chemotherapeutics, are small molecules, can be obtained
commercially, can be chemically synthesized, or can be obtained
from random or combinatorial libraries as described below (see,
e.g., Werner et al., (2006) Brief Funct. Genomic Proteomic
5(1):32-6).
[0243] Non-limiting examples of FGFR fusion molecule inhibitors
include the FGFR inhibitors AZD4547 (see Gavine et al., (2012)
Cancer Res, 72(8); 2045-56; see also PCT Application Publication
No. WO 2008/075068, each of which are hereby incorporated by
reference in their entireties); NVP-BGJ398 (see Guagnano et al.,
(2011) J. Med. Chem., 54:7066-7083; see also U.S. Patent
Application Publication No. 2008-0312248 A1, each of which are
hereby incorporated by reference in their entireties); PD173074
(see Guagnano et al., (2011) J. Med. Chem., 54:7066-7083; see also
Mohammadi et al., (1998) EMBO J., 17:5896-5904, each of which are
hereby incorporated by reference in their entireties); NF449 (EMD
Millipore (Billerica, Mass.) Cat. No. 480420; see also Krejci,
(2010) the Journal of Biological Chemistry, 285(27):20644-20653,
which is hereby incorporated by reference in its entirety);
LY2874455 (Active Biochem; see Zhao et al. (2011) Mol Cancer Ther.
(11):2200-10; see also PCT Application Publication No. WO
2010129509, each of which are hereby incorporated by reference in
their entireties); TKI258 (Dovitinib); BIBF-1120
(Intedanib-Vargatef); BMS-582664 (Brivanib alaninate); AZD-2171
(Cediranib); TSU-68 (Orantinib); AB-1010 (Masitinib); AP-24534
(Ponatinib); and E-7080 (by Eisai). A non-limiting example of an
FGFR fusion molecule inhibitor includes the TACC inhibitor KHS101
(Wurdak et al., (2010) PNAS, 107(38): 16542-47, which is hereby
incorporated by reference in its entirety).
[0244] Structures of FGFR fusion molecule inhibitors useful for the
invention include, but are not limited to: the FGFR inhibitor
AZD4547,
##STR00110##
the FGFR inhibitor NVP-BGJ398,
##STR00111##
the FGFR inhibitor PD173074,
##STR00112##
the FGFR inhibitor LY2874455
##STR00113##
and the FGFR inhibitor NF449 (EMD Millipore (Billerica, Mass.) Cat.
No. 480420),
##STR00114##
[0245] Other FGFR inhibitors include, but are not limited to:
##STR00115## ##STR00116##
[0246] In other embodiments, the FGFR fusion molecule inhibitor
comprises an oral pan-FGFR tyrosine kinase inhibitor. In other
embodiments, the FGFR fusion molecule inhibitor comprises
JNJ-42756493. Structures of FGFR fusion molecule inhibitors useful
for the invention include, but are not limited to: the FGFR
inhibitor JNJ-42756493.
[0247] A structure of an FGFR fusion molecule inhibitor useful for
the invention include, but is not limited to the TACC inhibitor
KHS101,
##STR00117##
Assessment and Therapeutic Treatment
[0248] The invention provides a method of decreasing the growth of
a solid tumor in a subject. The tumor is associated with, but not
limited to, glioblastoma multiforme, breast cancer, lung cancer,
prostate cancer, or colorectal carcinoma. In another embodiment,
the tumor is associated with, but not limited to, bladder
carcinoma, squamous lung carcinoma and head and neck carcinoma. In
one embodiment, the tumor is associated with, but not limited to,
glioma. In one embodiment, the tumor is associated with, but not
limited to, grade II or III glioma. In one embodiment, the tumor is
associated with, but not limited to, IDH wild-type grade II or III
glioma. In one embodiment, the method comprises detecting the
presence of a FGFR fusion molecule in a sample obtained from a
subject. In some embodiments, the sample is incubated with an agent
that binds to an FGFR fusion molecule, such as an antibody, a
probe, a nucleic acid primer, and the like. In further embodiments,
the method comprises administering to the subject an effective
amount of a FGFR fusion molecule inhibitor, wherein the inhibitor
decreases the size of the solid tumor. In further embodiments, the
method comprises further detecting the presence of IDH1 mutations,
EGFR amplification, CDK4 amplification, or MDM2 amplification. In
further embodiments, a FGFR fusion molecule inhibitor can be
administered in combination with CDK4 inhibitors, MDM2 inhibitors,
or a combination thereof.
[0249] The invention also provides a method for treating or
preventing a gene-fusion associated cancer in a subject. In one
embodiment, the gene-fusion associated cancer comprises an
epithelial cancer. In one embodiment, the gene-fusion associated
cancer comprises glioblastoma multiforme, breast cancer, lung
cancer, prostate cancer, or colorectal carcinoma. In some
embodiments, the epithelial cancer comprises bladder urothelial
carcinoma, breast carcinoma, colorectal cancer, prostate carcinoma,
lung squamous cell carcinoma, head and neck squamous cell
carcinoma, or a combination of the epithelial cancers described. In
one embodiment, the gene-fusion associated cancer comprises glioma.
In one embodiment, the gene-fusion associated cancer comprises
grade II or III glioma. In one embodiment, the gene-fusion
associated cancer comprises IDH wild-type grade II or III glioma.
In one embodiment, the method comprises detecting the presence of a
FGFR fusion molecule in a sample obtained from a subject, the
presence of the fusion being indicative of a gene-fusion associated
cancer, and, administering to the subject in need a therapeutic
treatment against a gene-fusion associated cancer. In some
embodiments, the sample is incubated with an agent that binds to an
FGFR fusion molecule, such as an antibody, a probe, a nucleic acid
primer, and the like. In further embodiments, the method comprises
further detecting the presence of IDH1 mutations, EGFR
amplification, CDK4 amplification, or MDM2 amplification. In
further embodiments, an agent that binds to an FGFR fusion molecule
can be administered in combination with CDK4 inhibitors, MDM2
inhibitors, or a combination thereof.
[0250] The invention also provides a method for decreasing in a
subject in need thereof the expression level or activity of a
fusion protein comprising the tyrosine kinase domain of an FGFR
protein fused to a polypeptide that constitutively activates the
tyrosine kinase domain of the FGFR protein. In some embodiments,
the method comprises obtaining a biological sample from the
subject. In some embodiments, the sample is incubated with an agent
that binds to an FGFR fusion molecule, such as an antibody, a
probe, a nucleic acid primer, and the like. In some embodiments,
the method comprises administering to the subject a therapeutic
amount of a composition comprising an admixture of a
pharmaceutically acceptable carrier an inhibitor of the fusion
protein of the invention. In one embodiment, the inhibitor is
JNJ-42756493. In another embodiment, the method further comprises
determining the fusion protein expression level or activity. In
another embodiment, the method further comprises detecting whether
the fusion protein expression level or activity is decreased as
compared to the fusion protein expression level or activity prior
to administration of the composition, thereby decreasing the
expression level or activity of the fusion protein. In some
embodiments, the fusion protein is an FGFR-TACC fusion protein. In
further embodiments, the method comprises further detecting the
presence of IDH1 mutations, EGFR amplification, CDK4 amplification,
or MDM2 amplification.
[0251] The administering step in each of the claimed methods can
comprise a drug administration, such as FGFR fusion molecule
inhibitor (for example, a pharmaceutical composition comprising an
antibody that specifically binds to a FGFR fusion molecule or a
fragment thereof; a small molecule that specifically binds to a
FGFR protein; a small molecule that specifically binds to a TACC
protein; an antisense RNA or antisense DNA that decreases
expression of a FGFR fusion molecule; a siRNA that specifically
targets a gene encoding a FGFR fusion molecule; a small molecule
such as JNJ-42756493; or a combination thereof). In one embodiment,
the therapeutic molecule to be administered comprises a polypeptide
of a FGFR fusion molecule, comprising at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
93%, at least about 95%, at least about 97%, at least about 98%, at
least about 99%, or 100% of the amino acid sequence of SEQ ID NOS:
79, 88, 150, 158-161, or 539-547 and exhibits the function of
decreasing expression of such a protein, thus treating a gene
fusion-associated cancer. In another embodiment, administration of
the therapeutic molecule decreases the size of the solid tumor
associated with glioblastoma multiforme, breast cancer, lung
cancer, prostate cancer, colorectal carcinoma, bladder carcinoma,
squamous lung carcinoma and head and neck carcinoma, glioma, grade
II or III glioma, or IDH wild-type grade II or III glioma. In
further embodiments, the therapeutic molecule can be administered
in combination with CDK4 inhibitors, MDM2 inhibitors, or a
combination thereof.
[0252] In another embodiment, the therapeutic molecule to be
administered comprises an siRNA directed to a human nucleic acid
sequence comprising a FGFR fusion molecule. In one embodiment, the
siRNA is directed to any one of SEQ ID NOS: 80-82, 84, 94-145, 515,
517, 519-527, or 530-538. In another embodiment, the siRNA is
directed to any one of SEQ ID NOS: 1-77, 80-82, 84-145, 515, 517,
519-527, or 530-538. In a further embodiment, the therapeutic
molecule to be administered comprises an antibody or binding
fragment thereof, which is directed against SEQ ID NOS: 79, 88,
150, 158-161, or 539-547. In some embodiments, the therapeutic
molecule to be administered comprises a small molecule that
specifically binds to a FGFR protein, such as AZD4547, NVP-BGJ398,
PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68,
AB1010, AP24534, E-7080, or LY2874455. In some embodiments, the
therapeutic molecule to be administered is JNJ-42756493. In other
embodiments, the therapeutic molecule to be administered comprises
a small molecule that specifically binds to a TACC protein, such as
KHS101.
[0253] In one embodiment, the invention provides for the detection
of a chromosomal rearrangement at given chromosomal coordinates. In
another embodiment, the detection or determination comprises
nucleic acid sequencing, selective hybridization, selective
amplification, gene expression analysis, or a combination thereof.
In another embodiment, the detection or determination comprises
protein expression analysis, for example by western blot analysis,
immunostaining, ELISA, or other antibody detection methods.
[0254] In one embodiment, the biological sample comprises neuronal
cells, serum, bone marrow, blood, peripheral blood, lymph nodes,
cerebro-spinal fluid, urine, a saliva sample, a buccal swab, a
serum sample, a sputum sample, a lacrimal secretion sample, a semen
sample, a vaginal secretion sample, a fetal tissue sample, or a
combination thereof. In some embodiments the sample is a tissue
sample. In some embodiments, the sample is a paraffin embedded
tissue section. In some embodiments, the tissue sample is a tumor
sample.
[0255] A FGFR fusion molecule, for example, a fusion between FGFR1,
FGFR2, FGFR3, or any other FGFR, and TACC1, TACC2, TACC3 or any
other TACC, can be determined at the level of the DNA, RNA, or
polypeptide. Optionally, detection can be determined by performing
an oligonucleotide ligation assay, a confirmation based assay, a
hybridization assay, a sequencing assay, an allele-specific
amplification assay, a microsequencing assay, a melting curve
analysis, a denaturing high performance liquid chromatography
(DHPLC) assay (for example, see Jones et al, (2000) Hum Genet.,
106(6):663-8), or a combination thereof. In one embodiment, the
detection is performed by sequencing all or part of a FGFR fusion
molecule (e.g., a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other
FGFR-TACC nucleic acid, or a FGFR1, TACC1, FGFR2, TACC2, FGFR3,
TACC3 or other FGFR or TACC nucleic acid), or by selective
hybridization or amplification of all or part of a FGFR fusion
molecule (e.g., a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other
FGFR-TACC nucleic acid, or a FGFR1, TACC1, FGFR2, TACC2, FGFR3,
TACC3 or other FGFR or TACC nucleic acid). A FGFR fusion molecule
specific amplification (e.g., a FGFR1-TACC1, FGFR2-TACC2,
FGFR3-TACC3 or other FGFR-TACC nucleic acid specific amplification)
can be carried out before the fusion identification step.
[0256] The invention provides for a method of detecting a
chromosomal alteration in a subject afflicted with a gene-fusion
associated cancer. In one embodiment, the chromosomal alteration is
an in-frame fused transcript described herein, for example an FGFR
fusion molecule. In some embodiments, the chromosomal alteration is
a chromosomal translocation, for example an FGFR fusion molecule.
An alteration in a chromosome region occupied by a FGFR fusion
molecule, such as a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other
FGFR-TACC nucleic acid, can be any form of mutation(s),
deletion(s), rearrangement(s) and/or insertions in the coding
and/or non-coding region of the locus, alone or in various
combination(s). Mutations can include point mutations. Insertions
can encompass the addition of one or several residues in a coding
or non-coding portion of the gene locus. Insertions can comprise an
addition of between 1 and 50 base pairs in the gene locus.
Deletions can encompass any region of one, two or more residues in
a coding or non-coding portion of the gene locus, such as from two
residues up to the entire gene or locus. Deletions can affect
smaller regions, such as domains (introns) or repeated sequences or
fragments of less than about 50 consecutive base pairs, although
larger deletions can occur as well. Rearrangement includes
inversion of sequences. The alteration in a chromosome region
occupied by a FGFR fusion molecule, e.g., a FGFR1-TACC1,
FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid, can
result in amino acid substitutions, RNA splicing or processing,
product instability, the creation of stop codons, production of
oncogenic fusion proteins, frame-shift mutations, and/or truncated
polypeptide production. The alteration can result in the production
of a FGFR fusion molecule, for example, one encoded by a
FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic
acid, with altered function, stability, targeting or structure. The
alteration can also cause a reduction, or even an increase in
protein expression. In one embodiment, the alteration in the
chromosome region occupied by a FGFR fusion molecule can comprise a
chromosomal rearrangement resulting in the production of a FGFR
fusion molecule, such as a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or
other FGFR-TACC fusion. This alteration can be determined at the
level of the DNA, RNA, or polypeptide. In another embodiment, the
detection or determination comprises nucleic acid sequencing,
selective hybridization, selective amplification, gene expression
analysis, or a combination thereof. In another embodiment, the
detection or determination comprises protein expression analysis,
for example by western blot analysis, ELISA, immunostaining or
other antibody detection methods. In one embodiment, the
coordinates comprising FGFR1 translocations comprise
chr8:38,268,656-38,325,363. In another embodiment, the coordinates
comprising FGFR2 translocations comprise
chr10:123,237,844-123,357,972. In a further embodiment, the
coordinates comprising FGFR3 translocations comprise
chr4:1,795,039-1,810,599. In yet another embodiment, the
coordinates comprising FGFR4 translocations comprise
chr5:176,513,921-176,525,126. In one embodiment, the coordinates
comprising TACC1 translocations comprise
chr8:38,644,722-38,710,546. In another embodiment, the coordinates
comprising TACC2 translocations comprise
chr10:123,748,689-124,014,057. In a further embodiment, the
coordinates comprising TACC3 translocations comprise
chr4:1,723,217-1,746,905.
[0257] The present invention provides a method for treating a
gene-fusion associated cancer in a subject in need thereof. In one
embodiment, the method comprises obtaining a sample from the
subject to determine the level of expression of an FGFR fusion
molecule in the subject. In some embodiments, the sample is
incubated with an agent that binds to an FGFR fusion molecule, such
as an antibody, a probe, a nucleic acid primer, and the like. In
another embodiment, the detection or determination comprises
nucleic acid sequencing, selective hybridization, selective
amplification, gene expression analysis, or a combination thereof.
In another embodiment, the detection or determination comprises
protein expression analysis, for example by western blot analysis,
immunostaining, ELISA, or other antibody detection methods. In some
embodiments, the method further comprises assessing whether to
administer a FGFR fusion molecule inhibitor based on the expression
pattern of the subject. In further embodiments, the method
comprises administering a FGFR fusion molecule inhibitor to the
subject. In one embodiment, the FGFR fusion molecule inhibitor is
JNJ-42756493. In one embodiment, the gene-fusion associated cancer
comprises an epithelial cancer. In one embodiment, the gene-fusion
associated cancer comprises glioblastoma multiforme, breast cancer,
lung cancer, prostate cancer, or colorectal carcinoma. In some
embodiments, the epithelial cancer comprises bladder urothelial
carcinoma, breast carcinoma, colorectal cancer, prostate carcinoma,
lung squamous cell carcinoma, head and neck squamous cell
carcinoma, or a combination of the epithelial cancers described. In
one embodiment, the gene-fusion associated cancer comprises glioma,
grade II or III glioma, or IDH wild-type grade II or III glioma. In
further embodiments, the method comprises further detecting the
presence of IDH1 mutations, EGFR amplification, CDK4 amplification,
or MDM2 amplification. In further embodiments, a FGFR fusion
molecule inhibitor can be administered in combination with CDK4
inhibitors, MDM2 inhibitors, or a combination thereof.
[0258] In one embodiment, the invention provides for a method of
detecting the presence of altered RNA expression of an FGFR fusion
molecule in a subject, for example one afflicted with a gene-fusion
associated cancer. In another embodiment, the invention provides
for a method of detecting the presence of an FGFR fusion molecule
in a subject. In some embodiments, the method comprises obtaining a
sample from the subject to determine whether the subject expresses
an FGFR fusion molecule. In some embodiments, the sample is
incubated with an agent that binds to an FGFR fusion molecule, such
as an antibody, a probe, a nucleic acid primer, and the like. In
other embodiments, the detection or determination comprises nucleic
acid sequencing, selective hybridization, selective amplification,
gene expression analysis, or a combination thereof. In another
embodiment, the detection or determination comprises protein
expression analysis, for example by western blot analysis, ELISA,
or other antibody detection methods. In some embodiments, the
method further comprises assessing whether to administer a FGFR
fusion molecule inhibitor based on the expression pattern of the
subject. In further embodiments, the method comprises administering
a FGFR fusion molecule inhibitor to the subject. In one embodiment,
the FGFR fusion molecule inhibitor is JNJ-42756493. Altered RNA
expression includes the presence of an altered RNA sequence, the
presence of an altered RNA splicing or processing, or the presence
of an altered quantity of RNA. These can be detected by various
techniques known in the art, including sequencing all or part of
the RNA or by selective hybridization or selective amplification of
all or part of the RNA. In a further embodiment, the method can
comprise detecting the presence or expression of a FGFR fusion
molecule, such as one encoded by a FGFR1-TACC1, FGFR2-TACC2,
FGFR3-TACC3 or other FGFR-TACC nucleic acid. Altered polypeptide
expression includes the presence of an altered polypeptide
sequence, the presence of an altered quantity of polypeptide, or
the presence of an altered tissue distribution. These can be
detected by various techniques known in the art, including by
sequencing and/or binding to specific ligands (such as antibodies).
In one embodiment, the detecting comprises using a northern blot;
real time PCR and primers directed to SEQ ID NOS: 80-82, 84,
94-145, 515, 517, 519-527, or 530-538; a ribonuclease protection
assay; a hybridization, amplification, or sequencing technique to
detect an FGFR fusion molecule, such as one comprising SEQ ID NOS:
80-82, 84, 94-145, 515, 517, 519-527, or 530-538; or a combination
thereof. In another embodiment, the PCR primers comprise SEQ ID
NOS: 162, 163, 164, 165 166, 167, 168, 169, 495, 496, 497, 498,
507, 508, 509, 510, 511, 512, 513, or 514. In a further embodiment,
primers used for the screening of FGFR fusion molecules, such as
FGFR-TACC fusions, comprise SEQ ID NOS: 166, 167, 168, 169, 495,
496, 497, 498, 507, 508, 509, or 510. In some embodiments, primers
used for genomic detection of an FGFR3-TACC3 fusion comprise SEQ ID
NOS: 170 171, 499, 500, 501, 502, 503, 504, 505, or 506.
[0259] In some aspects of the invention, the method comprises
further detecting the presence of IDH1 mutations, EGFR
amplification, CDK4 amplification, or MDM2 amplification. MDM2
encodes a nuclear-localized E3 ubiquitin ligase. Alternative
splicing results in a multitude of transcript variants, many of
which may be expressed only in tumor cells. EGFR (epidermal growth
factor receptor) is a transmembrane glycoprotein that is a member
of the protein kinase superfamily. This protein is a receptor for
members of the epidermal growth factor family. EGFR is a cell
surface protein that binds to epidermal growth factor. Multiple
alternatively spliced transcript variants that encode different
protein isoforms have been found for this gene. CDK4 (cyclin
dependent kinase 4) is a member of the Ser/Thr protein kinase
family. It is a catalytic subunit of the protein kinase complex
that is important for cell cycle G1 phase progression. The activity
of this kinase is restricted to the G1-S phase, which is controlled
by the regulatory subunits D-type cyclins and CDK inhibitor
p16(INK4a). Multiple polyadenylation sites of this gene have been
reported. IDH1 (isocitrate dehydrogenase 1 (NADP+), soluble)
catalyzes the oxidative decarboxylation of isocitrate to
2-oxoglutarate. Alternatively spliced transcript variants encoding
the same protein have been found for this gene. IDH1 mutations,
EGFR amplification, CDK4 amplification, or MDM2 amplification can
be detected using various techniques know in the art, including,
but not limited to sequencing and qPCR.
[0260] Various techniques known in the art can be used to detect or
quantify altered gene or RNA expression or nucleic acid sequences,
which include, but are not limited to, hybridization, sequencing,
amplification, and/or binding to specific ligands (such as
antibodies). Other suitable methods include allele-specific
oligonucleotide (ASO), oligonucleotide ligation, allele-specific
amplification, Southern blot (for DNAs), Northern blot (for RNAs),
single-stranded conformation analysis (SSCA), PFGE, fluorescent in
situ hybridization (FISH), gel migration, clamped denaturing gel
electrophoresis, denaturing HLPC, melting curve analysis,
heteroduplex analysis, RNase protection, chemical or enzymatic
mismatch cleavage, ELISA, radio-immunoassays (RIA) and
immuno-enzymatic assays (IEMA).
[0261] Some of these approaches (such as SSCA and constant gradient
gel electrophoresis (CGGE)) are based on a change in
electrophoretic mobility of the nucleic acids, as a result of the
presence of an altered sequence. According to these techniques, the
altered sequence is visualized by a shift in mobility on gels. The
fragments can then be sequenced to confirm the alteration. Some
other approaches are based on specific hybridization between
nucleic acids from the subject and a probe specific for wild type
or altered gene or RNA. The probe can be in suspension or
immobilized on a substrate. The probe can be labeled to facilitate
detection of hybrids. Some of these approaches are suited for
assessing a polypeptide sequence or expression level, such as
Northern blot, ELISA and RIA. These latter require the use of a
ligand specific for the polypeptide, for example, the use of a
specific antibody.
[0262] Hybridization.
[0263] Hybridization detection methods are based on the formation
of specific hybrids between complementary nucleic acid sequences
that serve to detect nucleic acid sequence alteration(s). A
detection technique involves the use of a nucleic acid probe
specific for a wild type or altered gene or RNA, followed by the
detection of the presence of a hybrid. The probe can be in
suspension or immobilized on a substrate or support (for example,
as in nucleic acid array or chips technologies). The probe can be
labeled to facilitate detection of hybrids. In one embodiment, the
probe according to the invention can comprise a nucleic acid
directed to SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or
530-538. For example, a sample from the subject can be contacted
with a nucleic acid probe specific for a gene encoding a FGFR
fusion molecule, and the formation of a hybrid can be subsequently
assessed. In one embodiment, the method comprises contacting
simultaneously the sample with a set of probes that are specific
for an FGFR fusion molecule. Also, various samples from various
subjects can be investigated in parallel.
[0264] According to the invention, a probe can be a polynucleotide
sequence which is complementary to and specifically hybridizes with
a, or a target portion of a, gene or RNA corresponding to a FGFR
fusion molecule. Useful probes are those that are complementary to
the gene, RNA, or target portion thereof. Probes can comprise
single-stranded nucleic acids of between 8 to 1000 nucleotides in
length, for instance between 10 and 800, between 15 and 700, or
between 20 and 500. Longer probes can be used as well. A useful
probe of the invention is a single stranded nucleic acid molecule
of between 8 to 500 nucleotides in length, which can specifically
hybridize to a region of a gene or RNA that corresponds to a FGFR
fusion molecule.
[0265] The sequence of the probes can be derived from the sequences
of the FGFR fusion genes provided herein. Nucleotide substitutions
can be performed, as well as chemical modifications of the probe.
Such chemical modifications can be accomplished to increase the
stability of hybrids (e.g., intercalating groups) or to label the
probe. Some examples of labels include, without limitation,
radioactivity, fluorescence, luminescence, and enzymatic
labeling.
[0266] A guide to the hybridization of nucleic acids is found in
e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual
(3.sup.rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989;
Current Protocols In Molecular Biology, Ausubel, ed. John Wiley
& Sons, Inc., New York, 2001; Laboratory Techniques In
Biochemistry And Molecular Biology: Hybridization With Nucleic Acid
Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.
Elsevier, N.Y., 1993.
[0267] Sequencing.
[0268] Sequencing can be carried out using techniques well known in
the art, using automatic sequencers. The sequencing can be
performed on the complete FGFR fusion molecule or on specific
domains thereof.
[0269] Amplification.
[0270] Amplification is based on the formation of specific hybrids
between complementary nucleic acid sequences that serve to initiate
nucleic acid reproduction. Amplification can be performed according
to various techniques known in the art, such as by polymerase chain
reaction (PCR), ligase chain reaction (LCR), strand displacement
amplification (SDA) and nucleic acid sequence based amplification
(NASBA). These techniques can be performed using commercially
available reagents and protocols. Useful techniques in the art
encompass real-time PCR, allele-specific PCR, or PCR based
single-strand conformational polymorphism (SSCP). Amplification
usually requires the use of specific nucleic acid primers, to
initiate the reaction. For example, nucleic acid primers useful for
amplifying sequences corresponding to a FGFR fusion molecule are
able to specifically hybridize with a portion of the gene locus
that flanks a target region of the locus. In one embodiment,
amplification comprises using forward and reverse PCR primers
directed to SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or
530-538. Nucleic acid primers useful for amplifying sequences from
a FGFR fusion molecule (e.g., a FGFR1-TACC1, FGFR2-TACC2,
FGFR3-TACC3 or other FGFR-TACC nucleic acid); the primers
specifically hybridize with a portion of an FGFR fusion molecule.
In certain subjects, the presence of an FGFR fusion molecule
corresponds to a subject with a gene fusion-associated cancer. In
one embodiment, amplification can comprise using forward and
reverse PCR primers comprising nucleotide sequences of SEQ ID NOS:
80-82, 84, 94-145, 515, 517, 519-527, or 530-538. In one
embodiment, amplification can comprise using forward and reverse
PCR primers comprising nucleotide sequences of SEQ ID NOS: 162-169,
or 495-514.
[0271] Non-limiting amplification methods include, e.g., polymerase
chain reaction, PCR (PCR Protocols, A Guide To Methods And
Applications, ed. Innis, Academic Press, N.Y., 1990 and PCR
Strategies, 1995, ed. Innis, Academic Press, Inc., N.Y.); ligase
chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988)
Science 241:1077; Barringer (1990) Gene 89:117); transcription
amplification (Kwoh (1989) PNAS 86:1173); and, self-sustained
sequence replication (Guatelli (1990) PNAS 87:1874); Q Beta
replicase amplification (Smith (1997) J. Clin. Microbiol.
35:1477-1491), automated Q-beta replicase amplification assay (Burg
(1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase
mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario;
see also Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. Nos.
4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology
13:563-564). All the references stated above are incorporated by
reference in their entireties.
[0272] The invention provides for a nucleic acid primer, wherein
the primer can be complementary to and hybridize specifically to a
portion of a FGFR fusion molecule, such as a FGFR1-TACC1,
FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid (e.g., DNA
or RNA) in certain subjects having a gene fusion-associated cancer.
In one embodiment, the gene-fusion associated cancer comprises
glioblastoma multiforme, breast cancer, lung cancer, prostate
cancer, or colorectal carcinoma. Primers of the invention can be
specific for fusion sequences in a FGFR1-TACC1, FGFR2-TACC2,
FGFR3-TACC3 or other FGFR-TACC nucleic acid (DNA or RNA). By using
such primers, the detection of an amplification product indicates
the presence of a fusion of a FGFR1 and TACC1, FGFR2 and TACC2,
FGFR3 and TACC3 or other FGFR and TACC nucleic acid. Examples of
primers of this invention can be single-stranded nucleic acid
molecules of about 5 to 60 nucleotides in length, or about 8 to
about 25 nucleotides in length. The sequence can be derived
directly from the sequence of a FGFR fusion molecule, e.g.
FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic
acid. Perfect complementarity is useful to ensure high specificity;
however, certain mismatch can be tolerated. For example, a nucleic
acid primer or a pair of nucleic acid primers as described above
can be used in a method for detecting the presence of a gene
fusion-associated cancer in a subject. In one embodiment, primers
can be used to detect an FGFR fusion molecule, such as a primer
comprising SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or
530-538; or a combination thereof. In another embodiment, the PCR
primers comprise SEQ ID NOS: 162, 163, 164, 165, 166, 167, 168,
169, 495, 496, 497, 498, 507, 508, 509, 510, 511, 512, 513, or 514.
Ina further embodiment, primers used for the screening of FGFR
fusion molecules, such as FGFR-TACC fusions, comprise SEQ ID NOS:
166, 167, 168, 169, 495, 496, 497, 498, 507, 508, 509, or 510. In
some embodiments, primers used for genomic detection of an
FGFR3-TACC3 fusion comprise SEQ ID NOS: 170, 171, 499, 500, 501,
502, 503, 504, 505, or 506. In one embodiment, the method can
comprise contacting a sample from the subject with primers specific
for a FGFR fusion molecule, and determining the presence of an PCR
product. In another embodiment, the method can comprise contacting
a sample from the subject with primer specific for a FGFR molecule,
or a TACC molecule, and determining the presence of a PCR product.
In another embodiment, the primers can recognize the nucleic acids
encoding a FGFR3 C-terminal region, or nucleic acids encoding a
TACC3 N-terminal region, or a combination thereof. In another
embodiment, the method can comprise contacting a sample from the
subject with primers specific for a FGFR molecule, or a TACC
molecule, or a FGFR fusion molecule, and determining the amount of
PCR product formed compared to the amount of PCR product formed in
non-tumor cells or tissue, wherein an increased amount of PCR
product indicates the presence of an FGFR fusion. In one
embodiment, primers and/or the PCR product are labeled to enable
detection of the PCR product. For example, nucleic acid primers
useful for amplifying sequences corresponding to a FGFR fusion
molecules can be labeled with fluorescent molecules, radioactive
molecules, chemiluminescent molecules, or affinity molecules (e.g.
biotin) which can then be detected by methods known in the art
(e.g. fluorescently labeled streptavidin). PCR products can also be
detected by using dyes that can be incorporated into newly formed
PCR products, such as, but not limited to, SYBR Green.
[0273] Specific Ligand Binding.
[0274] As discussed herein, a nucleic acid encoding a FGFR fusion
molecule or expression of a FGFR fusion molecule, can also be
detected by screening for alteration(s) in a sequence or expression
level of a polypeptide encoded by the same. Different types of
ligands can be used, such as specific antibodies. In one
embodiment, the sample is contacted with an antibody specific for a
polypeptide encoded by a FGFR fusion molecule and the formation of
an immune complex is subsequently determined Various methods for
detecting an immune complex can be used, such as ELISA,
immunostaining, radioimmunoassays (RIA) and immuno-enzymatic assays
(IEMA).
[0275] For example, an antibody can be a polyclonal antibody, a
monoclonal antibody, as well as fragments or derivatives thereof
having substantially the same antigen specificity. Fragments
include Fab, Fab'2, or CDR regions. Derivatives include
single-chain antibodies, humanized antibodies, or poly-functional
antibodies. An antibody specific for a polypeptide encoded by a
FGFR fusion molecule can be an antibody that selectively binds such
a polypeptide. In one embodiment, the antibody is raised against a
polypeptide encoded by a FGFR fusion molecule (such as FGFR1-TACC1,
FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC fusion) or an
epitope-containing fragment thereof. Although non-specific binding
towards other antigens can occur, binding to the target polypeptide
occurs with a higher affinity and can be reliably discriminated
from non-specific binding. In one embodiment, the method can
comprise contacting a sample from the subject with an antibody
specific for a FGFR fusion molecule, and determining the presence
of an immune complex. Optionally, the sample can be contacted to a
support coated with antibody specific for a FGFR fusion molecule.
In one embodiment, the sample can be contacted simultaneously, or
in parallel, or sequentially, with various antibodies specific for
different forms of a FGFR fusion molecule, e.g., FGFR1-TACC1,
FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC fusion.
[0276] In one embodiment, the method can comprise contacting a
sample from the subject with an antibody specific for a FGFR fusion
molecule, and determining the presence of an immune complex. In
another embodiment, the method can comprise contacting a sample
from the subject with an antibody specific for a FGFR molecule, or
a TACC molecule, and determining the presence of an immune complex.
In another embodiment, the antibody can recognize the FGFR3
C-terminal region, or the TACC3 N-terminal region, or a combination
thereof. In another embodiment, the antibody can recognize the
FGFR3 C-terminal region, or the TACC3 N-terminal region, or a
combination thereof. In another embodiment, the method can comprise
contacting a sample from the subject with an antibody specific for
a FGFR molecule, or a TACC molecule, or a FGFR fusion molecule, and
determining the amount of an immune complex formed compared to the
amount of immune complex formed in non-tumor cells or tissue,
wherein an increased amount of an immune complex indicates the
presence of an FGFR fusion.
[0277] Detection the formation of a complex between an antibody and
a protein can be performed by a variety of method known in the art.
For example, an antibody-protein complex can be detected by using
antibodies or secondary antibodies labeled with fluorescent
molecules, chromogenic molecules, chemiluminescent molecules,
radioactive isotopes, or affinity molecules (e.g. biotin) which can
then be detected by methods known in the art (e.g. fluorescently
labeled streptavidin).
[0278] The invention also provides for a diagnostic kit comprising
products and reagents for detecting in a sample from a subject the
presence of a FGFR fusion molecule. The kit can be useful for
determining whether a sample from a subject exhibits increased or
reduced expression of a FGFR fusion molecule. For example, the
diagnostic kit according to the present invention comprises any
primer, any pair of primers, any nucleic acid probe and/or any
ligand, or any antibody directed specifically to a FGFR fusion
molecule. The diagnostic kit according to the present invention can
further comprise reagents and/or protocols for performing a
hybridization, amplification, or antigen-antibody immune reaction.
In one embodiment, the kit can comprise nucleic acid primers that
specifically hybridize to and can prime a polymerase reaction from
a FGFR fusion molecule comprising SEQ ID NOS: 80-82, 84, 94-145,
515, 517, 519-527, or 530-538, or a combination thereof. In one
embodiment, primers can be used to detect a FGFR fusion molecule,
such as a primer directed to SEQ ID NOS: 80-82, 84, 94-145, 515,
517, 519-527, or 530-538; or a combination thereof. In another
embodiment, the PCR primer comprises SEQ ID NOS: 162, 163, 164,
165, 166, 167, 168, 169, 495, 496, 497, 498, 507, 508, 509, 510,
511, 512, 513, or 514. Ina further embodiment, primers used for the
screening of FGFR fusion molecules, such as FGFR-TACC fusions,
comprise SEQ ID NOS: 166, 167, 168, 169, 495, 496, 497, 498, 507,
508, 509, or 510. In some embodiments, primers used for genomic
detection of an FGFR3-TACC3 fusion comprise SEQ ID NOS: 170, 171,
499, 500, 501, 502, 503, 504, 505, or 506. In some embodiments, the
kit comprises an antibody that specifically binds to a FGFR fusion
molecule comprising SEQ ID NOS: 79, 85-89, 150, 158-161, or
539-547, wherein the antibody will recognize the protein only when
a FGFR fusion molecule is present. The diagnosis methods can be
performed in vitro, ex vivo, or in vivo. These methods utilize a
sample from the subject in order to assess the status of a FGFR
fusion molecule. The sample can be any biological sample derived
from a subject, which contains nucleic acids or polypeptides.
Examples of such samples include, but are not limited to, fluids,
tissues, cell samples, organs, and tissue biopsies. Non-limiting
examples of samples include blood, liver, plasma, serum, saliva,
urine, or seminal fluid. In some embodiments the sample is a tissue
sample. In some embodiments, the sample is a paraffin embedded
tissue section. In some embodiments, the tissue sample is a tumor
sample. The sample can be collected according to conventional
techniques and used directly for diagnosis or stored. The sample
can be treated prior to performing the method, in order to render
or improve availability of nucleic acids or polypeptides for
testing. Treatments include, for instance, lysis (e.g., mechanical,
physical, or chemical), centrifugation. The nucleic acids and/or
polypeptides can be pre-purified or enriched by conventional
techniques, and/or reduced in complexity. Nucleic acids and
polypeptides can also be treated with enzymes or other chemical or
physical treatments to produce fragments thereof. In one
embodiment, the sample is contacted with reagents, such as probes,
primers, or ligands, in order to assess the presence of a FGFR
fusion molecule. Contacting can be performed in any suitable
device, such as a plate, tube, well, or glass. In some embodiments,
the contacting is performed on a substrate coated with the reagent,
such as a nucleic acid array or a specific ligand array. The
substrate can be a solid or semi-solid substrate such as any
support comprising glass, plastic, nylon, paper, metal, or
polymers. The substrate can be of various forms and sizes, such as
a slide, a membrane, a bead, a column, or a gel. The contacting can
be made under any condition suitable for a complex to be formed
between the reagent and the nucleic acids or polypeptides of the
sample.
Nucleic Acid Delivery Methods
[0279] Delivery of nucleic acids into viable cells can be effected
ex vivo, in situ, or in vivo by use of vectors, such as viral
vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a
retrovirus), or ex vivo by use of physical DNA transfer methods
(e.g., liposomes or chemical treatments). Non-limiting techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, and the calcium
phosphate precipitation method (See, for example, Anderson, Nature,
1998) supplement to 392(6679):25( ). Introduction of a nucleic acid
or a gene encoding a polypeptide of the invention can also be
accomplished with extrachromosomal substrates (transient
expression) or artificial chromosomes (stable expression). Cells
can also be cultured ex vivo in the presence of therapeutic
compositions of the present invention in order to proliferate or to
produce a desired effect on or activity in such cells. Treated
cells can then be introduced in vivo for therapeutic purposes.
[0280] Nucleic acids can be inserted into vectors and used as gene
therapy vectors. A number of viruses have been used as gene
transfer vectors, including papovaviruses, e.g., SV40 (Madzak et
al., (1992) J Gen Virol. 73(Pt 6):1533-6), adenovirus (Berkner
(1992) Curr Top Microbiol Immunol. 158:39-66; Berkner (1988)
Biotechniques, 6(7):616-29; Gorziglia and Kapikian (1992) J Virol.
66(7):4407-12; Quantin et al., (1992) Proc Natl Acad Sci USA.
89(7):2581-4; Rosenfeld et al., (1992) Cell. 68(1):143-55;
Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9;
Stratford-Perricaudet et al., (1990) Hum Gene Ther. 1(3):241-56),
vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22),
adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol
Immunol. 158:97-129; Ohi et al., (1990) Gene. 89(2):279-82),
herpesviruses including HSV and EBV (Margolskee (1992) Curr Top
Microbiol Immunol. 158:67-95; Johnson et al., (1992) Brain Res Mol
Brain Res. 12(1-3):95-102; Fink et al., (1992) Hum Gene Ther.
3(1):11-9; Breakefield and Geller (1987) Mol Neurobiol.
1(4):339-71; Freese et al., (1990) Biochem Pharmacol.
40(10):2189-99), and retroviruses of avian (Bandyopadhyay and Temin
(1984) Mol Cell Biol. 4(4):749-54; Petropoulos et al., (1992) J
Virol. 66(6):3391-7), murine (Miller et al. (1992) Mol Cell Biol.
12(7):3262-72; Miller et al., (1985) J Virol. 55(3):521-6; Sorge et
al., (1984) Mol Cell Biol. 4(9):1730-7; Mann and Baltimore (1985) J
Virol. 54(2):401-7; Miller et al., (1988) J Virol. 62(11):4337-45),
and human origin (Shimada et al., (1991) J Clin Invest.
88(3):1043-7; Helseth et al., (1990) J Virol. 64(12):6314-8; Page
et al., (1990) J Virol. 64(11):5270-6; Buchschacher and Panganiban
(1992) J Virol. 66(5):2731-9).
[0281] Non-limiting examples of in vivo gene transfer techniques
include transfection with viral (e.g., retroviral) vectors (see
U.S. Pat. No. 5,252,479, which is incorporated by reference in its
entirety) and viral coat protein-liposome mediated transfection
(Dzau et al., (1993) Trends in Biotechnology 11:205-210),
incorporated entirely by reference). For example, naked DNA
vaccines are generally known in the art; see Brower, (1998) Nature
Biotechnology, 16:1304-1305, which is incorporated by reference in
its entirety. Gene therapy vectors can be delivered to a subject
by, for example, intravenous injection, local administration (see,
e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see,
e.g., Chen, et al., (1994) Proc. Natl. Acad. Sci. USA
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g., retroviral vectors, the pharmaceutical preparation can
include one or more cells that produce the gene delivery
system.
[0282] For reviews of nucleic acid delivery protocols and methods
see Anderson et al. (1992) Science 256:808-813; U.S. Pat. Nos.
5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847;
and U.S. Application Publication No. 2002/0077313, which are all
hereby incorporated by reference in their entireties. For
additional reviews, see Friedmann (1989) Science, 244:1275-1281;
Verma, Scientific American: 68-84 (1990); Miller (1992) Nature,
357: 455-460; Kikuchi et al. (2008) J Dermatol Sci. 50(2):87-98;
Isaka et al. (2007) Expert Opin Drug Deliv. 4(5):561-71; Jager et
al. (2007) Curr Gene Ther. 7(4):272-83; Waehler et al. (2007) Nat
Rev Genet. 8(8):573-87; Jensen et al. (2007) Ann Med. 39(2):108-15;
Herweijer et al. (2007) Gene Ther. 14(2):99-107; Eliyahu et al.
(2005) Molecules 10(1):34-64; and Altaras et al. (2005) Adv Biochem
Eng Biotechnol. 99:193-260, all of which are hereby incorporated by
reference in their entireties.
[0283] A FGFR fusion nucleic acid can also be delivered in a
controlled release system. For example, the FGFR fusion molecule
can be administered using intravenous infusion, an implantable
osmotic pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump can be used (see Sefton
(1987) Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507;
Saudek et al. (1989) N. Engl. J Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228:190;
During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J.
Neurosurg. 71:105). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)). Other controlled release systems are discussed in
the review by Langer (Science (1990) 249:1527-1533).
Pharmaceutical Compositions and Administration for Therapy
[0284] An inhibitor of the invention can be incorporated into
pharmaceutical compositions suitable for administration, for
example the inhibitor and a pharmaceutically acceptable carrier
[0285] A FGFR fusion molecule or inhibitor of the invention (e.g.
JNJ-42756493) can be administered to the subject once (e.g., as a
single injection or deposition). Alternatively, a FGFR fusion
molecule or inhibitor can be administered once or twice daily to a
subject in need thereof for a period of from about two to about
twenty-eight days, or from about seven to about ten days. A FGFR
fusion molecule or inhibitor can also be administered once or twice
daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 times per year, or a combination thereof. A FGFR fusion
molecule or inhibitor can also be administered in seven to ten day
repeating cycles (i.e. administration of a FGFR fusion molecule or
inhibitor for seven to ten days, followed by no administration of a
FGFR fusion molecule or inhibitor for seven to ten days).
Furthermore, a FGFR fusion molecule or inhibitor of the invention
can be co-administrated with another therapeutic. Where a dosage
regimen comprises multiple administrations, the effective amount of
the FGFR fusion molecule or inhibitor administered to the subject
can comprise the total amount of gene product administered over the
entire dosage regimen.
[0286] A FGFR fusion molecule or inhibitor can be administered to a
subject by any means suitable for delivering the FGFR fusion
molecule or inhibitor to cells of the subject, such as cancer
cells, e.g., glioblastoma multiforme, breast cancer, lung cancer,
prostate cancer, colorectal carcinoma, bladder carcinoma, squamous
lung carcinoma, head and neck carcinoma, glioma, grade II or III
glioma, or IDH wild-type grade II or III glioma. For example, a
FGFR fusion molecule or inhibitor can be administered by methods
suitable to transfect cells. Transfection methods for eukaryotic
cells are well known in the art, and include direct injection of
the nucleic acid into the nucleus or pronucleus of a cell;
electroporation; liposome transfer or transfer mediated by
lipophilic materials; receptor mediated nucleic acid delivery,
bioballistic or particle acceleration; calcium phosphate
precipitation, and transfection mediated by viral vectors.
[0287] The compositions of this invention can be formulated and
administered to reduce the symptoms associated with a gene
fusion-associated cancer, e.g., glioblastoma multiforme, breast
cancer, lung cancer, prostate cancer, colorectal carcinoma, bladder
carcinoma, squamous lung carcinoma, head and neck carcinoma,
glioma, grade II or III glioma, or IDH wild-type grade II or III
glioma, by any means that produces contact of the active ingredient
with the agent's site of action in the body of a subject, such as a
human or animal (e.g., a dog, cat, or horse). They can be
administered by any conventional means available for use in
conjunction with pharmaceuticals, either as individual therapeutic
active ingredients or in a combination of therapeutic active
ingredients. They can be administered alone, but are generally
administered with a pharmaceutical carrier selected on the basis of
the chosen route of administration and standard pharmaceutical
practice.
[0288] A therapeutically effective dose of FGFR fusion molecule or
inhibitor (e.g. JNJ-42756493) can depend upon a number of factors
known to those or ordinary skill in the art. The dose(s) of the
FGFR fusion molecule inhibitor can vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the a FGFR fusion molecule inhibitor
to have upon the nucleic acid or polypeptide of the invention. For
example, 12 mg of JNJ-42756493 can be orally administered daily.
JNJ-42756493 can be administered in seven to ten day repeating
cycles (i.e. administration of JNJ-42756493 for seven to ten days,
followed by no administration of JNJ-42756493 for seven to ten
days). These amounts can be readily determined by a skilled
artisan. Any of the therapeutic applications described herein can
be applied to any subject in need of such therapy, including, for
example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a
monkey, a pig, a sheep, a goat, or a human.
[0289] Pharmaceutical compositions for use in accordance with the
invention can be formulated in conventional manner using one or
more physiologically acceptable carriers or excipients. The
therapeutic compositions of the invention can be formulated for a
variety of routes of administration, including systemic and topical
or localized administration. Techniques and formulations generally
can be found in Remmington's Pharmaceutical Sciences, Meade
Publishing Co., Easton, Pa. (20.sup.th Ed., 2000), the entire
disclosure of which is herein incorporated by reference. For
systemic administration, an injection is useful, including
intramuscular, intravenous, intraperitoneal, and subcutaneous. For
injection, the therapeutic compositions of the invention can be
formulated in liquid solutions, for example in physiologically
compatible buffers such as Hank's solution or Ringer's solution. In
addition, the therapeutic compositions can be formulated in solid
form and redissolved or suspended immediately prior to use.
Lyophilized forms are also included. Pharmaceutical compositions of
the present invention are characterized as being at least sterile
and pyrogen-free. These pharmaceutical formulations include
formulations for human and veterinary use.
[0290] According to the invention, a pharmaceutically acceptable
carrier can comprise any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Any
conventional media or agent that is compatible with the active
compound can be used. Supplementary active compounds can also be
incorporated into the compositions.
[0291] A pharmaceutical composition containing FGFR fusion molecule
inhibitor can be administered in conjunction with a
pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed herein. Such pharmaceutical compositions can
comprise, for example antibodies directed to a FGFR fusion
molecule, or a variant thereof, or antagonists of a FGFR fusion
molecule, or JNJ-42756493. The compositions can be administered
alone or in combination with at least one other agent, such as a
stabilizing compound, which can be administered in any sterile,
biocompatible pharmaceutical carrier including, but not limited to,
saline, buffered saline, dextrose, and water. The compositions can
be administered to a patient alone, or in combination with other
agents, drugs or hormones.
[0292] Sterile injectable solutions can be prepared by
incorporating the FGFR fusion molecule inhibitor (e.g., a
polypeptide or antibody) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated herein,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated herein. In the
case of sterile powders for the preparation of sterile injectable
solutions, examples of useful preparation methods are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0293] In some embodiments, the FGFR fusion molecule inhibitor can
be applied via transdermal delivery systems, which slowly releases
the active compound for percutaneous absorption. Permeation
enhancers can be used to facilitate transdermal penetration of the
active factors in the conditioned media. Transdermal patches are
described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No.
5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S.
Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No.
5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S.
Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No.
4,921,475.
[0294] "Subcutaneous" administration can refer to administration
just beneath the skin (i.e., beneath the dermis). Generally, the
subcutaneous tissue is a layer of fat and connective tissue that
houses larger blood vessels and nerves. The size of this layer
varies throughout the body and from person to person. The interface
between the subcutaneous and muscle layers can be encompassed by
subcutaneous administration. This mode of administration can be
feasible where the subcutaneous layer is sufficiently thin so that
the factors present in the compositions can migrate or diffuse from
the locus of administration. Thus, where intradermal administration
is utilized, the bolus of composition administered is localized
proximate to the subcutaneous layer.
[0295] Administration of the cell aggregates (such as DP or DS
aggregates) is not restricted to a single route, but can encompass
administration by multiple routes. For instance, exemplary
administrations by multiple routes include, among others, a
combination of intradermal and intramuscular administration, or
intradermal and subcutaneous administration. Multiple
administrations can be sequential or concurrent. Other modes of
application by multiple routes will be apparent to the skilled
artisan.
[0296] In other embodiments, this implantation method will be a
one-time treatment for some subjects. In further embodiments of the
invention, multiple cell therapy implantations will be required. In
some embodiments, the cells used for implantation will generally be
subject-specific genetically engineered cells. In another
embodiment, cells obtained from a different species or another
individual of the same species can be used. Thus, using such cells
can require administering an immunosuppressant to prevent rejection
of the implanted cells. Such methods have also been described in
U.S. Pat. No. 7,419,661 and PCT application publication WO
2001/32840, and are hereby incorporated by reference.
[0297] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation or
ingestion), transdermal (topical), transmucosal, and rectal
administration. For example, JNJ-42756493 can be orally
administered. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0298] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, or phosphate buffered saline (PBS). In all
cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, a pharmaceutically
acceptable polyol like glycerol, propylene glycol, liquid
polyethylene glycol, and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, it can be useful to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0299] Sterile injectable solutions can be prepared by
incorporating the inhibitor (e.g., a polypeptide or antibody or
small molecule) of the invention in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated herein, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
herein. In the case of sterile powders for the preparation of
sterile injectable solutions, examples of useful preparation
methods are vacuum drying and freeze-drying which yields a powder
of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof.
[0300] Oral compositions (e.g. of JNJ-42756493) generally include
an inert diluent or an edible carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic administration, the active compound can be
incorporated with excipients and used in the form of tablets,
troches, or capsules. Oral compositions can also be prepared using
a fluid carrier and subsequently swallowed.
[0301] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0302] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0303] In some embodiments, the effective amount of the
administered FGFR fusion molecule inhibitor (e.g. JNJ-42756493) is
at least about 0.0001 .mu.g/kg body weight, at least about 0.00025
.mu.g/kg body weight, at least about 0.0005 .mu.g/kg body weight,
at least about 0.00075 .mu.g/kg body weight, at least about 0.001
.mu.g/kg body weight, at least about 0.0025 .mu.g/kg body weight,
at least about 0.005 .mu.g/kg body weight, at least about 0.0075
.mu.g/kg body weight, at least about 0.01 .mu.g/kg body weight, at
least about 0.025 .mu.g/kg body weight, at least about 0.05
.mu.g/kg body weight, at least about 0.075 .mu.g/kg body weight, at
least about 0.1 .mu.g/kg body weight, at least about 0.25 .mu.g/kg
body weight, at least about 0.5 .mu.g/kg body weight, at least
about 0.75 .mu.g/kg body weight, at least about 1 .mu.g/kg body
weight, at least about 5 .mu.g/kg body weight, at least about 10
.mu.g/kg body weight, at least about 25 .mu.g/kg body weight, at
least about 50 .mu.g/kg body weight, at least about 75 .mu.g/kg
body weight, at least about 100 .mu.g/kg body weight, at least
about 150 .mu.g/kg body weight, at least about 200 .mu.g/kg body
weight, at least about 250 .mu.g/kg body weight, at least about 300
.mu.g/kg body weight, at least about 350 .mu.g/kg body weight, at
least about 400 .mu.g/kg body weight, at least about 450 .mu.g/kg
body weight, at least about 500 .mu.g/kg body weight, at least
about 550 .mu.g/kg body weight, at least about 600 .mu.g/kg body
weight, at least about 650 .mu.g/kg body weight, at least about 700
.mu.g/kg body weight, at least about 750 .mu.g/kg body weight, at
least about 800 .mu.g/kg body weight, at least about 850 .mu.g/kg
body weight, at least about 900 .mu.g/kg body weight, at least
about 950 .mu.g/kg body weight, at least about 1,000 .mu.g/kg body
weight, at least about 2,000 .mu.g/kg body weight, at least about
3,000 .mu.g/kg body weight, at least about 4,000 .mu.g/kg body
weight, at least about 5,000 .mu.g/kg body weight, at least about
6,000 .mu.g/kg body weight, at least about 7,000 .mu.g/kg body
weight, at least about 8,000 .mu.g/kg body weight, at least about
9,500 .mu.g/kg body weight, or at least about 10,000 .mu.g/kg body
weight.
[0304] In some embodiments, the effective amount of the
administered FGFR fusion molecule inhibitor (e.g. JNJ-42756493) is
at least about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9
mg, 10 mg, 11 mg, 12 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg,
20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29
mg, 30 mg.
[0305] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention.
[0306] All publications and other references mentioned herein are
incorporated by reference in their entirety, as if each individual
publication or reference were specifically and individually
indicated to be incorporated by reference. Publications and
references cited herein are not admitted to be prior art.
EXAMPLES
[0307] Examples are provided below to facilitate a more complete
understanding of the invention. The following examples illustrate
the exemplary modes of making and practicing the invention.
However, the scope of the invention is not limited to specific
embodiments disclosed in these Examples, which are for purposes of
illustration only, since alternative methods can be utilized to
obtain similar results.
[0308] The invention is further illustrated in Singh et al.,
Science (2012), 337(6099):1231-5 (including the accompanying
Supplementary Information). The entire contents of Singh et al.,
Science (2012), 337(6099):1231-5, including the accompanying
"Supplementary Information," is expressly incorporated by
reference. The invention is also further illustrated in Di Stefano
et al., "Detection, characterization and inhibition of FGFR-TACC
fusions in IDH wild type glioma" Clin. Cancer Res. (2015), the
entire contents of which are expressly incorporated by
reference.
Example 1
Transforming and Recurrent Fusions of FGFR and TACC Gene in
Glioblastoma
[0309] The history of successful targeted therapy of cancer largely
coincides with the inactivation of recurrent, oncogenic and
addicting gene fusions in hematological malignancies and recently
in some types of epithelial cancer. Glioblastoma multiforme (GBM)
is among the most lethal forms of human cancer. Here, an integrated
gene fusion discovery pipeline was developed for the detection of
in-frame fused transcripts from RNA-seq and genomic fusions from
whole exome sequences. The application of the pipeline to human GBM
unraveled recurrent chromosomal translocations, which fuse in-frame
the tyrosine kinase domain of FGFR genes (FGFR1 or FGFR3) to the
TACC domain of TACC1 or TACC3, respectively. The frequency of
FGFR-TACC fusions is 3 of 97 GBM (3.1%). The FGFR-TACC fusion
protein displays strong oncogenic activity when introduced into
astrocytes or transduced by lentivirus-mediated stereotactic
delivery to the adult mouse brain. The FGFR-TACC fusion protein
mis-localizes over the mitotic spindle pole, has constitutive
tyrosine kinase activity and dysregulates the mitotic cycle with
delayed mitotic progression. The impaired mitotic fidelity triggers
chromatid cohesion defects, defective spindle checkpoint
activation, chromosomal mis-segregation, and rampant aneuploidy.
Inhibition of FGFR kinase corrects the aneuploidy and oral
administration of a specific FGFR tyrosine kinase inhibitor under
clinical investigation arrests tumor growth and prolongs survival
of mice harboring intracranial FGFR3-TACC3-initiated glioma.
FGFR-TACC fusions identify a subset of GBM patients who may benefit
from targeted inhibition of the tyrosine kinase activity of
FGFR.
[0310] Glioblastoma multiforme (GBM) is among the most difficult
forms of cancer to treat in humans (Ohgaki and Kleihues, 2005). So
far, the targeted therapeutic approaches that have been tested
against potentially important oncogenic drivers in GBM have met
limited success (Lo, 2010; Reardon et al., 2010; van den Bent et
al., 2009). Recurrent chromosomal translocations leading to
production of oncogenic fusion proteins are viewed as initiating
and addicting events in the pathogenesis of human cancer, thus
providing the most desirable molecular targets for cancer therapy
(Ablain et al., 2011; Mitelman et al., 2007). Chromosomal
rearrangements resulting in recurrent and oncogenic gene fusions
are hallmarks of hematological malignancies and recently they have
also been uncovered in subsets of solid tumors (breast, prostate,
lung and colorectal carcinoma), but they have not been found in GBM
(Bass et al., 2011; Prensner and Chinnaiyan, 2009). Important and
successful targeted therapeutic interventions for patients whose
tumors carry these rearrangements have stemmed from the discovery
of functional gene fusions, especially when the translocations
involve kinase-coding genes (BCR-ABL, EML4-ALK) (Druker, 2009;
Gerber and Minna, 2010).
[0311] A hallmark of GBM is rampant chromosomal instability (CIN),
which leads to aneuploidy (Furnari et al., 2007). CIN and
aneuploidy are early events in the pathogenesis of cancer (Cahill
et al., 1999). It has been suggested that genetic alterations
targeting mitotic fidelity might be responsible for mis-segregation
of chromosomes during mitosis, resulting in aneuploidy (Gordon et
al., 2012; Solomon et al., 2011). Here, the first cases of
recurrent and oncogenic gene fusions in human GBM are described.
The resulting fusion protein localizes to mitotic cells, disrupts
the normal control of chromosome segregation and induces
aneuploidy. A therapeutic strategy with FGFR tyrosine kinase
inhibitors is also reported for the targeted therapy of GBM
patients harboring these chromosomal rearrangements.
[0312] Identification of Recurrent Fusions of FGFR and TACC
Genes.
[0313] To identify genomic rearrangements in GBM that generate
functional fusion proteins and are recurrent, gene pairs discovered
as in-frame fused transcripts from the analysis of massively
parallel, paired-end sequencing of expressed transcripts (RNA-seq)
would also emerge as fused gene pairs from the genomic analysis of
human GBM. Towards this aim, two complementary gene fusion
discovery methods were devised and were applied to two GBM cohorts.
The first, TX-Fuse, is an algorithm for the discovery of candidate
fusion transcripts from RNA-seq (FIG. 8). The second, Exome-Fuse,
detects fusion genes from whole exome DNA sequences (FIG. 8). As
first step for the detection of fused transcripts, RNA-seq data was
generated from short-term cultures of glioma stem-like cells (GSCs)
freshly isolated from nine patients carrying primary GBM. The
culture of primary GBM tumors under serum-free conditions selects
cells that retain phenotypes and genotypes closely mirroring
primary tumor profiles as compared to serum-cultured glioma cell
lines that have largely lost their developmental identities (Lee et
al., 2006). Therefore, without being bound by theory, if glioma
cells carry gene fusions causally responsible for the most
aggressive hallmarks of GBM, they should be selected in GSCs.
RNA-seq generated an average of 60.3 million paired reads for each
GSC culture, of which over 80% were mapped to the reference
transcriptome and genome. TX-Fuse detects two main sources of
evidence: split reads and split inserts (see Experimental
Procedures). The application of TX-Fuse to the RNA-seq dataset from
nine GSCs led to the discovery of five candidate rearrangements
(all of which were intrachromosomal) that give rise to in-frame
fusion transcripts (Table 1).
TABLE-US-00043 TABLE 1 Predicted in-frame fusion proteins from
RNA-Seq of nine GSCs # Split # Split Ref Ref Tx Tx Inserts Reads
Sample Gene1 Gene2 Seq1 Seq2 Pos1 Pos2 294 76 GSC- FGFR3 TACC3
NM_000142 NM_006342 2530 1751 1123 37 54 GSC- POLR2A WRAP53
NM_000937 NM_001143990 479 798 0114 7 48 GSC- CAPZB UBR4
NM_001206540 NM_020765 228 12111 0114 8 29 GSC- ST8SIA4 PAM
NM_005668 NM_000919 1125 730 0517 6 17 GSC- PIGU NCOA6 NM_080476
NM_014071 729 6471 0308 1 6 GSC- IFNAR2 IL10RB NM_000874 NM_000628
1083 149 0127 #Split #Split Inserts Reads Sample Chr1 Strand1
hg19_GenPos1 Chr2 Strand2 hg19_GenPos2 294 76 GSC- 4 + 1808842 4 +
1737004 1123 37 54 GSC- 17 + 7399259 17 + 7604059 0114 7 48 GSC- 1
- 19712098 1 - 19433440 0114 8 29 GSC- 5 - 100147809 5 + 102260661
0517 6 17 GSC- 20 - 33203914 20 - 33303130 0308 1 6 GSC- 21 +
34632901 21 + 34640699 0127
[0314] Next, genomic rearrangements leading to gene fusions were
identified in GBM by applying Exome-Fuse to a dataset of paired-end
exome DNA sequences from 84 GBM samples from TCGA (Table 2).
[0315] This analysis detected 147 paired gene fusions, thus
producing an average of 1.75 gene fusion events per tumor (Table
3).
[0316] The FGFR and TACC families of genes were markedly enriched
among those recurrently involved in genomic fusions, with eight
tumors harboring FGFR rearrangements and seven tumors harboring
fusions that implicate TACC genes (FIG. 1A). The comparative
analysis of the TX-Fuse and Exon-Fuse outputs revealed that
FGFR3-TACC3 was the only fusion pair identified as either an
in-frame transcript by TX-Fuse and genomic fusions by Exome-Fuse
(Tables 1, 2 and 3).
[0317] Table 2 shows fusion breakpoint information of recurrent
gene fusions identified by Exome-fuse analysis of 84 GBM from TCGA.
As multiple junctions may exist in each fusion candidate,
information for all breakpoints is displayed. Column definitions
include: sample=TCGA sample ID,
virtForSplitReads/virtRevSplitReads/virtTotSplitReads=#
forward/reverse/total split reads, splitInserts=# split inserts,
dirA/dirB=forward (1) or reverse (0) direction of split read
portion mapping to gene A/B, dirAB_matepair=direction of mate pair
of split read, cosmicA+B=# recorded mutations of gene A+B in
COSMIC.
TABLE-US-00044 TABLE 2 Fusion breakpoint information of recurrent
gene fusions identified by Exome-fuse analysis of 84 GBM from TCGA.
cosmic Sample vlrtForSplitReads vlrtRevSplitReads vlrtForSplitReads
splitinserts geneA chrA senseA posA geneB chrB senseB posB chrA
chrB dirAB_matepair A + B TCGA-06-6390 10 9 19 8 FGFR3 chr4 +
1778521 TACC3 chr4 + 1708787 1 1 0 2803 TCGA-12-0826 5 6 11 5 FGFR3
chr4 + 1778502 TACC3 chr4 + 1707185 0 0 1 2803 TCGA-19-5958 3 0 3 2
FGFR3 chr4 + 1778539 TACC3 chr4 + 1707203 0 1 1 2803 TCGA-27-1835
11 1 12 4 FGFR3 chr4 + 1778595 TACC3 chr4 + 1709397 0 0 1 2803
TCGA-12-0820 7 2 9 4 FGFR3 chr4 + 1779184 PRKG2 chr4 - 82338347 1 1
0 2805 TCGA-12-1088 3 1 4 4 ABL1 chr9 + 132597569 TNFRSF10B chr8 -
22936252 0 0 1 892 TCGA-06-1802 7 1 8 8 ADAM12 chr10 - 127698245
PTPRD chr9 - 8596127 0 0 1 54 TCGA-06-1801 7 0 7 5 HIP1 chr7 -
75010010 PTPRD chr9 - 9387093 1 0 0 52 TCGA-12-1088 3 0 3 3
KIDINS220 chr2 - 8886300 PPP1R3A chr7 - 113305567 0 0 1 45
TCGA-12-1088 37 1 38 10 KIDINS220 chr2 - 8887075 PPP1R3A chr7 -
113305191 1 1 0 45 TCGA-32-2491 2 17 19 6 ODZ1 chrX - 123342503
STAG2 chrX + 123019118 0 1 0 36 TCGA-32-2491 11 1 12 10 ODZ1 chrX -
123526882 SASH3 chrX + 128749198 1 0 0 34 TCGA-12-0829 24 0 24 13
LRRK2 chr12 + 39032542 VSNL1 chr2 + 17630556 1 1 0 32 TCGA-12-0829
25 1 26 13 LRRK2 chr12 + 38975444 VSNL1 chr2 + 17639377 1 0 0 32
TCGA-12-0829 87 16 103 58 LRRK2 chr12 + 38975652 VSNL1 chr2 +
17639552 0 1 1 32 TCGA-19-0957 3 2 5 6 NUDT19 chr19 + 37891921 ODZ1
chrX - 123925223 1 0 0 32 TCGA-12-1088 12 1 13 5 GLI3 chr7 -
42031380 RIMBP2 chr12 - 129517282 1 0 0 31 TCGA-12-1088 5 0 5 1
GLI3 chr7 - 42031574 RIMBP2 chr12 - 129517455 0 1 1 31 TCGA-12-1089
10 0 10 5 AHNAK chr11 - 62056459 C21orf29 chr21 - 44923276 0 1 1 30
TCGA-06-1801 27 1 28 12 CROCC chr1 + 17171362 CSMD2 chr1 - 34381139
1 0 0 29 TCGA-12-1089 12 1 13 6 CLK3 chr15 + 72705401 LRP1 chr12 +
55880002 0 1 1 28 TCGA-12-1089 14 2 16 8 CLK3 chr15 + 72705248 LRP1
chr12 + 55879646 1 0 0 28 TCGA-12-1089 42 5 47 24 LAMA2 chr6 +
129836071 PDE10A chr6 - 165858426 1 0 0 28 TCGA-06-1802 48 9 57 27
LAMA2 chr6 + 129483265 SEC14L3 chr22 - 29193005 1 1 0 27
TCGA-19-0957 4 16 20 6 CSMD2 chr1 - 34115076 MDH2 chr7 + 75525221 0
0 1 27 TCGA-06-1801 21 1 22 4 FAM192A chr16 - 55757701 LRP1 chr12 +
55858598 0 0 1 26 TCGA-12-1089 27 0 27 2 FGFR4 chr5 + 176447670
LILRB1 chr19 + 59840807 1 0 0 25 TCGA-19-0957 0 1 1 4 EML1 chr14 +
99349006 NRXN3 chr14 + 79233969 1 0 0 24 TCGA-06-1801 19 133 152 51
NHSL2 chrX + 71082676 TAF1 chrX + 70520522 1 0 0 22 TCGA-06-1801 51
3 54 8 NHSL2 chrX + 71083319 TAF1 chrX + 70521607 1 0 1 22
TCGA-12-1089 9 0 9 4 CACNA1C chr12 + 2325330 ITGAV chr2 + 187195411
0 0 1 22 TCGA-19-0957 8 1 9 6 CDH11 chr16 - 63579650 RERE chr1 -
8588774 0 0 1 22 TCGA-12-0829 12 3 15 4 ENTPD2 chr9 - 139062591
FREM2 chr13 + 38318644 1 1 0 21 TCGA-12-0829 2 3 5 1 EFS chr14 -
22896776 NRXN3 ch14 + 78678529 1 0 1 21 TCGA-12-0829 56 6 62 14
DIS3L chr15 + 64377566 GLI3 chr7 - 42032535 1 0 TCGA-12-0829 8 2 10
3 EFS chr14 - 22896431 NRXN3 chr14 + 78678139 1 0 TCGA-12-0829 9 65
74 37 DIS3L chr15 + 64377398 GLI3 chr7 - 42032341 1 0 TCGA-27-1835
14 0 14 4 FAM19A2 chr12 - 60707200 GLI1 chr12 + 56146523 0 0
TCGA-06-1801 20 0 20 2 FREM2 chr13 + 38163882 RALYL chr8 + 85785432
1 1 TCGA-12-0827 2 0 2 2 ABCC12 chr16 - 46722685 FGFR4 chr5 +
176457194 1 1 TCGA-12-0829 35 0 35 7 ANXA7 chr10 - 74808655 CACNA1C
chr12 + 2458351 1 1 TCGA-06-2559 60 37 97 1 PLEKHM3 chr2 -
208426920 PTPRS chr19 - 5222592 0 0 TCGA-12-1088 2 0 2 2 PLCL1 chr2
+ 198630224 TACC2 chr10 + 123987513 1 1 TCGA-06-1801 10 0 10 4
FGFR4 chr5 + 176450528 WISP2 chr20 + 42782576 0 1 TCGA-06-1802 15 0
15 2 PDHA2 chr4 + 96980717 PDZRN4 chr12 + 39959553 0 1 TCGA-06-1802
4 0 4 2 PDHA2 chr4 + 96980509 PDZRN4 chr12 + 39959384 1 0
TCGA-06-6390 53 0 53 18 GPR182 chr12 + 55675639 PDZRN4 chr12 +
39957003 1 0 TCGA-12-0829 1121 252 1373 602 ADCY8 chr8 - 131886108
SSX3 chrX - 48091929 0 0 TCGA-12-0829 14 8 22 3 ADCY8 chr8 -
131886506 SSX3 chrX - 48091719 1 1 TCGA-12-0829 9 42 51 18 ADAM12
chr10 - 127733231 DAPK1 chr9 + 89454764 0 1 TCGA-12-3653 22 0 22 10
JOSD2 chr19 - 55705579 PTPRS chr19 - 5245999 0 1 TCGA-12-0829 100 0
100 20 COL14A1 chr8 + 121370990 MMP12 chr11 - 102242881 1 0
TCGA-12-0829 152 0 152 24 COL14A1 chr8 + 121371195 MMP12 chr11 -
102242953 0 1 TCGA-06-1802 11 47 58 19 MUSK chr9 + 112509906 SYNPO2
chr4 + 120172123 0 0 TCGA-06-1805 6 4 10 6 COL14A1 chr8 + 121332080
NCRNA0015 chr21 - 18174873 1 1 TCGA-12-0822 37 0 37 3 C7orf44 chr7
- 43683128 TACC2 chr10 + 123835337 1 0 TCGA-12-0829 0 2 2 365 GSTA3
chr6 - 52878492 TACC2 chr10 + 123884543 0 1 TCGA-12-0829 124 16 140
51 GSTA3 chr6 - 52878680 TACC2 chr10 + 123884705 0 1 TCGA-12-0829
21 7 28 10 HIP1 chr7 - 75022909 MASP1 chr3 - 188452372 0 0
TCGA-12-0829 268 123 391 242 HIP1 chr7 - 75022741 MASP1 chr3 -
188452581 1 1 TCGA-12-0829 36 641 677 365 GSTA3 chr6 - 52878496
TACC2 chr10 + 123884531 0 1 TCGA-12-1088 10 1 11 3 CAMTA1 chr1 +
7710762 TMPRSS3 chr21 - 42665918 0 1 TCGA-12-1088 65 0 65 6 ADCY10
chr1 - 166139873 DUSP27 chr1 + 165351555 0 0 TCGA-12-1088 8 1 9 4
CAMTA1 chr1 + 7714539 TMPRSS3 chr21 - 42666044 1 0 TCGA-27-1835 83
1 84 22 CMYA5 chr5 + 79120729 SRRM1 chr1 + 24870899 0 0
TCGA-06-1801 0 43 43 31 CAMTA1 chr1 + 7264935 GDPD2 chrX + 69563759
0 1 TCGA-06-1801 13 41 54 31 CAMTA1 chr1 + 7265429 GDPD2 chrX +
69563431 1 0 TCGA-06-1801 24 66 90 61 CAMTA1 chr1 + 7265556 GDPD2
chrX + 69563762 0 1 TCGA-12-0829 2 0 2 3 CCDC147 chr10 + 106165013
ISX chr22 + 33795708 0 1 TCGA-12-1088 7 1 8 5 CMYA5 chr5 + 79045621
STK24 chr13 - 97969547 1 0 TCGA-06-1801 7 1 8 4 DEPDC5 chr22 +
30619774 ROBO1 chr3 - 79802538 0 1 TCGA-12-0820 110 20 130 23
ABCA13 chr7 + 48597322 NHSL2 chrX + 71077547 1 0 TCGA-12-0820 29 4
33 3 ABCA13 chr7 + 48597477 NHSL2 chrX + 71077690 0 1 TCGA-12-0829
46 2 48 4 LIN9 chr1 - 224536835 NCOR1 chr17 - 15883585 0 0
TCGA-12-3644 3 0 3 1 EFHC1 chr6 + 52432073 LRBA chr4 - 151418615 1
0 TCGA-12-3644 3 10 13 3 EFHC1 chr6 + 52431890 LRBA chr4 -
151418438 1 0 TCGA-19-5958 6 6 12 7 DEPDC5 chr22 + 30504095 SLC5A4
chr22 - 30974671 0 1 TCGA-06-1801 4 4 8 5 KCND3 chr1 - 112227957
LY75 chr2 - 160443238 1 1 TCGA-12-0820 26 1 27 2 BBX chr3 +
108997451 CUL3 chr2 - 225108623 0 0 TCGA-12-0828 8 67 75 31 ADCY2
chr5 + 7558840 SDAD1 chr4 - 77096208 1 1 TCGA-12-0829 13 21 34 16
AGBL4 chr1 - 48902776 NUP188 chr9 + 130808425 0 0 TCGA-12-0829 64
308 372 197 EYS chr6 - 64513356 IL1RN chr2 + 113603712 0 1
TCGA-12-0829 7 25 32 11 AGBL4 chr1 - 48902600 NUP188 chr9 +
130808628 1 1 TCGA-12-0829 9 0 9 1 LRBA chr4 - 151790893 PSEN1
chr14 + 72707609 1 0 TCGA-12-1093 65 4 69 21 OSBPL10 chr3 -
31687272 TRAPPC9 chr8 - 140828099 1 1 TCGA-12-1600 9 0 9 5 5-Sep
chr22 + 18088018 NCOR1 chr17 - 15915170 0 1 TCGA-19-0957 18 1 19 7
ADCY10 chr1 - 166060645 AKT3 chr1 - 241743142 0 1 TCGA-19-0957 34 2
36 11 ADCY10 chr1 - 166060502 AKT3 chr1 - 241742588 1 0
TCGA-12-0822 0 1 1 16 ITGB2 chr21 - 45147805 SH3RF3 chr2 +
109430489 0 1 TCGA-12-0822 6 2 8 1 ITGB2 chr21 - 45147994 SH3RF3
chr2 + 109430669 0 1 TCGA-12-0827 25 3 28 8 CUL3 chr2 - 225126210
LY75 chr2 - 160455052 1 0 TCGA-12-0828 7 2 9 4 FH chr1 - 239743589
SRGAP1 chr12 + 62723692 0 1 TCGA-12-0829 24 0 24 9 ITGA9 chr3 +
37712050 SNX5 chr20 - 17885523 0 1 TCGA-12-1089 17 2 19 5 ABCC1
chr16 + 16077635 RNF216 chr7 - 5692038 1 1 TCGA-12-1089 6 0 6 8
CAMSAP1 chr9 - 137867066 NCF2 chr1 - 181799323 1 0 TCGA-19-0957 16
0 16 4 CCDC147 chr10 + 106114657 STK4 chr20 + 43111359 0 0
TCGA-06-1801 5 33 38 18 AP4S1 chr14 + 30611930 EYS chr6 - 64770011
1 0 TCGA-06-1805 3 14 17 9 CUL3 chr2 - 225064315 SLC44A2 chr19 +
10608393 1 0 TCGA-12-0829 14 27 41 23 ADCY2 chr5 + 7798046
C14orf174 chr14 + 76914809 1 0 TCGA-12-0829 59 7 66 18 NR3C1 chr5 -
142760085 SORCS2 chr4 + 7354165 0 0 TCGA-12-0829 9 40 49 28 ADCY2
chr5 + 7798641 C14orf174 chr14 + 76915034 0 1 TCGA-12-1093 20 0 20
4 GAPVD1 chr9 + 127104266 MAPKAP1 chr9 - 127490362 1 0 TCGA-12-1600
7 0 7 4 CILP chr15 - 63283865 PARP16 chr15 - 63350048 0 1 1 10
TCGA-19-0957 13 3 16 6 AQP2 chr12 + 48635567 CDH4 chr20 + 59413648
0 1 1 10 TCGA-19-0957 6 0 6 1 AQP2 chr12 + 48635406 CDH4 chr20 +
59413468 1 0 0 10 TCGA-06-0166 2 0 2 3 CCDC158 chr4 - 77541796 SNX5
chr20 - 17885346 0 1 1 9 TCGA-06-1802 30 0 30 9 RANBP2 chr2 +
108758804 SATB2 chr2 - 199895572 0 1 1 9 TCGA-06-1805 4 0 4 3 C2CD3
chr11 - 73430819 XRRA1 chr11 - 74309669 1 1 0 9 TCGA-06-1805 6 1 7
5 NEUROG1 chr5 - 134898853 PRKCH chr14 + 51027580 1 1 0 9
TCGA-12-0820 27 0 27 2 RANBP2 chr2 + 108749908 TTC27 chr2 +
32839367 0 1 1 9 TCGA-12-0820 58 7 65 15 RANBP2 chr2 + 108749412
TTC27 chr2 + 32837790 1 0 0 9 TCGA-12-0829 6 128 134 35 C2CD3 chr11
- 73529639 CAPZB chr1 - 19556435 0 0 1 9 TCGA-12-0829 84 443 527
227 C2CD3 chr11 - 73529293 CAPZB chr1 - 19556627 1 1 0 9
TCGA-12-1088 10 0 10 2 PACSIN1 chr6 + 34589431 TNC chr9 - 116884742
0 1 0 9 TCGA-12-1088 12 0 12 2 PACSIN1 chr6 + 34589619 TNC chr9 -
116884958 1 0 1 9 TCGA-19-0957 34 19 53 17 PRKCH chr14 + 61032978
ZFAND3 chr6 + 38228111 1 0 1 9 TCGA-19-0957 7 1 8 7 MAPKAP1 chr9 -
127348507 SLC9A1 chr1 - 27302334 0 1 0 9 TCGA-19-0957 8 39 47 21
PRKCH chr14 + 61032774 ZFAND3 chr6 + 38227949 1 0 0 9 TCGA-06-1801
5 11 16 4 MAOA chrX + 43486192 SH3RF3 chr2 + 109237058 1 0 1 8
TCGA-06-1802 10 12 22 12 DNM1L chr12 + 32736794 SYNPO2 chr4 +
120172271 0 1 1 8 TCGA-06-1802 18 42 60 24 MUC4 chr3 - 196982875
SMOC2 chr6 + 168676813 1 0 1 8 TCGA-12-0829 6 0 6 6 ATXN1 chr6 -
16669201 CACNA1G chr17 + 46004995 0 0 1 8 TCGA-12-0829 7 0 7 3
ATP6V0D2 chr8 + 87186716 RERE chr1 - 8336574 1 1 0 8 TCGA-12-1088
11 1 12 6 BCAS3 chr17 + 56321892 CACNA1G chr17 + 46010698 1 1 0 8
TCGA-12-1088 15 2 17 5 ABCC1 chr16 + 16135771 AGBL4 chr1 - 49315120
1 0 0 8 TCGA-12-1088 17 3 20 4 MST1R chr3 - 49910627 WDFY1 chr2 -
224512774 1 0 0 8 TCGA-12-1088 4 0 4 2 FBXL4 chr6 - 99431443 SYNPO2
chr4 + 120172560 0 0 1 8 TCGA-12-1092 39 4 43 12 CNTN2 chr1 +
203302926 DNAJC6 chr1 + 65591195 0 0 1 8 TCGA-12-1598 4 0 4 5 MPP1
chrX - 153673715 SRGAP1 chr12 + 62777947 0 0 1 8 TCGA-19-1786 5 19
24 7 ATP5B chr12 - 55320148 USP48 chr1 - 21920103 1 0 1 8
TCGA-19-2621 21 0 21 3 BCAS3 chr17 + 56731673 TTYH1 chr19 +
59638801 1 1 0 8 TCGA-06-1801 15 0 15 9 C15orf23 chr15 + 38469150
DMD chrX - 32092185 0 1 1 7 TCGA-06-1805 6 3 9 5 FAM19A2 chr12 -
60547321 POLM chr7 - 44082653 0 1 0 7 TCGA-12-0829 13 84 97 44
ATP5B chr12 - 55318484 PRC1 chr15 - 89330475 1 0 1 7 TCGA-12-0829
158 207 365 44 ATP5B chr12 - 55320850 PRC1 chr15 - 89334458 1 1 0 7
TCGA-12-0829 2 1 3 2 DDI2 chr1 + 15825507 KIDINS220 chr2 - 8805399
0 1 0 7 TCGA-12-0829 25 6 31 44 ATP5B chr12 - 55321832 PRC1 chr15 -
89335627 1 0 1 7 TCGA-12-0829 34 21 55 44 ATP5B chr12 - 55321200
PRC1 chr15 - 89335044 0 1 0 7 TCGA-12-0829 44 6 50 4 ABCC6 chr16 -
16204784 SUMF1 chr3 - 4470138 1 0 0 7
TCGA-12-0829 53 28 81 35 DDI2 chr1 + 15825941 KIDINS220 chr2 -
8805580 1 0 1 7 TCGA-12-0829 9 0 9 5 DMD chrX - 32013100 N4BP2L2
chr13 - 32008512 0 0 1 7 TCGA-12-1092 9 0 9 4 LRRC48 chr19 -
55754780 NR3C1 chr5 - 142660156 0 1 0 7 TCGA-19-2621 3 22 25 11
PCDH12 chr5 - 141309153 SLC36A2 chr5 - 150679274 1 1 0 7
TCGA-06-1802 8 4 12 6 BAHD1 chr15 + 38539023 OSBPL10 chr3 -
31729622 0 1 1 6 TCGA-12-0828 11 0 11 1 PLOD3 chr7 - 100646340
VSNL1 chr2 + 17638618 1 1 0 6 TCGA-12-0828 40 9 49 20 PLOD3 chr7 -
100646511 VSNL1 chr2 + 17637955 0 0 1 6 TCGA-12-0829 16 2 18 9
C21orf29 chr21 - 44922864 MYT1 chr20 + 62300829 0 0 1 6
TCGA-12-0829 196 0 196 37 IGFBP3 chr7 - 45922866 SMOC2 chr6 +
168722450 0 0 1 6 TCGA-12-0829 5 1 6 1 FAM168A chr11 - 72839771
NCF2 chr1 - 181826115 1 0 1 6 TCGA-12-0829 5 18 23 9 FAM168A chr11
- 72839534 NCF2 chr1 - 181825930 1 0 0 6 TCGA-12-1089 20 0 20 2
SLC44A2 chr19 + 10602997 XRCC4 chr5 + 82430803 1 0 0 6 TCGA-19-0957
17 1 18 4 PAX3 chr2 - 222778052 WDFY1 chr2 - 224453159 1 0 1 6
TCGA-06-1801 5 0 5 1 CAP2 chr6 + 17571234 DNAJC6 chr1 + 65602700 1
0 0 5 TCGA-06-1801 6 32 38 15 CAP2 chr6 + 17571666 DNAJC6 chr1 +
65603089 0 1 1 5 TCGA-06-1805 3 0 3 8 PLCL1 chr2 + 198578552 SURF6
chr9 - 135188818 0 1 0 5 TCGA-06-1805 7 2 9 4 PLCL1 chr2 +
198578671 SURF6 chr9 - 135189294 1 0 1 5 TCGA-12-0822 17 4 21 4
TAAR6 chr6 + 132933266 TTYH1 chr19 + 59629451 0 1 1 5 TCGA-12-0828
17 0 17 8 AQP2 chr12 + 48634800 ECE1 chr1 - 21515240 0 1 1 5
TCGA-12-0828 7 0 7 1 AQP2 chr12 + 48634610 ECE1 chr1 - 21515033 1 0
0 5 TCGA-12-0829 12 0 12 7 CACNA1G chr17 + 46039372 CNTNAP4 chr16 +
74873868 0 1 1 5 TCGA-19-0957 4 0 4 3 PCDH12 chr5 - 141316624
SH3BP5 chr3 - 15315567 0 0 1 5 TCGA-19-0957 8 2 10 2 PCDH12 chr5 -
141316405 SH3BP5 chr3 - 15315731 1 1 0 5 TCGA-06-1801 13 1 14 1
ABCC6 chr16 - 16205051 CMTM7 chr3 + 32443880 0 1 1 4 TCGA-06-1801
33 7 40 4 ABCC6 chr16 - 16204860 CMTM7 chr3 + 32443722 1 0 0 4
TCGA-06-1805 10 3 13 6 AGBL4 chr1 - 49449813 NOX4 chr11 - 88714996
0 0 1 4 TCGA-12-0829 11 0 11 4 FAM160A1 chr4 + 152595916 LY75 chr2
- 160440194 0 1 0 4 TCGA-12-0829 17 1 18 5 FAM160A1 chr4 +
152596097 LY75 chr2 - 160440376 1 0 1 4 TCGA-12-0829 487 83 570 249
CORO7 chr16 - 4375428 DYRK3 chr1 + 204876154 0 0 1 4 TCGA-12-1088 2
12 14 4 FAM172A chr5 - 93052315 TRIOBP chr22 + 36427382 0 1 1 4
TCGA-06-1801 30 7 37 18 DEPDC7 chr11 + 33003811 EIF2C2 chr8 -
141618836 1 0 0 3 TCGA-06-1801 40 28 68 33 MAP7 chr6 - 136728609
SH3RF3 chr2 + 109392677 0 0 TCGA-12-1093 6 15 21 8 CORO7 chr16 -
4398302 PLEK2 chr14 - 66934201 0 1 TCGA-12-3644 33 0 33 4 EDA chrX
+ 69073054 SSX3 chrX - 48094443 1 1 TCGA-12-3644 37 15 52 17
C15orf33 chr15 - 47424122 PARP16 chr15 - 63350289 0 0 TCGA-19-1791
14 3 17 8 PSEN1 chr14 + 72748293 ZNF410 chr14 + 73431112 0 0
TCGA-06-1802 35 2 37 17 CELF2 chr10 + 11352537 PLA2G2F chr1 +
20348173 1 1 TCGA-06-1802 63 26 89 25 CELF2 chr10 + 11352765
PLA2G2F chr1 + 20347997 0 0 TCGA-06-1802 8 2 10 8 LCLAT1 chr2 +
30535977 PACSIN1 chr6 + 34576195 1 0 TCGA-06-2562 6 0 6 4 SNTA1
chr20 - 31473415 TMEM80 chr11 + 689744 0 0 TCGA-12-0829 16 1 17 4
LASS6 chr2 + 169045211 NKAIN2 chr6 + 125021252 0 0 TCGA-12-0829 7 0
7 2 LASS6 chr2 + 169045333 NKAIN2 chr6 + 125021072 1 1 TCGA-14-0813
339 39 378 5 SNTA1 chr20 - 31481069 TMEM80 chr11 + 686739 0 0
TCGA-12-0820 49 3 52 11 CAMKK1 chr17 - 3712344 FAM184B chr4 -
17271273 0 1 TCGA-12-0826 8 18 26 13 CELF2 chr10 + 11406463 NME4
chr16 + 389427 1 1 TCGA-12-1089 17 4 21 10 C6orf170 chr6 -
121478035 NKAIN2 chr6 + 125083380 1 0 TCGA-12-1600 19 0 19 3
ATP6AP1L chr5 + 81649744 FAM172A chr5 - 93336459 1 0 TCGA-12-1600 5
35 40 6 ATP6AP1L chr5 + 81649902 FAM172A chr5 - 93336676 1 0
TCGA-19-1790 4 0 4 4 ARMC6 chr19 + 19026932 FAM184B chr4 - 17391210
1 0 TCGA-06-1802 12 0 12 8 EIF2C2 chr8 - 141648334 TNFRSF10B chr8 -
22940680 0 0 TCGA-14-0781 22 2 24 8 FAM160A1 chr4 + 152584637
UNC93B1 chr11 - 67523253 1 0
TABLE-US-00045 TABLE 3 Recurrent gene fusion pairs from Exome- fuse
analysis of 84 GBM from TCGA. Sample gene A gene B TCGA-12-0820
ABCA13 NHSL2 TCGA-12-1089 ABCC1 RNF216 TCGA-12-1088 ABCC1 AGBL4
TCGA-12-0827 ABCC12 FGFR4 TCGA-12-0829 ABCC6 SUMF1 TCGA-06-1801
ABCC6 CMTM7 TCGA-12-1088 ABL1 TNFRSF10B TCGA-06-1802 ADAM12 PTPRD
TCGA-12-0829 ADAM12 DAPK1 TCGA-12-1088 ADCY10 DUSP27 TCGA-19-0957
ADCY10 AKT3 TCGA-12-0828 ADCY2 SDAD1 TCGA-12-0829 ADCY2 C14orf174
TCGA-12-0829 ADCY8 SSX3 TCGA-12-0829 AGBL4 NUP188 TCGA-06-1805
AGBL4 NOX4 TCGA-12-1089 AHNAK C21orf29 TCGA-12-0829 ANXA7 CACNA1C
TCGA-06-1801 AP4S1 EYS TCGA-12-0828 AQP2 ECE1 TCGA-19-0957 AQP2
CDH4 TCGA-19-1790 ARMC6 FAM184B TCGA-19-1786 ATP5B USP48
TCGA-12-0829 ATP5B PRC1 TCGA-12-1600 ATP6AP1L FAM172A TCGA-12-0829
ATP6V0D2 RERE TCGA-12-0829 ATXN1 CACNA1G TCGA-06-1802 BAHD1 OSBPL10
TCGA-12-0820 BBX CUL3 TCGA-19-2621 BCAS3 TTYH1 TCGA-12-1088 BCAS3
CACNA1G TCGA-06-1801 C15orf23 DMD TCGA-12-3644 C15orf33 PARP16
TCGA-12-0829 C21orf29 MYT1 TCGA-06-1805 C2CD3 XRRA1 TCGA-12-0829
C2CD3 CAPZ8 TCGA-12-1089 C6orf170 NKAIN2 TCGA-12-0822 C7orf44 TACC2
TCGA-12-1089 CACNA1C ITGAV TCGA-12-0829 CACNA1G CNTNAP4
TCGA-12-0820 CAMKK1 FAM184B TCGA-12-1089 CAMSAP1 NCF2 TCGA-12-1088
CAMTA1 TMPRSS3 TCGA-06-1801 CAMTA1 GDPD2 TCGA-06-1801 CAP2 DNAJC6
TCGA-19-0957 CCDC147 STK4 TCGA-12-0829 CCDC147 ISX TCGA-06-0166
CCDC158 SNX5 TCGA-19-0957 CDH11 RERE TCGA-06-1802 CELF2 PLA2G2F
TCGA-12-0826 CELF2 NME4 TCGA-12-1600 CILP PARP16 TCGA-12-1089 CLK3
LRP1 TCGA-12-1088 CMYA5 STK24 TCGA-27-1835 CMYA5 SRRM1 TCGA-12-1092
CNTN2 DNAJC6 TCGA-06-1805 COL14A1 NCRNA00157 TCGA-12-0829 COL14A1
MMP12 TCGA-12-1093 CORO7 PLEK2 TCGA-12-0829 CORO7 DYRK3
TCGA-06-1801 CROCC CSMD2 TCGA-19-0957 CSMD2 MDH2 TCGA-06-1805 CUL3
SLC44A2 TCGA-12-0827 CUL3 LY75 TCGA-12-0829 DDI2 KIDINS220
TCGA-19-5958 DEPDC5 SLC5A4 TCGA-06-1801 DEPDC5 ROBO1 TCGA-06-1801
DEPDC7 EIF2C2 TCGA-12-0829 DIS3L GLI3 TCGA-12-0829 DMD N4BP2L2
TCGA-06-1802 DNM1L SYNPO2 TCGA-12-3644 EDA SSX3 TCGA-12-3644 EFHC1
LRBA TCGA-12-0829 EFS NRXN3 TCGA-06-1802 EIF2C2 TNFRSF10B
TCGA-19-0957 EML1 NRXN3 TCGA-12-0829 ENTPD2 FREM2 TCGA-12-0829 EYS
IL1RN TCGA-14-0781 FAM160A1 UNC93B1 TCGA-12-0829 FAM160A1 LY75
[0318] Table 3 above shows recurrent gene fusion pairs from
Exome-fuse analysis of 84 GBM from TCGA. Fusion candidates have
been nominated if they have at least two split inserts and at least
two split reads. To further filter the list on recurrence, any
fusion candidate was kept in which one of the genes is involved in
at least two fusions across different samples.
[0319] To experimentally validate the computational predictions
that emerged from TX-Fuse, the PCR products spanning the fusion
breakpoint were sequenced and validated each of the five in-frame
fusion predictions (FIGS. 1 and 9). In FIG. 1B, the prediction is
shown and in FIG. 1C, the cDNA sequence validation for the fusion
with the highest read support involving FGFR3 fused in-frame with
TACC3 in GSC-1123 is shown. The same FGFR3-TACC3 fusion transcript
was also detected in the primary GBM-1123 tumor specimen from which
the GSC-1123 culture was established (FIG. 1C). The amplified cDNA
contained an open reading frame for a protein of 1,048 amino acids
resulting from the fusion of a FGFR3 amino-terminal portion of
residues 1-758 with a TACC3 carboxy-terminal portion of residues
549-838 (FIG. 1D). FGFR3 is a member of the FGFR receptor tyrosine
kinase (TK) family that transduces intracellular signals after
binding to FGF ligands (Turner and Grose, 2010). TACC3 belongs to
the evolutionarily conserved TACC gene family, which also includes
TACC1 and TACC2. The distinctive feature of TACC proteins is the
presence of a coiled-coil domain at the C-terminus, known as the
TACC domain. Through the TACC domain, TACC proteins localize to the
mitotic spindle during metaphase and stabilize the microtubule
spindle network (Hood and Royle, 2011; Peset and Vernos, 2008). In
the predicted fusion protein the intracellular TK domain of FGFR3
is fused upstream of the TACC domain of TACC3 (FIG. 1D).
[0320] Exon-specific gene expression analysis from the RNA-seq
coverage in GSC-1123 demonstrated that the FGFR3 and TACC3 exons
implicated in the fusion are highly overexpressed compared with the
mRNA sequences not included in the fusion event (FIG. 10A).
Quantitative RT-PCR showed that the expression of the fused
FGFR3-TACC3 exons is significantly higher in GSC-1123 than other
GSCs and the normal brain (80 to 130-fold, FIG. 10B). Without being
bound by theory, functionally significant genetic rearrangements
may result in marked overexpression (outlier) of the genes
implicated in the fusion events (Tomlins et al., 2007; Tomlins et
al., 2005). The FGFR3-TACC3 fusion protein was also abundantly
expressed in GSC-1123 and in the primary tumor GBM-1123, as shown
by Western blot and immunohistochemistry (FIGS. 10C and 10D). On a
Western Blot, the FGFR3-TACC3 fusion protein migrated at a size of
.about.150 kD and immunoprecipitation followed by mass spectrometry
revealed the presence of FGFR3 and TACC3 peptides consistent with
the cDNA translation prediction (FIG. 10E). Using PCR, the genomic
breakpoint coordinates were mapped to chromosome 4 (#1,808,966 for
FGFR3 and #1,737,080 for TACC3, genome build GRCh37/hg19) falling
within FGFR3 exon 17 and TACC3 intron 7, which gives rise to a
transcript in which the 5' FGFR3 exon 16 is spliced to the 3' TACC3
exon 8. The DNA junctions of FGFR3 and TACC3 show microhomology
within a 10-base region, an observation consistent with results
previously reported for other chromosomal rearrangements in human
cancer (Bass et al., 2011; Stephens et al., 2009) (FIG. 1E).
[0321] The experimental validation of the inferred genomic fusions
was focused on FGFR3-TACC3. Exome-Fuse identified FGFR3-TACC3 gene
fusions in four GBM samples with breakpoints spanning invariably
within intron 16 of FGFR3 (which is downstream to the coding region
for the TK domain) and intron 7-10 of TACC3 (which is upstream to
the TACC domain) (FIG. 2A, Tables 4 and 5). Among the four positive
TCGA GBM specimens, two were available from TCGA centers for
molecular analysis (TCGA-27-1835 and TCGA-06-6390) and, by Sanger
sequencing, each of them were confirmed to carry an in-frame fusion
transcript that is consistent with the predicted genomic
breakpoints (FIGS. 2B and 2C). Thus, the frames of the FGFR3-TACC3
fusion proteins invariably result in juxtaposing the TK domain of
FGFR3 upstream of the TACC domain of TACC3. Consistent with the
abundant expression of FGFR3-TACC3 in GSC-1123 and GBM-1123, the
mRNA expression analysis of the TCGA tumors revealed that the four
FGFR3-TACC3-positive GBM display marked co-outlier expression of
FGFR3 and TACC3 (FIG. 2D). Recurrent gene fusions can be associated
with local copy number variations (CNV) of the breakpoint regions
(Wang et al., 2009). Accordingly, the analysis of SNP arrays in the
TCGA dataset revealed the presence of microamplification events of
the FGFR3 and TACC3 genes in all four FGFR3-TACC3-positive GBM
(FIG. 2E).
TABLE-US-00046 TABLE 4 List of split inserts supporting the
identification of FGFR3-TACC3 fusion genes in four GBM samples from
the ATLAS-TCGA exome collection (SEQ ID NOS 187-224, respectively,
in order of appearance) gene1 read read hg18 hg18 bit TCGAsampleID
gene1 length readID %identity length mismatch gap start end genome
genome e-value score read2 fasta TCGA-06-6390 FGFR3 76
C01PRACXX110828:1:1301:1934:116558 100 76 0 0 1 76 1778372 1778447
8E-40 151 GTGCTGGCATGCCGCGCC CTCCCAGAGGCCCACCTT CAAGCAGCTGGTGGAGGA
CCTGGACCGTGTCCTTAC CGTG TCGA-06-6390 FGFR3 76
C01RDACXX110828:3:2305:4872:47002 98.68 76 1 0 1 76 1778364 1778439
2E-37 143 ATGCGGGAGTGCTGGCAT GACGCGCCCTCCCAGAGG CCCACCTTCAAGCAGCTG
GTGGAGGACCTGGACCGT GTCC TCGA-06-6390 FGFR3 76
D03U9ACXX110625:6:1203:16178:138219 100 76 0 0 1 76 1778413 1778488
8E-40 151 AGCTGGTGGAGGACCTGG ACCGTGTCCTTACCGTGA CGTCCACCGACGTGAGTG
CTGGCTCTGGCCTGGTGC CACC TCGA-06-6390 TACC3 76
C01PRACXX110829:2:1102:13552:120312 100 76 0 0 1 76 1708918 1708843
8E-40 151 CCCTTAAAACAACTCGTT CCCTCAGACCACACACAA GACAGTTCAAGAGGGACT
CAAGGACTTACAGGAATG TCCA TCGA-06-6390 TACC3 76
C01PRACXX11D628:8:2308:6515:60354 100 76 0 0 1 76 1709956 1708881
8E-40 151 AACCAAAGGCTCAGACCC CCAGGAATAGAAAATATA GGCCCTTAAAACAACTCG
TTCCCTCAGACCACACAC AAGA TCGA-06-6390 TACC3 76
C01RDACXX110628:6:1305:16843:57213 98.68 76 1 0 1 76 1708865
1708780 2E-37 143 TCAAGGACTTACAGGAAT GTCCAGTGCTCCCAAGAA
ATCGAACTCCACAAGCTT GGCTTCCCGCGCACGTCC TGAG TCGA-06-6390 TACC3 75
D03U9ACXX110625:2:1202:19578:90281 100 75 0 0 1 75 1708861 1708787
3E-39 149 GGACTTACAGGAATGTCC AGTGCTCCCAAGAAATCG AACTCCACAAGCTTGGCT
TCCCGCGGACGTCCTGAG GGAT TCGA-06-6390 TACC3 76
D03U9ACXX110625:4:2306:2024:174970 100 76 0 0 1 76 1708869 1708821
8E-40 151 CAGACCACACACAAGACA GTTCAAGAGGGACTCAAG GACTTACAGGAATGTCCA
GTGCTCCCAAGAAATCGA ACTC TCGA-12-0826 FGFR3 72
61C59AAXX100217:4:21:17613:20556 98.61 72 0 1 1 71 1778439 1778510
2E-34 133 CTTACCGTGACGTCCACC GACGTGAGTGCTGGCTCT GGCCTGGTGCCACCCGCC
TATGCCCCTCCCCTGCCC TTAG TCGA-12-0826 TACC3 75
42MJNAAXX090813:5:30:1412:128060 100 75 0 0 2 76 1707299 1707225
3E-39 149 AAACTTGAGGTATAAGGA CTGCTTCCTCAAGGCCGA CTCCTTAAACTGGGGACA
AGAGGGCAAGTGATCAGG TCTG TCGA-12-0826 TACC3 76
61C59AAXX1002:17:4:2:4279:6948 100 76 0 0 1 76 1707209 1707224
8E-40 151 AACTTGAGGTATAAGGAC TGCTTGGTGAAGGCCGAC TCCTTAAACTGGGGACAA
GAGGGCAAGTGATCAGGT CTGA TCGA-12-0826 FGFR3 76
42MJNAAXX090813:5:37:435:1250#0 100 76 0 0 1 76 1778340 1778421
8E-40 151 GCCCGCAGGTACATGATC ATGCGGGAGTGCTGGCAT GCCGCGCCCTCCCAGAGG
CCCACCTTCAAGCAGCTG GTGG TCGA-12-0826 FGFR3 51
61C59AAXX100217:5:52:7727:2557 98.04 51 1 0 1 51 1778443 1778426
4E-24 93.7 ACCGTGACGTCCACCGAC GTGAGTGCTGGCTCTGGC CTGGTGCGACCCGCCGAT
CTCTCTCCCCTGTCCTTT TCCT TCGA-19-5958 TACC3 62
D03U9ACXX110825:4:2208:9451:114169 90.32 62 6 0 1 62 1707141
1707202 4E-17 75.6 TGGGAGGGTGCGGGGGGC CGGGGGGGGGAGTGTGCA
GGTGAGCTCCCTGGCCCT TGGCCCCCTGCCCTCTGG GGGG TCGA-19-5958 TACC3 74
D03U9ACXX110825:1:2204:20084:21192 98.95 74 3 0 1 74 1707097
1707170 5E-33 123 CTGGGAATGGTGGTGTCT CGGGCAGGGTTGTGGGTG
ACCGGGGGTGGGAGGGTG CGGGGGACCGGGGGGGGG AGGG TCGA-27-1835 FGFR3 76
C00HWAEXX110325:7:2202:17660:110656 100 76 0 0 1 76 1778338 1778413
8E-40 151 AGCGCCCTGCCCGCAGGT ACATGATCATGCGGGAGT GCTGGCATGCCGCGCCCT
CCCAGAGGCCCACCTTCA AGCA TCGA-27-1835 TACC3 76
C00HWAEXX110325:7:1104:10731:5183 100 76 0 0 1 76 1709417 1709417
8E-40 151 GCCAACGCCATGCCCAGG CCGGAGAGTCCCGGGGAG GCTGCTGGTGGGCAGCTG
ACTGCGGGGACACTGGGT GGAA TCGA-27-1835 TACC3 60
B0972ABXX11D408:2:2201:5911:24541 100 60 0 0 1 60 1709445 1709445
3E-30 119 AGGCCACCAGAGGCCAAC GCCATGCCCAGGCCGGAG AGTCCCGGGGAGGCTGCT
GGTGGGGAGGCGAACGCG GGGA TCGA-27-1835 TACC3 61
B097UABXX110405:4:2102:15742:63594 91.8 61 5 0 1 61 1709422 1709422
6E-19 91.9 TGCCCAGGCCGGAGAGTC CCGGGGCGGCTGCTGGGG GGGAGCTGACTGGGGGGG
CACTGGGGGGGAGACCCG GGCC hg18 hg18 gene2 read read genome genome bit
TCGAsampleID gene2 length readID %identity length mismatch gap
start end start end e-value score read2 fasta TCGA-06-6390 TACC3 76
C01FRACXX110628:1:1301:1934:116558 100 76 0 0 1 76 1708922 1708847
8E-40 151 TAGGCCCTTAAAACAACT CGTTCCCTCAGACCACAC ACAAGACAGTTCAAGAGG
GACTCAAGGACTTACAGG AATG TCGA-06-6390 TACC3 76
C01RDACXX110629:3:2305:4872:47008 100 76 0 0 1 76 1708867 1708792
8E-40 151 ACTCAAGGACTTACAGGA ATGTCCAGTGCTCCCAAG AAATCGAACTCCACAAGC
TTGGCTTCCCGCGGACGT CCTG TCGA-06-6390 TACC3 76
D03U9ACXX110625:6:1203:16178:138219 100 76 0 0 1 76 1708921 1708846
8E-40 151 AGGCCCTTAAAACAACTC GTTCCCTCAGACCACACA CAAGACAGTTCAAGAGGG
ACTCAAGGACTTACAGGA ATGT TCGA-06-6390 FGFR3 76
C01PRACXX110828:2:1102:13552:120312 100 76 0 0 1 76 1778387 1778462
8E-40 151 GCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGA GGACCTGGACCGTGTCCT
TACCGTGACGTCCACCGA CGTG TCGA-06-6390 FGFR3 76
C01PRACXX110528:8:2308:6515:60354 100 76 0 0 1 76 1778382 1778457
8E-40 151 GCCGCGCCCTCCCAGAGG CCCACCTTCAAGCAGCTG GTGGAGGACCTGGACCGT
GTCCTTACCGTGACGTCC ACCG TCGA-06-6390 FGFR3 76
C01RDACXX110628:6:1305:16843:57213 96.05 76 3 0 1 76 1778417
1778492 1E-32 127 GGTGGAGGACCTGGACCG TGACCTTACCGGGACGTC
CACCGACGGGAGTGCTGG CTCTGGCCTGGTGCCACC CGCC TCGA-06-6390 FGFR3 76
D03U9ACXX110525:2:1202:19578:90281 100 76 0 0 1 76 1778447 1776522
8E-40 151 GACGTCCACCGACGTGAG TGCTGGCTCTGGCCTGGT GCCACCCGCCTATGCCCC
TCCCCCTGCCGTCCCCGG CCAT TCGA-06-6390 FGFR3 76
D03U9ACXX110525:4:2306:2694:174970 100 76 0 0 1 76 1778435 1778510
8E-40 151 TGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGG CTCTGGCCTGGTGCCACC
CGCCTATGCCCCTCCCCC TGCC TCGA-12-0826 TACC3 72
61C59AAXX100217:4:21:17613:20886 95.83 72 3 0 1 72 1707352 1707221
3E-30 119 TACCTGCTGGTCTCGGTG GCCACGGGCACTGGTCTA CCAGGGCTGTCCCTCCGG
AGGGGGTCAAACTTGAGG GATA TCGA-12-0826 FGFR3 76
42MJNAAXX090813:5:30:1412:1280#0 98.68 76 1 0 1 76 1778427 1778502
2E-37 143 CTGGACCGTGTCCTTACC GTGACGTCCACCGACGTG AGTGCTGGCTCTGGCCTG
GTGCCACCCGCCCATGCC CCTC TCGA-12-0826 FGFR3 76
61C59AAXX100217:4:2:4279:6949 98.68 75 0 1 1 75 1778435 1776510
8E-37 141 TGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGG CTCTGGCCTGGTGCCACC
CGCCTATGCCCCTCCCCT GCCC TCGA-12-0826 TACC3 67
42MJNAAXX090813:5:37:435:1250#0 98.51 67 1 0 1 67 1707635 1707569
5E-32 125 AAAAGATTTAAGTTTAGA TCTTTAATATACCTAGAA CGGTGGCTGTAACCAGCA
AGGCAGGAGCCCTTTGTG TTGG TCGA-12-0826 TACC3 75
61C59AAXX100217:5:69:7727:2557 97.33 76 2 0 2 76 1707306 1707232
5E-36 133 TGGGTCAAACTTGAGGTA TAAGGACTGCTTCCTCAA GGCCGACTCCTTATACTG
GGGACAAGAGGGCAAGTG ATCA TCGA-19-5958 FGFR3 76
D03U9ACXX110625:4:2206:9451:114168 98.68 76 1 0 1 76 1778462
1776537 2E-37 143 GAGTGCTGGCTCTGGCCT GGTGCCACCCGCCTATGC
CCCTCCCCCTGGCGTCCC CGGCCATCCTGCCCCCCA GAGT TCGA-19-5958 FGFR3 76
D03U9ACXX110625:1:2204:20064:21192 100 76 0 0 1 76 1778462 1776537
2E-41 151 GAGTGCTGGCTCTGGCCT
GGTGCCACCCGCCTATGC CCCTCCCCCTGCCGTCCC CGGCCATCCTGCCCCCCA GAGT
TCGA-27-1835 TACC3 76 C00HWABXX110325:7:2202:17680:110566 96.05 76
3 0 1 76 1709492 1709417 1E-32 127 GCCAACGCCATGCCCAGG
CCGGAGAGTCCCGGGGAG GCTGCTGGTGGGGAGCTG ACTTCGGGGACACTGGGG GGAA
TCGA-27-1835 FGFR3 76 C00HWABXX110325:7:1104:10731:5183 96.05 76 3
0 1 76 1778363 1776438 1E-32 127 CATGCGGGAGTGCTGGCA
TGGCGCGCCCTCCCAGCG GCCCACCTTCAAGCAGCT GGTGGGGGACCTGGACCG TGTC
TCGA-27-1835 FGFR3 76 B09V2ABXX110408:2:2291:5811:24541 100 76 0 0
1 76 1778458 1778533 8E-40 151 ACGTGAGTGCTGGCTCTG
GCCTGGTGCCACCCGCCT ATGCCCCTCCCCCTGCCG TCCCCGGCCATCCTGCCC CCCA
TCGA-27-1835 FGFR3 76 B097UABXX110405:4:2102:15742:63594 100 76 0 0
1 76 1778388 1776453 8E-40 151 CCCTCCCAGAGGCCCACC
TTCAAGCAGCTGGTGGAG GACCTGGACCGTGTCCTT ACCGTGACGTCCACCGAC GTGA
TABLE-US-00047 TABLE 5 List of split reads supporting the
identification of FGFR3-TACC3 fusion genes in four GBM samples from
the ATLAS-TCGA exome collection (SEQ ID NOS 225-318, respectively,
in order of appearance) sample genesplit1 readID directionsplit
hg18startsplit1 hg18stopsplit1 length1 mismatch1 gap1 seqsplit
TCGA-06-6390 TACC3 D03U9ACXX110025:2:1202:19578:90281 R 1778521
1778521 1 0 0 GGACTTACAGGAATGTCCAG TGCTCCCAAGAAATCGAACT
CCACAAGCTTGGCTTCCCGC GGACGTCCTGAGGGA***T TCGA-06-6390 FGFR3
C01PRACXX11025:3:1104:10052:66371 F 1778520 1778521 2 0 0
CA***TCCCTCAGGACGTCC GCGGGAAGCCAAGCTTGTGG AGTTCGATTTCTTGGGAGCA
CTGGACATTCCTGTAAGTC TCGA-06-6390 FGFR3
C01PRACXX11028:5:1108:3119:22892 F 1778520 1778521 2 0 0
CA***TCCCTCAGGACGTCC GCGGGAAGCCAAGCTTGTGG AGTTCGATTTCTTGGGAGCA
CTGGACATTCCTGTAAGTC TCGA-06-6390 FGFR3
D03I9ACXX110025:8:2304:13007:108832 F 1778520 1778521 2 0 0
CA***TCCCTCAGGACGTCC GCGGGAAGCCAAGCTTGTGG AGTTCGATTTCTTGGGAGCA
CTGGACATTCCTGTAAGTC TCGA-06-6390 FGFR3
C01PRACXX11028:5:2108:1999:91559 F 1778518 1778521 4 0 0
GCCA***TCCCTCAGGACGT CCGCGGGAAGCCAAGCTTGT GGAGTTCGATTTCTTGGGAG
CACTGGACATTCCTGTAAG TCGA-06-6390 FGFR3
C01RDACXX110529:3:1336:1446:68211 F 1778515 1778521 7 0 0
CCGGCCA***TCCCTGAGGA CGTCCGCGGGAAGCCAAGCT TGTGGAGTTCGATTTCTTGG
GAGCACTGGACATTCCTGT TCGA-06-6390 TACC3
D03U9ACXX110605:5:2205:12523:196352 R 1778514 1778521 8 1 0
CAGGAATGTCCAGTGCTACC AAGAAATCGAACTCCACAAG CTTGGGTTCCCGCGGACGTC
CTCCGGGA***TGGCCGTG TCGA-06-6390 TACC3
C01PRACXX110628:5:2103:6315:17943 R 1778514 1778521 6 0 0
CAGGAATGTCCAGTGCTCCC AAGAAATCGAACTCCACAAG CTTGGCTTCCCGCGGACGTC
CTGAGGGA***TGGCCGGG TCGA-06-6390 FGFR3
C01PRACXX110629:3:1204:10831:2928 F 1778512 1778521 10 0 0
TCCCCGGCCA***TCCCTCA GGACGTCCGCGGGAAGCCAA GCTTGTGGAGTTCGATTTCT
TGGGAGCACTGGACATTCC TCGA-06-6390 FGFR3
C01PRACXX110828:5:2209:6732:191360 F 1778512 1778521 10 0 0
TCCCCGGCCA***TCCCTCA GGAAGTCCGCGGGAAGCCAA GCTTGTGGAGTTCGATTTCT
TGGGAGCACTGGACATTCC TCGA-06-6390 FGFR3
C01PRACXX110628:8:1308:2911:20590 F 1778511 1778521 11 0 0
GTCCCCGGCCA***TCCCTC AGGACGTCCGCGGGAAGCCA AGCTTGTGGAGTTCGATTTC
TTGGGAGCACTGGACATTC TCGA-06-6390 FGFR3
C01PRACXX110628:8:2207:4588:64017 F 1778509 1778521 13 0 0
CCGTCCCCGGCCA***TCCC TCAGGACGTCCGCGGGAAGC CAAGCTTGTGGAGTTCGATT
TCTTGGGAGCACTGGACAT TCGA-06-6390 TACC3
C01PRACXX110628::2205:11925:39734 R 1778501 1778521 21 0 0
TGCTCCCAAGAAATCGAACT CCACAAGCTTGGCTTCCCGC GGACGTCCTGAGGGA***TG
GCCGGGGACGGCAGGGGGA TCGA-06-6390 TACC3
C01PRACXX110528:6:1105:12159:179489 R 1778494 1778521 28 0 0
AAGAAATCGAACTCCACAAG CTTGGCTTCCCGCGGACGTC CTGAGGGA***TGGCCGGGG
ACGGCAGGGGGAGGGGCAT TCGA-06-6390 TACC3
D03U9ACXX110625:4:2209:12501:40382 R 1778491 1778521 31 1 0
AAATCGAACTCCACAAGCTT GGCTTCCCGCGGACGTCCTG AGGGA***TGGCCGGGGGCG
GCAGGGGGAGGGGCATAGG TCGA-06-6390 FGFR3
C01PRACXX110628:3:1305:3044:13239 F 1778473 1778521 49 0 0
CTGGCCTGGTGCCACCCGCC TATGCCCCTCCCCCTGCCGT CCCCGGCCA***TCCCTCAG
GACGTCCGCGGGAAGCCAA TCGA-06-6390 TACC3
D03U9ACXX110625:5:2205:12523:195352 R 1778470 1778521 52 4 0
TCGTCCCGCGGACTTCCTGA TGGA***TCGCCGGGGACGG CAGGGGGAGGGGCATAGGCG
TGTGGCACCAGGCCAGCTC TCGA-06-6390 TACC3
C01PRACXX110528:7:2205:11825:39734 R 1778469 1778521 53 1 0
CTTCCCGCGGACGTCCTGAG GGA***TGGCCGGGGACGGA AGGGGGAGGGGCATAGGCGG
GTGGCACCAGGCCAGAGCC TCGA-06-6390 FGFR3
D03U9ACXX110525:7:2100:4492:193350 F 1778464 1778521 58 0 0
GTGCTGGCTCTGGCCTGGTG CCACCCGCCTATGCCCCTCC CCCTGCCGTCCCCGGCCA**
*TCCCTCAGGACGTCCGCG TCGA-06-6390 TACC3
C01PRACXX110628:5:2103:6815:17943 R 1778452 1778521 70 0 0
GAGGGA***TGGCCGGGGAC GGCAGGGGGAGGGGCATAGG CGGGTGGCACCAGGCCAGAG
CCAGCACTCACGTCGGTGG TCGA-12-0826 TACC3
B1C5RAAXX1D0217:4:93:15133:6133 R 1778495 1778502 6 0 0
GGACAAGAGGGCAAGTGATC AGGTCTGACTGCCATCCCCT AACACACACAGGGGGGCTAA
GGGCAGGG***GAGGGGCA TCGA-12-0826 TACC3
B1C59AAXX100217:5:107:10875:15040 R 1778495 1778502 8 0 0
GGACAAGAGGGCAAGTGATC AGGTCTGACTGCCATCCCCT AACACACACAGGGGGGCTAA
GGGCAGGG***GAGGGGCA TCGA-12-0826 FGFR3
B1C59AAXX100217:5:108:1909:11295 F 1778494 1778502 9 0 0
ATGCCCCTC***CCCTGCCC TTAGCCCCCCTGTGTGTGTT AGGGGATGGCAGTCAGACCT
GATCACTTGCCCTCTTGTC TCGA-12-0826 FGFR3
B1C59AAXX100217:5:82:13129:10637 F 1778490 1778502 13 0 0
GCCTATGCCCCTC***CCCT GCCCTTAGCCCCCCTGTGTG TGTTAGGGGATGGCAGTCAG
ACCTGATCACTTGCCCTCT TCGA-12-0826 FGFR3
42MJNAAXX090813:5:80:891:1877#0 F 1778481 1778502 22 0 0
GTGCCACCCGCCTATGCCCC TC***CCCTGCCCTTAGCCC CCCTGTGTGTGTTAGGGGAT
GGCAGTCAGACCTGATCAC TCGA-12-0826 TACC3
B1C59AAXX100217:3:75:30598:12001 R 1778470 1778502 33 1 0
TGACTGCCATCCCCTAACAC ACACAGGGGGGCTAAGGGCA GGG***GAGGGGCATAGGCG
GGGGGCACCAGGCCAGAGC TCGA-12-0826 TACC3
B1C59AAXX100217:4:114:5844:3101 R 1778470 1778502 33 1 0
TGACTGCCATCCCCTAACAC ACACAGGGGGGCTAAGGGCA GGG***GAGGGGCATAGGCG
GGGGGCACCAGGCCAGAGC TCGA-12-0826 TACC3
42MJNAAXX0908136:70:652:108#0 R 1778458 1778502 37 3 0
TGCCATCCCCTAACACACAC AGGGGGGCTAAGGGCAGGG* **GAGGGGCATAGGCGGGGG
GCACCAGGACAGAGGCAGC TCGA-12-0826 TACC3
B1C59AAXX100217:3:55:4955:15075 R 1778451 1778502 52 5 0
CACACAGGGGGGCTAAGGGC AGGG***GAGGGGCATAGGC GGGGGGGACCAGGCCCGAGC
CAGCACTCACGTCGGGGGG TCGA-12-0826 FGFR3
42MJNAAXX090813:5:23:158:1180#0 F 1778447 1778502 56 0 0
GACGTCCACCGACGTGAGTG CTGGCTCTGGCCTGGTGCCA CCCGCCTATGCCCCTCC***
CCCTGCCCTTAGACCCCCTG TCGA-12-0826 FGFR3
B1C59AAXX100217:4:21:17013:20886 F 1778439 1778502 64 0 0
CTTACCGTGACGTCCACCGA CGTGAGTGCTGGCTCTGGCC TGGTGCCACCCGCCTATGCC
CCTC***CCCTGCCCTTAG TCGA-12-0826 FGFR3
B1C59AAXX100217:4:2:4279:5949 F 1778438 1778502 68 0 0
TGTCCTTACCGTGACGTCCA CCGACGTGAGTGCTGGCTCT GGCCTGGTGCCACCCGCCTA
TGCCCCTC***CCCTGCCC TCGA-19-5958 TACC3
C01RDACXX110528:6:1102:11157:101952 R 1778533 1778539 7 0 0
CGGGGGTGGGAGTGTGCGGG TGACCGGGGGTGGGAGTGTG CAGGTGACCTCCCTGGCCCT
TAGCCCCCT***GCACTCT TCGA-19-5958 TACC3
C01REACXX110629:2:2104:6009:99392 R 1778517 1778539 23 0 0
CGGGTGACCGGGGGAGGGAG TGTGCAGGGGACCTCCCTGA GGGTTAGCCCCCT***GCAC
TCTGGGGGGCAGGATGGCC TCGA-19-5958 TACC3
C01PRACXX110022:7:2103:12434:91988 R 1778501 1778539 39 0 0
GGAGTGTGCAGGTGACCTCC CTGGCCCTTAGCCCCCT*** GCACTCTGGGGGGCAGGATG
GCCGGGGACGGCAGGGGGA TCGA-27-1835 TACC3
B05UCABXX110322:5:1103:9252:48754 R 1778558 1778598 10 1 0
GGGGAGGCTGCTGGTGGGCA GCTGACTGCGGGGACACTGG GAGGAAGCCTGGACCCTCAG
CGAACT***TCGCCCAGCC TCGA-27-1835 FGFR3
C00HWABXX110325:4:1201:20830:90877 F 1778557 1778598 20 0 0
ACAGCCTGGGCACAGAGGTG GCTGTGCGA***AGGTCGCT GAGGGTCCAGGCTTCCACCC
AGTGTCCCCGCAGTCAGCT TCGA-27-1835 TACC3
B05UCABXX110322:5:1108:14043:83287 R 1778554 1778598 32 0 0
TGACTGCGGGGACACTGGGT GGAAGCCTGGACCCTCAGCG ACCT***TCGCACAGCCACC
TCTGTGCCCAGGCTGTGCC TCGA-27-1835 TACC3
B097UABXX110405:4:2204:19443:99453 R 1778558 1778598 38 0 0
CGGGGACACTGGGTGGAAGC CTGGACCCTCAGCGACCT** *TCGCACAGCCACCTCTGTG
CCCAGGCTGTGCCCCAGAA TCGA-27-1835 TACC3
B097UABXX110405:4:2201:20648:44401 R 1778557 1778598 39 2 0
GGGGACACTGGGTGGAAGCC TGGACCCTCAGCGACCT*** TCGCACAGCCACCTCTGTGG
CCAGGCTGTGCCACAGAAG
TCGA-27-1835 TACC3 B097UABXX110405:2:2104:15688:71022 R 1778555
1778598 41 0 0 GGACACTGGGTGGAAGCCTG GACCCTCAGCGACCT***TC
GCACAGCCACCTCTGTGCCC AGGCTGTGCCCCAGAAGGC TCGA-27-1835 TACC3
C00HWABXX110325:5:2102:20394:42427 R 1778543 1778598 53 3 0
GAAGCCTGGACCCTCAGCGA CCT***TCGCACAGCCACCT CTGTGCCCCGGCTGTGCCCC
AGCCGGCCCGCCCCACACC TCGA-27-1835 TACC3
B09V2ABXX110408:6:1203:19187:141962 R 1778543 1778598 53 0 0
GAAGCCTGGACCCTCAGCGA CCT***TCGCACAGCCACCT CTGTGCCCAGGCTGTGCCCC
AGAAGGCCCGCCCCACACC TCGA-27-1835 TACC3
B09V2ABXX110408:8:1205:4774:91604 R 1778537 1778598 59 0 0
TGGACCCTCAGCGACCT*** TCGCACAGCCACCTCTGTGC CCAGGCTGTGCCCCAGAAGG
CCCGCCCCACACCTCAGCA TCGA-27-1835 TACC3
C00HWABXX110325:2:1107:16165:23614 R 1778535 1778598 61 0 0
GACCCTCAGCGACCT***TC GCACAGCCACCTCTGTGCCC AGGCTGTGCCCCAGAAGGCC
CGCCCCACACCTCAGCACT TCGA-27-1835 TACC3
C00HWABXX110325:7:2107:1225:187363 R 1778530 1778598 66 0 0
TCAGCGACCT***TCGCACA GCCACCTCTGTGCCCAGGCT GTGCCCCAGAAGGCCCGCCC
CACACCTCAGCACTCTGGG TCGA-27-1835 TACC3
B097UABXX110405:2:214:15656:71022 R 1778523 1778598 73 0 0
CCT***TCGCACAGCCACCT CTGTGCCCAGGCTGTGCCCC AGAAGGCCCGCCCCACACCT
CAGCACTCTGGGGGGCAGG sample genesplit2 readID directionsplit
hg18startsplit2 hg18stopsplit2 length2 mismatch2 gap2 seqmate
TCGA-06-6390 FGFR3 D03U9ACXX110025:2:1202:19578:90281 R 1708787
1708851 75 0 0 GACGTCCACCGACGTGAGTG CTGGCTCTGGCCTGGTGCCA
CCCGCCTATGCCCCTCCCCC TGCCGTCCCCGGCCAT TCGA-06-6390 TACC3
C01PRACXX11025:3:1104:10052:66371 F 1708787 1708850 74 0 0
CAAGAGGGACTCAAGGACTT ACAGGAATGTCCAGTGCTCC CAAGAAATCGAACTCCACAA
GCTTGGCTTCCCGCGG TCGA-06-6390 TACC3
C01PRACXX11028:5:1108:3119:22892 F 1708787 1708850 74 0 0
CAAGAGGGACTCAAGGACTT ACAGGAATGTCCAGTGCTCC CAAGAAATCGAACTCCACAA
GCTTGGCTTCCCGCGG TCGA-06-6390 TACC3
D03I9ACXX110025:8:2304:13007:108832 F 1708787 1708850 74 0 0
ATAGGCCCTTAAAACAACTC GTTCCCTCAGACCACACACA AGACAGTTCAAGAGGGACTC
AAGGACTTACAGGAAT TCGA-06-6390 TACC3
C01PRACXX11028:5:2108:1999:91559 F 1708787 1708858 72 0 0
TCAAGAGGGACTCAAGGACT TACAGGAATGTCCAGTGCTC CCAAGAAATCGAACTCCACA
AGCTTGGCTTCCCGCG TCGA-06-6390 TACC3
C01RDACXX110529:3:1336:1446:68211 F 1708787 1705855 69 0 0
ACCACACACAAGACAGTTCA AGAGGGACTCAAGGACTTAC AGGAATGTCCAGTGCTCCCA
AGAAATCGAACTCCAC TCGA-06-6390 FGFR3
D03U9ACXX110605:5:2205:12523:196352 R 1708787 1705854 68 4 0
GAGCTGGCCTGGTGCCACAC GCCTATGCCCCTCCCCCTGC CGTCCCCGGCGATCCATCAG
GAAGTCCGCGGGACGA TCGA-06-6390 FGFR3
C01PRACXX110628:5:2103:6315:17943 R 1708787 1705854 68 0 0
CCACCGACGTGAGTGCTGGC TCTGGCCTGGTGCCACCCGC CTATGCCCCTCCCCCTGCCG
TCCCCGGCCATCCCTC TCGA-06-6390 TACC3
C01PRACXX110629:3:1204:10831:2928 F 1708787 1703852 66 0 0
CAAGAGCCTCAGACAGTGCA TGAGGGACCCGAGACAGTGC GGCGAGGGAACAGCACAGCG
GCCCCATGCCCCCAAC TCGA-06-6390 TACC3
C01PRACXX110828:5:2209:6732:191360 F 1708787 1703852 66 1 0
CAAGAGCCTCAGACAGTGCA TGAGGGACCCGAGACAGTGC GGCGAGGGAACAGCACAGGG
GCCCCATGCCCCCAAC TCGA-06-6390 TACC3
C01PRACXX110628:8:1308:2911:20590 F 1708787 1708851 65 0 0
CGTTCCCTCAGACCACACAC AAGACAGTTCAAGAGGGACT CAAGGACTTACAGGAATGTC
CAGTGCTCCCAAGAGA TCGA-06-6390 TACC3
C01PRACXX110628:8:2207:4588:64017 F 1708787 1708840 63 0 0
CCAGGAATAGAAAATATAGG CCCTTAAAACAACTCGTTCC CTCAGACCACACACAAGACA
GTTCAAGAGGGACTCA TCGA-06-6390 FGFR3
C01PRACXX110628::2205:11925:39734 R 1708787 1708841 55 0 0
GGCTCTGGCCTGGTGCCACC CGCCTATGCCCCTCCCCCTT CCGTCCCCGGCCATCCCTCA
GGACGTCCGCGGGAAG TCGA-06-6390 FGFR3
C01PRACXX110528:6:1105:12159:179489 R 1708787 1708834 48 0 0
GCCCTGCCCGCAGGTACATG ATCATGCGGGAGTGCTGGCA TGCCGCGCCCTCCCAGAGGC
CCACCTTCAAGCAGCT TCGA-06-6390 FGFR3
D03U9ACXX110625:4:2209:12501:40382 R 1708787 1708831 45 0 0
CTGGCATGCCGCGCCCTCCC AGAGGCCCACCTTTAAGCAG CTGGTAGAGGGCCTGGACCG
TGTCCTTACCGTGACG TCGA-06-6390 TACC3
C01PRACXX110628:3:1305:3044:13239 F 1708787 1708813 27 0 0
TAAAACAACTCGTTCCCTCA GACCACACACAAGACAGTTC AAGAGGGACTCAAGGACTTA
CAGGAATGTCCAGTGC TCGA-06-6390 FGFR3
D03U9ACXX110625:5:2205:12523:195352 R 1708787 1708810 24 4 0
CACGGCCATCCCGGAGGACG TCCGCGGGAACCCAAGCTTG TGGAGTTCGATTTCTTGGTA
GCACTGGACATTCCTG TCGA-06-6390 FGFR3
C01PRACXX110528:7:2205:11825:39734 R 1708787 1708809 23 0 0
TCCCCCTGCCGTCCCCGGCC ATCCCTCAGGACGTCCGCGG GAAGCCAAGCTTGTGGAGTT
CGATTTCTTGGGAGCA TCGA-06-6390 TACC3
D03U9ACXX110525:7:2100:4492:193350 F 1708787 1705804 18 0 0
AGACCACACACAAGACAGTT CAAGAGGGACTCAAGGACTT ACAGGAATGTCCAGTGCTCC
CAAGAAATCGAACTCC TCGA-06-6390 FGFR3
C01PRACXX110628:5:2103:6815:17943 R 1708787 1708702 6 0 0
CCCGGCCATCCCTCAGGACG TCCGCGGGAAGCCAAGCTTG TGGAGTTCGATTTCTTGGGA
GCACTGGACATTCCTG TCGA-12-0826 FGFR3 B1C5RAAXX1D0217:4:93:15133:6133
R 1707185 1707253 69 2 1 GGCATGCCGCGCCCTCCCAG AGGCCCACCTTCAAGCAGCT
GGTGGAGGACCTGGACCGTG TCCTTACCGTGACGTC TCGA-12-0826 FGFR3
B1C59AAXX100217:5:107:10875:15040 R 1707185 1707253 69 2 1
GGCATGCCGCGCCCTCCCAG AGGCCCACCTTCAAGCAGCT GGTGGAGGACCTGGACCGTG
TCCTTACCGTGACGTC TCGA-12-0826 TACC3
B1C59AAXX100217:5:108:1909:11295 F 1707185 1707252 68 2 1
CGGCGCACATACCTGCTGGT CTCGGTGGCCACGGGCACTG GTCTACCAGGACTGTCCCTC
AGGAGGGGGTCAAACT TCGA-12-0826 TACC3
B1C59AAXX100217:5:82:13129:10637 F 1707185 1707248 64 2 1
ATACCTGCTGGTCTCGGTGG CCACGGGCACTGGTCTACCA GGACTGTCCCTCAGGAGGGG
GTCAAACTTGAGGTAT TCGA-12-0826 TACC3 42MJNAAXX090813:5:80:891:1877#0
F 1707185 1707239 55 2 1 AGGTATAAGGACTGCTTCCT CAAGGCCGACTCCTTAAACT
GGGGACAAGAGGGCAAGTGA TCAGGTCTGACTGCCA TCGA-12-0826 FGFR3
B1C59AAXX100217:3:75:30598:12001 R 1707185 1707228 44 2 1
GGAGGACCTGGACTGTGTCC TTACCGTGACGTCCACCGAC GTGAGTGCTGGCTCTGGCCT
GGTGCCACCCGCCTAT TCGA-12-0826 FGFR3 B1C59AAXX100217:4:114:5844:3101
R 1707185 1707228 44 2 1 GGAGGACCTGGACCGTGTCC TTACCGTGACGTCCACCGAC
GTGAGTGCTGGCTCTGGCCT GGTGCCACCCGCCTAT TCGA-12-0826 FGFR3
42MJNAAXX0908136:70:652:108#0 R 1707185 1707224 40 2 1
CAAGCAGCTGGTGGAGGACC TGGACCGTGTCCTTACCGTG ACGTCCACCGACGTGAGTGC
TGGCTCTGGCCTGGTG TCGA-12-0826 FGFR3 B1C59AAXX100217:3:55:4955:15075
R 1707185 1707209 25 2 1 ACCTTCAAGCAGCTGGTGGA GGACCTGGACCGTGTCCTTA
CCGTGACGTCCACCGACGTG AGTGCTGGCTCTGGCC TCGA-12-0826 TACC3
42MJNAAXX090813:5:23:158:1180#0 F 1707185 1707205 21 2 1
CAAACTTGAGGTATAAGGAC TGCTTCCTCAAGGCCGACTC CTTAAACTGGGGACAAGAGG
GCAAGTGATCAGGTCT TCGA-12-0826 TACC3
B1C59AAXX100217:4:21:17013:20886 F 1707185 1707197 13 0 1
TACCTGCTGGTCTCGGTGGC CACGGGCACTGGTCTACCAG GGCTGTCCCTCCGGAGGGGG
TCAAACTTGAGGGATA TCGA-12-0826 TACC3 B1C59AAXX100217:4:2:4279:5949 F
1707185 1707193 8 0 1 AACTTGAGGTATAAGGACTG CTTCCTCAAGGCCGACTCCT
TAAACTGGGGACAAGAGGGC AAGTGATCAGGTCTGA TCGA-19-5958 FGFR3
C01RDACXX110528:6:1102:11157:101952 R 1707202 1707270 69 1 0
AGCTGGTGGAGGACCTGGAC CGTGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGGCT
CTGGCCTGGTGCCACC TCGA-19-5958 FGFR3
C01REACXX110629:2:2104:6009:99392 R 1707202 1707254 53 3 0
GCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGG ACCTGGACCGTGTCCTTACC
GTGACGTCCACCGACG TCGA-19-5958 FGFR3
C01PRACXX110022:7:2103:12434:91988 R 1707202 1707238 37 1 0
GCGGGAGTGCTGGCATGCCG
CGCCCTCCCAGAGGCCCACC TTCAAGCAGCTGGTGGAGGA CCTGGACCGTGTCCTT
TCGA-27-1835 FGFR3 B05UCABXX110322:5:1103:9252:48754 R 1709397
1709452 66 2 0 CCTCCACTGGGTCCTCAGGG GTGGGGGTCCCTCCGGGGCT
GGGCGGGGGAGGGACTGGCA GGCCTGCAGGGGGGTT TCGA-27-1835 TACC3
C00HWABXX110325:4:1201:20830:90877 F 1709397 1709443 47 0 0
TCACGGCAGCAAGAACCACA CTCACTGCTGCAAGGCCACC AGAGGCCAACGCCATGCCCA
GGCCGGAGAGTCCCGG TCGA-27-1835 FGFR3
B05UCABXX110322:5:1108:14043:83287 R 1709397 1709440 44 0 0
TACATGATCATGCGGGAGGG CTGGCATGCCGCGCCCTCCC AGAGGCCCACCTTCAAGCAG
CTGGTGGAGGGCCGGG TCGA-27-1835 FGFR3
B097UABXX110405:4:2204:19443:99453 R 1709397 1709434 38 0 0
GGTGGGAAGCGGCGGGGCTC ACTCCTGAGCGCCCTGCCCG CAGGGACATGATCATGCGGG
GGTGCTGGCCTTGCGG TCGA-27-1835 FGFR3
B097UABXX110405:4:2201:20648:44401 R 1709397 1709433 37 0 0
GCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGG ACCTGGACCGTGTCCTTACC
GTGACGTCCACCGACG TCGA-27-1835 FGFR3
B097UABXX110405:2:2104:15688:71022 R 1709397 1709431 35 0 0
CCTGCCCCCCAGAGTGCTGA GGTGTGGGGCGGGCCTTCTG GGGCACAGCCTGGGCACAGA
GGTGGCTGTGCGAAGG TCGA-27-1835 FGFR3
C00HWABXX110325:5:2102:20394:42427 R 1709397 1709419 23 0 0
GCAGGTACATGATCATGCGG GAGTGCCGGCATTTCGGGAC CTTCCCTCGGGCCACCCTCT
TCCGGTTGTTGTGGGC TCGA-27-1835 FGFR3
B09V2ABXX110408:6:1203:19187:141962 R 1709397 1709419 23 0 0
GCAGGTACATGATCATGCGG GAGTGCTGGCATGCCGCGCC CTCCCCGAGGACCACCTTCC
AGCAGCCGGGGGAGGG TCGA-27-1835 FGFR3
B09V2ABXX110408:8:1205:4774:91604 R 1709397 1709413 17 0 0
CCCGAATAAGGTGGGAAGCG GCGGGGCTCACTCCTGAGCG CCCTGACCGCAGGTACATGA
GCATGCGGGAGTGGCG TCGA-27-1835 FGFR3
C00HWABXX110325:2:1107:16165:23614 R 1709397 1709411 16 0 0
CGTGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGGCT CTGGCCTGGTGCCACCCGCC
TATGCCCCTCCCCCTG TCGA-27-1835 FGFR3
C00HWABXX110325:7:2107:1225:187363 R 1709397 1709400 10 0 0
ACATGATCATGCGGGAGTGC TGGCATGCCGCGCCCCCCCA GAGGCCCACCTTCAAGCAGC
TGGTGGAGGACCTGGA TCGA-27-1835 FGFR3
B097UABXX110405:2:214:15656:71022 R 1709397 1709390 3 0 0
GCCTTCTGGGGCACAGCCTG GGCACAGAGGTGGCTGTGCG AAGGTCGCTGAGGGTCCAGG
CTTCCACCCAGTGTCC
[0322] The FGFR3 and TACC3 genes are located 48-Kb apart on human
chromosome 4p16. The other members of the FGFR and TACC families
retain the close physical association of FGFR3 and TACC3, with
FGFR1 and TACC1 paired on chromosome 8p11 and FGFR2 and TACC2
paired on chromosome 10q26. Without being bound by theory, the
ancestral FGFR and TACC genes were physically linked and that this
tandem gene cluster was duplicated at least twice to generate the
FGFR1-TACC1, FGFR2-TACC2 and FGFR3-TACC3 pairs that mark mammalian
evolution (Still et al., 1999). The highly conserved TK domains
among FGFR genes and TACC domains among TACC genes together with
their invariable fusion in the FGFR3-TACC3 rearrangements prompted
to ask whether other intrachromosomal FGFR-TACC fusion combinations
exist in human GBM.
[0323] cDNA from a panel of 88 primary GBM were screened using
pairs of upstream PCR primers that bind the amino-terminal coding
region of the TK domains of FGFR1, FGFR2 and FGFR3 and downstream
primers that bind to the carboxy-terminal coding region of the TACC
domains of TACC1, TACC2 and TACC3 genes, respectively. The
screening resulted in the identification of intrachromosomal
FGFR-TACC fusions in two additional cases (one harboring
FGFR1-TACC1 and one FGFR3-TACC3), corresponding to three of 97
total GBM (3.1%), including the GBM-1123 case. The FGFR1-TACC1
fusion breakpoint in GBM-51 joined in-frame exon 17 of FGFR1 to
exon 7 of TACC1, resulting in a novel protein in which the TK
domain of FGFR1 is fused upstream of the TACC domain of TACC1 (FIG.
2F). The same structure was conserved again in GBM-22 in which exon
16 of FGFR3 is joined in-frame to exon 10 of TACC3 (FIG. 2G). None
of the tumors harboring FGFR-TACC fusions had mutations in IDH1 or
IDH2 genes, thus indicating that FGFR-TACC-positive GBM mark an
independent subgroup of patients from those carrying IDH mutations
(Table 6) (Yan et al., 2009). The constant linkage of the FGFR-TK
to the TACC domain created in each of the seven GBM harboring
FGFR-TACC rearrangements suggests that FGFR-TACC fusion proteins
may generate important functional consequences for oncogenesis in
the brain.
TABLE-US-00048 TABLE 6 Age at initial IDH1-2 status IDH1-2 status
Samples Type Time Status pathologic diagnosis (Sanger) (exome)
TCGA-12-0826 FGFR3-TACC3 845 DECEASED 38 WT WT TCGA-27-1635
FGFR3-TACC3 646 DECEASED 53 NA WT TCGA-19-5958 FGFR3-TACC3 164
LIVING 56 NA WT TCGA-06-6390 FGFR3-TACC3 163 DECEASED 58 WT WT
GBM-22 FGFR3-TACC3 390 DECEASED 60 WT NA GBM-1123 FGFR3-TACC3 NA
DECEASED 62 WT NA GBM-51 FGFR1-TACC1 NA NA NA WT NA Time = Survival
(days after diagnosis) Sanger = analysis done by Sanger sequencing
of genomic DNA Exome = ainalysis done by the SAVI (Statistical
Algorithm for Variant Identification), an algorithm developed to
detect point mutation in cancer (BRAF Mutations in Hairy-Cell
Leukemia, Tiacci E et al. The New England Journal of Medicine 2011
Jun 16; 364(24): 2305-15) NA = Not Available WT = Wild type
sequence for R132 and R172 of IDH1 and IDH2, respectively
[0324] Transforming Activity of FGFR-TACC Fusions.
[0325] To test the functional importance of the FGFR-TACC fusions
in GBM, the FGFR3-TACC3 cDNA was cloned from GSC-1123 and
recombinant lentiviruses were prepared expressing FGFR3-TACC3,
FGFR1-TACC1, a kinase-dead FGFR3-TACC3 protein (FGFR3-TACC3-K508M),
wild type FGFR3 and wild type TACC3. Transduction of Rat1A
fibroblasts and Ink4A;Arf-/- astrocytes with the FGFR3-TACC3
lentivirus resulted in the expression of the fusion protein at
levels comparable to those present in GSC-1123 (FIG. 11). Having
reconstituted in non-transformed cells the endogenous level of the
FGFR-TACC protein that accumulates in GBM cells, it was determined
whether it was sufficient to initiate oncogenic transformation in
vitro and in vivo. Rat1A cells expressing FGFR3-TACC3 and
FGFR1-TACC1 but not those expressing FGFR3-TACC3-K508M, FGFR3,
TACC3 or the empty lentivirus acquired the ability to grow in
anchorage-independent conditions in soft agar (FIG. 3A).
Transduction of the same lentiviruses in primary Ink4A;Arf-/-
astrocytes followed by subcutaneous injection into immunodeficient
mice revealed that only astrocytes expressing FGFR3-TACC3 and
FGFR1-TACC1 formed tumors. The tumors emerged in 100% of the mice
injected with astrocytes expressing the fusion proteins and were
glioma-like lesions with strong positivity for Ki67,
phospho-histone H3, nestin, GFAP and Olig2 (FIG. 3B).
[0326] Next, it was determined whether the FGFR3-TACC3 fusion
protein is oncogenic when transduced to a small number of cells
directly into the brain of immunocompetent animals. A recently
described mouse glioma model was used in which brain tumors are
initiated by lentiviral transduction of oncogenes and inactivation
of p53 in the mouse brain (Marumoto et al., 2009). To target adult
NSCs, the adult mouse hippocampus was stereotactically transduced
with purified lentivirus expressing the FGFR3-TACC3 protein and
shRNA against p53 (pTomo-FGFR3-TACC3-shp53). Seven of eight mice
(87.5%) transduced with FGFR3-TACC3 succumbed from malignant brain
tumors within 240 days (FIG. 3C). None of the mice transduced with
a lentivirus expressing the most frequent gain-of-function mutation
in GBM (the constitutively active EGFRvIII, pTomo-EGFRvIII-shp53)
or the pTomo-shp53 control lentivirus died or developed clinical
signs of brain tumors (FIG. 3C). The FGFR3-TACC3 tumors were
high-grade glioma with strong propensity to invade the normal brain
and stained positive for the glioma stem cell markers nestin and
Olig2 and the glial marker GFAP. They were also highly positive for
Ki67 and phospho-histone H3, thus displaying rapid tumor growth
(FIG. 3D). The expression of FGFR3-TACC3 in the xenograft and
intracranial tumor models was comparable to the expression of the
endogenous protein in the human GSCs and tumor (FIGS. 11D, 11E and
11F).
[0327] These data show that FGFR-TACC fusion proteins possess
transforming activity in two independent cellular models and this
activity is not the result of the overexpression of individual FGFR
and TACC genes. They also show that direct transduction of the
FGFR3-TACC3 protein to the adult mouse brain leads to efficient
development of malignant glioma.
[0328] The FGFR-TACC Fusions Interfere with Mitotic Progression and
Induce Chromosome Missegregation and Aneuploidy.
[0329] To elucidate the mechanism by which the FGFR-TACC fusion
drives oncogenesis, it was explored whether it activates downstream
FGFR signaling. FGFR3-TACC3 failed to hyperactivate the canonical
signaling events downstream of FGFR (pERK and pAKT) in the presence
or absence of the ligands FGF-1, FGF-2 or FGF-8 (Wesche et al.,
2011) (FIGS. 12A, 12B and 12C). However, FGFR3-TACC3 displayed
constitutive phosphorylation of its TK domain and the adaptor
protein FRS2, both of which were abolished by the specific
inhibitor of FGFR-associated TK activity PD173074 (Mohammadi et
al., 1998) or the K508M mutation (FIG. 4A). Thus, FGFR3-TACC3 gains
constitutive kinase activity that is essential for oncogenic
transformation but the downstream signaling of this aberrant
activity is distinct from the canonical signaling events downstream
to FGFR. By driving the localization of the fusion protein, the
TACC domain can create entirely novel TK-dependent functions. The
TACC domain is essential for the localization of TACC proteins to
the mitotic spindle (Hood and Royle, 2011; Peset and Vernos, 2008).
Confocal imaging showed that FGFR3-TACC3 designed an arc-shaped
structure bending over and encasing the metaphase spindle poles,
frequently displaying asymmetry towards one of the two poles and
relocated to the midbody as cells progressed into the late stages
of mitosis (telophase and cytokinesis) (FIGS. 4B and 12D).
Conversely, the localization of TACC3 was restricted to spindle
microtubules and did not relocalize to the midbody (FIG. 12E). Wild
type FGFR3 lacked discrete localization patterns in mitosis (FIG.
12F).
[0330] The mitotic localization of FGFR3-TACC3 indicates that it
may impact the fidelity of mitosis and perturb the accurate
delivery of the diploid chromosomal content to daughter cells, thus
generating aneuploidy. Mitotic progression of individual cells was
examined in vector-transduced and FGFR3-TACC3 expressing cells
co-expressing histone H2B-GFP by time-lapse microscopy. The average
time from nuclear envelope breakdown to anaphase onset was
increased in cells expressing FGFR3-TACC3 in comparison with
control cells. The mitotic delay was further exacerbated by
difficulties in completing cytokinesis (FIGS. 4C and 4D).
[0331] Next, it was determined whether the expressions of the
FGFR-TACC fusion proteins induce defects of chromosomal
segregation. Quantitative analyses of mitoses revealed that cells
expressing FGFR3-TACC3 or FGFR1-TACC1 exhibit a three to five fold
increase of chromosomal segregation errors than control cells. The
most frequent mitotic aberrations triggered by the fusion proteins
were misaligned chromosomes during metaphase, lagging chromosomes
at anaphase and chromosome bridges that impaired cytokinesis and
generated micronuclei in the daughter cells (FIGS. 4E, 4F and 13A).
Aberrations at the metaphase-anaphase transition frequently lead to
the inability of mitotic cells to maintain a metaphase arrest after
treatment with a spindle poison. Over 18% of cells expressing
FGFR3-TACC3 displayed prematurely separated sister chromatids in
contrast with less than 3% in control, FGFR3 or TACC3-expressing
cells (FIGS. 13B and 13C). Accordingly, cells expressing the fusion
protein were unable to efficiently arrest in metaphase after
nocodazole treatment (FIG. 13D).
[0332] The above findings indicate that expression of the
FGFR3-TACC3 fusion protein may spark aneuploidy. Karyotype analysis
revealed that FGFR3-TACC3 increased over 2.5 fold the percent of
aneuploidy and led to the accumulation of cells with broad
distribution of chromosome counts in comparison with cells
transduced with empty vector, FGFR3 or TACC3 (FIG. 5A).
Accordingly, GSC-1123 contained aneuploid modal number of
chromosomes (49) and manifested a broad distribution of chromosome
counts characterized by 60% of metaphase spreads that deviate from
the mode (Table 7)
TABLE-US-00049 TABLE 7 Chromosome analysis by SKY of 20 cells from
the GSC-1123 culture Cell # Chr # +1 +2 +3 t(3:14) +4 (-4) del(4)
+5 +6 +7 del(7) +8 -9 +9 -10 +10 +11 1 97 2 2 2 2 1 2 2 4 2 2 2 2
51 1 1 3 49 1 1 4 50 1 1 5 49 1 1 6 86 2 2 2 2 2 3 1 1 2 7 95 2 2 2
2 1 2 4 2 2 2 8 98 2 3 2 1 2 1 2 4 3 1 2 9 86 2 1 1 1 1 1 1 6 2 3 1
1 10 44 1 1 1 1 1 1 1 11 49 1 1 1 12 49 1 1 13 98 2 2 2 2 2 2 4 2 2
2 14 49 1 1 15 48 1 1 16 51 1 1 17 49 1 1 18 50 1 1 1 19 49 1 1 20
49 1 1 Cell # Chr # +12 +13 del(13) -14 +14 +15 -16 +16 +17 +18 +19
+20 -21 +21 -22 +22 +X 1 97 2 2 2 2 2 2 2 3 4 4 2 2 2 2 51 2 2 1 1
1 3 49 1 1 1 1 4 50 1 1 1 1 1 5 49 1 1 1 1 6 86 2 2 2 2 2 2 4 3 3 1
2 7 95 2 2 2 2 2 2 2 4 2 4 2 2 2 8 98 2 2 2 2 2 2 2 3 4 4 3 2 2 9
86 2 2 2 2 1 4 3 1 2 1 2 10 44 1 1 1 1 1 11 49 1 1 1 12 49 1 1 1 1
13 98 2 2 2 2 2 2 4 4 4 2 2 2 14 49 1 1 1 1 15 48 1 1 1 1 16 51 1 1
1 2 1 17 49 1 1 1 1 18 50 1 1 1 1 1 19 49 1 1 1 20 49 1 1 1 1
[0333] Next, it was determined whether aneuploidy is a direct
consequence of FGFR3-TACC3 expression and is induced in human
diploid neural cells. Primary human astrocytes analyzed six days
after transduction with the FGFR3-TACC3 lentivirus exhibited a
5-fold increase of the rate of aneuploidy and a significantly wider
distribution of chromosome counts (FIGS. 5B, 5C and 5D). Consistent
that aneuploidy is detrimental to cellular fitness, acute
expression of FGFR3-TACC3 compromised the proliferation capacity of
human astrocytes. However, continuous culture of
FGFR3-TACC3-expressing human astrocytes led to progressive gain of
proliferative capacity that overrode that of control cells (FIGS.
14A, 14B). Thus, the acute expression of FGFR3-TACC3 in primary
normal human cells from the central nervous system causes CIN and
aneuploidy with an acute fitness cost manifested by slower
proliferation.
[0334] It was also determined whether the CIN and aneuploidy caused
by FGFR3-TACC3 requires the TK activity of FGFR3 and can be
corrected. Treatment with PD173074 rescued the aneuploidy caused by
FGFR3-TACC3 by over 80%, restored the narrow distribution of
chromosome counts typical of control cells and largely corrected
the cohesion defect (FIGS. 6A, 6B and 6C). Together, these findings
indicate that the CIN and aneuploidy caused by rearrangements of
FGFR and TACC genes are reversible and suggest that specific FGFR
kinase inhibition may be a valuable therapeutic strategy in tumor
cells expressing FGFR-TACC fusion proteins.
[0335] FGFR-TACC Fusion Proteins are New Therapeutic Targets in
GBM.
[0336] Driver genetic alterations trigger a state of oncogene
addiction in the cancer cells harboring them that can be exploited
therapeutically. To ask whether FGFR-TACC fusions confer addiction
to FGFR-TK activity, cell growth was analyzed in the presence of
PD173074, AZD4547 or BGJ398, the latter being two highly specific
inhibitors of FGFR-TK under clinical investigation (Gavine et al.,
2012; Guagnano et al., 2011). Each of the three drugs inhibited
growth of cells expressing FGFR3-TACC3 and FGFR1-TACC1 at
concentrations<10 nM whereas they were ineffective at
concentrations as high as 1 .mu.M in cells transduced with vector,
FGFR3, TACC3 and the FGFR3-TACC3-K508M mutant (FIGS. 7A, 14C and
14D). These findings underscore the elevated degree of specificity
for FGFR kinase inhibition towards cells carrying the fusion
protein. The growth of GSC-1123 cells, which naturally harbor the
FGFR3-TACC3 translocation, was also abolished by nanomolar
concentrations of FGFR-TK inhibitors (FIG. 7B). Targeting of the
fusion gene by FGFR3 shRNA inhibited the growth of cells
ectopically expressing FGFR3-TACC3 and GSC-1123 proportionally to
the silencing efficiency of FGFR3-TACC3 (FIGS. 7C and 14E).
[0337] Finally, it was determined whether treatment with PD173074
of mice bearing glioma xenografts of FGFR3-TACC3 transformed
astrocytes inhibits tumor growth. Twelve days after injection of
tumor cells, subcutaneous tumors were present in all animals. The
mice were randomized in two cohorts and treated with PD173074 or
vehicle. PD173074 elicited a potent growth inhibition of
FGFR3-TACC3 glioma (FIG. 7D). To confirm the efficacy of a
clinically meaningful FGFR-TK inhibitor using a more anatomically
relevant model, the AZD4547 FGFR inhibitor, a compound under
clinical investigation (Gavine et al., 2012), was used against
intracranial luciferase-expressing FGFR3-TACC3-driven glioma
xenografts. After an engraftment period, tumor-bearing animals were
treated with either AZD4547 or vehicle. Oral administration of
AZD4547 markedly prolonged survival (FIG. 7E). Taken together, the
data provide a strong rationale for a clinical trial based on FGFR
inhibitors in GBM harboring FGFR-TACC rearrangements.
[0338] Discussion
[0339] This work has established that recurrent, oncogenic and
addicting gene fusions identify a subset of GBM patients. The
functional characterization of FGFR-TACC fusions indicates that the
constitutively active FGFR-TK and the TACC domain of the fusion
protein are both essential for oncogenesis. The TACC-dependent
mis-localization to mitotic cells of the FGFR kinase results in
aberrant compartmentalization of a constitutively active TK to the
mitotic spindle pole, thus providing a mechanistic explanation for
the impaired mitotic fidelity, chromosome mis-segregation and
aneuploidy instigated by the fusion protein.
[0340] Without being bound by theory, mutation of the genes that
control chromosome segregation during mitosis can explain the high
rate of CIN and aneuploidy, which is typical of most solid tumors
including GBM (Gordon et al., 2012). A few examples of mutational
inactivation of candidate genes have been reported in human cancer
(Solomon et al., 2011; Thompson et al., 2010). However,
gain-of-function mutations causally implicated in the control of
mitotic fidelity have not been described. This clashes with the
classical observation from cell fusion experiments that the
underlying mechanisms that cause CIN behave as dominant traits,
indicating that the CIN phenotype results from gain-of-function
events rather than gene inactivation (Lengauer et al., 1997, 1998).
The FGFR-TACC gene fusion is a novel mechanism for the initiation
of CIN and provides a clue to the nature of dominant mutations
responsible for aneuploidy in human cancer.
[0341] The rapid emergence of mitotic defects and aneuploid cell
populations triggered by the fusion protein in normal human
astrocytes, combined with the correction of aneuploidy after short
inhibition of FGFR-TK activity indicate that aneuploidy is a key
event in tumor induction by the FGFR-TACC gene fusions. Induction
of aneuploidy per se is detrimental to cellular fitness (Sheltzer
and Amon, 2011). Full-blown tumorigenesis requires cooperation
between aneuploidy and genetic lesions that confer growth advantage
and protect cells against the detrimental effects of aneuploidy
(Coschi and Dick, 2012; Holland and Cleveland, 2009; Weaver and
Cleveland, 2009). Therefore, the potent tumor-initiating activity
of FGFR-TACC fusions shows that the novel oncoproteins have
growth-promoting signaling functions that complement the loss of
mitotic fidelity with ensuing karyotypic alterations (Sheltzer and
Amon, 2011).
[0342] Targeted therapies against common genetic alterations in GBM
have not changed the dismal clinical outcome of the disease, most
likely because they have systematically failed to eradicate the
truly addicting oncoprotein activities of GBM. The highly specific
anti-tumor effects and the correction of aneuploidy precipitated by
FGFR-TK inhibition of FGFR-TACC-driven GBM provide a strong
rationale for clinical trials based on FGFR inhibitors in patients
harboring FGFR-TACC rearrangements. The computational gene fusion
discovery pipeline reported here detected other GBM cases in which
FGFR family genes are implicated in additional gene fusions beyond
the FGFR-TACC rearrangements. Therefore, the frequency of 3.1% is
likely to be an underestimate of the target GBM patient population
that may benefit from FGFR-TK inhibition.
[0343] Experimental Procedures
[0344] Cell Culture and Isolation and Maintenance of GSCs.
[0345] Rat1A, mouse astrocytes Ink4A;Arf-/-, and human astrocytes
were cultured in DMEM supplemented with 10% FBS. Isolation and
culture of GSCs was performed as described (Carro et al., 2010).
For treatment in vitro with PD173074, AZD4547 or BJG398, cells
infected with vector control, FGFR3, TACC3, FGFR-TACC fusions or
FGFR3-TACC3-K508M were seeded in 96-well plates and treated with
increasing concentrations of FGFR inhibitors. After 72-120 h,
growth rate was measured using the
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay. Data were expressed as mean.+-.SD. Proliferation rate in
GSC-1123 infected with FGFR3 shRNA lentivirus was determined by
plating dissociated gliomaspheres at 2.times.10.sup.4 cells/well in
twelve-well plates 5 days after infection. The number of viable
cells was determined by trypan blue exclusion in triplicate
cultures obtained from triplicate independent infections. Cell
number was scored every other day.
[0346] DNA, RNA Preparation, Genomic and Real-Time Quantitative PCR
(qRT-PCR).
[0347] The validation of fusion transcripts was performed using
both genomic and RT-PCR with forward and reverse primer
combinations designed within the margins of the paired-end read
sequences detected by RNA-seq. DNA, RNA preparation and qRT-PCR
were performed as described (Carro et al., 2010; Zhao et al.,
2008). To identify novel fusion transcripts within the GBM cohort,
PCR primers pairs were designed to bind upstream to the TK domain
of the FGFR genes and inside or downstream the Coiled Coil domain
of the TACC genes. Expressed fusion transcript variants were
subjected to direct sequencing to confirm sequence and frame.
Primer sequences are included below.
[0348] Subcutaneous Xenografts and Drug Treatment.
[0349] Rat1A or Ink4A;Arf-/- astrocytes (5.times.10.sup.5)
transduced with different lentiviral constructs were suspended in
150 .mu.l of PBS, together with 30 .mu.l of Matrigel (BD
Biosciences), and injected subcutaneously in the flank of athymic
nude (Nu/Nu) mice (Charles River Laboratories, Wilmington, Mass.).
For experiments with FGFR inhibitors, mice carrying .about.200-300
mm.sup.3 subcutaneous tumors derived from Ink4A;Arf-/- astrocytes
were randomized to receive 50 mg/kg PD173074 in 0.05 M lactate
buffer (pH 5) or an equal volume of lactate buffer by oral gavage.
Treatment was administered for three cycles consisting of four
consecutive days followed by two days of rest. Tumor diameters were
measured with caliper, and tumor volumes estimated using the
formula: 0.5.times.length.times.width. Data are expressed as
mean.+-.SE. Mice were sacrificed when tumors in the control group
reached the maximal size allowed.
[0350] Orthotopic Transplantation and Drug Treatment.
[0351] Ink4A;Arf-/- astrocytes carrying a luciferase expressing
vector were transduced with FGFR3-TACC3 lentivitus.
1.times.10.sup.3 cells in 2 .mu.l of saline were injected in the
caudate-putamen of 4-6 week old male athymic nude (Nu/Nu) mice
using a stereotaxic frame (coordinates relative to bregma: 0.5 mm
anterior; 1.1 mm lateral; 3.0 mm ventral) and a 26 gauge Hamilton
syringe. Six days after injection, mice underwent bioluminescence
imaging using a Xenogen CCD apparatus and were randomized to
receive 50 mg/kg AZD4547 in 1% Tween 80 (treatment group) or DMSO
in an equal volume of vehicle by oral gavage (control group).
AZD4547 was administered daily for two cycles of 10 days with a two
day interval. Mice were monitored daily and sacrificed when
neurological symptoms appeared. Kaplan-Meier survival curve was
generated using the DNA Statview software package (AbacusConcepts,
Berkeley, Calif.). Log-rank analysis was performed on the
Kaplan-Meier survival curve to determine statistical
significance.
[0352] Intracranial Injections of Lentiviruses.
[0353] Intracranial injection of FGFR3-TACC3-shp53, EGFRvIII-shp53
or shp53 pTomo lentiviruses was performed in 4 week-old C57/BL/6J
mice in accordance with guidelines of IACUC Committee. Briefly, 1.8
.mu.l of purified lentiviral particles in PBS (1.times.10.sup.9/ml)
were injected into the dentate gyms using a stereotaxic frame
(coordinates relative to bregma: 1.45 mm posterior; 1.65 mm
lateral; 2.4 mm ventral) and a 26 gauge Hamilton syringe. Mice were
monitored daily and sacrificed when neurological symptoms appeared.
Mouse brain was analyzed histopathologically and by
immunofluorescence staining.
[0354] Histology and Immunostaining.
[0355] Tissue preparation and immunohistochemistry on brain tumors
and immunofluorescence staining were performed as previously
described (Carro et al., 2010; Zhao et al., 2009; Zhao et al.,
2008). Antibodies used in immunostaining and immunoblotting are
listed below.
[0356] Cloning and Lentiviral Production.
[0357] Lentivirus preparation and infections were performed as
described (Carro et al., 2010) and are detailed in Extended
Experimental Procedures.
[0358] Karyotype Analysis.
[0359] Cultured cells were colcemid (20 ng/ml) treated for 90
minutes before harvesting for karyotopic analysis as detailed in
Extended Experimental procedures. At least one hundred cells in
metaphase were examined for chromosome count. PMSCS was scored in
cells where a majority of the sister chromosomes were no longer
associated. Two-tailed unpaired t-tests with Welch's correction
were performed for comparison of means analysis.
[0360] Immunofluorescence and Live-Cell Microscopy.
[0361] Immunofluorescence microscopy was performed on cells fixed
with 4% PFA in PHEM (60 mM Pipes, 27 mM Hepes, 10 mM EGTA, 4 mM
MgSO.sub.4, pH 7.0). Cells were permeabilized using 1% Triton
X-100. Mitotic spindles were visualized by anti-.alpha.-tubulin
antibody (Sigma). Secondary antibodies conjugated to Alexa
Fluor-488/-594 (Molecular Probes) were used. All staining with
multiple antibodies were performed in a sequential manner. DNA was
stained by DAPI (Sigma). Fluorescence microscopy was performed on a
Nikon A1R MP microscope.
[0362] Identification of Gene Fusions from Whole Transcriptome
(RNA-Seq) and Exome Sequencing.
[0363] RNA-Sequencing was performed from total RNA extracted from
GSC cultures isolated from nine GBM patients using Illumina HiSeq
2000, producing roughly 60.3 million paired reads per sample. Using
the global alignment software Burrows-Wheeler Aligner (BWA) (Li and
Durbin, 2009) with modified Mott's trimming, an initial seed length
of 32, maximum edit distance of 2 and a maximum gap number of 1, on
average 43.1 million reads were mapped properly to the RefSeq
transcriptome and, of the remaining, 8.6 million were mapped to the
hg19 genome per sample. The remaining 14.3% of paired
reads--including those that failed to map to either transcriptome
or genome with proper forward-reverse (F-R) orientation, within
expected insert size, and with minimal soft clipping (unmapped
portions at the ends of a read)--were considered to be appropriate
for gene fusion analysis.
[0364] A novel computational pipeline was constructed called
TX-Fuse that identifies two sources of evidence for the presence of
a gene fusion: 1. Split inserts, in which each read of a mate pair
maps entirely to one side of a breakpoint, and 2. Individual split
reads that span a breakpoint. Split inserts are readily detected
from BWA mapping. On the other hand, split reads demand precision
alignment of smaller nucleotide stretches. To that end, the
pipeline employs the local alignment package BLAST with word size
of 20, identity cutoff of 95%, expectation cutoff of 10.sup.-4, and
soft filtering to map raw paired reads against the RefSeq
transcriptome. From this procedure, a list of potential split reads
were obtained that were filtered to ensure maintenance of coding
frame in the predicted fusion transcript given the proper F-R
orientation in the read pair. False positive candidates produced
from paralogous gene pairs were also screened out using the
Duplicated Genes Database and the EnsemblCompara GeneTrees (Vilella
et al., 2009). Pseudogenes in the candidate list were annotated
using the list from HUGO Gene Nomenclature Committee (HGNC)
database (Seal et al., 2011) and given lower priority. For each
remaining gene fusion candidate, a virtual reference was created
based on the predicted fusion transcript and re-mapped all unmapped
reads using BLAST with word size of 16, identity cutoff of 85%,
query coverage greater than 85%, and expectation cutoff of
10.sup.-4 to obtain a final count of split reads and inserts.
Moreover, sequencing depth per base of the virtual reference was
calculated to corroborate that components of each gene
participating in the gene fusion were highly expressed.
[0365] To establish the recurrence of the initial panel of gene
fusion candidates, the gene fusion discovery pipeline was modified
to produce EXome-Fuse, which probes for fusions within the
available dataset of paired-read exome DNA sequencing of 84 matched
GBM samples from TCGA. To increase sensitivity for gene fusion
identification, reads unmapped by BWA were aligned to the gene pair
participating in each fusion candidate using a BLAST word size of
24 for split inserts and 16 for split read and split insert
discovery. Given that the breakpoint detected in DNA cannot
directly indicate the resulting breakpoint in the transcribed RNA,
no restriction was made on split insert orientation. For split
reads, it was only required that the component of the split read
mapped to the same gene as its mate maintained F-R
directionality.
[0366] Co-Outlier Expression and CNV Analysis from TCGA GBM
Samples.
[0367] Tomlins et al. (Tomlins et al., 2005) reported that outlier
gene expression from microarray datasets identifies candidate
oncogenic gene fusions. Wang et al. (Wang et al., 2009) suggested a
"breakpoint principle" for intragenic copy number aberrations in
fusion partners. The two principles (outlier expression and
intragenic CNV) were combined to identify candidate gene fusions in
GBM samples from Atlas-TCGA. Genomic and expression data sets were
downloaded from TCGA public data portal as available on Dec. 1,
2011, where a description of TCGA data types, platforms, and
analyses is also available (2008). Specific data sources were
(according to Data Levels and Data Types) as follows: Expression
data, "Level 2" normalized signals per probe set (Affymetrix
HT_HG-U133A) of 84 samples; Copy number data, "Level 1" raw signals
per probe (Affymetrix Genome-Wide Human SNP Array 6.0) of the 4
FGFR3-TACC3 gene fusion positive samples (tumor and matched normal
control).
[0368] The gene expression analysis was performed first using
R.sup.3. The median absolute deviation (MAD) was calculated and
then a gene was labeled as an outlier according to the following
formula: Z.sub.i,j=0.6745(x.sub.i,j-mean(x.sub.i))/MAD.sub.i>3.5
(Iglewicz and Hoaglin, 1993). Samples were identified as ECFS
(expression candidate fusion sample) if both genes of interest (e.
g. FGFR3 and TACC3) displayed outlier behavior (co-outliers). Next,
ECFS were analyzed for CNV using pennCNV (Wang et al., 2007).
Tumors samples were paired to their normal controls to obtain the
log ratio values and the VEGA algorithm was used to obtain a more
accurate segmentation (Morganella et al., 2010).
[0369] Karyotypic Analysis.
[0370] The colcemid treated cells were trypsinized, centrifuged for
7 minutes at 200.times.g, and the cell pellet re-suspended in
warmed hypotonic solution and incubated at 37.degree. C. for 13
minutes. The swollen cells were then centrifuged and the pellet
re-suspended in 8 ml of Carnoy's fixative (3:1 methanol:glacial
acetic acid). The cell suspension was centrifuged and washed twice
in Carnoy's fixative. After the last centrifugation, the cells were
resuspended in 0.5 to 1 ml of freshly prepared fixative to produce
an opalescent cell suspension. Drops of the final cell suspension
were placed on clean slides and air-dried. Slides were stained with
DAPI and metaphases were analyzed under a fluorescent
microscope.
[0371] Cloning and Lentiviral Production.
[0372] Lentiviral expression vectors, pLOC-GFP (Open Biosystems)
and pTomo-shp53, were used to clone FGFR3, TACC3, FGFR3-TACC3,
FGFR3-TACC3-K508M, and FGFR1-TACC1. pTomo-shp53 was a gift of Inder
Verma and Dinorah Friedman-Morvinski (Salk Institute, San Diego).
The FGFR3-TACC3-K508M mutant was generated using the Phusion Site
Direct Mutagenesis kit (NEB, USA). MISSION shRNAs clones (pLKO.1
lentiviral expression vectors) against FGFR3 were purchased from
Sigma. The hairpin sequences targeting the FGFR3 gene are--
TABLE-US-00050 (#TRCN0000000372; Sh#2) (SEQ ID NO: 182)
5'-TGCGTCGTGGAGAACAAGTTT-3'; (#TRCN0000430673; Sh#3) (SEQ ID NO:
183) 5'-GTTCCACTGCAAGGTGTACAG-3'; (#TRCN0000000374; Sh#4) (SEQ ID
NO: 184) 5'-GCACAACCTCGACTACTACAA-3'.
[0373] Genomic and mRNA RT-PCR.
[0374] Total RNA was extracted from cells by using RNeasy Mini Kit
(QIAGEN), following the manufacturer instructions. 500 ng of total
RNA was retro-transcribed by using the Superscript III kit
(Invitrogen), following the manufacturer instructions. The cDNAs
obtained after the retro-transcription was used as templates for
qPCR. The reaction was performed with a Roche480 thermal cycler, by
using the Absolute Blue QPCR SYBR Green Mix from Thermo Scientific.
The relative amount of specific mRNA was normalized to 18S. Results
are presented as the mean.+-.SD of triplicate amplifications.
[0375] Primers used are:
TABLE-US-00051 hFGFR3-RT-FW1: (SEQ ID NO: 162)
5'-GTAACCTGCGGGAGTTTCTG-3'; hFGFR3-RT-REV1: (SEQ ID NO: 163)
5'-ACACCAGGTCCTTGAAGGTG-3'; hTACC3-RT-FW2: (SEQ ID NO: 164)
5'-CCTGAGGGACAGTCCTGGTA-3'; hTACC3-RT-REV2: (SEQ ID NO: 165)
5'-AGTGCTCCCAAGAAATCGAA-3'; hWRAP53-RT-FW1: (SEQ ID NO: 180)
5'-AGAGGTGACCACCAATCAGC-3'; hWRAP53-RT-REV1: (SEQ ID NO: 181)
5'-CGTGTCCCACACAGAGACAG-3'.
[0376] Primers used for the screening of FGFR-TACC fusions are:
TABLE-US-00052 FGFR3-FW1: (SEQ ID NO: 166)
5'-CGTGAAGATGCTGAAAGACGATG-3'; TACC3-REV1: (SEQ ID NO: 167) 5'-
AAACGCTTGAAGAGGTCGGAG-3'; FGFR1-FW1: (SEQ ID NO: 168)
5'-ATGCTAGCAGGGGTCTCTGA-3'; TACC1-REV1: (SEQ ID NO: 169)
5'-CCCTTCCAGAACACCTTTCA-3'.
[0377] Primers used for genomic detection of FGFR3-TACC3 fusion in
GBM-1123 and GSC-1123 are:
TABLE-US-00053 Genomic FGFR3-FW1: (SEQ ID NO: 170)
5'-ATGATCATGCGGGAGTGC-3'; genomic TACC3-REV1: (SEQ ID NO: 171)
5'-GGGGGTCGAACTTGAGGTAT-3'.
[0378] Primers used to validate fusions detected by RNA-seq
are:
TABLE-US-00054 POLR2A-FW1: (SEQ ID NO: 172)
5'-CGCAGGCTTTTTGTAGTGAG-3'; WRAP53-REV1: (SEQ ID NO: 173)
5'-TGTAGGCGCGAAAGGAAG-3'; PIGU-FW1: (SEQ ID NO: 174)
5'-GAACTCATCCGGACCCCTAT-3'; NCOA6-REV1: (SEQ ID NO: 175)
5'-GCTTTCCCCATTGCACTTTA-3'; ST8SIA4-FW1: (SEQ ID NO: 176)
5'-GAGGAGAGAAGCACGTGGAG-3'; PAM-REV1: (SEQ ID NO: 177)
5'-GGCAGACGTGTGAGGTGTAA-3'; CAPZB-FW: (SEQ ID NO: 178)
5'-GTGATCAGCAGCTGGACTGT-3'; UBR4-REV1: (SEQ ID NO: 179)
5'-GAGCCTGGGCATGGATCT-3'.
[0379] Confocal Microscopy Imaging.
[0380] For immunofluorescence of fixed cells, images were recorded
with a Z-optical spacing of 0.25 .mu.m using a Nikon AIR MP and a
60X1.3 oil objective and analyzed using ImageJ software (National
Institute of Health). For live-cell analyses, Rat1A cells infected
with pLNCX-H2B retrovirus and transduced with lentiviral vector or
FGFR3-TACC3 fusion were seeded in glass bottom dishes in phenol red
free DMEM and followed by time-lapse microscopy using the Nikon AIR
MP biostation at 37.degree. C. and 5% CO.sub.2/95% air. Images with
a Z-optical spacing of 1 .mu.m were recorded every 4 min for 8 h.
Images of unchallenged mitosis from early prophase until
cytokinesis were processed using ImageJ software (National
Institute of Health). The time-point of nuclear envelope breakdown
(NEB) was defined as the first frame showing loss of smooth
appearance of chromatin and anaphase was the first frame when
chromosome movement towards the poles became apparent. Nuclear
envelope reconstitution (NER) was defined as the first frame
showing nuclei decondensation.
[0381] Box and whisker plots were calculated from image sequences
from at least 50 recorded cells. Two-tailed unpaired t-tests with
Welch's correction were performed for comparison of means analysis
using StatView software (AbacusConcepts, Berkeley, Calif.).
[0382] Immunofluorescence.
[0383] Antibodies and concentrations used in immunofluorescence
staining are:
TABLE-US-00055 Anti-Ki67 Rabbit 1:1000 Vector Labs Anti-pHH3 Rabbit
1:500 Millipore Anti-FGFR3 Mouse 1:1000 Santa Cruz Anti-Tacc3 Goat
1:1000 USBiological Anti-a-tubulin Mouse 1:1000 Sigma Anti-Nestin
Mouse 1:1000 BD Pharmingen Anti-Olig2 Rabbit 1:200 IBL Anti-GFAP
Rabbit 1:200 Dako Anti-ERK Rabbit 1:1000 Cell Signaling Anti-pERK
Rabbit 1:1000 Cell Signaling AntiFRS Rabbit 1:250 Santa Cruz
Anti-pFRS Rabbit 1:1000 Cell Signaling Anti-AKT Rabbit 1:1000 Cell
Signaling Anti-pAKT473 Rabbit 1:1000 Cell Signaling
REFERENCES
[0384] Ablain, J., Nasr, R., Bazarbachi, A., and de The, H. (2011).
The Drug-Induced Degradation of Oncoproteins: An Unexpected
Achilles' Heel of Cancer Cells? Cancer Discov. 1, 117-127. [0385]
Bass, A. J., Lawrence, M. S., Brace, L. E., Ramos, A. H., Drier,
Y., Cibulskis, K., Sougnez, C., Voet, D., Saksena, G., Sivachenko,
A., et al. (2011). Genomic sequencing of colorectal adenocarcinomas
identifies a recurrent VTI1A-TCF7L2 fusion. Nat. Genet. 43,
964-968. [0386] Cahill, D. P., Kinzler, K. W., Vogelstein, B., and
Lengauer, C. (1999). Genetic instability and darwinian selection in
tumours. Trends Cell. Biol. 9, M57-60. [0387] Carro, M. S., Lim, W.
K., Alvarez, M. J., Bollo, R. J., Zhao, X., Snyder, E. Y., Sulman,
E. P., Anne, S. L., Doetsch, F., Colman, H., et al. (2010). The
transcriptional network for mesenchymal transformation of brain
tumours. Nature 463, 318-325. [0388] Coschi, C. H., and Dick, F. A.
(2012). Chromosome instability and deregulated proliferation: an
unavoidable duo. Cell. Mol. Life Sci. 69, 2009-2024 [0389] Druker,
B. J. (2009). Perspectives on the development of imatinib and the
future of cancer research. Nat. Med. 15, 1149-1152. [0390] Furnari,
F. B., Fenton, T., Bachoo, R. M., Mukasa, A., Stommel, J. M.,
Stegh, A., Hahn, W. C., Ligon, K. L., Louis, D. N., Brennan, C., et
al. (2007). Malignant astrocytic glioma: genetics, biology, and
paths to treatment. Genes Dev. 21, 2683-2710. [0391] Gavine, P. R.,
Mooney, L., Kilgour, E., Thomas, A. P., Al-Kadhimi, K., Beck, S.,
Rooney, C., Coleman, T., Baker, D., Mellor, M. J., et al. (2012).
AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of
the Fibroblast Growth Factor Receptor Tyrosine Kinase Family.
Cancer Res. 72, 2045-2056. [0392] Gerber, D. E., and Minna, J. D.
(2010). ALK inhibition for non-small cell lung cancer: from
discovery to therapy in record time. Cancer Cell 18, 548-551.
[0393] Gordon, D. J., Resio, B., and Pellman, D. (2012). Causes and
consequences of aneuploidy in cancer. Nature reviews Genet. 13,
189-203. [0394] Guagnano, V., Furet, P., Spanka, C., Bordas, V., Le
Douget, M., Stamm, C., Brueggen, J., Jensen, M. R., Schnell, C.,
Schmid, H., et al. (2011). Discovery of
3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-ph-
enylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and
selective inhibitor of the fibroblast growth factor receptor family
of receptor tyrosine kinase. J. Med. Chem. 54, 7066-7083. [0395]
Holland, A. J., and Cleveland, D. W. (2009). Boveri revisited:
chromosomal instability, aneuploidy and tumorigenesis. Nat. Rev.
Mol. Cell. Biol. 10, 478-487. [0396] Hood, F. E., and Royle, S. J.
(2011). Pulling it together: The mitotic function of TACC3.
Bioarchitecture 1, 105-109. [0397] Lee, J., Kotliarova, S.,
Kotliarov, Y., Li, A., Su, Q., Donin, N. M., Pastorino, S., Purow,
B. W., Christopher, N., Zhang, W., et al. (2006). Tumor stem cells
derived from glioblastomas cultured in bFGF and EGF more closely
mirror the phenotype and genotype of primary tumors than do
serum-cultured cell lines. Cancer Cell 9, 391-403. [0398] Lengauer,
C., Kinzler, K. W., and Vogelstein, B. (1997). Genetic instability
in colorectal cancers. Nature 386, 623-627. [0399] Lengauer, C.,
Kinzler, K. W., and Vogelstein, B. (1998). Genetic instabilities in
human cancers. Nature 396, 643-649. [0400] Lo, H. W. (2010).
EGFR-targeted therapy in malignant glioma: novel aspects and
mechanisms of drug resistance. Curr. Mol. Pharmacol. 3, 37-52.
[0401] Marumoto, T., Tashiro, A., Friedmann-Morvinski, D., Scadeng,
M., Soda, Y., Gage, F. H., and Verma, I. M. (2009). Development of
a novel mouse glioma model using lentiviral vectors. Nat. Med. 15,
110-116. [0402] Mitelman, F., Johansson, B., and Mertens, F.
(2007). The impact of translocations and gene fusions on cancer
causation. Nat. Rev. Cancer 7, 233-245. [0403] Mohammadi, M.,
Froum, S., Hamby, J. M., Schroeder, M. C., Panek, R. L., Lu, G. H.,
Eliseenkova, A. V., Green, D., Schlessinger, J., and Hubbard, S. R.
(1998). Crystal structure of an angiogenesis inhibitor bound to the
FGF receptor tyrosine kinase domain. EMBO J. 17, 5896-5904. [0404]
Ohgaki, H., and Kleihues, P. (2005). Population-based studies on
incidence, survival rates, and genetic alterations in astrocytic
and oligodendroglial gliomas. J. Neuropathol. Exp. Neurol. 64,
479-489. [0405] Peset, I., and Vernos, I. (2008). The TACC
proteins: TACC-ling microtubule dynamics and centrosome function.
Trends Cell. Biol. 18, 379-388. [0406] Prensner, J. R., and
Chinnaiyan, A. M. (2009). Oncogenic gene fusions in epithelial
carcinomas. Curr Opin Genet. Dev. 19, 82-91. [0407] Reardon, D. A.,
Desjardins, A., Vredenburgh, J. J., Gururangan, S., Friedman, A.
H., Herndon, J. E., 2nd, Marcello, J., Norfleet, J. A., McLendon,
R. E., Sampson, J. H., et al. (2010). Phase 2 trial of erlotinib
plus sirolimus in adults with recurrent glioblastoma. J.
Neurooncol. 96, 219-230. [0408] Sheltzer, J. M., and Amon, A.
(2011). The aneuploidy paradox: costs and benefits of an incorrect
karyotype. Trends Genet. 27, 446-453. [0409] Solomon, D. A., Kim,
T., Diaz-Martinez, L. A., Fair, J., Elkahloun, A. G., Harris, B.
T., Toretsky, J. A., Rosenberg, S. A., Shukla, N., Ladanyi, M., et
al. (2011). Mutational inactivation of STAG2 causes aneuploidy in
human cancer. Science 333, 1039-1043. [0410] Stephens, P. J.,
McBride, D. J., Lin, M. L., Varela, I., Pleasance, E. D., Simpson,
J. T., Stebbings, L. A., Leroy, C., Edkins, S., Mudie, L. J., et
al. (2009). Complex landscapes of somatic rearrangement in human
breast cancer genomes. Nature 462, 1005-1010. [0411] Still, I. H.,
Vince, P., and Cowell, J. K. (1999). The third member of the
transforming acidic coiled coil-containing gene family, TACC3, maps
in 4p16, close to translocation breakpoints in multiple myeloma,
and is upregulated in various cancer cell lines. Genomics 58,
165-170. [0412] Thompson, S. L., Bakhoum, S. F., and Compton, D. A.
(2010). Mechanisms of chromosomal instability. Curr. Biol. 20,
R285-295. [0413] Tomlins, S. A., Laxman, B., Dhanasekaran, S. M.,
Helgeson, B. E., Cao, X., Morris, D. S., Menon, A., Jing, X., Cao,
Q., Han, B., et al. (2007). Distinct classes of chromosomal
rearrangements create oncogenic ETS gene fusions in prostate
cancer. Nature 448, 595-599. [0414] Tomlins, S. A., Rhodes, D. R.,
Perner, S., Dhanasekaran, S. M., Mehra, R., Sun, X. W., Varambally,
S., Cao, X., Tchinda, J., Kuefer, R., et al. (2005). Recurrent
fusion of TMPRSS2 and ETS transcription factor genes in prostate
cancer. Science 310, 644-648. [0415] Turner, N., and Grose, R.
(2010). Fibroblast growth factor signalling: from development to
cancer. Nat. Rev. Cancer 10, 116-129. [0416] van den Bent, M. J.,
Brandes, A. A., Rampling, R., Kouwenhoven, M. C., Kros, J. M.,
Carpentier, A. F., Clement, P. M., Frenay, M., Campone, M.,
Baurain, J. F., et al. (2009). Randomized phase II trial of
erlotinib versus temozolomide or carmustine in recurrent
glioblastoma: EORTC brain tumor group study 26034. J. Clin. Oncol.
27, 1268-1274. [0417] Wang, X. S., Prensner, J. R., Chen, G., Cao,
Q., Han, B., Dhanasekaran, S. M., Ponnala, R., Cao, X., Varambally,
S., Thomas, D. G., et al. (2009). An integrative approach to reveal
driver gene fusions from paired-end sequencing data in cancer. Nat.
Biotechnol. 27, 1005-1011. [0418] Weaver, B. A., and Cleveland, D.
W. (2009). The role of aneuploidy in promoting and suppressing
tumors. J. Cell. Biol. 185, 935-937. [0419] Wesche, J., Haglund,
K., and Haugsten, E. M. (2011). Fibroblast growth factors and their
receptors in cancer. Biochem. J. 437, 199-213. [0420] Yan, H.,
Parsons, D. W., Jin, G., McLendon, R., Rasheed, B. A., Yuan, W.,
Kos, I., Batinic-Haberle, I., Jones, S., Riggins, G. J., et al.
(2009). IDH1 and IDH2 mutations in gliomas. New Engl. J. Med. 360,
765-773. [0421] Zhao, X., D, D. A., Lim, W. K., Brahmachary, M.,
Carro, M. S., Ludwig, T., Cardo, C. C., Guillemot, F., Aldape, K.,
Califano, A., et al. (2009). The N-Myc-DLL3 cascade is suppressed
by the ubiquitin ligase Huwel to inhibit proliferation and promote
neurogenesis in the developing brain. Dev. Cell 17, 210-221. [0422]
Zhao, X., Heng, J. I., Guardavaccaro, D., Jiang, R., Pagano, M.,
Guillemot, F., Iavarone, A., and Lasorella, A. (2008). The
HECT-domain ubiquitin ligase Huwel controls neural differentiation
and proliferation by destabilizing the N-Myc oncoprotein. Nat Cell
Biol 10, 643-653. [0423] (2008). Comprehensive genomic
characterization defines human glioblastoma genes and core
pathways. Nature 455, 1061-1068. [0424] Iglewicz, B., and Hoaglin,
D. C. (1993). How to detect and handle outliers (Milwaukee, Wis.:
ASQC). [0425] Li, H., and Durbin, R. (2009). Fast and accurate
short read alignment with Burrows-Wheeler transform. Bioinformatics
25, 1754-1760. [0426] Morganella, S., Cerulo, L., Viglietto, G.,
and Ceccarelli, M. (2010). VEGA: variational segmentation for copy
number detection. Bioinformatics 26, 3020-3027. [0427] Seal, R. L.,
Gordon, S. M., Lush, M. J., Wright, M. W., and Bruford, E. A.
(2011). genenames.org: the HGNC resources in 2011. Nucleic Acids
Res 39, D514-519. [0428] Tomlins, S. A., Rhodes, D. R., Perner, S.,
Dhanasekaran, S. M., Mehra, R., Sun, X. W., Varambally, S., Cao,
X., Tchinda, J., Kuefer, R., et al. (2005). Recurrent fusion of
TMPRSS2 and ETS transcription factor genes in prostate cancer.
Science 310, 644-648. [0429] Vilella, A. J., Severin, J.,
Ureta-Vidal, A., Heng, L., Durbin, R., and Birney, E. (2009).
EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic
trees in vertebrates. Genome Res. 19, 327-335. [0430] Wang, K., Li,
M., Hadley, D., Liu, R., Glessner, J., Grant, S. F., Hakonarson,
H., and Bucan, M. (2007). PennCNV: an integrated hidden Markov
model designed for high-resolution copy number variation detection
in whole-genome SNP genotyping data. Genome Res. 17, 1665-1674.
[0431] Wang, X. S., Prensner, J. R., Chen, G., Cao, Q., Han, B.,
Dhanasekaran, S. M., Ponnala, R., Cao, X., Varambally, S., Thomas,
D. G., et al. (2009). An integrative approach to reveal driver gene
fusions from paired-end sequencing data in cancer. Nat. Biotechnol.
27, 1005-1011.
Example 2
Fusions in GBM
TABLE-US-00056 [0432] TABLE 8 Soft agar colony assay Cell line
Vector FGFR3 TACC3 F1-T1 Fusion F3-T3 Fusion F3-T3-K508M Fusion
Rat1 0 0 0 225.3 .+-. 10.0 198.7 .+-. 8.0 0 Balb 3T3 0 0 0 n.d.
45.5 .+-. 8.9 n.d. n.d.: not done
TABLE-US-00057 TABLE 9 Subcutaneous tumor xenografts Cell line
Vector FGFR3 TACC3 F1-T1 Fusion F3-T3 Fusion F3-T3-K508M Fusion
Rat1 0/5 0/5 0/5 n.d. 5/5 n.d. Ink4A/Arf-/- 0/9 0/5 0/5 8/8 12/12
0/8 Astrocytes n.d.: not done
TABLE-US-00058 TABLE 10 Analysis of chromosomal number in Rat1
cells Number of cells Percent Mean Average variation Cell line
counted aneuploidy Range number from mean number p-value Rat1A
Vector 100 27 35-43 41.2 1.2 Rat1A FGFR3 100 33 35-44 42.1 1.3 n.s.
Rat1A TACC3 100 41 34-46 40.7 1.1 n.s. Rat1A FGFR3-TACC3 100 69
35-73 43.6 3.1 <0.0001
TABLE-US-00059 TABLE 11 Analysis of chromosomal number in human
astrocytes Number of Percent Mean Average variation Cell line cells
counted aneuploidy Range number from mean number p-value Human
Astrocytes Vector 100 8 42-46 45.85 0.28 p = <0.001 Human
Astrocytes FGFR3-TACC3 100 42 28-48 42.24 3.33
Example 3
Fusions in Other Cancers
[0433] The inventors previously reported in Example 1 that 3.1% of
human glioblastoma harbor FGFR3-TACC3 and FGFR1-TACC1 gene fusions.
Tumors harboring FGFR3-TACC3 gene fusions are identified by the
presence of highly specific focal micro-amplification events of the
rearranged portions of the FGFR3 and TACC3 genes (See FIG. 2E).
Therefore, these micro-amplification events can be used as
distinctive marks for the presence of FGFR3-TACC3 gene fusions. It
was asked whether other types of human tumors also harbor
FGFR3-TACC3 gene fusions from the analysis of Copy Number
Variations (CNVs) of SNP arrays generated from the Atlas-TCGA
project. This analysis was performed using segmented CNVs data
visualized using the Integrated Genomic Viewers software. The
analysis revealed that the following tumors, shown in the FIGS.
31-35, display focal micro-amplification events of FGFR3 and TACC3
that indicate the presence of FGFR3-TACC3 gene fusions (in FIGS.
31-35, red indicates amplification (A), blue indicates deletion
(D); FIG. 31: Bladder Urothelial Carcinoma; FIG. 32: Breast
Carcinoma; FIG. 33: Colorectal Carcinoma; FIG. 34: Lung Squamous
Cell Carcinoma; FIG. 35: Head and Neck Squamous Cell
Carcinoma).
[0434] Taken together, these data indicate that the same
FGFR3-TACC3 gene fusions reported for the first time in
Glioblastoma also occur in several other types of human tumors.
Therefore, as for Glioblastoma and other epithelial cancers (such
as the human tumors discussed herein), the identification of
FGFR-TACC gene fusions also provides a new diagnostic and
therapeutic target for treatment with drugs that inhibit FGFR-TACC
gene fusions.
Example 4
Detection, Characterization and Inhibition of FGFR-TACC Fusions in
IDH Wild Type Glioma
[0435] Translational Relevance
[0436] Described herein is an unbiased screening assay for
FGFR-TACC fusions in glioma that overcomes the great variability of
variants that are generated by FGFR-TACC chromosomal translocation
in human cancer. FGFR-TACC fusions occur in grade II and III glioma
harboring wildtype IDH1 with frequency similar to glioblastoma
(GBM), therefore providing a clue to the aggressive clinical
behavior of this glioma subtype. The molecular characterization of
fusion-positive glioma revealed that FGFR-TACC is mutually
exclusive with EGFR amplification but co-occurs with CDK4
amplification. FGFR-TACC-positive glioma displays strikingly
uniform and strong expression of the fusion protein at the single
cell level. Preclinical experiments with FGFR3-TACC3-positive
glioma cells treated with the FGFR inhibitor JNJ-42756493 showed
strong antitumor effects and treatment of two patients with
recurrent GBM harboring FGFR3-TACC3 resulted in clinical
improvement and radiological tumor reduction. These findings
validate the treatment with FGFR inhibitors of glioma patients
harboring FGFR-TACC chromosomal translocations.
[0437] Abstract
[0438] Purpose.
[0439] Oncogenic fusions consisting of FGFR and TACC are present in
a subgroup of glioblastoma (GBM) and other human cancers and have
been proposed as new therapeutic targets. Frequency, molecular
features of FGFR-TACC fusions, and the therapeutic efficacy of
inhibiting FGFR kinase in GBM and grade-II-III glioma were
analyzed.
[0440] Experimental Design.
[0441] Overall, 795 gliomas (584 GBM, 85 grade-II-III with
wild-type and 126 with IDH1/2 mutation) were screened for FGFR-TACC
breakpoints and associated molecular profile. Expression of the
FGFR3 and TACC3 components of the fusions were also analyzed. The
effects of the specific FGFR inhibitor JNJ-42756493 for
FGFR3-TACC3-positive glioma were determined in preclinical
experiments. Two patients with advanced FGFR3-TACC3-positive GBM
received JNJ-42756493 and were assessed for therapeutic
response.
[0442] Results.
[0443] Three of 85 IDH1/2 wild type (3.5%) but none of 126 IDH1/2
mutant grade-II-III glioma harbored FGFR3-TACC3 fusions. FGFR-TACC
rearrangements were present in 17 of 584 GBM (2.9%). FGFR3-TACC3
fusions were associated with strong and homogeneous FGFR3
immunostaining. They are mutually exclusive with IDH1/2 mutations
and EGFR amplification whereas co-occur with CDK4 amplification.
JNJ-42756493 inhibited growth of glioma cells harboring FGFR3-TACC3
in vitro and in vivo. The two patients with FGFR3-TACC3
rearrangements who received JNJ-42756493 manifested clinical
improvement with stable disease and minor response,
respectively.
[0444] Conclusions.
[0445] RT-PCR-sequencing is a sensitive and specific method to
identify FGFR-TACC-positive patients. FGFR3-TACC3 fusions are
associated with uniform intra-tumor expression of the fusion
protein. The clinical response observed in the FGFR3-TACC3-positive
patients treated with a FGFR inhibitor supports clinical studies of
FGFR inhibition in FGFR-TACC-positive patients.
[0446] Introduction
[0447] The history of successful targeted therapy of cancer largely
coincides with the inactivation of recurrent, oncogenic and
addicting gene fusions in hematological malignancies and recently
in some types of epithelial cancer (1, 2). Glioblastoma multiforme
(GBM) is among the most lethal forms of human cancer and targeted
therapies against common genetic alterations in GBM have not
changed the dismal outcome of the disease (3, 4). Underlying
biological features including infiltrative growth behavior,
intratumoral heterogeneity, and adaptive resistance mechanisms
coupled with the unique challenges of intracranial location present
significant problems in its effective management. Despite surgery
and chemo-radiotherapy, most patients rapidly recur and no
effective treatment options are available at that stage. Beside
GBM, which features the highest grade of malignancy among glioma
(grade IV), lower grade glioma which include grade II and grade III
are a heterogeneous group of tumors in which specific molecular
features are associated with divergent clinical outcome. The
majority of grade II-III glioma (but only a small subgroup of GBM)
harbor mutations in IDH genes (IDH1 or IDH2), which confer a more
favorable clinical outcome. Conversely, the absence of IDH
mutations is associated with the worst prognosis (5).
[0448] Described herein is the identification of FGFR-TACC gene
fusions (mostly FGFR3-TACC3, and rarely FGFR1-TACC1) as the first
example of highly oncogenic and recurrent gene fusions in GBM. The
FGFR-TACC fusions that have been identified so far include the
Tyrosine Kinase (TK) domain of FGFR and the coiled-coil domain of
TACC proteins, both necessary for the oncogenic function of
FGFR-TACC fusions. Tumor dependency on FGFR-TACC fusions was also
tested in preclinical mouse models of FGFR-TACC glioma and observed
marked anti-tumor effects by FGFR inhibition (6). FGFR3-TACC3
fusions have been identified in pediatric and adult glioma, bladder
carcinoma, squamous lung carcinoma and head and neck carcinoma,
thus establishing FGFR-TACC fusions as one of the chromosomal
translocation most frequently found across multiple types of human
cancers (6-15).
[0449] From a mechanistic standpoint, the unexpected capacity of
FGFR-TACC fusions to trigger aberrant chromosome segregation during
mitosis, thus initiating chromosome instability (CIN) and
aneuploidy, two hallmarks of cancer, is described herein. However,
the full repertoire of the structural variants of FGFR-TACC fusions
occurring in GBM and lower grade glioma is not completely
understood. Furthermore, it remains unknown whether FGFR-TACC
fusions mark distinct grades of glioma and GBM subtypes.
[0450] To date eight variants of the FGFR3-TACC3 fusion have been
reported that mostly differ for the breakpoint in the TACC3 gene
(6-15). Because of the close proximity of FGFR3 and TACC3 (the two
genes map at a distance of 70 Kb on chromosome 4p16.3), detection
of FGFR3-TACC3 rearrangements by FISH is not a feasible option with
the currently available methods. Here a screening method for
FGFR-TACC fusions is reported that includes a RT-PCR assay designed
to identify the known and novel FGFR3-TACC3 fusion transcripts,
followed by confirmation of the inframe breakpoint by Sanger
sequencing. Using this assay, a dataset of 584 GBM and 211 grade II
and grade III gliomas has been analyzed.
[0451] A crucial question with fundamental clinical relevance for
any novel candidate target mutation is the frequency of the
alteration in the cancer cell population, thus discriminating
between a clonal or sub-clonal origin of the mutation. In fact, GBM
is characterized by a formidable degree of subclonal heterogeneity,
whereby neighboring cells display amplification and expression of
different Receptor Tyrosine Kinase (RTK)-coding genes (16-19). This
notion poses major therapeutic challenges for targeting any
individual RTK will result, at best, in the eradication of a
limited tumor sub-clone. Described herein, it was determined that
brain tumors harboring FGFR-TACC fusions manifest strong and
homogeneous intra-tumor expression of the FGFR3 and TACC3 component
invariably included in the fusion protein, when analyzed by
immunostaining. A significant clinical benefit following treatment
with a specific inhibitor of FGFR-TK is reported in two GBM
patients who harbored FGFR3-TACC3 rearrangement.
[0452] Materials and Methods
[0453] Patients and Tissue Samples.
[0454] This example includes a cohort of 746 untreated patients
with histologic diagnosis of glioma from 5 institutions. Forty-nine
recurrent gliomas from Pitie-Salp triere Hospital and one recurrent
glioma from the University of Calgary were also included. A summary
of the patient cohort is provided in Table 12.
TABLE-US-00060 TABLE 12 Frequency of FGFR3-TACC3 Fusions in GBM and
Grade II-III glioma. Distribution of the FGFR3-TACC3 fusions in GBM
(upper panel) and lower grade glioma (lower panel) samples
stratified according to the Institution of origin. The table
reports number of cases analyzed, number of tumors harboring
FGFR3-TACC3 fusion transcripts, and results of FGFR3
immunostaining. Lower grade glioma samples are further classified
according to IDH status (IDH1 and IDH2). The respective frequency
of FGFR3-TACC3 in GBM, Glioma grade II-III IDH wild type (wt), and
IDH mutant (Mut) glioma is reported in parentheses. Immunostaining
No of case No of detected FGFR3 positive/Sample Tumor sample source
(GBM) fusions analyzed Pitie-Salp triere Hospital 380 9 9/9 Besta
Neurological Institute 85 5 2/2 University of Calgary 60 +
1R.sup..sctn. 2 + 1R.sup..sctn. 1/1 + 1/1R.sup..sctn. Montreal
Neurological Institute 51 1 -- University of British Columbia 8 0
-- Total 584 (100%).sup..English Pound. 17 (2.9%) Immunostaining No
of cases No of detected FGFR3 positive/Sample Tumor sample source
IDH Status (Grade II-III) fusions analyzed Pitie-Salp triere
Hospital IDH wt 85* (100%) 3 (3.5%) 3/3 IDH1/IDH2 Mut 126 (100%) 0
(0%).sup. 0 R.sup..sctn.Recurrent GBM. .sup..English
Pound.Recurrent GBM from the University of Calgary Dataset is not
included in the total count of GBM. *25 cases out of 85 are unknown
for IDH2 status.
[0455] Tumor specimens, blood samples and clinico-pathological
information were collected with informed consent and relevant
ethical board approval in accordance with the tenets of the
Declaration of Helsinki. For the samples from the Pitie-Salp triere
Hospital, clinical data and follow-up are available in the
neuro-oncology database (Onconeurotek, GH Pitie-Salp triere,
Paris).
[0456] Two recurrent GBM patients harboring FGFR3-TACC3 were
enrolled in the dose escalation part of JNJ-42756493 trial at the
Gustave Roussy Institute.
[0457] Identification of Fusion Transcripts and Analysis of Genomic
Breakpoints.
[0458] Total RNA was extracted from frozen tissues using Trizol
(Invitrogen) according to manufacturer instructions. Two to three
hundred nanograms of total RNA were retro-transcribed with the
Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) or
SuperScript II (Invitrogen). RT-PCR was performed using AccuPrime
Taq DNA Polymerase (Invitrogen). Primer pairs used for the
FGFR3-TACC3 fusions screening were: FGFR3ex12-FW:
5'-CGTGAAGATGCTGAAAGACGATG-3 (SEQ ID NO: 495) and TACC3ex14-RV:
5'-AAACGCTTGAAGAGGTCGGAG-3 (SEQ ID NO: 496); amplification
conditions were 94.degree. C.-3 min, (94.degree. C.-30
sec/61.degree. C.-30 sec/68.degree. C.-1 min 40 sec) for 35 cycles,
68.degree. C.-7 min. FGFR1-TACC1 fusions were amplified with
FGFR1ex16-FW: 5'-TGCCTGTGGAGGAACTTTTCA-3' (SEQ ID NO: 497) and
TACC1ex13-RV: 5'-CCCAAACTCAGCAGCCTAAG-3' (SEQ ID NO: 498) primers
(94.degree. C.-30 sec/60.degree. C.-30 sec/68.degree. C.-1 min 40
sec for 35 cycles). PCR products were subjected to Sanger
sequencing.
[0459] FGFR3-TACC3 genomic breakpoints were analyzed in 6
FGFR3-TACC3 positive samples, 5 of which from the Pitie-Salp triere
Hospital and 1 from Montreal Neurological Institute. Three
additional samples (MB-22, TCGA 27-1835 and TCGA 06-6390) available
from the previous study (6) were also included in the analysis.
Fifty nanograms of genomic DNA were used in the PCR reaction,
performed with Accuprime Taq Polymerase (Invitrogen) and PCR
products were Sanger sequenced. Primers used in genomic PCR were
designed according to the breakpoint sequence in the mRNA; the list
of primers used are: FGFR3ex17-FW 5'-TGGACCGTGTCCTTACCGT-3' (SEQ ID
NO: 499) (PCR Samples 3048, 4373, 4867, 4451, MB-22, OPK-14,
06-6390, 27-1835 and Sequencing samples 3048, 4373, 4867, 4451,
MB-22, OPK14, 06-6390, 27-1835); FGFR3ex16-FW
5'-GGTCCTTTGGGGTCCTGCT-3' (SEQ ID NO: 500) (PCR and Sequencing
Sample 3808); TACC3ex6-RV 5'-CCTCTTTCAGCTCCAAGGCA-3' (SEQ ID NO:
501) (PCR and Sequencing Samples PCR 4451 and OPK-14); TACC3ex8-RV
5'-TCTACCAGGACTGTCCCTCAG-3' (SEQ ID NO: 502) (Sequencing Samples
3048 and 4373); TACC3ex9-RV 5'-GGGAGTCTCATTTGCACCGT-3' (SEQ ID NO:
503) (PCR Samples 3048, 4373, 4867 and Sequencing Sample 4867);
TACC3ex10-RV 5'-CTGCATCCAGGTCCTTCTGG-3' (SEQ ID NO: 504) (PCR and
Sequencing Samples MB-22 and 06-6390); TACC3ex11-RV
5'-CCAGTTCCAGGTTCTTCCCG-3' (SEQ ID NO: 505) (Sequencing Samples
27-1837 and 3808); TACC3ex12-RV 5'-CAACCTCTTCGAACCTGTCCA-3' (SEQ ID
NO: 506) (PCR and Sequencing Samples 27-1837 and 3808). PCR
conditions were 94.degree. C.-30 sec/60.degree. C.-30
sec/68.degree. C.-2 min 30 sec for 40 cycles. For amplifications
performed with the primer TACC3ex9-RV, the program was 94.degree.
C.-30 sec/56.degree. C.-30 sec/68.degree. C.-2 min 30 sec) for 40
cycles.
[0460] Quantitation of FGFR3 and TACC3 Transcripts in GBM.
[0461] The relative expression of FGFR3 and TACC3 regions included
in or excluded from the fusion transcript was assessed by qRT-PCR.
Primer pairs with comparable efficiency of amplification were
identified and efficiency was assessed using serial dilutions of
cDNA (20) prepared from OAW28 ovarian carcinoma cells that contain
wild type FGFR3 and TACC3 (21). Primers used are: N-terminal region
of FGFR3, FGFR3-N: Forward 5'-AAGACGATGCCACTGACAAG-3' (SEQ ID NO:
507), Reverse 5'-CCCAGCAGGTTGATGATGTTTTTG-3' (SEQ ID NO: 508);
C-terminal region of TACC3, TACC3-C: Forward
5'-TCCTTCTCCGACCTCTTCAAGC-3' (SEQ ID NO: 509), Reverse
5'-TAATCCTCCACGCACTTCTTCAG-3' (SEQ ID NO: 510). To amplify
transcripts in regions excluded from FGFR3-TACC3 fusion, primers
were designed in the C-terminal region of FGFR3, FGFR3-C: Forward
5'-TACCTGGACCTGTCGGCG-3' (SEQ ID NO: 511), Reverse
5'-TGGGCAAACACGGAGTCG-3' (SEQ ID NO: 512) and N-terminal domain of
TACC3, TACC3-N: Forward 5'-CCACAGACGCACAGGATTCTAAGTC-3' (SEQ ID NO:
513), Reverse 5'-TGAGTTTTCCAGTCCAAGGGTG-3' (SEQ ID NO: 514). All
reactions were performed in triplicate and the data are reported as
Fold Change.+-.Standard Deviation.
[0462] Immunofluorescence and Immunohistochemistry.
[0463] For immunofluorescence (IF) staining of FGFR3, 5 .mu.m FFPE
sections subjected to antigen retrieval with citrate buffer for 8
min. Primary antibodies were: FGFR3-N (1:400, sc-13121, Santa Cruz
Biotechnology), FGFR3-C (1:2000, sc-123, Santa Cruz Biotechnology),
TACC3-N (1:600, ab134153, Abcam), and TACC3-C (1:300, NBP1-01032,
Novus Biological). Secondary biotinylated antibodies were used at
1:50,000 followed by streptavidin and TSA Cy3-conjugated. Nuclei
were counterstained with DAPI. For immunohistochemical analysis
(IHC) of FGFR3 expression, antigen retrieval was performed for 12
min and FGFR-3 antibody (sc-13121, Santa Cruz Biotechnology) was
diluted 1:500. Biotinylated anti-mouse antibody (1:30,000) and
streptavidin were added before incubation with the chromogen.
Nuclei were counterstaining with hematoxylin.
[0464] Molecular Characterization of Tumor Samples.
[0465] Mutational status of IDH1, IDH2, TERT promoter, as well as
the methylation status of the MGMT promoter was analyzed in the
Pitie-Salp triere cohort. Expression of IDH1-R132H mutant was
analyzed by IHC in 500 cases as previously described (22). IDH1 and
IDH2 gene mutations were identified by Sanger sequencing in 464 and
388 gliomas, respectively (5). IDH wild-type tumors are defined
according to the absence of IDH1-R132H immunopositivity and/or
mutations in IDH1 and IDH2 genes. TERT promoter status was
determined by the same technique in 277 samples (23).
Hyper-methylation of the MGMT promoter was tested in 242 samples by
bisulfate pyro-sequencing (24). The presence of EGFRvIII was
evaluated by RT-PCR in 118 samples using EGFR-FW
5'-CTTCGGGGAGCAGCGATGCGAC-3' (SEQ ID NO: 515) and EGFR-RV
5'CTGTCCATCCAGAGG AGGAGTA-3' (SEQ ID NO: 516) primers (25).
[0466] Copy number variations analyses have been performed on 192
tissue samples using CGH arrays using BAC arrays (N=187), Agilent
4x180K (N=2), Nimblegen 3x720K (N=2), Agilent 8x60K (N=1). Results
were normalized using control DNA from matched blood samples as
previously described (26). Additional analyses of 193 tumor
specimens were performed by SNP array, using Illumina Omni (N=110),
Illumina HumCore (N=32), Illumina 370K (N=27), or Illumina 610K
(N=24), as previously described (27). Array processing was
outsourced to Integragen. Raw copy numbers were estimated at each
of the SNP and copy-number markers. Biodiscovery property
SNP-FASST2 algorithm was then used to segment copy number data.
Segments were mapped to hg18 genome assembly (28). Copy number
alterations (CAN) magnitudes called log-R ratio (LRR) were
classified using simple thresholds: deletion (x.ltoreq.-1), loss
(-1<x.ltoreq.-0.2), gain (0.2.ltoreq.x<1) or amplification
(x.gtoreq.1) according to default Nexus 7.5 software. For
additional 56 gliomas, 10q loss was assessed on tumor and blood DNA
by microsatellite analysis, while amplification of EGFR, MDM2 and
CDK4, and deletion of CDKN2A gene, were determined by qPCR, as
previously reported (29, 30).
[0467] The molecular profiles obtained in Pitie-Salp triere dataset
were combined with those available in the TCGA dataportal. TCGA GBM
segmented copy number variation profile was downloaded from The
UCSC Cancer Genomics Browser (31). Copy Number Variations (CNVs)
were measured experimentally using the Affymetrix Genome-Wide Human
SNP Array 6.0 platform at the Broad TCGA genome characterization
center (32). Raw copy numbers were estimated at each of the SNP and
copy-number markers. Circular binary segmentation was then used to
segment the copy number data (28). Segments are mapped to hg18
genome assembly at Broad.
[0468] For CNV analysis of the regions across FGFR3 and TACC3
genes, samples for which RNAseq and CNV data were available or
samples for which only CNV data were available and
RT-PCR-sequencing of FGFR3-TACC3 fusion had been performed were
considered. Overall, 158 GBM (all with a wild type IDH1 gene)
satisfied these criteria. Among them, 5 harbored an FGFR3-TACC3
fusion whereas 153 were FGFR-TACC-negative. The CNV magnitudes,
called log-R ratio (LRR), were classified using the following
thresholds: deletion (x<-1), loss (-1.ltoreq.x.ltoreq.-0.2),
gain (0.2.ltoreq.x.ltoreq.1) or amplification (x>1), according
to the Atlas-TCGA (32). The analysis of the genomic regions
encompassing EGFR, MDM2, CDK4, CDKN2A, 7p, 10q, according to hg18
genome assembly, was performed to evaluate their CNV. EGFRvIII
mutation status was inferred according to Brennan et al. (32). The
frequencies of the aberrations of these genes in FGFR3-TACC3
positive and negative samples were calculated and the obtained data
were then combined with the Pitie-Salp triere Hospital dataset.
[0469] Statistical Analysis.
[0470] Differences in the distribution on categorical variables
were analyzed using Fisher Exact test. The p-values were adjusted
for multiple testing according to Benjamini and Hochberg false
discovery rate (FDR). A q-value.ltoreq.0.05 (two-sided) was
considered to be statistically significant.
[0471] Overall survival (OS) was defined as the time between the
diagnosis and death or last follow-up. Patients who were still
alive at the last follow-up were considered as censored events in
the analysis. Progression-free survival (PFS) was defined as the
time between the diagnosis and recurrence or last follow-up.
Patients who were recurrence-free at the last follow-up were
considered as censored events in the analysis. Survival curves were
calculated by the Kaplan-Meier method and differences between
curves assessed using the Log-Rank test. A Log-Rank test
p-value.ltoreq.0.05 (two-sided) was considered to be statistically
significant.
[0472] Cell Culture and Cell Growth Assay.
[0473] GIC-1123 gliomaspheres were cultured in neurobasal medium
(Invitrogen) supplemented with B27, N2 (Invitrogen), EGF and FGF2
(20 ng/ml, PeproTech). Mouse astrocytes Ink4A-Arf-/- were cultured
in DMEM supplemented with 10% Fetal Bovine Serum. Cells were seeded
at 1,000 cells/well in a 96-well plate and treated with
JNJ-42756493. After 72 hours cell viability was assessed using the
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay. Data are mean.+-.SEM of six replicates. Experiments were
performed three times.
[0474] Subcutaneous Xenografts and Drug Treatment.
[0475] GIC-1123 cells (5.times.10.sup.5) were injected
subcutaneously in the flank of athymic nude (Nu/Nu) mice (Charles
River Laboratories). Mice carrying .about.200 mm.sup.3 subcutaneous
tumors were randomized to receive 12 mg/kg JNJ-42756493 or DMSO in
1% Tween 80 by oral gavage. Tumor diameters were measured with
caliper and tumor volumes estimated using the formula:
0.5.times.length.times.width. Data are mean.+-.SD of nine mice in
each group. Mice were sacrificed when tumors in the control group
reached the maximal size allowed by the IACUC Committee at Columbia
University.
[0476] MRI Imaging and Evaluation of Clinical Response to
JNJ-42756493.
[0477] Baseline and follow-up imaging assessments were performed on
1.5 Tesla MR imaging systems, including at least axial T1 weighted
images before gadolinium injection, Axial or 3D FLAIR
(Fluid-Attenuated Inversion-Recovery), dynamic susceptibility
contrast MR perfusion (0.1 mmol/kg of gadobutrol), axial and 3D T1
weighted images after gadolinium injection. Tumor response was
assessed according to the RANO criteria (33). Contrast-enhancing
lesion volume was assessed with the help of a semi-automated
volumetry tool (SegmentiX), based on shape-detection and
thresholding, with control and manual correction of edges when
necessary. Since exclusion of cystic or necrotic portions of the
lesion may be affected by operator subjectivity, both were included
for volumetric and axial measurements.
[0478] DSC (dynamic susceptibility contrast) perfusion datasets
were processed with vendor's software suite (Neuroperfusion,
Philips), including coregistration and rCBV (relative cerebral
blood volume) parametric maps generation with 3 different
algorithms (Gamma-variate fitting, Arterial Input Function based
deconvolution and Model Free).
[0479] Results
[0480] Detection of FGFR1-TACC1 and FGFR3-TACC3 Fusions in GBM and
Grade II-III Glioma.
[0481] To determine the frequency and molecular features of
FGFR-TACC fusions in human glioma patients, a cohort of 584 GBM and
211 grade II-III glioma treated at five Neurooncology centers
(Table 12) were screened. 108 were grade III (49 IDH wild type, 52
IDH1 mutant and 7 IDH2 mutant) and 103 were grade II (36 IDH wild
type, 63 IDH1 mutant and 4 IDH2 mutant). The IDH mutational status
of 333 GBM was also established and it was determined that 303
harbored wild type IDH1/2 and 30 were mutated at codon 132 of IDH1.
A RT-PCR assay was designed for the detection of all known and
possibly new variants of FGFR1-TACC1 and FGFR3-TACC3 fusions that
retain the mRNA sequences coding for the key FGFR-TK and TACC
domains required for the oncogenic activity of the fusion protein
(FIG. 36 and FIGS. 37A-D). Overall, 20 tumors with an FGFR3-TACC3
fusion were found, of which 17 were GBM (2.9% positives) and 3
lower grade glioma harboring wild type IDH1/2 genes (3.5%
positives). The size of the FGFR3-TACC3 RT-PCR amplicons ranged
from 928 bp (for FGFR3ex18-TACC3ex13) to 1706 bp (for
FGFR3ex18-TACC3ex4). The FGFR1-TACC1 fusion was detected in one
grade II IDH wild type glioma (FIG. 36). Conversely, an IDH1/2
mutant glioma harboring FGFR-TACC fusions (p<0.02) was not
found. Sanger sequencing of the fusion amplicons revealed that each
FGFR-TACC cDNA joined in-frame the sequence coding for the entire
TK domain upstream of TACC-coding sequences that invariably include
the coiled-coil TACC domain (FIG. 36). However, a notable
variability among FGFR3-TACC3 fusion isoforms was detected, whereby
5 of the identified variants occurred only in individual cases
(FIG. 36). Furthermore, 6 fusion transcripts emerged as new
variants that have not been reported before in human cancer (marked
in red in FIG. 36).
[0482] Next, suitable PCR primers were designed to map the genomic
breakpoint coordinates for 9 FGFR3-TACC3-positive samples for which
genomic DNA was available (FIGS. 40 and 41). The genomic
breakpoints were successfully reconstructed by Sanger sequencing
and found that they differ for each of the 9 positive cases.
Interestingly, even cases harboring the same FGFR3-TACC3 transcript
splice variants (#4451 and #OPK-14 joining exon 17 of FGFR3 to exon
6 of TACC3; #3048 and #4373 joining exon 17 of FGFR3 to exon 8 of
TACC3; #3808 and #27-1835 joining exon 17 of FGFR3 to exon 11 of
TACC3) had different genomic breakpoints (FIG. 41). Taken together,
the above findings indicate that the noticeable variability among
FGFR3-TACC3 fusion transcripts and genomic breakpoints is
efficiently resolved by the RT-PCR screening assay.
[0483] Immunostaining Analysis of FGFR3-TACC3-Positive Tumors.
[0484] The expression of the FGFR3 fusion protein was analyzed by
IHC or IF using an antibody that recognizes the N-terminal region
of FGFR3 (FGFR3-N) in 12 GBM and 3 lower grade glioma harboring
FGFR3-TACC3 fusions for which sufficient tissue was available.
Remarkably, each of the 15 positive tumors but none of those that
had scored negative in the RT-PCR assay, displayed strong
positivity for FGFR3 in the vast majority of tumor cells but not
endothelial cells throughout the analyzed tumor section (FIGS.
37A-H). Notably, IF using an antibody that recognizes an epitope at
the C-terminus of TACC3, which is invariably retained within
FGFR3-TACC3 variants (TACC3-C), reproduced the staining pattern of
the FGFR3-N antibody in FGFR3-TACC3 positive tumors. Conversely,
negative or very weak staining was obtained in FGFR3-TACC3-positive
tumors with antibodies recognizing the regions of FGFR3 (FGFR3
C-terminal region, FGFR3-C) and TACC3 (TACC3 N-terminal region,
TACC3-N) constantly excluded from FGFR3-TACC3 fusion proteins (FIG.
42A). Consistently, quantitative RT-PCR of GBM harboring
FGFR3-TACC3 fusions showed that the expression of the N-terminal
coding region of FGFR3 and the C-terminal coding region of TACC3
(which are included in the fusion genes) is markedly higher than
the expression of the C-terminal coding region of FGFR3 and the
N-terminal coding region of TACC3, which are excluded from the
fusion transcripts (FIG. 42B). One recurrent GBM from a patient
whose tumor had been found positive for FGFR3-TACC3 at the initial
diagnosis and who had recurred after concurrent radiotherapy and
temozolomide treatment was analyzed. The recurrent tumor retained
the same FGFR3-TACC3 fusion gene and protein that was present in
the untreated GBM as determined by RT-PCR-sequencing and FGFR3 IF,
respectively (FIG. 43A-C). Although this requires additional
evaluation, the retained uniform positivity for FGFR3 in this
recurrent GBM suggests that targeting the FGFR3-TACC3 fusion
protein at relapse is a valid therapeutic strategy.
[0485] Clinical and Molecular Characteristics of Glioma Patients
with FGFR3-TACC3 Fusions.
[0486] Clinical and molecular profiling data were available for 591
patients including 380 GBM (9 with FGFR3-TACC3 fusions) and all 211
lower grade glioma (3 with FGFR3-TACC3 fusions). Of these 12
patients 5 are males and 7 females, aged 48y to 82y (median=61y).
The molecular profile of FGFR3-TACC3-positive glioma was
determined. To do so, the analysis of CNVs and somatic mutations of
key GBM genes in the dataset was combined with the SNP6.0
high-density genomic array analysis of 158 TCGA-derived GBM samples
fully annotated for FGFR3-TACC3 fusion genes (the RNA-seq and/or
RT-PCR analysis of these samples had revealed that 5 of them harbor
FGFR3-TACC3 fusions) (6). Patients with FGFR3-TACC3 fusions
displayed unique characteristics (Table 13). FGFR3-TACC3 fusions
were mutually exclusive with EGFR amplification (0/16 vs. 166/411;
p=0.0004, FDR q-value corrected for multiple comparisons=0.0012)
and showed a clear trend against the presence of the EGFRvIII
transcript variant (0/16 vs. 37/219; p=0.083). Conversely, CDK4
amplification was significantly more frequent in
FGFR3-TACC3-positive tumors (7/16 vs 41/408, p=0.0008; FDR
q-value=0.0024). A less significant association of FGFR3-TACC3
fusions was also seen with amplification of MDM2, which as CDK4,
maps to chromosome 12q (4/16 vs 24/408, p=0.016; FDR
q-value=0.048). No statistical association between FGFR3-TACC3
fusions and other genetic and epigenetic alterations that commonly
occur in gliomas harboring wild type IDH genes was found (CDKN2A
deletion, TERT promoter mutations, gain of chromosome 7p, loss of
chromosome 10q and methylation of the MGMT promoter, Table 13).
When compared with the IDH wild type patient population of grade II
and grade III glioma and GBM, there was no significant difference
in progression free survival (PFS) or overall survival (OS) between
patients positive or negative for FGFR3-TACC3 (FIGS. 44A-B).
Finally, it was established whether the CNV analysis of the FGFR3
and TACC3 genomic loci could be used to predict positivity for
FGFR3-TACC3 fusions. The analysis of high-density SNP6.0 arrays of
the 158 GBM samples from the Atlas-TCGA revealed that 10 samples
displayed different degrees of copy number gains encompassing the
entire FGFR3 and TACC3 loci (FIG. 45). However, none of them
harbored FGFR3-TACC3 fusions. Conversely, the 5
FGFR3-TACC3-positive samples in the dataset harbor
micro-amplification events involving only the exons of the FGFR3
gene that are included in the fusion breakpoint. This finding
suggests that any CNV survey that is less accurate than
high-density SNP arrays, could fail to identify the genomic marks
associated with true FGFR3-TACC3-positive cases.
TABLE-US-00061 TABLE 13 Molecular alterations in IDH wild type
glioma harboring FGFR3-TACC3 fusions. The table reports the
absolute number and frequency (percentage) of individual
glioma-specific molecular alterations in tumors scoring positive or
negative for FGFR3-TACC3 fusions. The analysis is done on the Union
dataset (TCGA and "Pitie- Salp triere Hospital" datasets, see
methods for details). Statistically significant associations are
indicated in bold (Fisher Exact test, q-values adjusted with FDR).
N of % of N of % of FGFR3-TACC3 FGFR3-TACC3 FGFR3-TACC3 FGFR3-TACC
P-value q-value Positive Positive Negative Negative (Fisher test)
(FDR) EGFR amplification 0/16 0.0% 166/411 40.4% 4.E-04 0.0012 CDK4
amplification 7/16 43.7% 41/408 10.0% 8.E-04 0.0024 MDM2
amplification 4/16 25.0% 24/408 5.9% 0.016 0.048 EGFRvIII 0/16 0.0%
37/219 16.9% 0.083 0.25 CDKN2A deletion 4/16 25.0% 188/411 45.7%
0.13 0.39 Chr. 7p gain 12/15 80.0% 242/374 64.7% 0.28 0.84 Chr. 10q
deletion 12/16 75.0% 253/420 60.2% 0.3 0.9 TERT promoter 9/11 81.8%
128/163 78.5% 0.8 1 mutation MGMT promoter 6/12 50.0% 73/160 45.6%
0.7 1 hypermethylation
[0487] Preclinical and Clinical Relevance of Targeting FGFR3-TACC3
Fusions.
[0488] JNJ-42756493 is a potent, oral pan-FGFR tyrosine kinase
inhibitor with IC50 values in the low nanomolar range for all
members of the FGFR family. It has demonstrated potent antitumor
activities in nonclinical models with FGFR aberrations including
squamous non-small cell lung cancer, gastric, breast,
hepatocellular cancer (HCC), endometrial, and bladder (34, 35). To
ask whether JNJ-42756493 is effective in targeting specifically
FGFR-TACC-positive cells, mouse astrocytes expressing FGFR3-TACC3,
FGFR3-TACC3 containing a mutation that inactivates the kinase
activity of FGFR3 (FGFR3-TACC3-KD), or the empty vector were
treated with JNJ-42756493. The effect of JNJ-42756493 on human
glioma stem cells GIC-1123 that harbor the FGFR3-TACC3 gene fusion
(6) was also studied. These experiments revealed that both mouse
astrocytes and GIC-1123 that express FGFR3-TACC3 but not cells
expressing the KD mutant fusion or the empty vector are highly
sensitive to FGFR inhibition by JNJ-42756493 with an IC50 of 3.03
nM and 1.55 nM, respectively (FIGS. 38A-B). Next, the effect of
oral treatment with JNJ-42756493 of mice bearing xenografts of
human GIC-1123 affects tumor growth was tested. Mice were
randomized to receive vehicle or JNJ-42756493 (12 mg/kg). Mirroring
the in vitro results, JNJ-42756493 elicited a potent growth
inhibition of GIC-1123 tumor xenografts (FIGS. 38C-D) with a
statistically significant tumor regression after two weeks (p-value
of the slope calculated from the treatment starting point=0.04).
The above findings provide a strong foundation for the treatment of
GBM patients harboring FGFR-TACC rearrangements with
JNJ-42756493.
[0489] Two patients with recurrent GBM harboring FGFR3-TACC3
fusions were treated with JNJ-42756493 in a first-in-man phase I
trial. Patient 1, male aged 52, underwent partial surgical
resection of a right parietal GBM, followed by fractionated
radiotherapy and concomitant temozolomide (TMZ) as first line
treatment (36). The RT-PCR-sequencing analysis of the GBM specimen
revealed positivity for the FGFR3-TACC3 fusion
(FGFR3-exon17-TACC3-exon 6, sample 4451, FIGS. 40 and 41) and the
immunostaining using FGFR3 antibody on paraffin embedded sections
showed strong positivity in a large fraction of tumor cells. After
5 cycles of TMZ, the patient presented with dizziness and headache
and brain MRI revealed tumor progression (FIG. 39A). At this time
the patient was enrolled in the JNJ-42756493 trial and received
JNJ-42756493 (12 mg/day administered in cycles of 7 days followed
by 7 days off treatment). After 3 weeks the patient reported a
marked clinical improvement (complete regression of dizziness and
headache). On MRI, the sum of product diameters (RANO criteria,
FIG. 39B) and volumetry (FIG. 39C) measured without excluding
cystic and necrotic components showed disease stabilization.
However, the tumor mass underwent significant decrease of the
enhancing parenchyma (-44%) with formation of a cystic portion in
the central core (33). The objective response was further
corroborated by the marked reduction of the extent of tumor
vascularity estimated by quantitative analysis of rCBV (relative
cerebral blood volume) from dynamic susceptibility MR perfusion
maps (37) (FIG. 39D). Stabilization lasted for 115 days. During
JNJ-42756493 treatment mild and manageable toxicity was observed
(grade I hyperphosphatemia, asthenia, dysgueusia, dry mouth,
keratitis, and grade II nail changes). After 4 months, tumor
progressed on MRI locally both on T1 contrast-enhanced area and
T2/FLAIR hypersignal. The patient was re-operated and subsequently
treated with CCNU. He is still alive, but in progression after 21
months from diagnosis and 287 days from the start of the anti-FGFR
therapy.
[0490] Patient 2 is a 64 years old woman, affected by left parietal
GBM, diagnosed by stereotactic biopsy. The tumor was positive for
FGFR3-TACC3 gene fusion by RT-PCR-sequencing and showed diffuse
FGFR3 expression in most tumor cells (FIGS. 37A, 37C, 37E, sample
4620). The patient received as first line treatment fractionated
radiotherapy and TMZ according to the Stupp protocol (36), but
after 2 cycles of monthly TMZ she presented with clinical
deterioration including progressive headaches, right homonymous
hemianopsia and memory impairment. Brain MRI performed 3 and 4
months after the completion of concomitant chemo-radiotherapy
revealed tumor progression with increase of the left parietal mass
and the appearance of a small contralateral lesion (FIG. 49E). The
patient was thus enrolled in the JNJ-42756493 trial (12 mg/day
administered in cycles of 7 days followed by 7 days off treatment)
and showed clinical improvement after 4 weeks (regression of
headaches, visual field defect and memory impairment). Best
response was observed after 104 days of treatment with a 22%
reduction of tumor size according to the RANO criteria (FIG. 39F)
and 28% according to volumetry (FIG. 39G). Grade I
hyperphosphatemia, nail changes, and mucositis were observed.
Clinical status remained stable until disease progression occurring
134 days after the start of the anti-FGFR. The patient is still
alive and is receiving a third-line chemotherapy with nitrosoureas
and bevacizumab.
TABLE-US-00062 TABLE 14 Summary of FGFR-TACC fusion transcripts
identified in all cancer types. FGFR3-TACC3 fusion variants are
ranked according to their prevalence across any cancer type. The
number of FGFR-TACC fusions identified in each tumor type,
including those identified in the present study, is also indicated.
FGFR-TACC Fusion Variants N Cases Tumor Type FGFR3-TACC3
FGFR3exon17-TACC3exon11 30 Brain Tumors, N = 10 (N = 2,.sup.8; N =
2,.sup.9,15; N = 6, Present Study). Bladder Cancer, N = 6 (N =
3,.sup.12,15; N = 3,.sup.11). Lung Cancer, N = 13 (N =
4,.sup.12,15; N = 9,.sup.10). Renal Carcinoma, N = 1,.sup.15.
FGFR3exon17-TACC3exon10 18 Brain Tumors, N = 5 (N = 1,.sup.8; N =
1,.sup.9; N = 3, Present study). Oral Cancer, N = 1,.sup.12,. Head
and Neck Cancer, N = 2,.sup.12,15. Bladder Cancer, N = 3,.sup.7.
Lung Cancer, N = 7 (N = 4,.sup.8; N = 2,.sup.10; N = 1,.sup.14).
FGFR3exon17-TACC3exon8 8 Brain Tumors, N = 6 (N = 2,.sup.8. N = 4,
Present study). Lung Cancer, N = 2 (N = 1,.sup.10; N = 1,.sup.14).
FGFR3exon17-TACC3exon4 4 Brain Tumors, N = 2 (N = 1,.sup.9; N =
1,.sup.10). Bladder Cancer, N = 1.sup.9. Lung Cancer, N = 1.sup.14.
FGFR3exon17-TACC3exon6 2 Brain Tumors, N = 2, Present study.
FGFR3exon18-TACC3exon4 1 Brain Tumors, N = 1, Present study.
FGFR3exon17-TACC3exon9 INS63bp 1 Brain Tumors, N = 1,.sup.5.
FGFR3exon18-TACC3exon9 INS66bp 1 Brain Tumors, N = 1, Present
study. FGFR3exon18-TACC3exon5 1 Brain Tumors, N = 1, Present study.
FGFR3exon18-TACC3exon5 INS33bp 1 Brain Tumors, N = 1, Present
study. INS71bp 1 Lung Cancer, N = 1,.sup.10.
FGFR3exon18-TACC3exon13 1 Brain Tumors, N = 1, Present study.
FGFR3exon18-TACC3exon11 1 Lung Cancer, N = 1,.sup.10. FGFR1-TACC1
FGFR1exon17-TACC1exon7 5 Brain Tumors, N = 5 (N = 1,.sup.5; N =
3,.sup.13; N = 1, Present study). FGFR2-TACC2 1 Stomach
Adenocarcinoma, N = 1.sup.15.
[0491] Discussion
[0492] FGFR-TACC fusions are potent oncogenic events that when
present in brain tumor cells confer sensitivity to FGFR inhibitors
(6). Since the original identification of recurrent FGFR-TACC
fusions in GBM, small subgroups of patients harboring FGFR-TACC
translocations have been identified in several other tumor types
(7-15). Here, an unbiased RT-PCR-sequencing analysis for the
identification of all possible functional FGFR-TACC fusion
transcripts is reported. The screening of a large glioma dataset
from multiple Institutions not only confirmed that FGFR-TACC
rearrangements occur in .about.3% of human GBM but also revealed
that FGFR-TACC fusions are present in the subgroup of IDH wild type
lower grade glioma (grade with prevalence similar to that of GBM.
IDH wild type grade II and III glioma have a significantly worse
clinical outcome than the IDH mutant glioma and manifests molecular
and clinical features that resemble GBM (5). The finding that
FGFR-TACC fusions occur in IDH wild type but not IDH mutant glioma
provides an important clue for the molecular characterization of
this glioma subtype. Furthermore, the clustering of such potent
oncogenic events in IDH wild type glioma underscores the
particularly aggressive nature of this group of glioma. While it
was shown that FGFR-TACC fusions cluster within the poor clinical
outcome subgroup of IDH wild type glioma, these translocations do
not seem to carry prognostic value within the IDH wild type
subgroup of glioma patients. Without being bound by theory, the
sample size of patients harboring FGFR-TACC fusions is too small to
draw definitive conclusions with respect to the impact on survival
and larger studies may be necessary to clarify the prognostic role
of FGFR-TACC fusions in IDH wild type glioma.
[0493] Beside mutual exclusivity between IDH1 mutations and
FGFR-TACC fusions, the results showed that patients with
FGFR3-TACC3 rearrangements lack EGFR amplification and EGFRvIII but
are significantly enriched for amplification of CDK4 (and MDM2 to a
lesser extent). Knowledge of these molecular characteristics will
help select those patients who most likely harbor FGFR-TACC
rearrangements and design combinatorial targeted therapies that
might be more effective in the FGFR-TACC-positive glioma
subgroup.
[0494] The molecular screen uncovered 6 new FGFR3-TACC3 fusion
events. Together with the previously identified variants, 12
distinct isoforms of FGFR3-TACC3 have been reported, thus revealing
a remarkable variability of FGFR3-TACC3 transcripts in human cancer
(see Table 14 summarizing the structure of all the FGFR-TACC
variants identified to date). The structural heterogeneity of
FGFR3-TACC3 fusions is yet more pronounced at the genomic level,
whereby each fusion event harbors distinct genomic breakpoints,
even for identical fusion transcripts. This finding underscores the
notion that targeted genomic analyses are unlikely to be suitable
approaches for the molecular diagnosis of FGFR3-TACC3 positivity.
Conversely, the unbiased identification of FGFR3-TACC3-positive
tumors with the RT-PCR-sequencing assay reported here overcomes the
limitations of screening only for previously identified FGFR3-TACC3
fusions and provides a simple molecular diagnostic assay.
[0495] Rather than displaying uniform amplifications of the FGFR3
and TACC3 genomic loci, FGFR3-TACC3-positive samples harbor small,
intragenic micro-amplification events typically encompassing only
the exons of the FGFR3 and TACC3 genes included in the breakpoint
(6). This finding is consistent with the notion that a "fusion
breakpoint principle" sustains the CNVs of driver gene fusions such
as FGFR3-TACC3 in which local CNVs target exclusively the
breakpoint region (38). It is noted that such small and irregular
CNVs may easily go undetected from CNV analyses performed using
platforms less sensitive than the high-density SNP6.0 genomic
arrays. Furthermore, the notion that FGFR3-TACC3-negative GBM may
harbor uniform amplifications across the FGFR3 and TACC3 loci
argues against the standard analysis of FGFR3 and/or TACC3 CNVs as
a method for the selection of FGFR3-TACC3-positive tumors.
[0496] There is a growing body of evidence supporting the notion
that GBM is a markedly heterogeneous tumor. The formidable degree
of intra-tumor heterogeneity of GBM is a potential cause of failure
of targeted therapies in these tumors. In particular, the
intra-tumor heterogeneity of GBM has previously been recognized in
light of the mosaic expression of the RTK genes EGFR, PDGFRA and
MET by neighboring cells (16-19). Thus, in the majority of GBM,
amplification or overexpression of individual RTK genes are present
in a sub-clonal fraction of tumor cells and co-exist with
amplification/expression of other RTK-coding genes within the tumor
mass. Therefore, it was essential to determine whether such
heterogeneity was also present in gliomas harboring FGFR-TACC
translocations. The immunostaining of FGFR3-TACC3-positive tumors
revealed that positive specimens manifest strong and uniform
expression of the fusion protein, which is also retained after
recurrence. This behavior is reminiscent of other driver chromosome
translocations (BCR-ABL, EML4-ALK) and is compatible with the
glioma-initiating functions of FGFR-TACC fusions (6). It is also
the scenario expected for a driver oncogene whose activity remains
essential for tumor maintenance regardless of secondary genetic
alterations that occur during tumor progression. The strong
antitumor effects obtained with JNJ-42756493 in glioma cells
harboring FGFR3-TACC3 fusions have built a compelling rationale for
the treatment of glioma patients positive for FGFR-TACC
rearrangements. JNJ-42756493 is an oral ATP-competitive pan-FGFR
selective inhibitor that inhibits tyrosine phosphorylation of
activated FGFR at nanomolar concentrations (34, 35). The enrollment
of two patients with recurrent FGFR3-TACC3-positive GBM in a phase
I trial with JNJ-42756493 showed that this treatment has tolerable
toxicity and clear anti-tumor activity, thus validating FGFR-TACC
as a therapeutic target. Therefore, targeted inhibition of FGFR-TK
in preselected IDH wild type FGFR-TACC-positive glioma may provide
clinical benefits for patients with recurrent glioma who currently
lack valuable therapeutic options. In conclusion, described herein
is the importance and feasibility of prospective genotyping for
FGFR-TACC fusions in glioma patients and provided a preliminary
evidence of clinical response that warrants the investigation of
the sensitivity of gliomas harboring FGFR-TACC rearrangements to
FGFR kinase inhibition in clinical trials.
REFERENCES
[0497] 1. Medves S, Demoulin J B. Tyrosine kinase gene fusions in
cancer: translating mechanisms into targeted therapies. J Cell Mol
Med 2012; 16:237-48. [0498] 2. Mitelman F, Johansson B, Mertens F.
The impact of translocations and gene fusions on cancer causation.
Nat Rev Cancer 2007; 7:233-45. [0499] 3. Weathers S P, Gilbert M R.
Advances in treating glioblastoma. F1000Prime Rep. 2014; 6:46.
[0500] 4. Omuro A, DeAngelis L M. Glioblastoma and other malignant
gliomas: a clinical review. JAMA 2013; 310:1842-50. [0501] 5.
Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F, et al.
Isocitrate dehydrogenase 1 codon 132 mutation is an important
prognostic biomarker in gliomas. J Clin Oncol 2009; 27:4150-4.
[0502] 6. Singh D, Chan J M, Zoppoli P, Niola F, Sullivan R,
Castano A, et al. Transforming fusions of FGFR and TACC genes in
human glioblastoma. Science 2012; 337:1231-5. [0503] 7. Cancer
Genome Atlas Research N. Comprehensive molecular characterization
of urothelial bladder carcinoma. Nature 2014; 507:315-22. [0504] 8.
Majewski I J, Mittempergher L, Davidson N M, Bosma A, Willems S M,
Horlings H M, et al. Identification of recurrent FGFR3 fusion genes
in lung cancer through kinome-centred RNA sequencing. J Pathol
2013; 230:270-6. [0505] 9. Parker B C, Annala M J, Cogdell D E,
Granberg K J, Sun Y, Ji P, et al. The tumorigenic FGFR3-TACC3 gene
fusion escapes miR-99a regulation in glioblastoma. J Clin Invest
2013; 123:855-65. [0506] 10. Wang R, Wang L, Li Y, Hu H, Shen L,
Shen X, et al. FGFR1/3 Tyrosine Kinase Fusions Define a Unique
Molecular Subtype of Non-Small Cell Lung Cancer. Clin Cancer Res
2014; 20:4107-14. [0507] 11. Williams S V, Hurst C D, Knowles M A.
Oncogenic FGFR3 gene fusions in bladder cancer. Hum Mol Genet 2013;
22:795-803. [0508] 12. Wu Y M, Su F, Kalyana-Sundaram S, Khazanov
N, Ateeq B, Cao X, et al. Identification of targetable FGFR gene
fusions in diverse cancers. Cancer Discov 2013; 3:636-47. [0509]
13. Zhang J, Wu G, Miller C P, Tatevossian R G, Dalton J D, Tang B,
et al. Whole-genome sequencing identifies genetic alterations in
pediatric low-grade gliomas. Nat Genet 2013; 45:602-12. [0510] 14.
Capelletti M, Dodge M E, Ercan D, Hammerman P S, Park S I, Kim J,
et al. Identification of Recurrent FGFR3-TACC3 Fusion Oncogenes
from Lung Adenocarcinoma. Clin Cancer Res 2014, DOI:
10.1158/1078-0432. CCR-14-1337; in press. [0511] 15. Stransky N,
Cerami E, Schalm S, Kim J L, Lengauer C. The landscape of kinase
fusions in cancer. Nat Commun 2014; 5:4846. [0512] 16. Inda M M,
Bonavia R, Mukasa A, Narita Y, Sah D W, Vandenberg S, et al. Tumor
heterogeneity is an active process maintained by a mutant
EGFR-induced cytokine circuit in glioblastoma. Genes Dev 2010;
24:1731-45. [0513] 17. Snuderl M, Fazlollahi L, Le L P, Nitta M,
Zhelyazkova B H, Davidson C J, et al. Mosaic amplification of
multiple receptor tyrosine kinase genes in glioblastoma. Cancer
Cell 2011; 20:810-7. [0514] 18. Ene C I, Fine H A. Many tumors in
one: a daunting therapeutic prospect. Cancer Cell 2011; 20:695-7.
[0515] 19. Sottoriva A, Spiteri I, Piccirillo S G, Touloumis A,
Collins V P, Marioni J C, et al. Intratumor heterogeneity in human
glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad
Sci USA 2013; 110:4009-14. [0516] 20. Kindich R, Florl A R, Jung V,
Engers R, Muller M, Schulz W A, et al.
[0517] Application of a modified real-time PCR technique for
relative gene copy number quantification to the determination of
the relationship between NKX3.1 loss and MYC gain in prostate
cancer. Clin Chem 2005; 51:649-52. [0518] 21. Bulusu K C, Tym J E,
Coker E A, Schierz A C, Al-Lazikani B. canSAR: updated cancer
research and drug discovery knowledgebase. Nucleic Acids Res 2014;
42:D1040-7. [0519] 22. Reyes-Botero G, Giry M, Mokhtari K,
Labussiere M, Idbaih A, Delattre J Y, et al. Molecular analysis of
diffuse intrinsic brainstem gliomas in adults. J Neurooncol 2014;
116:405-11. [0520] 23. Labussiere M B B, Mokhtari K, Di Stefano A
L, Rahimian A, Rossetto M, Ciccarino P, Saulnier O, Paterra R,
Marie Y, Finocchiaro G, Sanson M. Combined analysis of TERT, EGFR
and IDH status define distinct prognostic glioblastoma classes.
Neurology 2014; 83:1200-6. [0521] 24. Quillien V, Lavenu A,
Karayan-Tapon L, Carpentier C, Labussiere M, Lesimple T, et al.
Comparative assessment of 5 methods (methylation-specific
polymerase chain reaction, MethyLight, pyrosequencing,
methylation-sensitive high-resolution melting, and
immunohistochemistry) to analyze
O6-methylguanine-DNA-methyltranferase in a series of 100
glioblastoma patients. Cancer 2012; 118:4201-11. [0522] 25. Idbaih
A, Aimard J, Boisselier B, Marie Y, Paris S, Criniere E, et al.
Epidermal growth factor receptor extracellular domain mutations in
primary glioblastoma. Neuropathol Appl Neurobiol 2009; 35:208-13.
[0523] 26. Idbaih A, Marie Y, Lucchesi C, Pierron G, Manie E,
Raynal V, et al. BAC array CGH distinguishes mutually exclusive
alterations that define clinicogenetic subtypes of gliomas. Int J
Cancer 2008; 122:1778-86. [0524] 27. Gonzalez-Aguilar A, Idbaih A,
Boisselier B, Habbita N, Rossetto M, Laurenge A, et al. Recurrent
mutations of MYD88 and TBL1XR1 in primary central nervous system
lymphomas. Clin Cancer Res 2012; 18:5203-11. [0525] 28. Olshen A B,
Venkatraman E S, Lucito R, Wigler M. Circular binary segmentation
for the analysis of array-based DNA copy number data. Biostatistics
2004; 5:557-72. [0526] 29. Hoang-Xuan K, He J, Huguet S, Mokhtari
K, Marie Y, Kujas M, et al. Molecular heterogeneity of
oligodendrogliomas suggests alternative pathways in tumor
progression. Neurology 2001; 57:1278-81. [0527] 30. Houillier C,
Lejeune J, Benouaich-Amiel A, Laigle-Donadey F, Criniere E,
Mokhtari K, et al. Prognostic impact of molecular markers in a
series of 220 primary glioblastomas. Cancer 2006; 106:2218-23.
[0528] 31. Goldman M, Craft B, Swatloski T, Ellrott K, Cline M,
Diekhans M, et al. The UCSC Cancer Genomics Browser: update 2013.
Nucleic Acids Res 2013; 41:D949-54. [0529] 32. Brennan C W, Verhaak
R G, McKenna A, Campos B, Noushmehr H, Salama S R, et al. The
somatic genomic landscape of glioblastoma. Cell 2013; 155:462-77.
[0530] 33. Wen P Y, Macdonald D R, Reardon D A, Cloughesy T F,
Sorensen A G, Galanis E, et al. Updated response assessment
criteria for high-grade gliomas: response assessment in
neuro-oncology working group. J Clin Oncol 2010; 28:1963-72. [0531]
34. Bahleda R, Dienstmann R, Adamo B, Gazzah A, Infante J R, Zhong
B, et al. Phase 1 study of JNJ-42756493, a pan-fibroblast growth
factor receptor (FGFR) inhibitor, in patients with advanced solid
tumors. J Clin Oncol 2014; 32:suppl; abstr 2501. [0532] 35. Squires
M, Ward G, Saxty G, Berdini V, Cleasby A, King P, et al. Potent,
selective inhibitors of fibroblast growth factor receptor define
fibroblast growth factor dependence in preclinical cancer models.
Mol Cancer Ther 2011; 10:1542-52. [0533] 36. Stupp R, Mason W P,
van den Bent M J, Weller M, Fisher B, Taphoorn M J, et al.
Radiotherapy plus concomitant and adjuvant temozolomide for
glioblastoma. N Engl J Med 2005; 352:987-96. [0534] 37. Law M, Yang
S, Babb J S, Knopp E A, Golfinos J G, Zagzag D, et al. Comparison
of cerebral blood volume and vascular permeability from dynamic
susceptibility contrast-enhanced perfusion M R imaging with glioma
grade. AJNR Am J Neuroradiol 2004; 25:746-55. [0535] 38. Wang X S,
Prensner J R, Chen G, Cao Q, Han B, Dhanasekaran S M, et al. An
integrative approach to reveal driver gene fusions from paired-end
sequencing data in cancer. Nat Biotechnol 2009; 27:1005-11.
Sequence CWU 1
1
4941178DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1g tgc tgg cat gcc gcg ccc tcc cag agg ccc
acc ttc aag cag ctg gtg 49 Cys Trp His Ala Ala Pro Ser Gln Arg Pro
Thr Phe Lys Gln Leu Val 1 5 10 15 gag gac ctg gac cgt gtc ctt acc
gtg acg tcc acc gac ttt aag gag 97Glu Asp Leu Asp Arg Val Leu Thr
Val Thr Ser Thr Asp Phe Lys Glu 20 25 30 tcg gcc ttg agg aag cag
tcc tta tac ctc aag ttc gac ccc ctc ctg 145Ser Ala Leu Arg Lys Gln
Ser Leu Tyr Leu Lys Phe Asp Pro Leu Leu 35 40 45 agg gac agt cct
ggt aga cca gtg ccc gtg gcc 178Arg Asp Ser Pro Gly Arg Pro Val Pro
Val Ala 50 55 291DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 2ctttaaggag tcggccttga
ggaagcagtc cttatacctc aagttcgacc ccctcctgag 60ggacagtcct ggtagaccag
tgcccgtggc c 91391DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 3actttaagga gtcggccttg
aggaagcagt ccttatacct caagttcgac cccctcctga 60gggacagtcc tggtagacca
gtgcccgtgg g 91491DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 4gactttaagg agtcggcctt
gaggaagcag tccttatacc tcaagttcga ccccctcctg 60agggacagtc ctggtagacc
agtgcccgtg g 91591DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 5cgactttaag gagtcggcct
tgaggaagca gtccttatac ctcaagttcg accccctcct 60gagggacagt cctggtagac
cagtgcccgt t 91691DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 6ccgactttaa ggagtcggcc
ttgaggaagc agtccttata cctcaagttc gaccccctcc 60tgagggacag tcctggtaga
ccagtgcccg g 91791DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 7accgacttta aggagtcggc
cttgaggaag cagtccttat acctcaagtt cgaccccctc 60ctgagggaca gtcctggtag
accagtgccc c 91891DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 8caccgacttt aaggagtcgg
ccttgaggaa gcagtcctta tacctcaagt tcgaccccct 60cctgagggac agtcctggta
gaccagtgcc c 91991DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 9ccaccgactt taaggagtcg
gccttgagga agcagtcctt atacctcaag ttcgaccccc 60tcctgaggga cagtcctggt
agaccagtgc c 911091DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 10tccaccgact ttaaggagtc
ggccttgagg aagcagtcct tatacctcaa gttcgacccc 60ctcctgaggg acagtcctgg
tagaccagtg g 911191DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 11gtccaccgac tttaaggagt
cggccttgag gaagcagtcc ttatacctca agttcgaccc 60cctcctgagg gacagtcctg
gtagaccagt t 911291DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 12cgtccaccga ctttaaggag
tcggccttga ggaagcagtc cttatgcctc aagttcggcc 60ccctcctgag ggacagtcct
ggtagaccag g 911390DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 13gacgtccacc gactttaagg
agtcggcctt gaggaagcag tccttatacc tcaagttcga 60ccccctcctg agggacagtc
ctggtagacg 901491DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 14tgacgtccac cgactttaag
gagtcggcct tgaggaagca gtccttatac ctcaagttcg 60accccctcct gagggacagt
cctggtagac c 911591DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 15gtgacgtcca ccgactttaa
ggagtcggcc ttgaggaagc agtccttata cctcaagttc 60gaccccctcc tgagggacag
tcctggtaga a 911691DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 16cgtgacgtcc accgacttta
aggagtcggc cttgaggaag cagtccttat acctcaagtt 60cgaccccctc ctgagggaca
gtcctggtag g 911791DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 17ccgtgacgtc caccgacttt
aaggagtcgg ccttgaggaa gcagtcctta tacctcaagt 60tcgaccccct cctgagggac
agtcctggta a 911891DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 18cttaccgtga cgtccaccga
ctttaaggag tcggccttga ggaagcagtc cttatacctc 60aagttcgacc ccctcctgag
ggacagtcct t 911991DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 19gtccttaccg tgacgtccac
cgactttaag gagtcggcct tgaggaagca gtccttatac 60ctcaagttcg accccctcct
gagggacagt t 912091DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 20tgtccttacc gtgacgtcca
ccgactttaa ggagtcggcc ttgaggaagc agtccttata 60cctcaagttc gaccccctcc
tgagggacag g 912191DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 21gtgtccttac cgtgacgtcc
accgacttta aggagtcggc cttgaggaag cagtccttat 60acctcaagtt cgaccccctc
ctgagggaca a 912291DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 22cgtgtcctta ccgtgacgtc
caccgacttt aaggagtcgg ccttgaggaa gcagtcctta 60tacctcaagt tcgaccccct
cctgagggac c 912391DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 23accgtgtcct taccgtgacg
tccaccgact ttaaggagtc ggccttgagg aagcagtcct 60tatacctcaa gttcgacccc
ctcctgaggg g 912491DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 24gaccgtgtcc ttaccgtgac
gtccaccgac tttaaggagt cggccttgag gaagcagtcc 60ttatacctca agttcgaccc
cctcctgagg g 912591DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 25ggaccgtgtc cttaccgtga
cgtccaccga ctttaaggag tcggccttga ggaagcagtc 60cttatacctc aagttcgacc
ccctcctgag g 912691DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 26ctggaccgtg tccttaccgt
gacgtccacc gactttaagg agtcggcctt gaggaagcag 60tccttatacc tcaagttcga
ccccctcctg g 912791DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 27cctggaccgt gtccttaccg
tgacgtccac cgactttaag gagtcggcct tgaggaagca 60gtccttatac ctcaagttcg
accccctcct t 912891DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 28gacctggacc gtgtccttac
cgtgacgtcc accgacttta aggagtcggc cttgaggaag 60cagtccttat acctcaagtt
cgaccccctc c 912991DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 29ggacctggac cgtgtcctta
ccgtgacgtc caccgacttt aaggagtcgg ccttgaggaa 60gcagtcctta tacctcaagt
tcgaccccct t 913091DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 30aggacctgga ccgtgtcctt
accgtgacgt ccaccgactt taaggagtcg gccttgagga 60agcagtcctt atacctcaag
ttcgaccccc c 913190DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 31gaggacctgg accgtgtcct
taccgtgacg tccaccgact ttaaggagtc ggccttgagg 60aagcagtcct tatacctcaa
gttcgacccc 903290DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 32ggaggacctg gaccgtgtcc
ttaccgtgac gtccaccgac tttaaggagt cggccttgag 60gaagcagtcc ttatacctca
agttcgaccc 903390DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 33tggaggacct ggaccgtgtc
cttaccgtga cgtccaccga ctttaaggag tcggccttga 60ggaagcagtc cttatacctc
aagttcgacc 903490DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 34gtggaggacc tggaccgtgt
ccttaccgtg acgtccaccg actttaagga gtcggccttg 60aggaagcagt ccttatacct
caagttcgac 903590DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 35ggtggaggac ctggaccgtg
tccttaccgt gacgtccacc gactttaagg agtcggcctt 60gaggaagcag tccttatacc
tcaagttcga 903690DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 36tggtggagga cctggaccgt
gtccttaccg tgacgtccac cgactttaag gagtcggcct 60tgaggaagca gtccttatac
ctcaagttcg 903790DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 37ctggtggagg acctggaccg
tgtccttacc gtgacgtcca ccgactttaa ggagtcggcc 60ttgaggaagc agtccttata
cctctaatca 903890DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 38gctggtggag gacctggacc
gtgtccttac cgtgacgtcc accggcttta aggagtcggc 60ctcgaggaag cagccctttt
acctcaagtt 903990DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 39agctggtgga ggacctggac
cgtgtcctta ccgtgacgtc caccgacttt aaggagtcgg 60ccttgaggaa gcagtcctta
tacctcaagt 904090DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 40cagctggtgg aggacctgga
ccgtgtcctt accgtgacgt ccaccgactt taaggagtcg 60gccttgagga agcagtcctt
atacctcaag 904190DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 41gcagctggtg gaggacctgg
accgtgtcct taccgtgacg tccaccgact ttaaggagtc 60ggccttgagg aagcagtcct
tatacctcaa 904290DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 42agcagctggt ggaggacctg
gaccgtgtcc ttaccgtgac gtccaccgac tttaaggagt 60cggccttgag gaagcagtcc
ttatacctca 904390DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 43aagcagctgg tggaggacct
ggaccgtgtc cttaccgtga cgtccaccga ctttaaggag 60tcggccttga ggaagcagtc
cctatacccc 904490DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 44caagcagctg gtggaggacc
tggaccgtgt ccttaccgtg acgtccaccg actttaagga 60gtcggccttg aggaagcagt
ccttatacct 904590DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 45tcaagcagct ggtggaggac
ctggaccgtg tccttaccgt gacgtccacc gactttaagg 60agtcggcctt gaggaagcag
tccttatacc 904690DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 46ttcaagcagc tggtggagga
cctggaccgt gtccttaccg tgacgtccac cgactttaag 60gagtcggcct tgaggaagca
gtccttatac 904790DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 47cttcaagcag ctggtggagg
acctggaccg tgtccttacc gtgacgtcca ccgactttaa 60ggagtcggcc ttgaggaagc
agtccttata 904890DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 48ccttcaagca gctggtggag
gacctggacc gtgtccttac cgtgacgtcc accgacttta 60aggagtcggc cttgaggaag
cagtccttat 904990DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 49accttcaagc agctggtgga
ggacctggac cgtgtcctta ccgtgacgtc caccgacttt 60aaggagtcgg ccttgaggaa
gcagtcctta 905090DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 50caccttcaag cagctggtgg
aggacctgga ccgtgtcctt accgtgacgt ccaccgactt 60taaggagtcg gccttgagga
agcagtcctt 905190DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 51ccaccttcaa gcagctggtg
gaggacctgg accgtgtcct taccgtgacg tccaccgact 60ttaaggagtc ggccttgagg
aagcagtcct 905290DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 52cccaccttca agcagctggt
ggaggacctg gaccgtgtcc ttaccgtgac gtccaccgac 60tttaaggagt cggccttgag
gaagcagtcc 905390DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 53ggcccacctt caagcagctg
gtggaggacc tggaccgtgt ccttaccgtg acgtccaccg 60actttaagga gtcggccttg
aggaagcagt 905490DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 54aggcccacct tcaagcagct
ggtggaggac ctggaccgtg tccttaccgt gacgtccacc 60gactttaagg agtcggcctt
gaggaagcag 905590DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 55gaggcccacc ttcaagcagc
tggtggagga cctggaccgt gtccttaccg tgacgtccac 60cgactttaag gagtcggcct
tgaggaagca 905690DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 56agaggcccac cttcaagcag
ctggtggagg tcctggaccg tgtccttacc gtgacgtcca 60ccgactttaa ggagtcggcc
ttgaggaagc 905790DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 57cagaggccca ccttcaagca
gctggtggag gacctggacc gtgtccttac cgtgacgtcc 60accgacttta aggagtcggc
cttgaggaag 905890DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 58ccagaggccc accttcaagc
agctggtgga ggacctggac cgtgtcctta ccgtgacgtc 60caccgacttt aaggagtcgg
ccttgaggaa 905990DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 59cccagaggcc caccttcaag
cagctggtgg aggacctgga ccgtgtcctt accgtgacgt 60ccaccgactt taaggagtcg
gccttgagga 906090DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 60tcccagaggc ccaccttcaa
gcagctggtg gaggacctgg accgtgtcct taccgtgacg 60tccaccgact ttaaggagtc
ggccttgagg 906190DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 61ctcccagagg cccaccttca
agcagctggt ggaggacctg gaccgtgtcc ttaccgtgac 60gtccaccgac tttaaggagt
cggccttgag 906290DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 62cctcccagag gcccaccttc
aagcagctgg tggaggacct ggaccgtgtc cttaccgtga 60cgtccaccga ctttaaggag
tcggccttga 906390DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 63ccctcccaga ggcccacctt
caagcagctg gtggaggacc tggaccgtgt ccttaccgtg 60acgtccaccg actttaagga
gtcggccttg 906490DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 64gcgccctccc agaggcccac
cttcaagcag ctggtggagg acctggaccg tgtccttacc 60gtgacgtcca ccgactttaa
ggagtcggcc 906590DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 65cgcgccctcc cagaggccca
ccttcaagca gctggtggag gacctggacc gtgtccttac 60cgtgacgtcc accgacttta
aggagtcggc 906690DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 66ccgcgccctc ccagaggccc
accttcaagc agctggtgga ggacctggac cgtgtcctta 60ccgtgacgtc caccgacttt
aaggagtcgg 906790DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 67gccgcgccct cccagaggcc
caccttcaag cagctggtgg aggacctgga ccgtgtcctt 60accgtgacgt ccaccgactt
taaggagtcg 906890DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 68tgccgcgccc tcccagaggc
ccaccttcaa gcagctggtg gaggacctgg accgtgtcct 60taccgtgacg tccaccgact
ttaaggagtc 906990DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 69atgccgcgcc ctcccagagg
cccaccttca agcagctggt ggaggacctg gaccgtgtcc 60ttaccgtgac gtccaccgac
tttaaggagt 907090DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 70catgccgcgc
cctcccagag gcccaccttc aagcagctgg tggaggacct ggaccgtgtc 60cttaccgtga
cgtccaccga ctttaaggag 907190DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 71gcatgccgcg
ccctcccaga ggcccacctt caagcagctg gtggaggacc tggaccgtgt 60ccttaccgtg
acgtccaccg actttaagga 907290DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 72ggcatgccgc
gccctcccag aggcccacct tcaagcagct ggtggaggac ctggaccgtg 60tccttaccgt
gacgtccacc gactttaagg 907390DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 73tggcatgccg
cgccctccca gaggcccacc ttcaagcagc tggtggagga cctggaccgt 60gtccttaccg
tgacgtccac cgactttaag 907490DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 74ctggcatgcc
gcgccctccc agaggcccac cttcaagcag ctggtggagg acctggaccg 60tgtccttacc
gtgacgtcca ccgactttaa 907590DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 75gctggcatgc
cgcgccctcc cagaggccca ccttcaagca gctggtggag gacctggacc 60gtgtccttac
cgtgacgtcc accgacttta 907690DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 76tgctggcatg
ccgcgccctc ccagaggccc accttcaagc agctggtgga ggacctggac 60cgtgtcctta
ccgtgacgtc caccgacttt 907790DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 77gtgctggcat
gccgcgccct cccagaggcc caccttcaag cagctggtgg aggacctgga 60ccgtgtcctt
accgtgacgt ccaccgactt 907859PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 78Cys Trp His Ala Ala Pro
Ser Gln Arg Pro Thr Phe Lys Gln Leu Val 1 5 10 15 Glu Asp Leu Asp
Arg Val Leu Thr Val Thr Ser Thr Asp Phe Lys Glu 20 25 30 Ser Ala
Leu Arg Lys Gln Ser Leu Tyr Leu Lys Phe Asp Pro Leu Leu 35 40 45
Arg Asp Ser Pro Gly Arg Pro Val Pro Val Ala 50 55
791048PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys
Val Ala Val Ala Ile 1 5 10 15 Val Ala Gly Ala Ser Ser Glu Ser Leu
Gly Thr Glu Gln Arg Val Val 20 25 30 Gly Arg Ala Ala Glu Val Pro
Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40 45 Leu Val Phe Gly Ser
Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro 50 55 60 Gly Gly Gly
Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly 65 70 75 80 Leu
Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90
95 Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg
100 105 110 Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr
Asp Ala 115 120 125 Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu
Ala Glu Asp Thr 130 135 140 Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr
Arg Pro Glu Arg Met Asp 145 150 155 160 Lys Lys Leu Leu Ala Val Pro
Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170 175 Pro Ala Ala Gly Asn
Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly 180 185 190 Arg Glu Phe
Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205 Gln
Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215
220 Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr
225 230 235 240 Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro
Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu
Gly Ser Asp Val Glu 260 265 270 Phe His Cys Lys Val Tyr Ser Asp Ala
Gln Pro His Ile Gln Trp Leu 275 280 285 Lys His Val Glu Val Asn Gly
Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295 300 Tyr Val Thr Val Leu
Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu 305 310 315 320 Leu Glu
Val Leu Ser Leu His Asn Val Thr Phe Glu Asp Ala Gly Glu 325 330 335
Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Phe Ser His His Ser Ala 340
345 350 Trp Leu Val Val Leu Pro Ala Glu Glu Glu Leu Val Glu Ala Asp
Glu 355 360 365 Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val
Gly Phe Phe 370 375 380 Leu Phe Ile Leu Val Val Ala Ala Val Thr Leu
Cys Arg Leu Arg Ser 385 390 395 400 Pro Pro Lys Lys Gly Leu Gly Ser
Pro Thr Val His Lys Ile Ser Arg 405 410 415 Phe Pro Leu Lys Arg Gln
Val Ser Leu Glu Ser Asn Ala Ser Met Ser 420 425 430 Ser Asn Thr Pro
Leu Val Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly 435 440 445 Pro Thr
Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys 450 455 460
Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu 465
470 475 480 Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile
Asp Lys 485 490 495 Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys
Met Leu Lys Asp 500 505 510 Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu
Val Ser Glu Met Glu Met 515 520 525 Met Lys Met Ile Gly Lys His Lys
Asn Ile Ile Asn Leu Leu Gly Ala 530 535 540 Cys Thr Gln Gly Gly Pro
Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys 545 550 555 560 Gly Asn Leu
Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp 565 570 575 Tyr
Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys 580 585
590 Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu
595 600 605 Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn
Val Leu 610 615 620 Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe
Gly Leu Ala Arg 625 630 635 640 Asp Val His Asn Leu Asp Tyr Tyr Lys
Lys Thr Thr Asn Gly Arg Leu 645 650 655 Pro Val Lys Trp Met Ala Pro
Glu Ala Leu Phe Asp Arg Val Tyr Thr 660 665 670 His Gln Ser Asp Val
Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe 675 680 685 Thr Leu Gly
Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 690 695 700 Lys
Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr 705 710
715 720 His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro
Ser 725 730 735 Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp
Arg Val Leu 740 745 750 Thr Val Thr Ser Thr Asp Phe Lys Glu Ser Ala
Leu Arg Lys Gln Ser 755 760 765 Leu Tyr Leu Lys Phe Asp Pro Leu Leu
Arg Asp Ser Pro Gly Arg Pro 770 775 780 Val Pro Val Ala Thr Glu Thr
Ser Ser Met His Gly Ala Asn Glu Thr 785 790 795 800 Pro Ser Gly Arg
Pro Arg Glu Ala Lys Leu Val Glu Phe Asp Phe Leu 805 810 815 Gly Ala
Leu Asp Ile Pro Val Pro Gly Pro Pro Pro Gly Val Pro Ala 820 825 830
Pro Gly Gly Pro Pro Leu Ser Thr Gly Pro Ile Val Asp Leu Leu Gln 835
840 845 Tyr Ser Gln Lys Asp Leu Asp Ala Val Val Lys Ala Thr Gln Glu
Glu 850 855 860 Asn Arg Glu Leu Arg Ser Arg Cys Glu Glu Leu His Gly
Lys Asn Leu 865 870 875 880 Glu Leu Gly Lys Ile Met Asp Arg Phe Glu
Glu Val Val Tyr Gln Ala 885 890 895 Met Glu Glu Val Gln Lys Gln Lys
Glu Leu Ser Lys Ala Glu Ile Gln 900 905 910 Lys Val Leu Lys Glu Lys
Asp Gln Leu Thr Thr Asp Leu Asn Ser Met 915 920 925 Glu Lys Ser Phe
Ser Asp Leu Phe Lys Arg Phe Glu Lys Gln Lys Glu 930 935 940 Val Ile
Glu Gly Tyr Arg Lys Asn Glu Glu Ser Leu Lys Lys Cys Val 945 950 955
960 Glu Asp Tyr Leu Ala Arg Ile Thr Gln Glu Gly Gln Arg Tyr Gln Ala
965 970 975 Leu Lys Ala His Ala Glu Glu Lys Leu Gln Leu Ala Asn Glu
Glu Ile 980 985 990 Ala Gln Val Arg Ser Lys Ala Gln Ala Glu Ala Leu
Ala Leu Gln Ala 995 1000 1005 Ser Leu Arg Lys Glu Gln Met Arg Ile
Gln Ser Leu Glu Lys Thr 1010 1015 1020 Val Glu Gln Lys Thr Lys Glu
Asn Glu Glu Leu Thr Arg Ile Cys 1025 1030 1035 Asp Asp Leu Ile Ser
Lys Met Glu Lys Ile 1040 1045 8026DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 80g acg tcc acc
gac ttt aag gag tcg g 26 Thr Ser Thr Asp Phe Lys Glu Ser 1 5
8127DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 81g acg tcc acc gac gta aag gcg aca ca 27
Thr Ser Thr Asp Val Lys Ala Thr 1 5 8227DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 82gcc gtc ccc ggc cat ccc tca gga cgt 27Ala Val Pro
Gly His Pro Ser Gly Arg 1 5 8326DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 83g acc tcc aac
cag ggg ctg ctg gag t 26 Thr Ser Asn Gln Gly Leu Leu Glu 1 5
8427DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 84g acg tcc acc gac gtg cca ggc cca cc 27
Thr Ser Thr Asp Val Pro Gly Pro 1 5 858PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Thr
Ser Thr Asp Phe Lys Glu Ser 1 5 868PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Thr
Ser Thr Asp Val Lys Ala Thr 1 5 879PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 87Ala
Val Pro Gly His Pro Ser Gly Arg 1 5 888PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Thr
Ser Asn Gln Gly Leu Leu Glu 1 5 898PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 89Thr
Ser Thr Asp Val Pro Gly Pro 1 5 90806PRTHomo sapiens 90Met Gly Ala
Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile 1 5 10 15 Val
Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25
30 Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln
35 40 45 Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro
Pro Pro 50 55 60 Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys
Asp Gly Thr Gly 65 70 75 80 Leu Val Pro Ser Glu Arg Val Leu Val Gly
Pro Gln Arg Leu Gln Val 85 90 95 Leu Asn Ala Ser His Glu Asp Ser
Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110 Leu Thr Gln Arg Val Leu
Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125 Pro Ser Ser Gly
Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140 Gly Val
Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp 145 150 155
160 Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys
165 170 175 Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys
Asn Gly 180 185 190 Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile
Lys Leu Arg His 195 200 205 Gln Gln Trp Ser Leu Val Met Glu Ser Val
Val Pro Ser Asp Arg Gly 210 215 220 Asn Tyr Thr Cys Val Val Glu Asn
Lys Phe Gly Ser Ile Arg Gln Thr 225 230 235 240 Tyr Thr Leu Asp Val
Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu
Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270 Phe
His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280
285 Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro
290 295 300 Tyr Val Thr Val Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp
Lys Glu 305 310 315 320 Leu Glu Val Leu Ser Leu His Asn Val Thr Phe
Glu Asp Ala Gly Glu 325 330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile
Gly Phe Ser His His Ser Ala 340 345 350 Trp Leu Val Val Leu Pro Ala
Glu Glu Glu Leu Val Glu Ala Asp Glu 355 360 365 Ala Gly Ser Val Tyr
Ala Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe 370 375 380 Leu Phe Ile
Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser 385 390 395 400
Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile Ser Arg 405
410 415 Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser Met
Ser 420 425 430 Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser
Gly Glu Gly 435 440 445 Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu
Pro Ala Asp Pro Lys 450 455 460 Trp Glu Leu Ser Arg Ala Arg Leu Thr
Leu Gly Lys Pro Leu Gly Glu 465 470 475 480 Gly Cys Phe Gly Gln Val
Val Met Ala Glu Ala Ile Gly Ile Asp Lys 485 490 495 Asp Arg Ala Ala
Lys Pro Val Thr Val Ala Val Lys Met Leu Lys Asp 500 505 510 Asp Ala
Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 515 520 525
Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 530
535 540 Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala
Lys 545 550 555 560 Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro
Pro Gly Leu Asp 565 570 575 Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu
Glu Gln Leu Thr Phe Lys 580 585 590 Asp Leu Val Ser Cys Ala Tyr Gln
Val Ala Arg Gly Met Glu Tyr Leu 595 600 605 Ala Ser Gln Lys Cys Ile
His Arg Asp Leu Ala Ala Arg Asn Val Leu 610 615 620 Val Thr Glu Asp
Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg 625
630 635 640 Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly
Arg Leu 645 650 655 Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp
Arg Val Tyr Thr 660 665 670 His Gln Ser Asp Val Trp Ser Phe Gly Val
Leu Leu Trp Glu Ile Phe 675 680 685 Thr Leu Gly Gly Ser Pro Tyr Pro
Gly Ile Pro Val Glu Glu Leu Phe 690 695 700 Lys Leu Leu Lys Glu Gly
His Arg Met Asp Lys Pro Ala Asn Cys Thr 705 710 715 720 His Asp Leu
Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser 725 730 735 Gln
Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu 740 745
750 Thr Val Thr Ser Thr Asp Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu
755 760 765 Gln Tyr Ser Pro Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser
Ser Gly 770 775 780 Asp Asp Ser Val Phe Ala His Asp Leu Leu Pro Pro
Ala Pro Pro Ser 785 790 795 800 Ser Gly Gly Ser Arg Thr 805
914304DNAHomo sapiens 91gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt
cgcacggacg caccggcggg 60ccgccggccg gagggacggg gcgggagctg ggcccgcgga
cagcgagccg gagcgggagc 120cgcgcgtagc gagccgggct ccggcgctcg
ccagtctccc gagcggcgcc cgcctcccgc 180cggtgcccgc gccgggccgt
ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 240ggcccccgcc
cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc
300catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg
tggggcgagc 360ggcagaagtc ccgggcccag agcccggcca gcaggagcag
ttggtcttcg gcagcgggga 420tgctgtggag ctgagctgtc ccccgcccgg
gggtggtccc atggggccca ctgtctgggt 480caaggatggc acagggctgg
tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 540ggtgctgaat
gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca
600gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg
gagatgacga 660agacggggag gacgaggctg aggacacagg tgtggacaca
ggggcccctt actggacacg 720gcccgagcgg atggacaaga agctgctggc
cgtgccggcc gccaacaccg tccgcttccg 780ctgcccagcc gctggcaacc
ccactccctc catctcctgg ctgaagaacg gcagggagtt 840ccgcggcgag
caccgcattg gaggcatcaa gctgcggcat cagcagtgga gcctggtcat
900ggaaagcgtg gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga
acaagtttgg 960cagcatccgg cagacgtaca cgctggacgt gctggagcgc
tccccgcacc ggcccatcct 1020gcaggcgggg ctgccggcca accagacggc
ggtgctgggc agcgacgtgg agttccactg 1080caaggtgtac agtgacgcac
agccccacat ccagtggctc aagcacgtgg aggtgaatgg 1140cagcaaggtg
ggcccggacg gcacacccta cgttaccgtg ctcaagacgg cgggcgctaa
1200caccaccgac aaggagctag aggttctctc cttgcacaac gtcacctttg
aggacgccgg 1260ggagtacacc tgcctggcgg gcaattctat tgggttttct
catcactctg cgtggctggt 1320ggtgctgcca gccgaggagg agctggtgga
ggctgacgag gcgggcagtg tgtatgcagg 1380catcctcagc tacggggtgg
gcttcttcct gttcatcctg gtggtggcgg ctgtgacgct 1440ctgccgcctg
cgcagccccc ccaagaaagg cctgggctcc cccaccgtgc acaagatctc
1500ccgcttcccg ctcaagcgac aggtgtccct ggagtccaac gcgtccatga
gctccaacac 1560accactggtg cgcatcgcaa ggctgtcctc aggggagggc
cccacgctgg ccaatgtctc 1620cgagctcgag ctgcctgccg accccaaatg
ggagctgtct cgggcccggc tgaccctggg 1680caagcccctt ggggagggct
gcttcggcca ggtggtcatg gcggaggcca tcggcattga 1740caaggaccgg
gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag acgatgccac
1800tgacaaggac ctgtcggacc tggtgtctga gatggagatg atgaagatga
tcgggaaaca 1860caaaaacatc atcaacctgc tgggcgcctg cacgcagggc
gggcccctgt acgtgctggt 1920ggagtacgcg gccaagggta acctgcggga
gtttctgcgg gcgcggcggc ccccgggcct 1980ggactactcc ttcgacacct
gcaagccgcc cgaggagcag ctcaccttca aggacctggt 2040gtcctgtgcc
taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca
2100cagggacctg gctgcccgca atgtgctggt gaccgaggac aacgtgatga
agatcgcaga 2160cttcgggctg gcccgggacg tgcacaacct cgactactac
aagaagacaa ccaacggccg 2220gctgcccgtg aagtggatgg cgcctgaggc
cttgtttgac cgagtctaca ctcaccagag 2280tgacgtctgg tcctttgggg
tcctgctctg ggagatcttc acgctggggg gctccccgta 2340ccccggcatc
cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa
2400gcccgccaac tgcacacacg acctgtacat gatcatgcgg gagtgctggc
atgccgcgcc 2460ctcccagagg cccaccttca agcagctggt ggaggacctg
gaccgtgtcc ttaccgtgac 2520gtccaccgac gagtacctgg acctgtcggc
gcctttcgag cagtactccc cgggtggcca 2580ggacaccccc agctccagct
cctcagggga cgactccgtg tttgcccacg acctgctgcc 2640cccggcccca
cccagcagtg ggggctcgcg gacgtgaagg gccactggtc cccaacaatg
2700tgaggggtcc ctagcagccc accctgctgc tggtgcacag ccactccccg
gcatgagact 2760cagtgcagat ggagagacag ctacacagag ctttggtctg
tgtgtgtgtg tgtgcgtgtg 2820tgtgtgtgtg tgtgcacatc cgcgtgtgcc
tgtgtgcgtg cgcatcttgc ctccaggtgc 2880agaggtaccc tgggtgtccc
cgctgctgtg caacggtctc ctgactggtg ctgcagcacc 2940gaggggcctt
tgttctgggg ggacccagtg cagaatgtaa gtgggcccac ccggtgggac
3000ccccgtgggg cagggagctg ggcccgacat ggctccggcc tctgcctttg
caccacggga 3060catcacaggg tgggcctcgg cccctcccac acccaaagct
gagcctgcag ggaagcccca 3120catgtccagc accttgtgcc tggggtgtta
gtggcaccgc ctccccacct ccaggctttc 3180ccacttccca ccctgcccct
cagagactga aattacgggt acctgaagat gggagccttt 3240accttttatg
caaaaggttt attccggaaa ctagtgtaca tttctataaa tagatgctgt
3300gtatatggta tatatacata tatatatata acatatatgg aagaggaaaa
ggctggtaca 3360acggaggcct gcgaccctgg gggcacagga ggcaggcatg
gccctgggcg gggcgtgggg 3420gggcgtggag ggaggcccca gggggtctca
cccatgcaag cagaggacca gggccttttc 3480tggcaccgca gttttgtttt
aaaactggac ctgtatattt gtaaagctat ttatgggccc 3540ctggcactct
tgttcccaca ccccaacact tccagcattt agctggccac atggcggaga
3600gttttaattt ttaacttatt gacaaccgag aaggtttatc ccgccgatag
agggacggcc 3660aagaatgtac gtccagcctg ccccggagct ggaggatccc
ctccaagcct aaaaggttgt 3720taatagttgg aggtgattcc agtgaagata
ttttatttcc tttgtccttt ttcaggagaa 3780ttagatttct ataggatttt
tctttaggag atttattttt tggacttcaa agcaagctgg 3840tattttcata
caaattcttc taattgctgt gtgtcccagg cagggagacg gtttccaggg
3900aggggccggc cctgtgtgca ggttccgatg ttattagatg ttacaagttt
atatatatct 3960atatatataa tttattgagt ttttacaaga tgtatttgtt
gtagacttaa cacttcttac 4020gcaatgcttc tagagtttta tagcctggac
tgctaccttt caaagcttgg agggaagccg 4080tgaattcagt tggttcgttc
tgtactgtta ctgggccctg agtctgggca gctgtccctt 4140gcttgcctgc
agggccatgg ctcagggtgg tctcttcttg gggcccagtg catggtggcc
4200agaggtgtca cccaaaccgg caggtgcgat tttgttaacc cagcgacgaa
ctttccgaaa 4260aataaagaca cctggttgct aacctggaaa aaaaaaaaaa aaaa
430492838PRTHomo sapiens 92Met Ser Leu Gln Val Leu Asn Asp Lys Asn
Val Ser Asn Glu Lys Asn 1 5 10 15 Thr Glu Asn Cys Asp Phe Leu Phe
Ser Pro Pro Glu Val Thr Gly Arg 20 25 30 Ser Ser Val Leu Arg Val
Ser Gln Lys Glu Asn Val Pro Pro Lys Asn 35 40 45 Leu Ala Lys Ala
Met Lys Val Thr Phe Gln Thr Pro Leu Arg Asp Pro 50 55 60 Gln Thr
His Arg Ile Leu Ser Pro Ser Met Ala Ser Lys Leu Glu Ala 65 70 75 80
Pro Phe Thr Gln Asp Asp Thr Leu Gly Leu Glu Asn Ser His Pro Val 85
90 95 Trp Thr Gln Lys Glu Asn Gln Gln Leu Ile Lys Glu Val Asp Ala
Lys 100 105 110 Thr Thr His Gly Ile Leu Gln Lys Pro Val Glu Ala Asp
Thr Asp Leu 115 120 125 Leu Gly Asp Ala Ser Pro Ala Phe Gly Ser Gly
Ser Ser Ser Glu Ser 130 135 140 Gly Pro Gly Ala Leu Ala Asp Leu Asp
Cys Ser Ser Ser Ser Gln Ser 145 150 155 160 Pro Gly Ser Ser Glu Asn
Gln Met Val Ser Pro Gly Lys Val Ser Gly 165 170 175 Ser Pro Glu Gln
Ala Val Glu Glu Asn Leu Ser Ser Tyr Ser Leu Asp 180 185 190 Arg Arg
Val Thr Pro Ala Ser Glu Thr Leu Glu Asp Pro Cys Arg Thr 195 200 205
Glu Ser Gln His Lys Ala Glu Thr Pro His Gly Ala Glu Glu Glu Cys 210
215 220 Lys Ala Glu Thr Pro His Gly Ala Glu Glu Glu Cys Arg His Gly
Gly 225 230 235 240 Val Cys Ala Pro Ala Ala Val Ala Thr Ser Pro Pro
Gly Ala Ile Pro 245 250 255 Lys Glu Ala Cys Gly Gly Ala Pro Leu Gln
Gly Leu Pro Gly Glu Ala 260 265 270 Leu Gly Cys Pro Ala Gly Val Gly
Thr Pro Val Pro Ala Asp Gly Thr 275 280 285 Gln Thr Leu Thr Cys Ala
His Thr Ser Ala Pro Glu Ser Thr Ala Pro 290 295 300 Thr Asn His Leu
Val Ala Gly Arg Ala Met Thr Leu Ser Pro Gln Glu 305 310 315 320 Glu
Val Ala Ala Gly Gln Met Ala Ser Ser Ser Arg Ser Gly Pro Val 325 330
335 Lys Leu Glu Phe Asp Val Ser Asp Gly Ala Thr Ser Lys Arg Ala Pro
340 345 350 Pro Pro Arg Arg Leu Gly Glu Arg Ser Gly Leu Lys Pro Pro
Leu Arg 355 360 365 Lys Ala Ala Val Arg Gln Gln Lys Ala Pro Gln Glu
Val Glu Glu Asp 370 375 380 Asp Gly Arg Ser Gly Ala Gly Glu Asp Pro
Pro Met Pro Ala Ser Arg 385 390 395 400 Gly Ser Tyr His Leu Asp Trp
Asp Lys Met Asp Asp Pro Asn Phe Ile 405 410 415 Pro Phe Gly Gly Asp
Thr Lys Ser Gly Cys Ser Glu Ala Gln Pro Pro 420 425 430 Glu Ser Pro
Glu Thr Arg Leu Gly Gln Pro Ala Ala Glu Gln Leu His 435 440 445 Ala
Gly Pro Ala Thr Glu Glu Pro Gly Pro Cys Leu Ser Gln Gln Leu 450 455
460 His Ser Ala Ser Ala Glu Asp Thr Pro Val Val Gln Leu Ala Ala Glu
465 470 475 480 Thr Pro Thr Ala Glu Ser Lys Glu Arg Ala Leu Asn Ser
Ala Ser Thr 485 490 495 Ser Leu Pro Thr Ser Cys Pro Gly Ser Glu Pro
Val Pro Thr His Gln 500 505 510 Gln Gly Gln Pro Ala Leu Glu Leu Lys
Glu Glu Ser Phe Arg Asp Pro 515 520 525 Ala Glu Val Leu Gly Thr Gly
Ala Glu Val Asp Tyr Leu Glu Gln Phe 530 535 540 Gly Thr Ser Ser Phe
Lys Glu Ser Ala Leu Arg Lys Gln Ser Leu Tyr 545 550 555 560 Leu Lys
Phe Asp Pro Leu Leu Arg Asp Ser Pro Gly Arg Pro Val Pro 565 570 575
Val Ala Thr Glu Thr Ser Ser Met His Gly Ala Asn Glu Thr Pro Ser 580
585 590 Gly Arg Pro Arg Glu Ala Lys Leu Val Glu Phe Asp Phe Leu Gly
Ala 595 600 605 Leu Asp Ile Pro Val Pro Gly Pro Pro Pro Gly Val Pro
Ala Pro Gly 610 615 620 Gly Pro Pro Leu Ser Thr Gly Pro Ile Val Asp
Leu Leu Gln Tyr Ser 625 630 635 640 Gln Lys Asp Leu Asp Ala Val Val
Lys Ala Thr Gln Glu Glu Asn Arg 645 650 655 Glu Leu Arg Ser Arg Cys
Glu Glu Leu His Gly Lys Asn Leu Glu Leu 660 665 670 Gly Lys Ile Met
Asp Arg Phe Glu Glu Val Val Tyr Gln Ala Met Glu 675 680 685 Glu Val
Gln Lys Gln Lys Glu Leu Ser Lys Ala Glu Ile Gln Lys Val 690 695 700
Leu Lys Glu Lys Asp Gln Leu Thr Thr Asp Leu Asn Ser Met Glu Lys 705
710 715 720 Ser Phe Ser Asp Leu Phe Lys Arg Phe Glu Lys Gln Lys Glu
Val Ile 725 730 735 Glu Gly Tyr Arg Lys Asn Glu Glu Ser Leu Lys Lys
Cys Val Glu Asp 740 745 750 Tyr Leu Ala Arg Ile Thr Gln Glu Gly Gln
Arg Tyr Gln Ala Leu Lys 755 760 765 Ala His Ala Glu Glu Lys Leu Gln
Leu Ala Asn Glu Glu Ile Ala Gln 770 775 780 Val Arg Ser Lys Ala Gln
Ala Glu Ala Leu Ala Leu Gln Ala Ser Leu 785 790 795 800 Arg Lys Glu
Gln Met Arg Ile Gln Ser Leu Glu Lys Thr Val Glu Gln 805 810 815 Lys
Thr Lys Glu Asn Glu Glu Leu Thr Arg Ile Cys Asp Asp Leu Ile 820 825
830 Ser Lys Met Glu Lys Ile 835 932847DNAHomo sapiens 93gcgtttgaaa
ctccggcgcg ccggcggcca tcaagggcta gaagcgcgac ggcggtagca 60gctaggcttg
gcccccggcg tggagcagac gcggacccct ccttcctggc ggcggcggcg
120cgggctcaga gcccggcaac gggcgggcgg gcagaatgag tctgcaggtc
ttaaacgaca 180aaaatgtcag caatgaaaaa aatacagaaa attgcgactt
cctgttttcg ccaccagaag 240ttaccggaag atcgtctgtt cttcgtgtgt
cacagaaaga aaatgtgcca cccaagaacc 300tggccaaagc tatgaaggtg
acttttcaga cacctctgcg ggatccacag acgcacagga 360ttctaagtcc
tagcatggcc agcaaacttg aggctccttt cactcaggat gacacccttg
420gactggaaaa ctcacacccg gtctggacac agaaagagaa ccaacagctc
atcaaggaag 480tggatgccaa aactactcat ggaattctac agaaaccagt
ggaggctgac accgacctcc 540tgggggatgc aagcccagcc tttgggagtg
gcagctccag cgagtctggc ccaggtgccc 600tggctgacct ggactgctca
agctcttccc agagcccagg aagttctgag aaccaaatgg 660tgtctccagg
aaaagtgtct ggcagccctg agcaagccgt ggaggaaaac cttagttcct
720attccttaga cagaagagtg acacccgcct ctgagaccct agaagaccct
tgcaggacag 780agtcccagca caaagcggag actccgcacg gagccgagga
agaatgcaaa gcggagactc 840cgcacggagc cgaggaggaa tgccggcacg
gtggggtctg tgctcccgca gcagtggcca 900cttcgcctcc tggtgcaatc
cctaaggaag cctgcggagg agcacccctg cagggtctgc 960ctggcgaagc
cctgggctgc cctgcgggtg tgggcacccc cgtgccagca gatggcactc
1020agacccttac ctgtgcacac acctctgctc ctgagagcac agccccaacc
aaccacctgg 1080tggctggcag ggccatgacc ctgagtcctc aggaagaagt
ggctgcaggc caaatggcca 1140gctcctcgag gagcggacct gtaaaactag
aatttgatgt atctgatggc gccaccagca 1200aaagggcacc cccaccaagg
agactgggag agaggtccgg cctcaagcct cccttgagga 1260aagcagcagt
gaggcagcaa aaggccccgc aggaggtgga ggaggacgac ggtaggagcg
1320gagcaggaga ggaccccccc atgccagctt ctcggggctc ttaccacctc
gactgggaca 1380aaatggatga cccaaacttc atcccgttcg gaggtgacac
caagtctggt tgcagtgagg 1440cccagccccc agaaagccct gagaccaggc
tgggccagcc agcggctgaa cagttgcatg 1500ctgggcctgc cacggaggag
ccaggtccct gtctgagcca gcagctgcat tcagcctcag 1560cggaggacac
gcctgtggtg cagttggcag ccgagacccc aacagcagag agcaaggaga
1620gagccttgaa ctctgccagc acctcgcttc ccacaagctg tccaggcagt
gagccagtgc 1680ccacccatca gcaggggcag cctgccttgg agctgaaaga
ggagagcttc agagaccccg 1740ctgaggttct aggcacgggc gcggaggtgg
attacctgga gcagtttgga acttcctcgt 1800ttaaggagtc ggccttgagg
aagcagtcct tatacctcaa gttcgacccc ctcctgaggg 1860acagtcctgg
tagaccagtg cccgtggcca ccgagaccag cagcatgcac ggtgcaaatg
1920agactccctc aggacgtccg cgggaagcca agcttgtgga gttcgatttc
ttgggagcac 1980tggacattcc tgtgccaggc ccacccccag gtgttcccgc
gcctgggggc ccacccctgt 2040ccaccggacc tatagtggac ctgctccagt
acagccagaa ggacctggat gcagtggtaa 2100aggcgacaca ggaggagaac
cgggagctga ggagcaggtg tgaggagctc cacgggaaga 2160acctggaact
ggggaagatc atggacaggt tcgaagaggt tgtgtaccag gccatggagg
2220aagttcagaa gcagaaggaa ctttccaaag ctgaaatcca gaaagttcta
aaagaaaaag 2280accaacttac cacagatctg aactccatgg agaagtcctt
ctccgacctc ttcaagcgtt 2340ttgagaaaca gaaagaggtg atcgagggct
accgcaagaa cgaagagtca ctgaagaagt 2400gcgtggagga ttacctggca
aggatcaccc aggagggcca gaggtaccaa gccctgaagg 2460cccacgcgga
ggagaagctg cagctggcaa acgaggagat cgcccaggtc cggagcaagg
2520cccaggcgga agcgttggcc ctccaggcca gcctgaggaa ggagcagatg
cgcatccagt 2580cgctggagaa gacagtggag cagaagacta aagagaacga
ggagctgacc aggatctgcg 2640acgacctcat ctccaagatg gagaagatct
gacctccacg gagccgctgt ccccgccccc 2700ctgctcccgt ctgtctgtcc
tgtctgattc tcttaggtgt catgttcttt tttctgtctt 2760gtcttcaact
tttttaaaaa ctagattgct ttgaaaacat gactcaataa aagtttcctt
2820tcaatttaaa cactgaaaaa aaaaaaa 2847943578DNAHomo sapiens
94gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg caccggcggg
60ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg gagcgggagc
120cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc gagcggcgcc
cgcctcccgc 180cggtgcccgc gccgggccgt ggggggcagc atgcccgcgc
gcgctgcctg aggacgccgc 240ggcccccgcc cccgccatgg gcgcccctgc
ctgcgccctc gcgctctgcg tggccgtggc 300catcgtggcc ggcgcctcct
cggagtcctt ggggacggag cagcgcgtcg tggggcgagc 360ggcagaagtc
ccgggcccag agcccggcca gcaggagcag ttggtcttcg gcagcgggga
420tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc atggggccca
ctgtctgggt 480caaggatggc acagggctgg tgccctcgga gcgtgtcctg
gtggggcccc agcggctgca 540ggtgctgaat gcctcccacg aggactccgg
ggcctacagc tgccggcagc ggctcacgca 600gcgcgtactg tgccacttca
gtgtgcgggt gacagacgct ccatcctcgg gagatgacga 660agacggggag
gacgaggctg aggacacagg tgtggacaca ggggcccctt actggacacg
720gcccgagcgg atggacaaga agctgctggc cgtgccggcc gccaacaccg
tccgcttccg 780ctgcccagcc gctggcaacc ccactccctc catctcctgg
ctgaagaacg gcagggagtt 840ccgcggcgag caccgcattg gaggcatcaa
gctgcggcat cagcagtgga gcctggtcat 900ggaaagcgtg gtgccctcgg
accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 960cagcatccgg
cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct
1020gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg
agttccactg 1080caaggtgtac agtgacgcac agccccacat ccagtggctc
aagcacgtgg aggtgaatgg 1140cagcaaggtg ggcccggacg
gcacacccta cgttaccgtg ctcaagacgg cgggcgctaa 1200caccaccgac
aaggagctag aggttctctc cttgcacaac gtcacctttg aggacgccgg
1260ggagtacacc tgcctggcgg gcaattctat tgggttttct catcactctg
cgtggctggt 1320ggtgctgcca gccgaggagg agctggtgga ggctgacgag
gcgggcagtg tgtatgcagg 1380catcctcagc tacggggtgg gcttcttcct
gttcatcctg gtggtggcgg ctgtgacgct 1440ctgccgcctg cgcagccccc
ccaagaaagg cctgggctcc cccaccgtgc acaagatctc 1500ccgcttcccg
ctcaagcgac aggtgtccct ggagtccaac gcgtccatga gctccaacac
1560accactggtg cgcatcgcaa ggctgtcctc aggggagggc cccacgctgg
ccaatgtctc 1620cgagctcgag ctgcctgccg accccaaatg ggagctgtct
cgggcccggc tgaccctggg 1680caagcccctt ggggagggct gcttcggcca
ggtggtcatg gcggaggcca tcggcattga 1740caaggaccgg gccgccaagc
ctgtcaccgt agccgtgaag atgctgaaag acgatgccac 1800tgacaaggac
ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca
1860caaaaacatc atcaacctgc tgggcgcctg cacgcagggc gggcccctgt
acgtgctggt 1920ggagtacgcg gccaagggta acctgcggga gtttctgcgg
gcgcggcggc ccccgggcct 1980ggactactcc ttcgacacct gcaagccgcc
cgaggagcag ctcaccttca aggacctggt 2040gtcctgtgcc taccaggtgg
cccggggcat ggagtacttg gcctcccaga agtgcatcca 2100cagggacctg
gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga
2160cttcgggctg gcccgggacg tgcacaacct cgactactac aagaagacaa
ccaacggccg 2220gctgcccgtg aagtggatgg cgcctgaggc cttgtttgac
cgagtctaca ctcaccagag 2280tgacgtctgg tcctttgggg tcctgctctg
ggagatcttc acgctggggg gctccccgta 2340ccccggcatc cctgtggagg
agctcttcaa gctgctgaag gagggccacc gcatggacaa 2400gcccgccaac
tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc
2460ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc
ttaccgtgac 2520gtccaccgac tttaaggagt cggccttgag gaagcagtcc
ttatacctca agttcgaccc 2580cctcctgagg gacagtcctg gtagaccagt
gcccgtggcc accgagacca gcagcatgca 2640cggtgcaaat gagactccct
caggacgtcc gcgggaagcc aagcttgtgg agttcgattt 2700cttgggagca
ctggacattc ctgtgccagg cccaccccca ggtgttcccg cgcctggggg
2760cccacccctg tccaccggac ctatagtgga cctgctccag tacagccaga
aggacctgga 2820tgcagtggta aaggcgacac aggaggagaa ccgggagctg
aggagcaggt gtgaggagct 2880ccacgggaag aacctggaac tggggaagat
catggacagg ttcgaagagg ttgtgtacca 2940ggccatggag gaagttcaga
agcagaagga actttccaaa gctgaaatcc agaaagttct 3000aaaagaaaaa
gaccaactta ccacagatct gaactccatg gagaagtcct tctccgacct
3060cttcaagcgt tttgagaaac agaaagaggt gatcgagggc taccgcaaga
acgaagagtc 3120actgaagaag tgcgtggagg attacctggc aaggatcacc
caggagggcc agaggtacca 3180agccctgaag gcccacgcgg aggagaagct
gcagctggca aacgaggaga tcgcccaggt 3240ccggagcaag gcccaggcgg
aagcgttggc cctccaggcc agcctgagga aggagcagat 3300gcgcatccag
tcgctggaga agacagtgga gcagaagact aaagagaacg aggagctgac
3360caggatctgc gacgacctca tctccaagat ggagaagatc tgacctccac
ggagccgctg 3420tccccgcccc cctgctcccg tctgtctgtc ctgtctgatt
ctcttaggtg tcatgttctt 3480ttttctgtct tgtcttcaac ttttttaaaa
actagattgc tttgaaaaca tgactcaata 3540aaagtttcct ttcaatttaa
acactgaaaa aaaaaaaa 357895152DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 95catcctgccc
cccagagtgc tgaggtgtgg ggcgggcctt ctggggcaca gcctgggcac 60agaggtggct
gtgcgaaggt cgctgagggt ccaggcttcc acccagtgtc cccgcagtca
120gctgcccacc agcagcctcc ccgggactct cc 1529677DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 96ggctgggcga agttcgctga gggtccaggc ttcctcccag
tgtccccgca gtcagctgcc 60caccagcagc ctccccc 779777DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 97acagcctggg cacagaggtg gctgtgcgaa ggtcgctgag
ggtccaggct tccacccagt 60gtccccgcag tcagctt 779877DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 98ggcacagcct gggcacagag gtggctgtgc gaaggtcgct
gagggtccag gcttccaccc 60agtgtccccg cagtcaa 779977DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 99ttctggggca cagcctgggc acagaggtgg ctgtgcgaag
gtcgctgagg gtccaggctt 60ccacccagtg tccccgg 7710077DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 100cttctgtggc acagcctggc cacagaggtg gctgtgcgaa
ggtcgctgag ggtccaggct 60tccacccagt gtccccc 7710177DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 101gccttctggg gcacagcctg ggcacagagg tggctgtgcg
aaggtcgctg agggtccagg 60cttccaccca gtgtccc 7710277DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 102ggtgtggggc gggccggctg gggcacagcc ggggcacaga
ggtggctgtg cgaaggtcgc 60tgagggtcca ggcttcc 7710377DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 103ggtgtggggc gggccttctg gggcacagcc tgggcacaga
ggtggctgtg cgaaggtcgc 60tgagggtcca ggcttcc 7710477DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 104tgctgaggtg tggggcgggc cttctggggc acagcctggg
cacagaggtg gctgtgcgaa 60ggtcgctgag ggtccaa 7710577DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 105agtgctgagg tgtggggcgg gccttctggg gcacagcctg
ggcacagagg tggctgtgcg 60aaggtcgctg agggtcc 7710677DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 106cccagagtgc tgaggtgtgg ggcgggcctt ctggggcaca
gcctgggcac agaggtggct 60gtgcgaaggt cgctgaa 7710776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 107cctgcccccc agagtgctga ggtgtggggc gggccttctg
gggcacagcc tgggcacaga 60ggtggctgtg cgaagg 76108153DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
108gtgctggctc tggcctggtg ccacccgcct atgcccctcc ccctgccgtc
cccggccatc 60ctgcccccca gagtgccggg ggctaagggc cagggaggtc acctgcacac
tcccaccccc 120ggtcacccgc acactcccac ccccggtcac cca
15310976DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 109agagtgcagg gggctaaggg ccagggaggt
cacctgcaca ctcccacccc cggtcacccg 60cacactccca cccccg
7611076DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 110ggccatcctg ccccccagag tgcagggggc
taagggccag ggaggtcccc tgcacactcc 60ctcccccggt cacccg
7611176DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111tccccctgcc gtccccggcc atcctgcccc
ccagagtgca gggggctaag ggccagggag 60gtcacctgca cactcc
76112152DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 112tgacgtccac cgacgtgagt gctggctctg
gcctggtgcc acccgcctat gcccctcccc 60ctgccgtccc cggccatccc tcaggacgtc
cgcgggaagc caagcttgtg gagttcgatt 120tcttgggagc actggacatt
cctgtaagtc ct 15211376DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 113atccctcagg
acgtccgcgg gaagccaagc ttgtggagtt cgatttcttg ggagcactgg 60acattcctgt
aagtcc 7611476DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 114catccctcag gacgtccgcg
ggaagccaag cttgtggagt tcgatttctt gggagcactg 60gacattcctg taagtc
7611576DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 115catccctcag gacgtccgcg ggaagccaag
cttgtggagt tcgatttctt gggagcactg 60gacattcctg taagtc
7611676DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 116gccatccctc aggacgtccg cgggaagcca
agcttgtgga gttcgatttc ttgggagcac 60tggacattcc tgtaag
7611776DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 117ccggccatcc ctcaggacgt ccgcgggaag
ccaagcttgt ggagttcgat ttcttgggag 60cactggacat tcctgt
7611876DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 118cacggccatc ccggaggacg tccgcgggaa
cccaagcttg tggagttcga tttcttggta 60gcactggaca ttcctg
7611976DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 119cccggccatc cctcaggacg tccgcgggaa
gccaagcttg tggagttcga tttcttggga 60gcactggaca ttcctg
7612076DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 120tccccggcca tccctcagga cgtccgcggg
aagccaagct tgtggagttc gatttcttgg 60gagcactgga cattcc
7612176DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 121tccccggcca tccctcagga ngtccgcggg
aagccaagct tgtggagttc gatttcttgg 60gagcactgga cattcc
7612276DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 122gtccccggcc atccctcagg acgtccgcgg
gaagccaagc ttgtggagtt cgatttcttg 60ggagcactgg acattc
7612376DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 123ccgtccccgg ccatccctca ggacgtccgc
gggaagccaa gcttgtggag ttcgatttct 60tgggagcact ggacat
7612476DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 124tccccctgcc gtccccggcc atccctcagg
acgtccgcgg gaagccaagc ttgtggagtt 60cgatttcttg ggagca
7612576DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 125atgcccctcc ccctgccgtc cccggccatc
cctcaggacg tccgcgggaa gccaagcttg 60tggagttcga tttctt
7612676DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 126cctatgcccc tccccctgcc gcccccggcc
atccctcagg acgtccgcgg gaagccaagc 60ttgtggagtt cgattt
7612776DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 127ctggcctggt gccacccgcc tatgcccctc
cccctgccgt ccccggccat ccctcaggac 60gtccgcggga agccaa
7612876DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 128gagctggcct ggtgccacac gcctatgccc
ctccccctgc cgtccccggc gatccatcag 60gaagtccgcg ggacga
7612976DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 129ggctctggcc tggtgccacc cgcctatgcc
cctccccctn ccgtccccgg ccatccctca 60ggacgtccgc gggaag
7613076DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 130gtgctggctc tggcctggtg ccacccgcct
atgcccctcc ccctgccgtc cccggccatc 60cctcaggacg tccgcg
7613176DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 131ccaccgacgt gagtgctggc tctggcctgg
tgccacccgc ctatgcccct ccccctgccg 60tccccggcca tccctc
76132151DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 132tggaccgtgt ccttaccgtg acgtccaccg
acgtgagtgc tggctctggc ctggtgccac 60ccgcctatgc ccctcccctg gcccttagcc
cccgtgtgtg ttaggggatg gcagtcagac 120ctgatcactt gccctcttgt
ccccagttta a 15113380DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 133tgcccctccc
ctgcccttag cccccctgtg tgtgttaggg gatggcagtc agacctgatc 60acttgccctc
ttgtccgtcc 8013480DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 134atgcccctcc cctgccctta
gcccccctgt gtgtgttagg ggatggcagt cagacctgat 60cacttgccct cttgtctgtc
8013580DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 135gcctatgccc ctcccctgcc cttagccccc
ctgtgtgtgt taggggatgg cagtcagacc 60tgatcacttg ccctctctct
8013680DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 136gtgccacccg cctatgcccc tcccctgccc
ttagcccccc tgtgtgtgtt aggggatggc 60agtcagacct gatcactcac
8013780DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 137gctctggcct ggtgcccccc gcctatgccc
ctcccctgcc cttagccccc ctgtgtgtgt 60taggggatgg cagtcagtca
8013880DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 138gctctggcct ggtgcccccc gcctatgccc
ctcccctgcc cttagccccc ctgtgtgtgt 60taggggatgg cagtcagtca
8013981DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 139gctgcctctg tcctggtgcc ccccgcctat
gcccctcccc tgcccttagc ccccctgtgt 60gtgttagggg atggcatggc a
8114081DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 140cacccgccgg gggtgcgggc tctggcctgg
tgccccccgc ctatgcccct cccctgccct 60tagcccccct gtgtgttgtg t
8114180DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 141ccccccgacg tgagtgctgg ctcgggcctg
gtcccccccg cctatgcccc tcccctgccc 60ttagcccccc tgtgtgtgtg
8014282DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 142gacgtccacc gacgtgagtg ctggctctgg
cctggtgcca cccgcctatg cccctcccct 60gcccttagac cccctgcccc tg
8214384DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 143cttaccgtga cgtccaccga cgtgagtgct
ggctctggcc tggtgccacc cgcctatgcc 60cctcccctgc ccttaggccc ttag
8414482DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 144cccttcaggt cccccccccc gacgtgagtg
ctggctctgg cctggtgcca cccgcctatg 60cccctcccct gcccttgccc tt
8214582DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 145tgtccttacc gtgacgtcca ccgacgtgag
tgctggctct ggcctggtgc cacccgccta 60tgcccctccc ctgcccctgc cc
82146820PRTHomo sapiens 146Met Trp Ser Trp Lys Cys Leu Leu Phe Trp
Ala Val Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr Ala Arg Pro Ser
Pro Thr Leu Pro Glu Gln Ala Gln 20 25 30 Pro Trp Gly Ala Pro Val
Glu Val Glu Ser Phe Leu Val His Pro Gly 35 40 45 Asp Leu Leu Gln
Leu Arg Cys Arg Leu Arg Asp Asp Val Gln Ser Ile 50 55 60 Asn Trp
Leu Arg Asp Gly Val Gln Leu Ala Glu Ser Asn Arg Thr Arg 65 70 75 80
Ile Thr Gly Glu Glu Val Glu Val Gln Asp Ser Val Pro Ala Asp Ser 85
90 95 Gly Leu Tyr Ala Cys Val Thr Ser Ser Pro Ser Gly Ser Asp Thr
Thr 100 105 110 Tyr Phe Ser Val Asn Val Ser Asp Ala Leu Pro Ser Ser
Glu Asp Asp 115 120 125 Asp Asp Asp Asp Asp Ser Ser Ser Glu Glu Lys
Glu Thr Asp Asn Thr 130 135 140 Lys Pro Asn Arg Met Pro Val Ala Pro
Tyr Trp Thr Ser Pro Glu Lys 145 150 155 160 Met Glu Lys Lys Leu His
Ala Val Pro Ala Ala Lys Thr Val Lys Phe 165 170 175 Lys Cys Pro Ser
Ser Gly Thr Pro Asn Pro Thr Leu Arg Trp Leu Lys 180 185 190 Asn Gly
Lys Glu Phe Lys Pro Asp His Arg Ile Gly Gly Tyr Lys Val 195 200 205
Arg Tyr Ala Thr Trp Ser Ile Ile Met Asp Ser Val Val Pro Ser Asp 210
215 220 Lys Gly Asn Tyr Thr Cys Ile Val Glu Asn Glu Tyr Gly Ser Ile
Asn 225 230 235 240 His Thr Tyr Gln Leu Asp Val Val Glu Arg Ser Pro
His Arg Pro Ile 245 250
255 Leu Gln Ala Gly Leu Pro Ala Asn Lys Thr Val Ala Leu Gly Ser Asn
260 265 270 Val Glu Phe Met Cys Lys Val Tyr Ser Asp Pro Gln Pro His
Ile Gln 275 280 285 Trp Leu Lys His Ile Glu Val Asn Gly Ser Lys Ile
Gly Pro Asp Asn 290 295 300 Leu Pro Tyr Val Gln Ile Leu Lys Thr Ala
Gly Val Asn Thr Thr Asp 305 310 315 320 Lys Glu Met Glu Val Leu His
Leu Arg Asn Val Ser Phe Glu Asp Ala 325 330 335 Gly Glu Tyr Thr Cys
Leu Ala Gly Asn Ser Ile Gly Leu Ser His His 340 345 350 Ser Ala Trp
Leu Thr Val Leu Glu Ala Leu Glu Glu Arg Pro Ala Val 355 360 365 Met
Thr Ser Pro Leu Tyr Leu Glu Ile Ile Ile Tyr Cys Thr Gly Ala 370 375
380 Phe Leu Ile Ser Cys Met Val Gly Ser Val Ile Val Tyr Lys Met Lys
385 390 395 400 Ser Gly Thr Lys Lys Ser Asp Phe His Ser Gln Met Ala
Val His Lys 405 410 415 Leu Ala Lys Ser Ile Pro Leu Arg Arg Gln Val
Ser Ala Asp Ser Ser 420 425 430 Ala Ser Met Asn Ser Gly Val Leu Leu
Val Arg Pro Ser Arg Leu Ser 435 440 445 Ser Ser Gly Thr Pro Met Leu
Ala Gly Val Ser Glu Tyr Glu Leu Pro 450 455 460 Glu Asp Pro Arg Trp
Glu Leu Pro Arg Asp Arg Leu Val Leu Gly Lys 465 470 475 480 Pro Leu
Gly Glu Gly Cys Phe Gly Gln Val Val Leu Ala Glu Ala Ile 485 490 495
Gly Leu Asp Lys Asp Lys Pro Asn Arg Val Thr Lys Val Ala Val Lys 500
505 510 Met Leu Lys Ser Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Ile
Ser 515 520 525 Glu Met Glu Met Met Lys Met Ile Gly Lys His Lys Asn
Ile Ile Asn 530 535 540 Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu
Tyr Val Ile Val Glu 545 550 555 560 Tyr Ala Ser Lys Gly Asn Leu Arg
Glu Tyr Leu Gln Ala Arg Arg Pro 565 570 575 Pro Gly Leu Glu Tyr Cys
Tyr Asn Pro Ser His Asn Pro Glu Glu Gln 580 585 590 Leu Ser Ser Lys
Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly 595 600 605 Met Glu
Tyr Leu Ala Ser Lys Lys Cys Ile His Arg Asp Leu Ala Ala 610 615 620
Arg Asn Val Leu Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe 625
630 635 640 Gly Leu Ala Arg Asp Ile His His Ile Asp Tyr Tyr Lys Lys
Thr Thr 645 650 655 Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu
Ala Leu Phe Asp 660 665 670 Arg Ile Tyr Thr His Gln Ser Asp Val Trp
Ser Phe Gly Val Leu Leu 675 680 685 Trp Glu Ile Phe Thr Leu Gly Gly
Ser Pro Tyr Pro Gly Val Pro Val 690 695 700 Glu Glu Leu Phe Lys Leu
Leu Lys Glu Gly His Arg Met Asp Lys Pro 705 710 715 720 Ser Asn Cys
Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His 725 730 735 Ala
Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu 740 745
750 Asp Arg Ile Val Ala Leu Thr Ser Asn Gln Glu Tyr Leu Asp Leu Ser
755 760 765 Met Pro Leu Asp Gln Tyr Ser Pro Ser Phe Pro Asp Thr Arg
Ser Ser 770 775 780 Thr Cys Ser Ser Gly Glu Asp Ser Val Phe Ser His
Glu Pro Leu Pro 785 790 795 800 Glu Glu Pro Cys Leu Pro Arg His Pro
Ala Gln Leu Ala Asn Gly Gly 805 810 815 Leu Lys Arg Arg 820
1475895DNAHomo sapiens 147agatgcaggg gcgcaaacgc caaaggagac
caggctgtag gaagagaagg gcagagcgcc 60ggacagctcg gcccgctccc cgtcctttgg
ggccgcggct ggggaactac aaggcccagc 120aggcagctgc agggggcgga
ggcggaggag ggaccagcgc gggtgggagt gagagagcga 180gccctcgcgc
cccgccggcg catagcgctc ggagcgctct tgcggccaca ggcgcggcgt
240cctcggcggc gggcggcagc tagcgggagc cgggacgccg gtgcagccgc
agcgcgcgga 300ggaacccggg tgtgccggga gctgggcggc cacgtccgga
cgggaccgag acccctcgta 360gcgcattgcg gcgacctcgc cttccccggc
cgcgagcgcg ccgctgcttg aaaagccgcg 420gaacccaagg acttttctcc
ggtccgagct cggggcgccc cgcagggcgc acggtacccg 480tgctgcagtc
gggcacgccg cggcgccggg gcctccgcag ggcgatggag cccggtctgc
540aaggaaagtg aggcgccgcc gctgcgttct ggaggagggg ggcacaaggt
ctggagaccc 600cgggtggcgg acgggagccc tccccccgcc ccgcctccgg
ggcaccagct ccggctccat 660tgttcccgcc cgggctggag gcgccgagca
ccgagcgccg ccgggagtcg agcgccggcc 720gcggagctct tgcgaccccg
ccaggacccg aacagagccc gggggcggcg ggccggagcc 780ggggacgcgg
gcacacgccc gctcgcacaa gccacggcgg actctcccga ggcggaacct
840ccacgccgag cgagggtcag tttgaaaagg aggatcgagc tcactgtgga
gtatccatgg 900agatgtggag ccttgtcacc aacctctaac tgcagaactg
ggatgtggag ctggaagtgc 960ctcctcttct gggctgtgct ggtcacagcc
acactctgca ccgctaggcc gtccccgacc 1020ttgcctgaac aagcccagcc
ctggggagcc cctgtggaag tggagtcctt cctggtccac 1080cccggtgacc
tgctgcagct tcgctgtcgg ctgcgggacg atgtgcagag catcaactgg
1140ctgcgggacg gggtgcagct ggcggaaagc aaccgcaccc gcatcacagg
ggaggaggtg 1200gaggtgcagg actccgtgcc cgcagactcc ggcctctatg
cttgcgtaac cagcagcccc 1260tcgggcagtg acaccaccta cttctccgtc
aatgtttcag atgctctccc ctcctcggag 1320gatgatgatg atgatgatga
ctcctcttca gaggagaaag aaacagataa caccaaacca 1380aaccgtatgc
ccgtagctcc atattggaca tccccagaaa agatggaaaa gaaattgcat
1440gcagtgccgg ctgccaagac agtgaagttc aaatgccctt ccagtgggac
cccaaacccc 1500acactgcgct ggttgaaaaa tggcaaagaa ttcaaacctg
accacagaat tggaggctac 1560aaggtccgtt atgccacctg gagcatcata
atggactctg tggtgccctc tgacaagggc 1620aactacacct gcattgtgga
gaatgagtac ggcagcatca accacacata ccagctggat 1680gtcgtggagc
ggtcccctca ccggcccatc ctgcaagcag ggttgcccgc caacaaaaca
1740gtggccctgg gtagcaacgt ggagttcatg tgtaaggtgt acagtgaccc
gcagccgcac 1800atccagtggc taaagcacat cgaggtgaat gggagcaaga
ttggcccaga caacctgcct 1860tatgtccaga tcttgaagac tgctggagtt
aataccaccg acaaagagat ggaggtgctt 1920cacttaagaa atgtctcctt
tgaggacgca ggggagtata cgtgcttggc gggtaactct 1980atcggactct
cccatcactc tgcatggttg accgttctgg aagccctgga agagaggccg
2040gcagtgatga cctcgcccct gtacctggag atcatcatct attgcacagg
ggccttcctc 2100atctcctgca tggtggggtc ggtcatcgtc tacaagatga
agagtggtac caagaagagt 2160gacttccaca gccagatggc tgtgcacaag
ctggccaaga gcatccctct gcgcagacag 2220gtgtctgctg actccagtgc
atccatgaac tctggggttc ttctggttcg gccatcacgg 2280ctctcctcca
gtgggactcc catgctagca ggggtctctg agtatgagct tcccgaagac
2340cctcgctggg agctgcctcg ggacagactg gtcttaggca aacccctggg
agagggctgc 2400tttgggcagg tggtgttggc agaggctatc gggctggaca
aggacaaacc caaccgtgtg 2460accaaagtgg ctgtgaagat gttgaagtcg
gacgcaacag agaaagactt gtcagacctg 2520atctcagaaa tggagatgat
gaagatgatc gggaagcata agaatatcat caacctgctg 2580ggggcctgca
cgcaggatgg tcccttgtat gtcatcgtgg agtatgcctc caagggcaac
2640ctgcgggagt acctgcaggc ccggaggccc ccagggctgg aatactgcta
caaccccagc 2700cacaacccag aggagcagct ctcctccaag gacctggtgt
cctgcgccta ccaggtggcc 2760cgaggcatgg agtatctggc ctccaagaag
tgcatacacc gagacctggc agccaggaat 2820gtcctggtga cagaggacaa
tgtgatgaag atagcagact ttggcctcgc acgggacatt 2880caccacatcg
actactataa aaagacaacc aacggccgac tgcctgtgaa gtggatggca
2940cccgaggcat tatttgaccg gatctacacc caccagagtg atgtgtggtc
tttcggggtg 3000ctcctgtggg agatcttcac tctgggcggc tccccatacc
ccggtgtgcc tgtggaggaa 3060cttttcaagc tgctgaagga gggtcaccgc
atggacaagc ccagtaactg caccaacgag 3120ctgtacatga tgatgcggga
ctgctggcat gcagtgccct cacagagacc caccttcaag 3180cagctggtgg
aagacctgga ccgcatcgtg gccttgacct ccaaccagga gtacctggac
3240ctgtccatgc ccctggacca gtactccccc agctttcccg acacccggag
ctctacgtgc 3300tcctcagggg aggattccgt cttctctcat gagccgctgc
ccgaggagcc ctgcctgccc 3360cgacacccag cccagcttgc caatggcgga
ctcaaacgcc gctgactgcc acccacacgc 3420cctccccaga ctccaccgtc
agctgtaacc ctcacccaca gcccctgctg ggcccaccac 3480ctgtccgtcc
ctgtcccctt tcctgctggc aggagccggc tgcctaccag gggccttcct
3540gtgtggcctg ccttcacccc actcagctca cctctccctc cacctcctct
ccacctgctg 3600gtgagaggtg caaagaggca gatctttgct gccagccact
tcatcccctc ccagatgttg 3660gaccaacacc cctccctgcc accaggcact
gcctggaggg cagggagtgg gagccaatga 3720acaggcatgc aagtgagagc
ttcctgagct ttctcctgtc ggtttggtct gttttgcctt 3780cacccataag
cccctcgcac tctggtggca ggtgccttgt cctcagggct acagcagtag
3840ggaggtcagt gcttcgtgcc tcgattgaag gtgacctctg ccccagatag
gtggtgccag 3900tggcttatta attccgatac tagtttgctt tgctgaccaa
atgcctggta ccagaggatg 3960gtgaggcgaa ggccaggttg ggggcagtgt
tgtggccctg gggcccagcc ccaaactggg 4020ggctctgtat atagctatga
agaaaacaca aagtgtataa atctgagtat atatttacat 4080gtctttttaa
aagggtcgtt accagagatt tacccatcgg gtaagatgct cctggtggct
4140gggaggcatc agttgctata tattaaaaac aaaaaagaaa aaaaaggaaa
atgtttttaa 4200aaaggtcata tattttttgc tacttttgct gttttatttt
tttaaattat gttctaaacc 4260tattttcagt ttaggtccct caataaaaat
tgctgctgct tcatttatct atgggctgta 4320tgaaaagggt gggaatgtcc
actggaaaga agggacaccc acgggccctg gggctaggtc 4380tgtcccgagg
gcaccgcatg ctcccggcgc aggttccttg taacctcttc ttcctaggtc
4440ctgcacccag acctcacgac gcacctcctg cctctccgct gcttttggaa
agtcagaaaa 4500agaagatgtc tgcttcgagg gcaggaaccc catccatgca
gtagaggcgc tgggcagaga 4560gtcaaggccc agcagccatc gaccatggat
ggtttcctcc aaggaaaccg gtggggttgg 4620gctggggagg gggcacctac
ctaggaatag ccacggggta gagctacagt gattaagagg 4680aaagcaaggg
cgcggttgct cacgcctgta atcccagcac tttgggacac cgaggtgggc
4740agatcacttc aggtcaggag tttgagacca gcctggccaa cttagtgaaa
ccccatctct 4800actaaaaatg caaaaattat ccaggcatgg tggcacacgc
ctgtaatccc agctccacag 4860gaggctgagg cagaatccct tgaagctggg
aggcggaggt tgcagtgagc cgagattgcg 4920ccattgcact ccagcctggg
caacagagaa aacaaaaagg aaaacaaatg atgaaggtct 4980gcagaaactg
aaacccagac atgtgtctgc cccctctatg tgggcatggt tttgccagtg
5040cttctaagtg caggagaaca tgtcacctga ggctagtttt gcattcaggt
ccctggcttc 5100gtttcttgtt ggtatgcctc cccagatcgt ccttcctgta
tccatgtgac cagactgtat 5160ttgttgggac tgtcgcagat cttggcttct
tacagttctt cctgtccaaa ctccatcctg 5220tccctcagga acggggggaa
aattctccga atgtttttgg ttttttggct gcttggaatt 5280tacttctgcc
acctgctggt catcactgtc ctcactaagt ggattctggc tcccccgtac
5340ctcatggctc aaactaccac tcctcagtcg ctatattaaa gcttatattt
tgctggatta 5400ctgctaaata caaaagaaag ttcaatatgt tttcatttct
gtagggaaaa tgggattgct 5460gctttaaatt tctgagctag ggattttttg
gcagctgcag tgttggcgac tattgtaaaa 5520ttctctttgt ttctctctgt
aaatagcacc tgctaacatt acaatttgta tttatgttta 5580aagaaggcat
catttggtga acagaactag gaaatgaatt tttagctctt aaaagcattt
5640gctttgagac cgcacaggag tgtctttcct tgtaaaacag tgatgataat
ttctgccttg 5700gccctacctt gaagcaatgt tgtgtgaagg gatgaagaat
ctaaaagtct tcataagtcc 5760ttgggagagg tgctagaaaa atataaggca
ctatcataat tacagtgatg tccttgctgt 5820tactactcaa atcacccaca
aatttcccca aagactgcgc tagctgtcaa ataaaagaca 5880gtgaaattga cctga
5895148805PRTHomo sapiens 148Met Ala Phe Ser Pro Trp Gln Ile Leu
Ser Pro Val Gln Trp Ala Lys 1 5 10 15 Trp Thr Trp Ser Ala Val Arg
Gly Gly Ala Ala Gly Glu Asp Glu Ala 20 25 30 Gly Gly Pro Glu Gly
Asp Pro Glu Glu Glu Asp Ser Gln Ala Glu Thr 35 40 45 Lys Ser Leu
Ser Phe Ser Ser Asp Ser Glu Gly Asn Phe Glu Thr Pro 50 55 60 Glu
Ala Glu Thr Pro Ile Arg Ser Pro Phe Lys Glu Ser Cys Asp Pro 65 70
75 80 Ser Leu Gly Leu Ala Gly Pro Gly Ala Lys Ser Gln Glu Ser Gln
Glu 85 90 95 Ala Asp Glu Gln Leu Val Ala Glu Val Val Glu Lys Cys
Ser Ser Lys 100 105 110 Thr Cys Ser Lys Pro Ser Glu Asn Glu Val Pro
Gln Gln Ala Ile Asp 115 120 125 Ser His Ser Val Lys Asn Phe Arg Glu
Glu Pro Glu His Asp Phe Ser 130 135 140 Lys Ile Ser Ile Val Arg Pro
Phe Ser Ile Glu Thr Lys Asp Ser Thr 145 150 155 160 Asp Ile Ser Ala
Val Leu Gly Thr Lys Ala Ala His Gly Cys Val Thr 165 170 175 Ala Val
Ser Gly Lys Ala Leu Pro Ser Ser Pro Pro Asp Ala Leu Gln 180 185 190
Asp Glu Ala Met Thr Glu Gly Ser Met Gly Val Thr Leu Glu Ala Ser 195
200 205 Ala Glu Ala Asp Leu Lys Ala Gly Asn Ser Cys Pro Glu Leu Val
Pro 210 215 220 Ser Arg Arg Ser Lys Leu Arg Lys Pro Lys Pro Val Pro
Leu Arg Lys 225 230 235 240 Lys Ala Ile Gly Gly Glu Phe Ser Asp Thr
Asn Ala Ala Val Glu Gly 245 250 255 Thr Pro Leu Pro Lys Ala Ser Tyr
His Phe Ser Pro Glu Glu Leu Asp 260 265 270 Glu Asn Thr Ser Pro Leu
Leu Gly Asp Ala Arg Phe Gln Lys Ser Pro 275 280 285 Pro Asp Leu Lys
Glu Thr Pro Gly Thr Leu Ser Ser Asp Thr Asn Asp 290 295 300 Ser Gly
Val Glu Leu Gly Glu Glu Ser Arg Ser Ser Pro Leu Lys Leu 305 310 315
320 Glu Phe Asp Phe Thr Glu Asp Thr Gly Asn Ile Glu Ala Arg Lys Ala
325 330 335 Leu Pro Arg Lys Leu Gly Arg Lys Leu Gly Ser Thr Leu Thr
Pro Lys 340 345 350 Ile Gln Lys Asp Gly Ile Ser Lys Ser Ala Gly Leu
Glu Gln Pro Thr 355 360 365 Asp Pro Val Ala Arg Asp Gly Pro Leu Ser
Gln Thr Ser Ser Lys Pro 370 375 380 Asp Pro Ser Gln Trp Glu Ser Pro
Ser Phe Asn Pro Phe Gly Ser His 385 390 395 400 Ser Val Leu Gln Asn
Ser Pro Pro Leu Ser Ser Glu Gly Ser Tyr His 405 410 415 Phe Asp Pro
Asp Asn Phe Asp Glu Ser Met Asp Pro Phe Lys Pro Thr 420 425 430 Thr
Thr Leu Thr Ser Ser Asp Phe Cys Ser Pro Thr Gly Asn His Val 435 440
445 Asn Glu Ile Leu Glu Ser Pro Lys Lys Ala Lys Ser Arg Leu Ile Thr
450 455 460 Ser Gly Cys Lys Val Lys Lys His Glu Thr Gln Ser Leu Ala
Leu Asp 465 470 475 480 Ala Cys Ser Arg Asp Glu Gly Ala Val Ile Ser
Gln Ile Ser Asp Ile 485 490 495 Ser Asn Arg Asp Gly His Ala Thr Asp
Glu Glu Lys Leu Ala Ser Thr 500 505 510 Ser Cys Gly Gln Lys Ser Ala
Gly Ala Glu Val Lys Gly Glu Pro Glu 515 520 525 Glu Asp Leu Glu Tyr
Phe Glu Cys Ser Asn Val Pro Val Ser Thr Ile 530 535 540 Asn His Ala
Phe Ser Ser Ser Glu Ala Gly Ile Glu Lys Glu Thr Cys 545 550 555 560
Gln Lys Met Glu Glu Asp Gly Ser Thr Val Leu Gly Leu Leu Glu Ser 565
570 575 Ser Ala Glu Lys Ala Pro Val Ser Val Ser Cys Gly Gly Glu Ser
Pro 580 585 590 Leu Asp Gly Ile Cys Leu Ser Glu Ser Asp Lys Thr Ala
Val Leu Thr 595 600 605 Leu Ile Arg Glu Glu Ile Ile Thr Lys Glu Ile
Glu Ala Asn Glu Trp 610 615 620 Lys Lys Lys Tyr Glu Glu Thr Arg Gln
Glu Val Leu Glu Met Arg Lys 625 630 635 640 Ile Val Ala Glu Tyr Glu
Lys Thr Ile Ala Gln Met Ile Glu Asp Glu 645 650 655 Gln Arg Thr Ser
Met Thr Ser Gln Lys Ser Phe Gln Gln Leu Thr Met 660 665 670 Glu Lys
Glu Gln Ala Leu Ala Asp Leu Asn Ser Val Glu Arg Ser Leu 675 680 685
Ser Asp Leu Phe Arg Arg Tyr Glu Asn Leu Lys Gly Val Leu Glu Gly 690
695 700 Phe Lys Lys Asn Glu Glu Ala Leu Lys Lys Cys Ala Gln Asp Tyr
Leu 705 710 715 720 Ala Arg Val Lys Gln Glu Glu Gln Arg Tyr Gln Ala
Leu Lys Ile His 725 730 735 Ala Glu Glu Lys Leu Asp Lys Ala Asn Glu
Glu Ile Ala Gln Val Arg 740 745 750 Thr Lys Ala Lys Ala Glu Ser Ala
Ala Leu His Ala Gly Leu Arg Lys 755 760 765 Glu Gln Met Lys Val Glu
Ser Leu Glu Arg Ala Leu Gln Gln Lys Asn 770 775 780 Gln Glu Ile Glu
Glu Leu Thr Lys Ile Cys Asp Glu Leu Ile Ala Lys 785 790 795 800 Leu
Gly Lys Thr Asp 805 1497802DNAHomo sapiens 149agctgatgcg cgccccgccg
gccgggaggc gggagtccgc gagccgggag cgggagcagc 60agaggtctag cagccgggcg
ccgcgggccg ggggcctgag
gaggccacag gacgggcgtc 120ttcccggcta gtggagcccg gcgcggggcc
cgctgcggcc gcaccgtgag gggaggaggc 180cgaggaggac gcagcgccgg
ctgccggcgg gaggaagcgc tccaccaggg cccccgacgg 240cactcgttta
accacatccg cgcctctgct ggaaacgctt gctggcgcct gtcaccggtt
300ccctccattt tgaaagggaa aaaggctctc cccacccatt cccctgcccc
taggagctgg 360agccggagga gccgcgctca tggcgttcag cccgtggcag
atcctgtccc ccgtgcagtg 420ggcgaaatgg acgtggtctg cggtacgcgg
cggggccgcc ggcgaggacg aggctggcgg 480gcccgagggc gaccccgagg
aggaggattc gcaagccgag accaaatcct tgagtttcag 540ctcggattct
gaaggtaatt ttgagactcc tgaagctgaa accccgatcc gatcaccttt
600caaggagtcc tgtgatccat cactcggatt ggcaggacct ggggccaaaa
gccaagaatc 660acaagaagct gatgaacagc ttgtagcaga agtggttgaa
aaatgttcat ctaagacttg 720ttctaaacct tcagaaaatg aagtgccaca
gcaggccatt gactctcact cagtcaagaa 780tttcagagaa gaacctgaac
atgattttag caaaatttcc atcgtgaggc cattttcaat 840agaaacgaag
gattccacgg atatctcggc agtcctcgga acaaaagcag ctcatggctg
900tgtaactgca gtctcaggca aggctctgcc ttccagcccg ccagacgccc
tccaggacga 960ggcgatgaca gaaggcagca tgggggtcac cctcgaggcc
tccgcagaag ctgatctaaa 1020agctggcaac tcctgtccag agcttgtgcc
cagcagaaga agcaagctga gaaagcccaa 1080gcctgtcccc ctgaggaaga
aagcaattgg aggagagttc tcagacacca acgctgctgt 1140ggagggcaca
cctctcccca aggcatccta tcacttcagt cctgaagagt tggatgagaa
1200cacaagtcct ttgctaggag atgccaggtt ccagaagtct ccccctgacc
ttaaagaaac 1260tcccggcact ctcagtagtg acaccaacga ctcaggggtt
gagctggggg aggagtcgag 1320gagctcacct ctcaagcttg agtttgattt
cacagaagat acaggaaaca tagaggccag 1380gaaagccctt ccaaggaagc
ttggcaggaa actgggtagc acactgactc ccaagataca 1440aaaagatggc
atcagtaagt cagcaggttt agaacagcct acagacccag tggcacgaga
1500cgggcctctc tcccaaacat cttccaagcc agatcctagt cagtgggaaa
gccccagctt 1560caaccccttt gggagccact ctgttctgca gaactcccca
cccctctctt ctgagggctc 1620ctaccacttt gacccagata actttgacga
atccatggat ccctttaaac caactacgac 1680cttaacaagc agtgactttt
gttctcccac tggtaatcac gttaatgaaa tcttagaatc 1740acccaagaag
gcaaagtcgc gtttaataac gagtggctgt aaggtgaaga agcatgaaac
1800tcagtctctc gccctggatg catgttctcg ggatgaaggg gcagtgatct
cccagatttc 1860agacatttct aatagggatg gccatgctac tgatgaggag
aaactggcat ccacgtcatg 1920tggtcagaaa tcagctggtg ccgaggtgaa
aggtgagcca gaggaagacc tggagtactt 1980tgaatgttcc aatgttcctg
tgtctaccat aaatcatgcg ttttcatcct cagaagcagg 2040catagagaag
gagacgtgcc agaagatgga agaagacggg tccactgtgc ttgggctgct
2100ggagtcctct gcagagaagg cccctgtgtc ggtgtcctgt ggaggtgaga
gccccctgga 2160tgggatctgc ctcagcgaat cagacaagac agccgtgctc
accttaataa gagaagagat 2220aattactaaa gagattgaag caaatgaatg
gaagaagaaa tacgaagaga cccggcaaga 2280agttttggag atgaggaaaa
ttgtagctga atatgaaaag actattgctc aaatgattga 2340agatgaacaa
aggacaagta tgacctctca gaagagcttc cagcaactga ccatggagaa
2400ggaacaggcc ctggctgacc ttaactctgt ggaaaggtcc ctttctgatc
tcttcaggag 2460atatgagaac ctgaaaggtg ttctggaagg gttcaagaag
aatgaagaag ccttgaagaa 2520atgtgctcag gattacttag ccagagttaa
acaagaggag cagcgatacc aggccctgaa 2580aatccacgca gaagagaaac
tggacaaagc caatgaagag attgctcagg ttcgaacaaa 2640agcaaaggct
gagagtgcag ctctccatgc tggactccgc aaagagcaga tgaaggtgga
2700gtccctggaa agggccctgc agcagaagaa ccaagaaatt gaagaactga
caaaaatctg 2760tgatgagctg attgcaaagc tgggaaagac tgactgagac
actccccctg ttagctcaac 2820agatctgcat ttggctgctt ctcttgtgac
cacaattatc ttgccttatc caggaataat 2880tgcccctttg cagagaaaaa
aaaaaactta aaaaaagcac atgcctactg ctgcctgtcc 2940cgctttgctg
ccaatgcaac agccctggaa gaaaccctag agggttgcat agtctagaaa
3000ggagtgtgac ctgacagtgc tggagcctcc tagtttcccc ctatgaaggt
tcccttaggc 3060tgctgagttt gggtttgtga tttatcttta gtttgtttta
aagtcatctt tactttccca 3120aatgtgttaa atttgtaact cctctttggg
gtcttctcca ccacctgtct gatttttttg 3180tgatctgttt aatcttttaa
ttttttagta tcagtggttt tatttaagga gacagtttgg 3240cctattgtta
cttccaattt ataatcaaga aggggctctg gatccccttt taaattacac
3300acactctcac acacatacat gtatgtttat agatgctgct gctcttttcc
ctgaagcata 3360gtcaagtaag aactgctcta cagaaggaca tatttccttg
gatgtgagac cctattttga 3420aatagagtcc tgactcagaa caccaactta
agaatttggg ggattaaaga tgtgaagacc 3480acagtcttgg gttttcatat
ctggagaaga ctatttgcca tgacgttttg ttgccctggt 3540atttggacac
tcctcagctt taatgggtgt ggccccttta gggttagtcc tcagactaat
3600gatagtgtct gctttctgca tgaacggcaa tatgggactc cctccaagct
agggtttggc 3660aagtctgccc tagagtcatt tactctcctc tgcctccatt
tgttaataca gaatcaacat 3720ttagtcttca ttatcttttt tttttttttt
gagacagagt ttcgatctat tttaagtatg 3780tgaagaaaat ctacttgtaa
aaggctcaga tcttaattaa aaggtaattg tagcacatta 3840ccaattataa
ggtgaagaaa tgtttttttc ccaagtgtga tgcattgttc ttcagatgtt
3900gaaaagaaag caaaaaatac cttctaactt aagacagaat ttttaacaaa
atgagcagta 3960aaagtcacat gaaccactcc aaaaatcagt gcattttgca
tatttttaaa caaagacagc 4020ttgttgaata ctgagaagag gagtgcaagg
agaaggtctg tactaacaaa gccaaattcc 4080tcaagctctt actggactca
gttcagagtg gtgggccatt aaccccaaca tggaattttt 4140ccatataaat
ctcaatgaat tccctttcat ttgaataggc aaacccaaat ccatgcaagt
4200gttttaaagc actgtcctgt cttaatctta catgctgaaa gtcttcatgg
tgatatgcac 4260tatattcagt atacgtatgt tttcctactt ctcttgtaaa
actgttgcat gatccaactt 4320cagcaatgaa ttgtgcctag tggagaacct
ctatagatct taaaaaatga attattcttt 4380agcagtgtat tactcacatg
ggtgcaatct ttagccccag ggaggtcaat aatgtctttt 4440aaagccagaa
gtcacatttt accaatatgc atttatcata attggtgctt aggctgtata
4500ttcaagcctg ttgtcttaac attttgtata aaaaagaaca acagaaatta
tctgtcattt 4560gagaagtggc ttgacaatca tttgagcttt gaaagcagtc
actgtggtgt aatatgaatg 4620ctgtcctagt ggtcatagta ccaagggcac
gtgtctcccc ttggtataac tgatttcctt 4680tttagtcctc tactgctaaa
taagttaatt ttgcattttg cagaaagaaa cattgattgc 4740taaatctttt
tgctgctgtg ttttggtgtt ttcatgttta cttgttttat attgatctgt
4800tttaagtatg agaggcttat agtgccctcc attgtaaatc catagtcatc
tttttaagct 4860tattgtgttt aagaaagtag ctatgtgtta aacagaggtg
atggcagccc ttccctagca 4920cactggtgga agagacccct taagaacctg
accccagtga atgaagctga tgcacaggga 4980gcaccaaagg accttcgtta
agtgataatt gtcctggcct ctcagccatg accgttatga 5040ggaaatatcc
cccattcgaa cttaacagat gcctcctctc caaagagaat taaaatcgta
5100gcttgtacag atcaagagaa tatactgggc agaatgaagt atgtttgttt
atttttcttt 5160aaaaataaag gattttggaa ctctggagag taagaatata
gtatagagtt tgcctcaaca 5220catgtgaggg ccaaataacc tgctagctag
gcagtaataa actctgttac agaagagaaa 5280aagggccggg cacagtggct
tattcctgta atcccaacac tgtggaaggc cgaggcagga 5340ggatcacttg
agtccaggag tttgaaacct acctaggcaa catggtgaaa ccttgtctct
5400accaaaataa aaattagctg ggcatggtgg cacgtgcctg tggtcccagc
tacttgggag 5460gctgaggtgg gagcctggga ggtcaaggct gcagtgagcc
atgatcatgc cactgcactc 5520catcctgggt gacagcaaga tcttgtctca
aaaaaaaaaa aaaaaaaaaa aaaaccagga 5580gtgaaaaagg aaagtagaag
gcagctgctg gcctagatgt tggtttggga atattaggtg 5640atcctgttga
gattctggat ccagagcaat ttctttagct tttgactttg ccaaagtgta
5700gatagccttt atccagcagt attttaagtg gggaatgcaa cgtgaggcca
actgaacaat 5760tccccccgtg gctgcccaga tagtcacagt caaggttgga
gagtctcctt ccagccagtg 5820acctacccaa accttttgtt ctgtaaaact
gctctggaaa taccgggaag cccagttttc 5880tcacgtggtt tctagcttct
tcagactcag cccaaattag gaagtgcaga agcacatgat 5940ggtgaaaaac
ctaggatttg gcagccttcc agaatggtat ggaatctgag ggaagattta
6000tgtttcgttt tggaggatag ctcaagttga attttctttc cagccagtta
ccctttcaac 6060ctacccatac tttgtacaac tcttacacaa atacttagat
atttattaga tagccctgaa 6120ttcactctaa ttataaacag ggagtgtaaa
ctgcccccag atgttcctgg gctgggtaaa 6180agcagctgga gtgaagcact
cattttccat aaaggtaaca aagggcagct cagtggttac 6240tcaagctcaa
aagggttttt ttaagagcaa gcattggtta agtctgtgta tactgagttg
6300gaagtgattt cagcacattc ttttttagtg gagtgaaagt tctgaagccc
ccttttaact 6360tcctcttggt ttttcattat aattggtagc catctcatga
actgtctctg actgttgtct 6420ctttgtggtc atgtgattgt gagcttgctt
tctgacttgc atttctgact ttatcctgtt 6480gttaggaaga tagaaactag
gttttgaaag attacatgat tcaagcgagg gattttaaag 6540taaagatgta
tttattctga agaatctaaa agataacaga ttatttgctt atgaaagaac
6600aatatagtct gggaatccca gaatgtcaag ccaaaggtct aagaagtcat
ctccttcaaa 6660tactttaata aagaagtatt tcgaggagat atctgtccaa
aaaggtttga ctggcctcca 6720gattccagtt atttttaaaa agcaacttac
cactaaatcc ttgagtctcc atagagtaac 6780agtaaagaaa ctgatgtaac
agactctcct ctcaaaggat ctcctctgga agagactatc 6840agcggcagca
ttctccaggg aagacccatc ccctagtgcc agagcttgca tcctggagac
6900taaagattgc acttttttgt agttttttgt ccaaatgcaa tcccatttct
gtgcctctta 6960gcatgcagtt agatttggac aaacaagatt cctaaggaat
gactttatta actataatat 7020ggttacagct attatataaa tatatattct
ggttatagtt ctaatatgga gatgttgtgt 7080gcaatgctgg cctgtggtgg
tctgtgtaat gctttaactt gtatggagga ggccaggctc 7140agagctgaga
tgtggcctga accttccctg tatcgatcct ttaatttaga actgtcaaga
7200tgtcactttc tccccctctg ccttttagtg gtatctgaca tatactcaaa
acagtaattt 7260cctggtcaca tcattaactg ctaattctgt atttataaag
aattttcaga tggacatgta 7320caaatttgaa ctcaaaccat ccccagtcca
gatacagggc agcgtgtagg tgaccacacc 7380agagcctcag cctcggtcct
tctcagccgt cgggatagga tccaggcatt tcttttaaat 7440ctcagaggta
gcagtaaact tttcagtatt gctgttagca agtgtgtgtt tgccaataga
7500tacccattat actaatgtgc caagtaaatg ttcattgcac atctgcttcc
actgtgttcc 7560cacgggtgcc atgaagtgtg tgaggagccc ctcatctgga
gggatgagtg ctgcgttgac 7620tactgctatc aggattgtgt tgtgtggaat
attcatctac ataaatttta tatgcacagt 7680aatttccctt tttatatgtc
aagtaactat ttgtaaaagt tatactcaca aattattata 7740atgattacta
atatattttt tccatgtttc attgcctgaa taaaaactgt ttaccactgt 7800ta
7802150907PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 150Met Trp Ser Trp Lys Cys Leu Leu Phe Trp
Ala Val Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr Ala Arg Pro Ser
Pro Thr Leu Pro Glu Gln Asp Ala 20 25 30 Leu Pro Ser Ser Glu Asp
Asp Asp Asp Asp Asp Asp Ser Ser Ser Glu 35 40 45 Glu Lys Glu Thr
Asp Asn Thr Lys Pro Asn Pro Val Ala Pro Tyr Trp 50 55 60 Thr Ser
Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro Ala Ala 65 70 75 80
Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn Pro Thr 85
90 95 Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His Arg
Ile 100 105 110 Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile Ile
Met Asp Ser 115 120 125 Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys
Ile Val Glu Asn Glu 130 135 140 Tyr Gly Ser Ile Asn His Thr Tyr Gln
Leu Asp Val Val Glu Arg Ser 145 150 155 160 Pro His Arg Pro Ile Leu
Gln Ala Gly Leu Pro Ala Asn Lys Thr Val 165 170 175 Ala Leu Gly Ser
Asn Val Glu Phe Met Cys Lys Val Tyr Ser Asp Pro 180 185 190 Gln Pro
His Ile Gln Trp Leu Lys His Ile Glu Val Asn Gly Ser Lys 195 200 205
Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu Lys Thr Ala Gly 210
215 220 Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His Leu Arg Asn
Val 225 230 235 240 Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala
Gly Asn Ser Ile 245 250 255 Gly Leu Ser His His Ser Ala Trp Leu Thr
Val Leu Glu Ala Leu Glu 260 265 270 Glu Arg Pro Ala Val Met Thr Ser
Pro Leu Tyr Leu Glu Ile Ile Ile 275 280 285 Tyr Cys Thr Gly Ala Phe
Leu Ile Ser Cys Met Val Gly Ser Val Ile 290 295 300 Val Tyr Lys Met
Lys Ser Gly Thr Lys Lys Ser Asp Phe His Ser Gln 305 310 315 320 Met
Ala Val His Lys Leu Ala Lys Ser Ile Pro Leu Arg Arg Gln Val 325 330
335 Thr Val Ser Ala Asp Ser Ser Ala Ser Met Asn Ser Gly Val Leu Leu
340 345 350 Val Arg Pro Ser Arg Leu Ser Ser Ser Gly Thr Pro Met Leu
Ala Gly 355 360 365 Val Ser Glu Tyr Glu Leu Pro Glu Asp Pro Arg Trp
Glu Leu Pro Arg 370 375 380 Asp Arg Leu Val Leu Gly Lys Pro Leu Gly
Glu Gly Cys Phe Gly Gln 385 390 395 400 Val Val Leu Ala Glu Ala Ile
Gly Leu Asp Lys Asp Lys Pro Asn Arg 405 410 415 Val Thr Lys Val Ala
Val Lys Met Leu Lys Ser Asp Ala Thr Glu Lys 420 425 430 Asp Leu Ser
Asp Leu Ile Ser Glu Met Glu Met Met Lys Met Ile Gly 435 440 445 Lys
His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly 450 455
460 Pro Leu Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu
465 470 475 480 Tyr Leu Gln Ala Arg Arg Pro Pro Gly Leu Glu Tyr Cys
Tyr Asn Pro 485 490 495 Ser His Asn Pro Glu Glu Gln Leu Ser Ser Lys
Asp Leu Val Ser Cys 500 505 510 Ala Tyr Gln Val Ala Arg Gly Met Glu
Tyr Leu Ala Ser Lys Lys Cys 515 520 525 Ile His Arg Asp Leu Ala Ala
Arg Asn Val Leu Val Thr Glu Asp Asn 530 535 540 Val Met Lys Ile Ala
Asp Phe Gly Leu Ala Arg Asp Ile His His Ile 545 550 555 560 Asp Tyr
Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met 565 570 575
Ala Pro Glu Ala Leu Phe Asp Arg Ile Tyr Thr His Gln Ser Asp Val 580
585 590 Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly
Ser 595 600 605 Pro Tyr Pro Gly Val Pro Val Glu Glu Leu Phe Lys Leu
Leu Lys Glu 610 615 620 Gly His Arg Met Asp Lys Pro Ser Asn Cys Thr
Asn Glu Leu Tyr Met 625 630 635 640 Met Met Arg Asp Cys Trp His Ala
Val Pro Ser Gln Arg Pro Thr Phe 645 650 655 Lys Gln Leu Val Glu Asp
Leu Asp Arg Ile Val Ala Leu Thr Ser Asn 660 665 670 Gln Gly Leu Leu
Glu Ser Ser Ala Glu Lys Ala Pro Val Ser Val Ser 675 680 685 Cys Gly
Gly Glu Ser Pro Leu Asp Gly Ile Cys Leu Ser Glu Ser Asp 690 695 700
Lys Thr Ala Val Leu Thr Leu Ile Arg Glu Glu Ile Ile Thr Lys Glu 705
710 715 720 Ile Glu Ala Asn Glu Trp Lys Lys Lys Tyr Glu Glu Thr Arg
Gln Glu 725 730 735 Val Leu Glu Met Arg Lys Ile Val Ala Glu Tyr Glu
Lys Thr Ile Ala 740 745 750 Gln Met Ile Glu Asp Glu Gln Arg Thr Ser
Met Thr Ser Gln Lys Ser 755 760 765 Phe Gln Gln Leu Thr Met Glu Lys
Glu Gln Ala Leu Ala Asp Leu Asn 770 775 780 Ser Val Glu Arg Ser Leu
Ser Asp Leu Phe Arg Arg Tyr Glu Asn Leu 785 790 795 800 Lys Gly Val
Leu Glu Gly Phe Lys Lys Asn Glu Glu Ala Leu Lys Lys 805 810 815 Cys
Ala Gln Asp Tyr Leu Ala Arg Val Lys Gln Glu Glu Gln Arg Tyr 820 825
830 Gln Ala Leu Lys Ile His Ala Glu Glu Lys Leu Asp Lys Ala Asn Glu
835 840 845 Glu Ile Ala Gln Val Arg Thr Lys Ala Lys Ala Glu Ser Ala
Ala Leu 850 855 860 His Ala Gly Leu Arg Lys Glu Gln Met Lys Val Glu
Ser Leu Glu Arg 865 870 875 880 Ala Leu Gln Gln Lys Asn Gln Glu Ile
Glu Glu Leu Thr Lys Ile Cys 885 890 895 Asp Glu Leu Ile Ala Lys Leu
Gly Lys Thr Asp 900 905 1512989DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 151atgtggagct
ggaagtgcct cctcttctgg gctgtgctgg tcacagccac actctgcacc 60gctaggccgt
ccccgacctt gcctgaacaa gcccagccct ggggagcccc tgtggaagtg
120gagtccttcc tggtccaccc cggtgacctg ctgcagcttc gctgtcggct
gcgggacgat 180gtgcagagca tcaactggct gcgggacggg gtgcagctgg
cggaaagcaa ccgcacccgc 240atcacagggg aggaggtgga ggtgcaggac
tccgtgcccg cagactccgg cctctatgct 300tgcgtaacca gcagcccctc
gggcagtgac accacctact tctccgtcaa tgtttcagat 360gctctcccct
cctcggagga tgatgatgat gatgatgact cctcttcaga ggagaaagaa
420acagataaca ccaaaccaaa ccgtatgccc gtagctccat attggacatc
cccagaaaag 480atggaaaaga aattgcatgc agtgccggct gccaagacag
tgaagttcaa atgcccttcc 540agtgggaccc caaaccccac actgcgctgg
ttgaaaaatg gcaaagaatt caaacctgac 600cacagaattg gaggctacaa
ggtccgttat gccacctgga gcatcataat ggactctgtg 660gtgccctctg
acaagggcaa ctacacctgc attgtggaga atgagtacgg cagcatcaac
720cacacatacc agctggatgt cgtggagcgg tcccctcacc ggcccatcct
gcaagcaggg 780ttgcccgcca acaaaacagt ggccctgggt agcaacgtgg
agttcatgtg taaggtgtac 840agtgacccgc agccgcacat ccagtggcta
aagcacatcg aggtgaatgg gagcaagatt 900ggcccagaca acctgcctta
tgtccagatc ttgaagactg ctggagttaa taccaccgac 960aaagagatgg
aggtgcttca cttaagaaat gtctcctttg aggacgcagg ggagtatacg
1020tgcttggcgg gtaactctat cggactctcc catcactctg catggttgac
cgttctggaa 1080gccctggaag agaggccggc agtgatgacc tcgcccctgt
acctggagat catcatctat 1140tgcacagggg ccttcctcat ctcctgcatg
gtggggtcgg tcatcgtcta caagatgaag 1200agtggtacca
agaagagtga cttccacagc cagatggctg tgcacaagct ggccaagagc
1260atccctctgc gcagacaggt gtctgctgac tccagtgcat ccatgaactc
tggggttctt 1320ctggttcggc catcacggct ctcctccagt gggactccca
tgctagcagg ggtctctgag 1380tatgagcttc ccgaagaccc tcgctgggag
ctgcctcggg acagactggt cttaggcaaa 1440cccctgggag agggctgctt
tgggcaggtg gtgttggcag aggctatcgg gctggacaag 1500gacaaaccca
accgtgtgac caaagtggct gtgaagatgt tgaagtcgga cgcaacagag
1560aaagacttgt cagacctgat ctcagaaatg gagatgatga agatgatcgg
gaagcataag 1620aatatcatca acctgctggg ggcctgcacg caggatggtc
ccttgtatgt catcgtggag 1680tatgcctcca agggcaacct gcgggagtac
ctgcaggccc ggaggccccc agggctggaa 1740tactgctaca accccagcca
caacccagag gagcagctct cctccaagga cctggtgtcc 1800tgcgcctacc
aggtggcccg aggcatggag tatctggcct ccaagaagtg catacaccga
1860gacctggcag ccaggaatgt cctggtgaca gaggacaatg tgatgaagat
agcagacttt 1920ggcctcgcac gggacattca ccacatcgac tactataaaa
agacaaccaa cggccgactg 1980cctgtgaagt ggatggcacc cgaggcatta
tttgaccgga tctacaccca ccagagtgat 2040gtgtggtctt tcggggtgct
cctgtgggag atcttcactc tgggcggctc cccatacccc 2100ggtgtgcctg
tggaggaact tttcaagctg ctgaaggagg gtcaccgcat ggacaagccc
2160agtaactgca ccaacgagct gtacatgatg atgcgggact gctggcatgc
agtgccctca 2220cagagaccca ccttcaagca gctggtggaa gacctggacc
gcatcgtggc cttgacctcc 2280aaccagtggg ctgctggagt cctctgcaga
gaaggcccct gtgtcggtgt cctgtggagg 2340tgagagcccc ctggatggga
tctgcctcag cgaatcagac aagacagccg tgctcacctt 2400aataagagaa
gagataatta ctaaagagat tgaagcaaat gaatggaaga agaaatacga
2460agagacccgg caagaagttt tggagatgag gaaaattgta gctgaatatg
aaaagactat 2520tgctcaaatg attgaagatg aacaaaggac aagtatgacc
tctcagaaga gcttccagca 2580actgaccatg gagaaggaac aggccctggc
tgaccttaac tctgtggaaa ggtccctttc 2640tgatctcttc aggagatatg
agaacctgaa aggtgttctg gaagggttca agaagaatga 2700agaagccttg
aagaaatgtg ctcaggatta cttagccaga gttaaacaag aggagcagcg
2760ataccaggcc ctgaaaatcc acgcagaaga gaaactggac aaagccaatg
aagagattgc 2820tcaggttcga acaaaagcaa aggctgagag tgcagctctc
catgctggac tccgcaaaga 2880gcagatgaag gtggagtccc tggaaagggc
cctgcagcag aagaaccaag aaattgaaga 2940actgacaaaa atctgtgatg
agctgattgc aaagctggga aagactgac 2989152821PRTHomo sapiens 152Met
Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala 1 5 10
15 Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr
20 25 30 Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln
Pro Glu 35 40 45 Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val
Arg Cys Leu Leu 50 55 60 Lys Asp Ala Ala Val Ile Ser Trp Thr Lys
Asp Gly Val His Leu Gly 65 70 75 80 Pro Asn Asn Arg Thr Val Leu Ile
Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95 Ala Thr Pro Arg Asp Ser
Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110 Val Asp Ser Glu
Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125 Ser Ser
Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140
Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu 145
150 155 160 Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr
Val Lys 165 170 175 Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr
Met Arg Trp Leu 180 185 190 Lys Asn Gly Lys Glu Phe Lys Gln Glu His
Arg Ile Gly Gly Tyr Lys 195 200 205 Val Arg Asn Gln His Trp Ser Leu
Ile Met Glu Ser Val Val Pro Ser 210 215 220 Asp Lys Gly Asn Tyr Thr
Cys Val Val Glu Asn Glu Tyr Gly Ser Ile 225 230 235 240 Asn His Thr
Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255 Ile
Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265
270 Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile
275 280 285 Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly
Pro Asp 290 295 300 Gly Leu Pro Tyr Leu Lys Val Leu Lys Ala Ala Gly
Val Asn Thr Thr 305 310 315 320 Asp Lys Glu Ile Glu Val Leu Tyr Ile
Arg Asn Val Thr Phe Glu Asp 325 330 335 Ala Gly Glu Tyr Thr Cys Leu
Ala Gly Asn Ser Ile Gly Ile Ser Phe 340 345 350 His Ser Ala Trp Leu
Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu 355 360 365 Ile Thr Ala
Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly 370 375 380 Val
Phe Leu Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met 385 390
395 400 Lys Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val
His 405 410 415 Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr
Val Ser Ala 420 425 430 Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro
Leu Val Arg Ile Thr 435 440 445 Thr Arg Leu Ser Ser Thr Ala Asp Thr
Pro Met Leu Ala Gly Val Ser 450 455 460 Glu Tyr Glu Leu Pro Glu Asp
Pro Lys Trp Glu Phe Pro Arg Asp Lys 465 470 475 480 Leu Thr Leu Gly
Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val 485 490 495 Met Ala
Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val 500 505 510
Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu 515
520 525 Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys
His 530 535 540 Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp
Gly Pro Leu 545 550 555 560 Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly
Asn Leu Arg Glu Tyr Leu 565 570 575 Arg Ala Arg Arg Pro Pro Gly Met
Glu Tyr Ser Tyr Asp Ile Asn Arg 580 585 590 Val Pro Glu Glu Gln Met
Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr 595 600 605 Gln Leu Ala Arg
Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His 610 615 620 Arg Asp
Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met 625 630 635
640 Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr
645 650 655 Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met
Ala Pro 660 665 670 Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser
Asp Val Trp Ser 675 680 685 Phe Gly Val Leu Met Trp Glu Ile Phe Thr
Leu Gly Gly Ser Pro Tyr 690 695 700 Pro Gly Ile Pro Val Glu Glu Leu
Phe Lys Leu Leu Lys Glu Gly His 705 710 715 720 Arg Met Asp Lys Pro
Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met 725 730 735 Arg Asp Cys
Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln 740 745 750 Leu
Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu 755 760
765 Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro
770 775 780 Asp Thr Arg Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe
Ser Pro 785 790 795 800 Asp Pro Met Pro Tyr Glu Pro Cys Leu Pro Gln
Tyr Pro His Ile Asn 805 810 815 Gly Ser Val Lys Thr 820
1534654DNAHomo sapiens 153ggcggcggct ggaggagagc gcggtggaga
gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag
ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact
gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc
gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg
240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc
gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac
gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt
ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc
ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc
attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc
540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag
atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac
cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca
tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc
acattagagc cagaagagcc accaaccaaa taccaaatct 780ctcaaccaga
agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga
840aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc
aacaatagga 900cagtgcttat tggggagtac ttgcagataa agggcgccac
gcctagagac tccggcctct 960atgcttgtac tgccagtagg actgtagaca
gtgaaacttg gtacttcatg gtgaatgtca 1020cagatgccat ctcatccgga
gatgatgagg atgacaccga tggtgcggaa gattttgtca 1080gtgagaacag
taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc
1140ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc
ggggggaacc 1200caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt
taagcaggag catcgcattg 1260gaggctacaa ggtacgaaac cagcactgga
gcctcattat ggaaagtgtg gtcccatctg 1320acaagggaaa ttatacctgt
gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1380acctggatgt
tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa
1440atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac
agtgatgccc 1500agccccacat ccagtggatc aagcacgtgg aaaagaacgg
cagtaaatac gggcccgacg 1560ggctgcccta cctcaaggtt ctcaaggccg
ccggtgttaa caccacggac aaagagattg 1620aggttctcta tattcggaat
gtaacttttg aggacgctgg ggaatatacg tgcttggcgg 1680gtaattctat
tgggatatcc tttcactctg catggttgac agttctgcca gcgcctggaa
1740gagaaaagga gattacagct tccccagact acctggagat agccatttac
tgcatagggg 1800tcttcttaat cgcctgtatg gtggtaacag tcatcctgtg
ccgaatgaag aacacgacca 1860agaagccaga cttcagcagc cagccggctg
tgcacaagct gaccaaacgt atccccctgc 1920ggagacaggt aacagtttcg
gctgagtcca gctcctccat gaactccaac accccgctgg 1980tgaggataac
aacacgcctc tcttcaacgg cagacacccc catgctggca ggggtctccg
2040agtatgaact tccagaggac ccaaaatggg agtttccaag agataagctg
acactgggca 2100agcccctggg agaaggttgc tttgggcaag tggtcatggc
ggaagcagtg ggaattgaca 2160aagacaagcc caaggaggcg gtcaccgtgg
ccgtgaagat gttgaaagat gatgccacag 2220agaaagacct ttctgatctg
gtgtcagaga tggagatgat gaagatgatt gggaaacaca 2280agaatatcat
aaatcttctt ggagcctgca cacaggatgg gcctctctat gtcatagttg
2340agtatgcctc taaaggcaac ctccgagaat acctccgagc ccggaggcca
cccgggatgg 2400agtactccta tgacattaac cgtgttcctg aggagcagat
gaccttcaag gacttggtgt 2460catgcaccta ccagctggcc agaggcatgg
agtacttggc ttcccaaaaa tgtattcatc 2520gagatttagc agccagaaat
gttttggtaa cagaaaacaa tgtgatgaaa atagcagact 2580ttggactcgc
cagagatatc aacaatatag actattacaa aaagaccacc aatgggcggc
2640ttccagtcaa gtggatggct ccagaagccc tgtttgatag agtatacact
catcagagtg 2700atgtctggtc cttcggggtg ttaatgtggg agatcttcac
tttagggggc tcgccctacc 2760cagggattcc cgtggaggaa ctttttaagc
tgctgaagga aggacacaga atggataagc 2820cagccaactg caccaacgaa
ctgtacatga tgatgaggga ctgttggcat gcagtgccct 2880cccagagacc
aacgttcaag cagttggtag aagacttgga tcgaattctc actctcacaa
2940ccaatgagga atacttggac ctcagccaac ctctcgaaca gtattcacct
agttaccctg 3000acacaagaag ttcttgttct tcaggagatg attctgtttt
ttctccagac cccatgcctt 3060acgaaccatg ccttcctcag tatccacaca
taaacggcag tgttaaaaca tgaatgactg 3120tgtctgcctg tccccaaaca
ggacagcact gggaacctag ctacactgag cagggagacc 3180atgcctccca
gagcttgttg tctccacttg tatatatgga tcagaggagt aaataattgg
3240aaaagtaatc agcatatgtg taaagattta tacagttgaa aacttgtaat
cttccccagg 3300aggagaagaa ggtttctgga gcagtggact gccacaagcc
accatgtaac ccctctcacc 3360tgccgtgcgt actggctgtg gaccagtagg
actcaaggtg gacgtgcgtt ctgccttcct 3420tgttaatttt gtaataattg
gagaagattt atgtcagcac acacttacag agcacaaatg 3480cagtatatag
gtgctggatg tatgtaaata tattcaaatt atgtataaat atatattata
3540tatttacaag gagttatttt ttgtattgat tttaaatgga tgtcccaatg
cacctagaaa 3600attggtctct ctttttttaa tagctatttg ctaaatgctg
ttcttacaca taatttctta 3660attttcaccg agcagaggtg gaaaaatact
tttgctttca gggaaaatgg tataacgtta 3720atttattaat aaattggtaa
tatacaaaac aattaatcat ttatagtttt ttttgtaatt 3780taagtggcat
ttctatgcag gcagcacagc agactagtta atctattgct tggacttaac
3840tagttatcag atcctttgaa aagagaatat ttacaatata tgactaattt
ggggaaaatg 3900aagttttgat ttatttgtgt ttaaatgctg ctgtcagacg
attgttctta gacctcctaa 3960atgccccata ttaaaagaac tcattcatag
gaaggtgttt cattttggtg tgcaaccctg 4020tcattacgtc aacgcaacgt
ctaactggac ttcccaagat aaatggtacc agcgtcctct 4080taaaagatgc
cttaatccat tccttgagga cagaccttag ttgaaatgat agcagaatgt
4140gcttctctct ggcagctggc cttctgcttc tgagttgcac attaatcaga
ttagcctgta 4200ttctcttcag tgaattttga taatggcttc cagactcttt
ggcgttggag acgcctgtta 4260ggatcttcaa gtcccatcat agaaaattga
aacacagagt tgttctgctg atagttttgg 4320ggatacgtcc atctttttaa
gggattgctt tcatctaatt ctggcaggac ctcaccaaaa 4380gatccagcct
catacctaca tcagacaaaa tatcgccgtt gttccttctg tactaaagta
4440ttgtgttttg ctttggaaac acccactcac tttgcaatag ccgtgcaaga
tgaatgcaga 4500ttacactgat cttatgtgtt acaaaattgg agaaagtatt
taataaaacc tgttaatttt 4560tatactgaca ataaaaatgt ttctacagat
attaatgtta acaagacaaa ataaatgtca 4620cgcaacttat ttttttaata
aaaaaaaaaa aaaa 46541542948PRTHomo sapiens 154Met Gly Asn Glu Asn
Ser Thr Ser Asp Asn Gln Arg Thr Leu Ser Ala 1 5 10 15 Gln Thr Pro
Arg Ser Ala Gln Pro Pro Gly Asn Ser Gln Asn Ile Lys 20 25 30 Arg
Lys Gln Gln Asp Thr Pro Gly Ser Pro Asp His Arg Asp Ala Ser 35 40
45 Ser Ile Gly Ser Val Gly Leu Gly Gly Phe Cys Thr Ala Ser Glu Ser
50 55 60 Ser Ala Ser Leu Asp Pro Cys Leu Val Ser Pro Glu Val Thr
Glu Pro 65 70 75 80 Arg Lys Asp Pro Gln Gly Ala Arg Gly Pro Glu Gly
Ser Leu Leu Pro 85 90 95 Ser Pro Pro Pro Ser Gln Glu Arg Glu His
Pro Ser Ser Ser Met Pro 100 105 110 Phe Ala Glu Cys Pro Pro Glu Gly
Cys Leu Ala Ser Pro Ala Ala Ala 115 120 125 Pro Glu Asp Gly Pro Gln
Thr Gln Ser Pro Arg Arg Glu Pro Ala Pro 130 135 140 Asn Ala Pro Gly
Asp Ile Ala Ala Ala Phe Pro Ala Glu Arg Asp Ser 145 150 155 160 Ser
Thr Pro Tyr Gln Glu Ile Ala Ala Val Pro Ser Ala Gly Arg Glu 165 170
175 Arg Gln Pro Lys Glu Glu Gly Gln Lys Ser Ser Phe Ser Phe Ser Ser
180 185 190 Gly Ile Asp Gln Ser Pro Gly Met Ser Pro Val Pro Leu Arg
Glu Pro 195 200 205 Met Lys Ala Pro Leu Cys Gly Glu Gly Asp Gln Pro
Gly Gly Phe Glu 210 215 220 Ser Gln Glu Lys Glu Ala Ala Gly Gly Phe
Pro Pro Ala Glu Ser Arg 225 230 235 240 Gln Gly Val Ala Ser Val Gln
Val Thr Pro Glu Ala Pro Ala Ala Ala 245 250 255 Gln Gln Gly Thr Glu
Ser Ser Ala Val Leu Glu Lys Ser Pro Leu Lys 260 265 270 Pro Met Ala
Pro Ile Pro Gln Asp Pro Ala Pro Arg Ala Ser Asp Arg 275 280 285 Glu
Arg Gly Gln Gly Glu Ala Pro Pro Gln Tyr Leu Thr Asp Asp Leu 290 295
300 Glu Phe Leu Arg Ala Cys His Leu Pro Arg Ser Asn Ser Gly Ala Ala
305 310 315 320 Pro Glu Ala Glu Val Asn Ala Ala Ser Gln Glu Ser Cys
Gln Gln Pro 325 330 335 Val Gly Ala Tyr Leu Pro His Ala Glu Leu Pro
Trp Gly Leu Pro Ser 340 345 350 Pro Ala Leu Val Pro Glu Ala Gly Gly
Ser Gly Lys Glu Ala Leu Asp 355 360 365 Thr Ile Asp Val Gln Gly His
Pro Gln Thr Gly Met Arg Gly Thr Lys 370 375 380 Pro Asn Gln Val Val
Cys Val Ala Ala Gly Gly Gln Pro Glu Gly Gly 385 390 395 400 Leu Pro
Val Ser Pro Glu Pro Ser Leu Leu Thr Pro Thr Glu Glu Ala 405 410 415
His Pro Ala Ser Ser Leu Ala Ser Phe Pro Ala Ala Gln Ile Pro Ile 420
425 430 Ala Val Glu Glu Pro Gly Ser Ser Ser Arg Glu Ser Val Ser Lys
Ala 435 440 445 Gly Met Pro Val Ser Ala Asp Ala Ala Lys Glu Val Val
Asp Ala Gly 450 455 460 Leu Val Gly Leu Glu Arg Gln Val Ser Asp Leu
Gly Ser Lys Gly Glu 465 470 475 480 His Pro Glu Gly Asp Pro Gly Glu
Val
Pro Ala Pro Ser Pro Gln Glu 485 490 495 Arg Gly Glu His Leu Asn Thr
Glu Gln Ser His Glu Val Gln Pro Gly 500 505 510 Val Pro Pro Pro Pro
Leu Pro Lys Glu Gln Ser His Glu Val Gln Pro 515 520 525 Gly Ala Pro
Pro Pro Pro Leu Pro Lys Ala Pro Ser Glu Ser Ala Arg 530 535 540 Gly
Pro Pro Gly Pro Thr Asp Gly Ala Lys Val His Glu Asp Ser Thr 545 550
555 560 Ser Pro Ala Val Ala Lys Glu Gly Ser Arg Ser Pro Gly Asp Ser
Pro 565 570 575 Gly Gly Lys Glu Glu Ala Pro Glu Pro Pro Asp Gly Gly
Asp Pro Gly 580 585 590 Asn Leu Gln Gly Glu Asp Ser Gln Ala Phe Ser
Ser Lys Arg Asp Pro 595 600 605 Glu Val Gly Lys Asp Glu Leu Ser Lys
Pro Ser Ser Asp Ala Glu Ser 610 615 620 Arg Asp His Pro Ser Ser His
Ser Ala Gln Pro Pro Arg Lys Gly Gly 625 630 635 640 Ala Gly His Thr
Asp Gly Pro His Ser Gln Thr Ala Glu Ala Asp Ala 645 650 655 Ser Gly
Leu Pro His Lys Leu Gly Glu Glu Asp Pro Val Leu Pro Pro 660 665 670
Val Pro Asp Gly Ala Gly Glu Pro Thr Val Pro Glu Gly Ala Ile Trp 675
680 685 Glu Gly Ser Gly Leu Gln Pro Lys Cys Pro Asp Thr Leu Gln Ser
Arg 690 695 700 Glu Gly Leu Gly Arg Met Glu Ser Phe Leu Thr Leu Glu
Ser Glu Lys 705 710 715 720 Ser Asp Phe Pro Pro Thr Pro Val Ala Glu
Val Ala Pro Lys Ala Gln 725 730 735 Glu Gly Glu Ser Thr Leu Glu Ile
Arg Lys Met Gly Ser Cys Asp Gly 740 745 750 Glu Gly Leu Leu Thr Ser
Pro Asp Gln Pro Arg Gly Pro Ala Cys Asp 755 760 765 Ala Ser Arg Gln
Glu Phe His Ala Gly Val Pro His Pro Pro Gln Gly 770 775 780 Glu Asn
Leu Ala Ala Asp Leu Gly Leu Thr Ala Leu Ile Leu Asp Gln 785 790 795
800 Asp Gln Gln Gly Ile Pro Ser Cys Pro Gly Glu Gly Trp Ile Arg Gly
805 810 815 Ala Ala Ser Glu Trp Pro Leu Leu Ser Ser Glu Lys His Leu
Gln Pro 820 825 830 Ser Gln Ala Gln Pro Glu Thr Ser Ile Phe Asp Val
Leu Lys Glu Gln 835 840 845 Ala Gln Pro Pro Glu Asn Gly Lys Glu Thr
Ser Pro Ser His Pro Gly 850 855 860 Phe Lys Asp Gln Gly Ala Asp Ser
Ser Gln Ile His Val Pro Val Glu 865 870 875 880 Pro Gln Glu Asp Asn
Asn Leu Pro Thr His Gly Gly Gln Glu Gln Ala 885 890 895 Leu Gly Ser
Glu Leu Gln Ser Gln Leu Pro Lys Gly Thr Leu Ser Asp 900 905 910 Thr
Pro Thr Ser Ser Pro Thr Asp Met Val Trp Glu Ser Ser Leu Thr 915 920
925 Glu Glu Ser Glu Leu Ser Ala Pro Thr Arg Gln Lys Leu Pro Ala Leu
930 935 940 Gly Glu Lys Arg Pro Glu Gly Ala Cys Gly Asp Gly Gln Ser
Ser Arg 945 950 955 960 Val Ser Pro Pro Ala Ala Asp Val Leu Lys Asp
Phe Ser Leu Ala Gly 965 970 975 Asn Phe Ser Arg Lys Glu Thr Cys Cys
Thr Gly Gln Gly Pro Asn Lys 980 985 990 Ser Gln Gln Ala Leu Ala Asp
Ala Leu Glu Glu Gly Ser Gln His Glu 995 1000 1005 Glu Ala Cys Gln
Arg His Pro Gly Ala Ser Glu Ala Ala Asp Gly 1010 1015 1020 Cys Ser
Pro Leu Trp Gly Leu Ser Lys Arg Glu Met Ala Ser Gly 1025 1030 1035
Asn Thr Gly Glu Ala Pro Pro Cys Gln Pro Asp Ser Val Ala Leu 1040
1045 1050 Leu Asp Ala Val Pro Cys Leu Pro Ala Leu Ala Pro Ala Ser
Pro 1055 1060 1065 Gly Val Thr Pro Thr Gln Asp Ala Pro Glu Thr Glu
Ala Cys Asp 1070 1075 1080 Glu Thr Gln Glu Gly Arg Gln Gln Pro Val
Pro Ala Pro Gln Gln 1085 1090 1095 Lys Met Glu Cys Trp Ala Thr Ser
Asp Ala Glu Ser Pro Lys Leu 1100 1105 1110 Leu Ala Ser Phe Pro Ser
Ala Gly Glu Gln Gly Gly Glu Ala Gly 1115 1120 1125 Ala Ala Glu Thr
Gly Gly Ser Ala Gly Ala Gly Asp Pro Gly Lys 1130 1135 1140 Gln Gln
Ala Pro Glu Lys Pro Gly Glu Ala Thr Leu Ser Cys Gly 1145 1150 1155
Leu Leu Gln Thr Glu His Cys Leu Thr Ser Gly Glu Glu Ala Ser 1160
1165 1170 Thr Ser Ala Leu Arg Glu Ser Cys Gln Ala Glu His Pro Met
Ala 1175 1180 1185 Ser Cys Gln Asp Ala Leu Leu Pro Ala Arg Glu Leu
Gly Gly Ile 1190 1195 1200 Pro Arg Ser Thr Met Asp Phe Ser Thr His
Gln Ala Val Pro Asp 1205 1210 1215 Pro Lys Glu Leu Leu Leu Ser Gly
Pro Pro Glu Val Ala Ala Pro 1220 1225 1230 Asp Thr Pro Tyr Leu His
Val Asp Ser Ala Ala Gln Arg Gly Ala 1235 1240 1245 Glu Asp Ser Gly
Val Lys Ala Val Ser Ser Ala Asp Pro Arg Ala 1250 1255 1260 Pro Gly
Glu Ser Pro Cys Pro Val Gly Glu Pro Pro Leu Ala Leu 1265 1270 1275
Glu Asn Ala Ala Ser Leu Lys Leu Phe Ala Gly Ser Leu Ala Pro 1280
1285 1290 Leu Leu Gln Pro Gly Ala Ala Gly Gly Glu Ile Pro Ala Val
Gln 1295 1300 1305 Ala Ser Ser Gly Ser Pro Lys Ala Arg Thr Thr Glu
Gly Pro Val 1310 1315 1320 Asp Ser Met Pro Cys Leu Asp Arg Met Pro
Leu Leu Ala Lys Gly 1325 1330 1335 Lys Gln Ala Thr Gly Glu Glu Lys
Ala Ala Thr Ala Pro Gly Ala 1340 1345 1350 Gly Ala Lys Ala Ser Gly
Glu Gly Met Ala Gly Asp Ala Ala Gly 1355 1360 1365 Glu Thr Glu Gly
Ser Met Glu Arg Met Gly Glu Pro Ser Gln Asp 1370 1375 1380 Pro Lys
Gln Gly Thr Ser Gly Gly Val Asp Thr Ser Ser Glu Gln 1385 1390 1395
Ile Ala Thr Leu Thr Gly Phe Pro Asp Phe Arg Glu His Ile Ala 1400
1405 1410 Lys Ile Phe Glu Lys Pro Val Leu Gly Ala Leu Ala Thr Pro
Gly 1415 1420 1425 Glu Lys Ala Gly Ala Gly Arg Ser Ala Val Gly Lys
Asp Leu Thr 1430 1435 1440 Arg Pro Leu Gly Pro Glu Lys Leu Leu Asp
Gly Pro Pro Gly Val 1445 1450 1455 Asp Val Thr Leu Leu Pro Ala Pro
Pro Ala Arg Leu Gln Val Glu 1460 1465 1470 Lys Lys Gln Gln Leu Ala
Gly Glu Ala Glu Ile Ser His Leu Ala 1475 1480 1485 Leu Gln Asp Pro
Ala Ser Asp Lys Leu Leu Gly Pro Ala Gly Leu 1490 1495 1500 Thr Trp
Glu Arg Asn Leu Pro Gly Ala Gly Val Gly Lys Glu Met 1505 1510 1515
Ala Gly Val Pro Pro Thr Leu Arg Glu Asp Glu Arg Pro Glu Gly 1520
1525 1530 Pro Gly Ala Ala Trp Pro Gly Leu Glu Gly Gln Ala Tyr Ser
Gln 1535 1540 1545 Leu Glu Arg Ser Arg Gln Glu Leu Ala Ser Gly Leu
Pro Ser Pro 1550 1555 1560 Ala Ala Thr Gln Glu Leu Pro Val Glu Arg
Ala Ala Ala Phe Gln 1565 1570 1575 Val Ala Pro His Ser His Gly Glu
Glu Ala Val Ala Gln Asp Arg 1580 1585 1590 Ile Pro Ser Gly Lys Gln
His Gln Glu Thr Ser Ala Cys Asp Ser 1595 1600 1605 Pro His Gly Glu
Asp Gly Pro Gly Asp Phe Ala His Thr Gly Val 1610 1615 1620 Pro Gly
His Val Pro Arg Ser Thr Cys Ala Pro Ser Pro Gln Arg 1625 1630 1635
Glu Val Leu Thr Val Pro Glu Ala Asn Ser Glu Pro Trp Thr Leu 1640
1645 1650 Asp Thr Leu Gly Gly Glu Arg Arg Pro Gly Val Thr Ala Gly
Ile 1655 1660 1665 Leu Glu Met Arg Asn Ala Leu Gly Asn Gln Ser Thr
Pro Ala Pro 1670 1675 1680 Pro Thr Gly Glu Val Ala Asp Thr Pro Leu
Glu Pro Gly Lys Val 1685 1690 1695 Ala Gly Ala Ala Gly Glu Ala Glu
Gly Asp Ile Thr Leu Ser Thr 1700 1705 1710 Ala Glu Thr Gln Ala Cys
Ala Ser Gly Asp Leu Pro Glu Ala Gly 1715 1720 1725 Thr Thr Arg Thr
Phe Ser Val Val Ala Gly Asp Leu Val Leu Pro 1730 1735 1740 Gly Ser
Cys Gln Asp Pro Ala Cys Ser Asp Lys Ala Pro Gly Met 1745 1750 1755
Glu Gly Thr Ala Ala Leu His Gly Asp Ser Pro Ala Arg Pro Gln 1760
1765 1770 Gln Ala Lys Glu Gln Pro Gly Pro Glu Arg Pro Ile Pro Ala
Gly 1775 1780 1785 Asp Gly Lys Val Cys Val Ser Ser Pro Pro Glu Pro
Asp Glu Thr 1790 1795 1800 His Asp Pro Lys Leu Gln His Leu Ala Pro
Glu Glu Leu His Thr 1805 1810 1815 Asp Arg Glu Ser Pro Arg Pro Gly
Pro Ser Met Leu Pro Ser Val 1820 1825 1830 Pro Lys Lys Asp Ala Pro
Arg Val Met Asp Lys Val Thr Ser Asp 1835 1840 1845 Glu Thr Arg Gly
Ala Glu Gly Thr Glu Ser Ser Pro Val Ala Asp 1850 1855 1860 Asp Ile
Ile Gln Pro Ala Ala Pro Ala Asp Leu Glu Ser Pro Thr 1865 1870 1875
Leu Ala Ala Ser Ser Tyr His Gly Asp Val Val Gly Gln Val Ser 1880
1885 1890 Thr Asp Leu Ile Ala Gln Ser Ile Ser Pro Ala Ala Ala His
Ala 1895 1900 1905 Gly Leu Pro Pro Ser Ala Ala Glu His Ile Val Ser
Pro Ser Ala 1910 1915 1920 Pro Ala Gly Asp Arg Val Glu Ala Ser Thr
Pro Ser Cys Pro Asp 1925 1930 1935 Pro Ala Lys Asp Leu Ser Arg Ser
Ser Asp Ser Glu Glu Ala Phe 1940 1945 1950 Glu Thr Pro Glu Ser Thr
Thr Pro Val Lys Ala Pro Pro Ala Pro 1955 1960 1965 Pro Pro Pro Pro
Pro Glu Val Ile Pro Glu Pro Glu Val Ser Thr 1970 1975 1980 Gln Pro
Pro Pro Glu Glu Pro Gly Cys Gly Ser Glu Thr Val Pro 1985 1990 1995
Val Pro Asp Gly Pro Arg Ser Asp Ser Val Glu Gly Ser Pro Phe 2000
2005 2010 Arg Pro Pro Ser His Ser Phe Ser Ala Val Phe Asp Glu Asp
Lys 2015 2020 2025 Pro Ile Ala Ser Ser Gly Thr Tyr Asn Leu Asp Phe
Asp Asn Ile 2030 2035 2040 Glu Leu Val Asp Thr Phe Gln Thr Leu Glu
Pro Arg Ala Ser Asp 2045 2050 2055 Ala Lys Asn Gln Glu Gly Lys Val
Asn Thr Arg Arg Lys Ser Thr 2060 2065 2070 Asp Ser Val Pro Ile Ser
Lys Ser Thr Leu Ser Arg Ser Leu Ser 2075 2080 2085 Leu Gln Ala Ser
Asp Phe Asp Gly Ala Ser Ser Ser Gly Asn Pro 2090 2095 2100 Glu Ala
Val Ala Leu Ala Pro Asp Ala Tyr Ser Thr Gly Ser Ser 2105 2110 2115
Ser Ala Ser Ser Thr Leu Lys Arg Thr Lys Lys Pro Arg Pro Pro 2120
2125 2130 Ser Leu Lys Lys Lys Gln Thr Thr Lys Lys Pro Thr Glu Thr
Pro 2135 2140 2145 Pro Val Lys Glu Thr Gln Gln Glu Pro Asp Glu Glu
Ser Leu Val 2150 2155 2160 Pro Ser Gly Glu Asn Leu Ala Ser Glu Thr
Lys Thr Glu Ser Ala 2165 2170 2175 Lys Thr Glu Gly Pro Ser Pro Ala
Leu Leu Glu Glu Thr Pro Leu 2180 2185 2190 Glu Pro Ala Val Gly Pro
Lys Ala Ala Cys Pro Leu Asp Ser Glu 2195 2200 2205 Ser Ala Glu Gly
Val Val Pro Pro Ala Ser Gly Gly Gly Arg Val 2210 2215 2220 Gln Asn
Ser Pro Pro Val Gly Arg Lys Thr Leu Pro Leu Thr Thr 2225 2230 2235
Ala Pro Glu Ala Gly Glu Val Thr Pro Ser Asp Ser Gly Gly Gln 2240
2245 2250 Glu Asp Ser Pro Ala Lys Gly Leu Ser Val Arg Leu Glu Phe
Asp 2255 2260 2265 Tyr Ser Glu Asp Lys Ser Ser Trp Asp Asn Gln Gln
Glu Asn Pro 2270 2275 2280 Pro Pro Thr Lys Lys Ile Gly Lys Lys Pro
Val Ala Lys Met Pro 2285 2290 2295 Leu Arg Arg Pro Lys Met Lys Lys
Thr Pro Glu Lys Leu Asp Asn 2300 2305 2310 Thr Pro Ala Ser Pro Pro
Arg Ser Pro Ala Glu Pro Asn Asp Ile 2315 2320 2325 Pro Ile Ala Lys
Gly Thr Tyr Thr Phe Asp Ile Asp Lys Trp Asp 2330 2335 2340 Asp Pro
Asn Phe Asn Pro Phe Ser Ser Thr Ser Lys Met Gln Glu 2345 2350 2355
Ser Pro Lys Leu Pro Gln Gln Ser Tyr Asn Phe Asp Pro Asp Thr 2360
2365 2370 Cys Asp Glu Ser Val Asp Pro Phe Lys Thr Ser Ser Lys Thr
Pro 2375 2380 2385 Ser Ser Pro Ser Lys Ser Pro Ala Ser Phe Glu Ile
Pro Ala Ser 2390 2395 2400 Ala Met Glu Ala Asn Gly Val Asp Gly Asp
Gly Leu Asn Lys Pro 2405 2410 2415 Ala Lys Lys Lys Lys Thr Pro Leu
Lys Thr Asp Thr Phe Arg Val 2420 2425 2430 Lys Lys Ser Pro Lys Arg
Ser Pro Leu Ser Asp Pro Pro Ser Gln 2435 2440 2445 Asp Pro Thr Pro
Ala Ala Thr Pro Glu Thr Pro Pro Val Ile Ser 2450 2455 2460 Ala Val
Val His Ala Thr Asp Glu Glu Lys Leu Ala Val Thr Asn 2465 2470 2475
Gln Lys Trp Thr Cys Met Thr Val Asp Leu Glu Ala Asp Lys Gln 2480
2485 2490 Asp Tyr Pro Gln Pro Ser Asp Leu Ser Thr Phe Val Asn Glu
Thr 2495 2500 2505 Lys Phe Ser Ser Pro Thr Glu Glu Leu Asp Tyr Arg
Asn Ser Tyr 2510 2515 2520 Glu Ile Glu Tyr Met Glu Lys Ile Gly Ser
Ser Leu Pro Gln Asp 2525 2530 2535 Asp Asp Ala Pro Lys Lys Gln Ala
Leu Tyr Leu Met Phe Asp Thr 2540 2545 2550 Ser Gln Glu Ser Pro Val
Lys Ser Ser Pro Val Arg Met Ser Glu 2555 2560 2565 Ser Pro Thr Pro
Cys Ser Gly Ser Ser Phe Glu Glu Thr Glu Ala 2570 2575 2580 Leu Val
Asn Thr Ala Ala Lys Asn Gln His Pro Val Pro Arg Gly 2585 2590 2595
Leu Ala Pro Asn Gln Glu Ser His Leu Gln Val Pro Glu Lys Ser 2600
2605 2610 Ser Gln Lys Glu Leu Glu Ala Met Gly Leu Gly Thr Pro Ser
Glu 2615 2620 2625 Ala Ile Glu Ile Thr Ala Pro Glu Gly Ser Phe Ala
Ser Ala Asp 2630 2635 2640 Ala Leu Leu Ser Arg Leu Ala His Pro Val
Ser Leu Cys Gly Ala 2645 2650 2655 Leu Asp Tyr Leu Glu Pro Asp Leu
Ala Glu Lys Asn Pro Pro Leu 2660 2665 2670 Phe Ala Gln Lys Leu Gln
Glu Glu Leu Glu Phe Ala Ile Met Arg 2675 2680 2685 Ile Glu Ala Leu
Lys Leu Ala Arg Gln Ile Ala Leu Ala Ser Arg 2690 2695 2700 Ser
His
Gln Asp Ala Lys Arg Glu Ala Ala His Pro Thr Asp Val 2705 2710 2715
Ser Ile Ser Lys Thr Ala Leu Tyr Ser Arg Ile Gly Thr Ala Glu 2720
2725 2730 Val Glu Lys Pro Ala Gly Leu Leu Phe Gln Gln Pro Asp Leu
Asp 2735 2740 2745 Ser Ala Leu Gln Ile Ala Arg Ala Glu Ile Ile Thr
Lys Glu Arg 2750 2755 2760 Glu Val Ser Glu Trp Lys Asp Lys Tyr Glu
Glu Ser Arg Arg Glu 2765 2770 2775 Val Met Glu Met Arg Lys Ile Val
Ala Glu Tyr Glu Lys Thr Ile 2780 2785 2790 Ala Gln Met Ile Glu Asp
Glu Gln Arg Glu Lys Ser Val Ser His 2795 2800 2805 Gln Thr Val Gln
Gln Leu Val Leu Glu Lys Glu Gln Ala Leu Ala 2810 2815 2820 Asp Leu
Asn Ser Val Glu Lys Ser Leu Ala Asp Leu Phe Arg Arg 2825 2830 2835
Tyr Glu Lys Met Lys Glu Val Leu Glu Gly Phe Arg Lys Asn Glu 2840
2845 2850 Glu Val Leu Lys Arg Cys Ala Gln Glu Tyr Leu Ser Arg Val
Lys 2855 2860 2865 Lys Glu Glu Gln Arg Tyr Gln Ala Leu Lys Val His
Ala Glu Glu 2870 2875 2880 Lys Leu Asp Arg Ala Asn Ala Glu Ile Ala
Gln Val Arg Gly Lys 2885 2890 2895 Ala Gln Gln Glu Gln Ala Ala His
Gln Ala Ser Leu Arg Lys Glu 2900 2905 2910 Gln Leu Arg Val Asp Ala
Leu Glu Arg Thr Leu Glu Gln Lys Asn 2915 2920 2925 Lys Glu Ile Glu
Glu Leu Thr Lys Ile Cys Asp Glu Leu Ile Ala 2930 2935 2940 Lys Met
Gly Lys Ser 2945 1559706DNAHomo sapiens 155gcctgctcca agggaaggat
caggagagaa gaaacgcaaa tcccagaacc gtgccaacat 60ataaaacccc acattaaggg
ttgtacagtg cactgggatt tctcaagtca cccgcttggt 120cctcttccaa
gtatacttta cttcctttca ttcctctcta aaactttttt aaaaactttc
180actcctgctc taaaagttat cttggtttct tactctacct tatgcccctt
gggcgaattt 240tttcctctga ggagggaaga atagagttgc tgctgcagac
acatcagatt ccctactggt 300aacagctgga gtgcgtcacc tctgacaaaa
ttctggggac gctgggaaca ctgaatcaac 360atgggcaatg agaacagcac
ctcggacaac cagaggactt tatcagctca gactccaagg 420tccgcgcagc
cacccgggaa cagtcagaat ataaaaagga agcagcagga cacgcccgga
480agccctgacc acagagacgc gtccagcatt ggcagcgttg ggcttggagg
cttctgcacc 540gcttctgaga gttctgccag cctggatcca tgccttgtgt
ccccagaggt gactgagcca 600aggaaggacc cacagggagc cagggggcca
gaaggttctt tgctgcccag cccaccaccg 660tcccaggagc gagagcaccc
ctcgtcctcc atgccctttg ccgagtgtcc cccggaaggt 720tgcttggcaa
gtccagcagc ggcacctgaa gatggtcctc agactcagtc tcccaggagg
780gaacctgccc caaatgcccc aggagacatc gcggcggcat ttcccgctga
gagggacagc 840tctactccat accaagagat tgctgccgtc cccagtgctg
gaagagagag acagccgaag 900gaagaaggac agaagtcctc cttctccttc
tccagtggca tcgaccagtc acctggaatg 960tcgccagtac ccctcagaga
gccaatgaag gcaccgctgt gtggagaggg ggaccagcct 1020ggtggttttg
agtcccaaga gaaagaggct gcaggtggct ttccccctgc agagtccagg
1080cagggggtgg cttctgtgca agtgacccct gaggcccctg ctgcagccca
gcagggcaca 1140gaaagctcag cggtcttgga gaagtccccc ctaaaaccca
tggccccgat cccacaagat 1200ccagccccaa gagcctcaga cagagaaaga
ggccaagggg aggcgccgcc tcagtattta 1260acagatgact tggaattcct
cagggcctgc catctcccta ggagcaattc aggggctgcc 1320ccagaagcag
aagtgaatgc cgcttcccag gagagctgcc agcagccagt gggagcatat
1380ctgccgcacg cagagctgcc ctggggcttg ccaagtcctg ccctggtgcc
agaggctggg 1440ggctctggga aggaggctct ggacaccatt gatgttcagg
gtcacccaca gacagggatg 1500cgaggaacca agcccaatca agttgtctgt
gtggcagcag gcggccagcc cgaagggggt 1560ttgcctgtga gccctgaacc
ttccctgctc actccgactg aggaagcaca tccagcttca 1620agcctcgctt
cattcccagc tgctcagatt cctattgctg tagaagaacc tggatcatca
1680tccagggaat cagtttccaa ggctgggatg ccagtttctg cagatgcagc
caaagaggtg 1740gtggatgcag ggttggtggg actggagagg caggtgtcag
atcttggaag caagggagag 1800catccagaag gggaccctgg agaggttcct
gccccatcac cccaggagag gggagagcac 1860ttgaacacgg agcaaagcca
tgaggtccaa ccaggagtac caccccctcc tcttcccaag 1920gagcaaagcc
atgaggtcca accaggagca ccaccccctc ctcttcccaa ggcaccaagt
1980gaaagtgcca gagggccacc ggggccaacg gatggagcca aggtccatga
agattccaca 2040agcccagccg tggctaaaga aggaagcaga tcacctggtg
acagccctgg aggaaaggag 2100gaagccccag agccacctga tggtggagac
ccagggaacc tgcaaggaga ggactctcag 2160gctttcagca gcaagcgtga
tccagaagta ggcaaagatg agctttcaaa gccaagcagt 2220gatgcagaga
gcagagacca tcccagctca cactcagcac agccacccag aaaggggggt
2280gctgggcaca cggacgggcc ccactctcag acagcagagg ctgatgcatc
tggcctacca 2340cacaagctgg gtgaggagga ccccgtcctg ccccctgtgc
cagatggagc tggtgagccc 2400actgttcccg aaggagccat ctgggagggg
tcaggattgc agcccaaatg tcctgacacc 2460cttcagagca gggaaggatt
gggaagaatg gagtctttcc tgactttaga atcagagaaa 2520tcagattttc
caccaactcc tgttgcagag gttgcaccca aagcccagga aggtgagagc
2580acattggaaa taaggaagat gggcagctgt gatggggagg gcttgctgac
gtccccagat 2640caaccccgcg ggccggcgtg tgatgcgtcg agacaggaat
ttcatgctgg ggtgccacat 2700cccccccagg gggagaactt ggcagcagac
ctggggctca cggcactcat cctggaccaa 2760gatcagcagg gaatcccatc
ctgcccaggg gaaggctgga taagaggagc tgcatccgag 2820tggcccctac
tatcttctga gaagcatctc cagccatccc aggcacaacc agagacatcc
2880atctttgacg tgctcaagga gcaggcccag ccacctgaaa atgggaaaga
gacttctcca 2940agccatccag gttttaagga ccagggagca gattcttccc
aaatccatgt acctgtggaa 3000cctcaggaag ataacaactt gcccactcat
ggaggacagg agcaggcttt gggatcagaa 3060cttcaaagtc agctccccaa
aggcaccctg tctgatactc caacttcatc tcccactgac 3120atggtttggg
agagttctct gacagaagag tcagaattgt cagcaccaac gagacagaag
3180ttgcctgcac taggggagaa gcggccagag ggagcatgcg gtgatggtca
gtcctcgagg 3240gtctcgcctc cagcagcaga tgtcttaaaa gacttttctc
ttgcagggaa cttcagcaga 3300aaggaaactt gctgcactgg gcaggggcca
aacaagtctc aacaggcatt ggctgatgcc 3360ttggaagaag gcagccagca
tgaagaagca tgtcaaaggc atccaggagc ttctgaagca 3420gctgatggtt
gttccccact ctggggcttg agtaagaggg agatggcaag tggaaacaca
3480ggggaggccc caccttgtca gcctgactca gtagctctcc tggatgcagt
tccctgcctg 3540ccagccctgg cgcccgccag ccccggagtc acacccaccc
aggatgcccc agagacagag 3600gcatgtgatg aaacccagga aggcaggcag
caaccagtgc cggccccgca gcagaaaatg 3660gagtgctggg ccacttcgga
tgcagagtcc ccaaagcttc ttgcaagttt cccatcagct 3720ggggagcaag
gtggtgaagc cggggctgct gagactggtg gcagcgctgg tgcaggagac
3780ccaggaaagc agcaggctcc ggagaaacct ggagaagcta ctttgagttg
tggcctcctt 3840cagactgagc actgccttac ctccggggag gaagcttcta
cctctgccct acgtgagtcc 3900tgccaagctg agcaccccat ggccagctgc
caggatgcct tgctgccagc cagagagctg 3960ggtgggattc ccaggagcac
catggatttt tctacacacc aggctgtccc agacccaaag 4020gagctcctgc
tgtctgggcc accagaagtg gctgctcctg acacccctta cctgcatgtc
4080gacagtgctg cccagagagg agcagaagac agtggagtga aagctgtttc
ctctgcagac 4140cccagagctc ctggcgaaag cccctgtcct gtaggggagc
ccccacttgc cttggaaaat 4200gctgcctcct tgaagctgtt tgctggctcc
ctcgcccccc tgttgcaacc aggagctgca 4260ggtggggaaa tccctgcagt
gcaagccagc agtggtagtc ccaaagccag aaccactgag 4320ggaccagtgg
actccatgcc atgcctggac cggatgccac ttctggccaa gggcaagcag
4380gcaacagggg aagagaaagc agcaacagct ccaggtgcag gtgccaaggc
cagtggggag 4440ggcatggcag gtgatgcagc aggagagaca gagggcagca
tggagaggat gggagagcct 4500tcccaggacc caaagcaggg cacatcaggt
ggtgtggaca caagctctga gcaaatcgcc 4560accctcactg gcttcccaga
cttcagggag cacatcgcca agatcttcga gaagcctgtg 4620ctcggagccc
tggccacacc tggagaaaag gcaggagctg ggaggagtgc agtgggtaaa
4680gacctcacca ggccattggg cccagagaag cttctagatg ggcctccagg
agtggatgtc 4740acccttctcc ctgcacctcc tgctcgactc caggtggaga
agaagcaaca gttggctgga 4800gaggctgaga tttcccatct ggctctgcaa
gatccagctt cagacaagct tctgggtcca 4860gcagggctga cctgggagcg
gaacttgcca ggtgccggtg tggggaagga gatggcaggt 4920gtcccaccca
cactgaggga agacgagagg ccagaggggc ctggggcagc ctggccaggc
4980ctggaaggcc aggcttactc acagctggag aggagcaggc aggaattagc
ttcaggtctt 5040ccttcaccag cagctactca ggagctccct gtggagagag
ctgctgcctt ccaggtggct 5100ccccatagcc atggagaaga ggccgtggcc
caagacagaa ttccttctgg aaagcagcac 5160caggaaacat ctgcctgcga
cagtccacat ggagaagatg gtcccgggga ctttgctcac 5220acaggggttc
caggacatgt gccaaggtcc acgtgtgccc cttctcctca gagggaggtt
5280ttgactgtgc ctgaggccaa cagtgagccc tggacccttg acacgcttgg
gggtgaaagg 5340agacccggag tcactgctgg catcttggaa atgcgaaatg
ccctgggcaa ccagagcacc 5400cctgcaccac caactggaga agtggcagac
actcccctgg agcctggcaa ggtggcaggc 5460gctgctgggg aagcagaggg
tgacatcacc ctgagcacag ctgagacaca ggcatgtgcg 5520tccggtgatc
tgcctgaagc aggtactacg aggacattct ccgttgtggc aggtgacttg
5580gtgctgccag gaagctgtca ggacccagcc tgctctgaca aggctccggg
gatggagggt 5640acagctgccc ttcatgggga cagcccagcc aggccccagc
aggctaagga gcagccaggg 5700cctgagcgcc ccattccagc tggggatggg
aaggtgtgcg tctcctcacc tccagagcct 5760gacgaaactc acgacccgaa
gctgcaacat ttggctccag aagagctcca cactgacaga 5820gagagcccca
ggcctggccc atccatgtta ccttcggttc ctaagaagga tgctccaaga
5880gtcatggata aagtcacttc agatgagacc agaggtgcgg aaggaacaga
aagttcacct 5940gtggcagatg atatcatcca gcccgctgcc cccgcagacc
tggaaagccc aaccttagct 6000gcctcttcct accacggtga tgttgttggc
caggtctcta cggatctgat agcccagagc 6060atctccccag ctgctgccca
tgcgggtctt cctccctcgg ctgcagaaca catagtttcg 6120ccatctgccc
cagctggtga cagagtagaa gcttccactc cctcctgccc agatccggcc
6180aaggacctca gcaggagttc cgattctgaa gaggcatttg agaccccgga
gtcaacgacc 6240cctgtcaaag ctccgccagc tccaccccca ccaccccccg
aagtcatccc agaacccgag 6300gtcagcacac agccaccccc ggaagaacca
ggatgtggtt ctgagacagt ccctgtccct 6360gatggcccac ggagcgactc
ggtggaagga agtcccttcc gtcccccgtc acactccttc 6420tctgccgtct
tcgatgaaga caagccgata gccagcagtg ggacttacaa cttggacttt
6480gacaacattg agcttgtgga tacctttcag accttggagc ctcgtgcctc
agacgctaag 6540aatcaggagg gcaaagtgaa cacacggagg aagtccacgg
attccgtccc catctctaag 6600tctacactgt cccggtcgct cagcctgcaa
gccagtgact ttgatggtgc ttcttcctca 6660ggcaatcccg aggccgtggc
ccttgcccca gatgcatata gcacgggttc cagcagtgct 6720tctagtaccc
ttaagcgaac taaaaaaccg aggccgcctt ccttaaaaaa gaaacagacc
6780accaagaaac ccacagagac ccccccagtg aaggagacgc aacaggagcc
agatgaagag 6840agccttgtcc ccagtgggga gaatctagca tctgagacga
aaacggaatc tgccaagacg 6900gaaggtccta gcccagcctt attggaggag
acgccccttg agcccgctgt ggggcccaaa 6960gctgcctgcc ctctggactc
agagagtgca gaaggggttg tccccccggc ttctggaggt 7020ggcagagtgc
agaactcacc ccctgtcggg aggaaaacgc tgcctcttac cacggccccg
7080gaggcagggg aggtaacccc atcggatagc ggggggcaag aggactctcc
agccaaaggg 7140ctctccgtaa ggctggagtt tgactattct gaggacaaga
gtagttggga caaccagcag 7200gaaaaccccc ctcctaccaa aaagataggc
aaaaagccag ttgccaaaat gcccctgagg 7260aggccaaaga tgaaaaagac
acccgagaaa cttgacaaca ctcctgcctc acctcccaga 7320tcccctgctg
aacccaatga catccccatt gctaaaggta cttacacctt tgatattgac
7380aagtgggatg accccaattt taaccctttt tcttccacct caaaaatgca
ggagtctccc 7440aaactgcccc aacaatcata caactttgac ccagacacct
gtgatgagtc cgttgacccc 7500tttaagacat cctctaagac ccccagctca
ccttctaaat ccccagcctc ctttgagatc 7560ccagccagtg ctatggaagc
caatggagtg gacggggatg ggctaaacaa gcccgccaag 7620aagaagaaga
cgcccctaaa gactgacaca tttagggtga aaaagtcgcc aaaacggtct
7680cctctctctg atccaccttc ccaggacccc accccagctg ctacaccaga
aacaccacca 7740gtgatctctg cggtggtcca cgccacagat gaggaaaagc
tggcggtcac caaccagaag 7800tggacgtgca tgacagtgga cctagaggct
gacaaacagg actacccgca gccctcggac 7860ctgtccacct ttgtaaacga
gaccaaattc agttcaccca ctgaggagtt ggattacaga 7920aactcctatg
aaattgaata tatggagaaa attggctcct ccttacctca ggacgacgat
7980gccccgaaga agcaggcctt gtaccttatg tttgacactt ctcaggagag
ccctgtcaag 8040tcatctcccg tccgcatgtc agagtccccg acgccgtgtt
cagggtcaag ttttgaagag 8100actgaagccc ttgtgaacac tgctgcgaaa
aaccagcatc ctgtcccacg aggactggcc 8160cctaaccaag agtcacactt
gcaggtgcca gagaaatcct cccagaagga gctggaggcc 8220atgggcttgg
gcaccccttc agaagcgatt gaaattacag ctcccgaggg ctcctttgcc
8280tctgctgacg ccctcctcag caggctagct caccccgtct ctctctgtgg
tgcacttgac 8340tatctggagc ccgacttagc agaaaagaac cccccactat
tcgctcagaa actccaggag 8400gagttagagt ttgccatcat gcggatagaa
gccctgaagc tggccaggca gattgctttg 8460gcttcccgca gccaccagga
tgccaagaga gaggctgctc acccaacaga cgtctccatc 8520tccaaaacag
ccttgtactc ccgcatcggg accgctgagg tggagaaacc tgcaggcctt
8580ctgttccagc agcccgacct ggactctgcc ctccagatcg ccagagcaga
gatcataacc 8640aaggagagag aggtctcaga atggaaagat aaatatgaag
aaagcaggcg ggaagtgatg 8700gaaatgagga aaatagtggc cgagtatgag
aagaccatcg ctcagatgat agaggacgaa 8760cagagagaga agtcagtctc
ccaccagacg gtgcagcagc tggttctgga gaaggagcaa 8820gccctggccg
acctgaactc cgtggagaag tctctggccg acctcttcag aagatatgag
8880aagatgaagg aggtcctaga aggcttccgc aagaatgaag aggtgttgaa
gagatgtgcg 8940caggagtacc tgtcccgggt gaagaaggag gagcagaggt
accaggccct gaaggtgcac 9000gcggaggaga aactggacag ggccaatgct
gagattgctc aggttcgagg caaggcccag 9060caggagcaag ccgcccacca
ggccagcctg cggaaggagc agctgcgagt ggacgccctg 9120gaaaggacgc
tggagcagaa gaataaagaa atagaagaac tcaccaagat ttgtgacgaa
9180ctgattgcca aaatggggaa aagctaactc tgaaccgaat gttttggact
taactgttgc 9240gtgcaatatg accgtcggca cactgctgtt cctccagttc
catggacagg ttctgttttc 9300actttttcgt atgcactact gtatttcctt
tctaaataaa attgatttga ttgtatgcag 9360tactaaggag actatcagaa
tttcttgcta ttggtttgca ttttcctagt ataattcata 9420gcaagttgac
ctcagagttc ctgtatcagg gagattgtct gattctctaa taaaagacac
9480attgctgacc ttggccttgc cctttgtaca caagttccca gggtgagcag
cttttggatt 9540taatatgaac atgtacagcg tgcataggga ctcttgcctt
aaggagtgta aacttgatct 9600gcatttgctg atttgttttt aaaaaaacaa
gaaatgcatg tttcaaataa aattctctat 9660tgtaaataaa attttttctt
tggatcttgg caaaaaaaaa aaaaaa 970615613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 156Ala
Gln Ala Glu Ala Leu Ala Leu Gln Ala Ser Leu Arg 1 5 10
1576PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 157Phe Asp Pro Leu Leu Arg 1 5
1581048PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 158Met Gly Ala Pro Ala Cys Ala Leu Ala Leu
Cys Val Ala Val Ala Ile 1 5 10 15 Val Ala Gly Ala Ser Ser Glu Ser
Leu Gly Thr Glu Gln Arg Val Val 20 25 30 Gly Arg Ala Ala Glu Val
Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40 45 Leu Val Phe Gly
Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro 50 55 60 Gly Gly
Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly 65 70 75 80
Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85
90 95 Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln
Arg 100 105 110 Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val
Thr Asp Ala 115 120 125 Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp
Glu Ala Glu Asp Thr 130 135 140 Gly Val Asp Thr Gly Ala Pro Tyr Trp
Thr Arg Pro Glu Arg Met Asp 145 150 155 160 Lys Lys Leu Leu Ala Val
Pro Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170 175 Pro Ala Ala Gly
Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly 180 185 190 Arg Glu
Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205
Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210
215 220 Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln
Thr 225 230 235 240 Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg
Pro Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala Asn Gln Thr Ala Val
Leu Gly Ser Asp Val Glu 260 265 270 Phe His Cys Lys Val Tyr Ser Asp
Ala Gln Pro His Ile Gln Trp Leu 275 280 285 Lys His Val Glu Val Asn
Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295 300 Tyr Val Thr Val
Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu 305 310 315 320 Leu
Glu Val Leu Ser Leu His Asn Val Thr Phe Glu Asp Ala Gly Glu 325 330
335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Phe Ser His His Ser Ala
340 345 350 Trp Leu Val Val Leu Pro Ala Glu Glu Glu Leu Val Glu Ala
Asp Glu 355 360 365 Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly
Val Gly Phe Phe 370 375 380 Leu Phe Ile Leu Val Val Ala Ala Val Thr
Leu Cys Arg Leu Arg Ser 385 390 395 400 Pro Pro Lys Lys Gly Leu Gly
Ser Pro Thr Val His Lys Ile Ser Arg 405 410 415 Phe Pro Leu Lys Arg
Gln Val Ser Leu Glu Ser Asn Ala Ser Met Ser 420 425 430 Ser Asn Thr
Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly 435 440 445 Pro
Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys 450 455
460 Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu
465 470 475 480 Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly
Ile Asp Lys 485 490 495 Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val
Lys Met Leu Lys
Asp 500 505 510 Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu
Met Glu Met 515 520 525 Met Lys Met Ile Gly Lys His Lys Asn Ile Ile
Asn Leu Leu Gly Ala 530 535 540 Cys Thr Gln Gly Gly Pro Leu Tyr Val
Leu Val Glu Tyr Ala Ala Lys 545 550 555 560 Gly Asn Leu Arg Glu Phe
Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp 565 570 575 Tyr Ser Phe Asp
Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys 580 585 590 Asp Leu
Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu 595 600 605
Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu 610
615 620 Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala
Arg 625 630 635 640 Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr
Asn Gly Arg Leu 645 650 655 Pro Val Lys Trp Met Ala Pro Glu Ala Leu
Phe Asp Arg Val Tyr Thr 660 665 670 His Gln Ser Asp Val Trp Ser Phe
Gly Val Leu Leu Trp Glu Ile Phe 675 680 685 Thr Leu Gly Gly Ser Pro
Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 690 695 700 Lys Leu Leu Lys
Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr 705 710 715 720 His
Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser 725 730
735 Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu
740 745 750 Thr Val Thr Ser Thr Asp Phe Lys Glu Ser Ala Leu Arg Lys
Gln Ser 755 760 765 Leu Tyr Leu Lys Phe Asp Pro Leu Leu Arg Asp Ser
Pro Gly Arg Pro 770 775 780 Val Pro Val Ala Thr Glu Thr Ser Ser Met
His Gly Ala Asn Glu Thr 785 790 795 800 Pro Ser Gly Arg Pro Arg Glu
Ala Lys Leu Val Glu Phe Asp Phe Leu 805 810 815 Gly Ala Leu Asp Ile
Pro Val Pro Gly Pro Pro Pro Gly Val Pro Ala 820 825 830 Pro Gly Gly
Pro Pro Leu Ser Thr Gly Pro Ile Val Asp Leu Leu Gln 835 840 845 Tyr
Ser Gln Lys Asp Leu Asp Ala Val Val Lys Ala Thr Gln Glu Glu 850 855
860 Asn Arg Glu Leu Arg Ser Arg Cys Glu Glu Leu His Gly Lys Asn Leu
865 870 875 880 Glu Leu Gly Lys Ile Met Asp Arg Phe Glu Glu Val Val
Tyr Gln Ala 885 890 895 Met Glu Glu Val Gln Lys Gln Lys Glu Leu Ser
Lys Ala Glu Ile Gln 900 905 910 Lys Val Leu Lys Glu Lys Asp Gln Leu
Thr Thr Asp Leu Asn Ser Met 915 920 925 Glu Lys Ser Phe Ser Asp Leu
Phe Lys Arg Phe Glu Lys Gln Lys Glu 930 935 940 Val Ile Glu Gly Tyr
Arg Lys Asn Glu Glu Ser Leu Lys Lys Cys Val 945 950 955 960 Glu Asp
Tyr Leu Ala Arg Ile Thr Gln Glu Gly Gln Arg Tyr Gln Ala 965 970 975
Leu Lys Ala His Ala Glu Glu Lys Leu Gln Leu Ala Asn Glu Glu Ile 980
985 990 Ala Gln Val Arg Ser Lys Ala Gln Ala Glu Ala Leu Ala Leu Gln
Ala 995 1000 1005 Ser Leu Arg Lys Glu Gln Met Arg Ile Gln Ser Leu
Glu Lys Thr 1010 1015 1020 Val Glu Gln Lys Thr Lys Glu Asn Glu Glu
Leu Thr Arg Ile Cys 1025 1030 1035 Asp Asp Leu Ile Ser Lys Met Glu
Lys Ile 1040 1045 1591027PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 159Met Gly Ala Pro Ala
Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile 1 5 10 15 Val Ala Gly
Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25 30 Gly
Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40
45 Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro
50 55 60 Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly
Thr Gly 65 70 75 80 Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln
Arg Leu Gln Val 85 90 95 Leu Asn Ala Ser His Glu Asp Ser Gly Ala
Tyr Ser Cys Arg Gln Arg 100 105 110 Leu Thr Gln Arg Val Leu Cys His
Phe Ser Val Arg Val Thr Asp Ala 115 120 125 Pro Ser Ser Gly Asp Asp
Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140 Gly Val Asp Thr
Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp 145 150 155 160 Lys
Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170
175 Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly
180 185 190 Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu
Arg His 195 200 205 Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro
Ser Asp Arg Gly 210 215 220 Asn Tyr Thr Cys Val Val Glu Asn Lys Phe
Gly Ser Ile Arg Gln Thr 225 230 235 240 Tyr Thr Leu Asp Val Leu Glu
Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala
Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270 Phe His Cys
Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280 285 Lys
His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295
300 Tyr Val Thr Val Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu
305 310 315 320 Leu Glu Val Leu Ser Leu His Asn Val Thr Phe Glu Asp
Ala Gly Glu 325 330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Phe
Ser His His Ser Ala 340 345 350 Trp Leu Val Val Leu Pro Ala Glu Glu
Glu Leu Val Glu Ala Asp Glu 355 360 365 Ala Gly Ser Val Tyr Ala Gly
Ile Leu Ser Tyr Gly Val Gly Phe Phe 370 375 380 Leu Phe Ile Leu Val
Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser 385 390 395 400 Pro Pro
Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile Ser Arg 405 410 415
Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser Met Ser 420
425 430 Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly Glu
Gly 435 440 445 Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala
Asp Pro Lys 450 455 460 Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly
Lys Pro Leu Gly Glu 465 470 475 480 Gly Cys Phe Gly Gln Val Val Met
Ala Glu Ala Ile Gly Ile Asp Lys 485 490 495 Asp Arg Ala Ala Lys Pro
Val Thr Val Ala Val Lys Met Leu Lys Asp 500 505 510 Asp Ala Thr Asp
Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 515 520 525 Met Lys
Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 530 535 540
Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys 545
550 555 560 Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly
Leu Asp 565 570 575 Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln
Leu Thr Phe Lys 580 585 590 Asp Leu Val Ser Cys Ala Tyr Gln Val Ala
Arg Gly Met Glu Tyr Leu 595 600 605 Ala Ser Gln Lys Cys Ile His Arg
Asp Leu Ala Ala Arg Asn Val Leu 610 615 620 Val Thr Glu Asp Asn Val
Met Lys Ile Ala Asp Phe Gly Leu Ala Arg 625 630 635 640 Asp Val His
Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 645 650 655 Pro
Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr 660 665
670 His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe
675 680 685 Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu
Leu Phe 690 695 700 Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro
Ala Asn Cys Thr 705 710 715 720 His Asp Leu Tyr Met Ile Met Arg Glu
Cys Trp His Ala Ala Pro Ser 725 730 735 Gln Arg Pro Thr Phe Lys Gln
Leu Val Glu Asp Leu Asp Arg Val Leu 740 745 750 Thr Val Thr Ser Thr
Asp Val Ser Ala Gly Ser Gly Leu Val Pro Pro 755 760 765 Ala Tyr Ala
Pro Pro Pro Ala Val Pro Gly His Pro Ser Gly Arg Pro 770 775 780 Arg
Glu Ala Lys Leu Val Glu Phe Asp Phe Leu Gly Ala Leu Asp Ile 785 790
795 800 Pro Val Pro Gly Pro Pro Pro Gly Val Pro Ala Pro Gly Gly Pro
Pro 805 810 815 Leu Ser Thr Gly Pro Ile Val Asp Leu Leu Gln Tyr Ser
Gln Lys Asp 820 825 830 Leu Asp Ala Val Val Lys Ala Thr Gln Glu Glu
Asn Arg Glu Leu Arg 835 840 845 Ser Arg Cys Glu Glu Leu His Gly Lys
Asn Leu Glu Leu Gly Lys Ile 850 855 860 Met Asp Arg Phe Glu Glu Val
Val Tyr Gln Ala Met Glu Glu Val Gln 865 870 875 880 Lys Gln Lys Glu
Leu Ser Lys Ala Glu Ile Gln Lys Val Leu Lys Glu 885 890 895 Lys Asp
Gln Leu Thr Thr Asp Leu Asn Ser Met Glu Lys Ser Phe Ser 900 905 910
Asp Leu Phe Lys Arg Phe Glu Lys Gln Lys Glu Val Ile Glu Gly Tyr 915
920 925 Arg Lys Asn Glu Glu Ser Leu Lys Lys Cys Val Glu Asp Tyr Leu
Ala 930 935 940 Arg Ile Thr Gln Glu Gly Gln Arg Tyr Gln Ala Leu Lys
Ala His Ala 945 950 955 960 Glu Glu Lys Leu Gln Leu Ala Asn Glu Glu
Ile Ala Gln Val Arg Ser 965 970 975 Lys Ala Gln Ala Glu Ala Leu Ala
Leu Gln Ala Ser Leu Arg Lys Glu 980 985 990 Gln Met Arg Ile Gln Ser
Leu Glu Lys Thr Val Glu Gln Lys Thr Lys 995 1000 1005 Glu Asn Glu
Glu Leu Thr Arg Ile Cys Asp Asp Leu Ile Ser Lys 1010 1015 1020 Met
Glu Lys Ile 1025 160984PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 160Met Gly Ala Pro Ala
Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile 1 5 10 15 Val Ala Gly
Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25 30 Gly
Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40
45 Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro
50 55 60 Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly
Thr Gly 65 70 75 80 Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln
Arg Leu Gln Val 85 90 95 Leu Asn Ala Ser His Glu Asp Ser Gly Ala
Tyr Ser Cys Arg Gln Arg 100 105 110 Leu Thr Gln Arg Val Leu Cys His
Phe Ser Val Arg Val Thr Asp Ala 115 120 125 Pro Ser Ser Gly Asp Asp
Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140 Gly Val Asp Thr
Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp 145 150 155 160 Lys
Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170
175 Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly
180 185 190 Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu
Arg His 195 200 205 Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro
Ser Asp Arg Gly 210 215 220 Asn Tyr Thr Cys Val Val Glu Asn Lys Phe
Gly Ser Ile Arg Gln Thr 225 230 235 240 Tyr Thr Leu Asp Val Leu Glu
Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala
Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270 Phe His Cys
Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280 285 Lys
His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295
300 Tyr Val Thr Val Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu
305 310 315 320 Leu Glu Val Leu Ser Leu His Asn Val Thr Phe Glu Asp
Ala Gly Glu 325 330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Phe
Ser His His Ser Ala 340 345 350 Trp Leu Val Val Leu Pro Ala Glu Glu
Glu Leu Val Glu Ala Asp Glu 355 360 365 Ala Gly Ser Val Tyr Ala Gly
Ile Leu Ser Tyr Gly Val Gly Phe Phe 370 375 380 Leu Phe Ile Leu Val
Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser 385 390 395 400 Pro Pro
Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile Ser Arg 405 410 415
Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser Met Ser 420
425 430 Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly Glu
Gly 435 440 445 Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala
Asp Pro Lys 450 455 460 Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly
Lys Pro Leu Gly Glu 465 470 475 480 Gly Cys Phe Gly Gln Val Val Met
Ala Glu Ala Ile Gly Ile Asp Lys 485 490 495 Asp Arg Ala Ala Lys Pro
Val Thr Val Ala Val Lys Met Leu Lys Asp 500 505 510 Asp Ala Thr Asp
Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 515 520 525 Met Lys
Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 530 535 540
Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys 545
550 555 560 Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly
Leu Asp 565 570 575 Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln
Leu Thr Phe Lys 580 585 590 Asp Leu Val Ser Cys Ala Tyr Gln Val Ala
Arg Gly Met Glu Tyr Leu 595 600 605 Ala Ser Gln Lys Cys Ile His Arg
Asp Leu Ala Ala Arg Asn Val Leu 610 615 620 Val Thr Glu Asp Asn Val
Met Lys Ile Ala Asp Phe Gly Leu Ala Arg 625 630 635 640 Asp Val His
Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 645 650 655 Pro
Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr 660 665
670 His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe
675 680 685 Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu
Leu Phe 690 695 700 Lys Leu Leu Lys
Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr 705 710 715 720 His
Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser 725 730
735 Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu
740 745 750 Thr Val Thr Ser Thr Asp Val Pro Gly Pro Pro Pro Gly Val
Pro Ala 755 760 765 Pro Gly Gly Pro Pro Leu Ser Thr Gly Pro Ile Val
Asp Leu Leu Gln 770 775 780 Tyr Ser Gln Lys Asp Leu Asp Ala Val Val
Lys Ala Thr Gln Glu Glu 785 790 795 800 Asn Arg Glu Leu Arg Ser Arg
Cys Glu Glu Leu His Gly Lys Asn Leu 805 810 815 Glu Leu Gly Lys Ile
Met Asp Arg Phe Glu Glu Val Val Tyr Gln Ala 820 825 830 Met Glu Glu
Val Gln Lys Gln Lys Glu Leu Ser Lys Ala Glu Ile Gln 835 840 845 Lys
Val Leu Lys Glu Lys Asp Gln Leu Thr Thr Asp Leu Asn Ser Met 850 855
860 Glu Lys Ser Phe Ser Asp Leu Phe Lys Arg Phe Glu Lys Gln Lys Glu
865 870 875 880 Val Ile Glu Gly Tyr Arg Lys Asn Glu Glu Ser Leu Lys
Lys Cys Val 885 890 895 Glu Asp Tyr Leu Ala Arg Ile Thr Gln Glu Gly
Gln Arg Tyr Gln Ala 900 905 910 Leu Lys Ala His Ala Glu Glu Lys Leu
Gln Leu Ala Asn Glu Glu Ile 915 920 925 Ala Gln Val Arg Ser Lys Ala
Gln Ala Glu Ala Leu Ala Leu Gln Ala 930 935 940 Ser Leu Arg Lys Glu
Gln Met Arg Ile Gln Ser Leu Glu Lys Thr Val 945 950 955 960 Glu Gln
Lys Thr Lys Glu Asn Glu Glu Leu Thr Arg Ile Cys Asp Asp 965 970 975
Leu Ile Ser Lys Met Glu Lys Ile 980 161949PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
161Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile
1 5 10 15 Val Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg
Val Val 20 25 30 Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly
Gln Gln Glu Gln 35 40 45 Leu Val Phe Gly Ser Gly Asp Ala Val Glu
Leu Ser Cys Pro Pro Pro 50 55 60 Gly Gly Gly Pro Met Gly Pro Thr
Val Trp Val Lys Asp Gly Thr Gly 65 70 75 80 Leu Val Pro Ser Glu Arg
Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90 95 Leu Asn Ala Ser
His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110 Leu Thr
Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125
Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130
135 140 Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met
Asp 145 150 155 160 Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val
Arg Phe Arg Cys 165 170 175 Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile
Ser Trp Leu Lys Asn Gly 180 185 190 Arg Glu Phe Arg Gly Glu His Arg
Ile Gly Gly Ile Lys Leu Arg His 195 200 205 Gln Gln Trp Ser Leu Val
Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215 220 Asn Tyr Thr Cys
Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr 225 230 235 240 Tyr
Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250
255 Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu
260 265 270 Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln
Trp Leu 275 280 285 Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro
Asp Gly Thr Pro 290 295 300 Tyr Val Thr Val Leu Lys Thr Ala Gly Ala
Asn Thr Thr Asp Lys Glu 305 310 315 320 Leu Glu Val Leu Ser Leu His
Asn Val Thr Phe Glu Asp Ala Gly Glu 325 330 335 Tyr Thr Cys Leu Ala
Gly Asn Ser Ile Gly Phe Ser His His Ser Ala 340 345 350 Trp Leu Val
Val Leu Pro Ala Glu Glu Glu Leu Val Glu Ala Asp Glu 355 360 365 Ala
Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe 370 375
380 Leu Phe Ile Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser
385 390 395 400 Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys
Ile Ser Arg 405 410 415 Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser
Asn Ala Ser Met Ser 420 425 430 Ser Asn Thr Pro Leu Val Arg Ile Ala
Arg Leu Ser Ser Gly Glu Gly 435 440 445 Pro Thr Leu Ala Asn Val Ser
Glu Leu Glu Leu Pro Ala Asp Pro Lys 450 455 460 Trp Glu Leu Ser Arg
Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu 465 470 475 480 Gly Cys
Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile Asp Lys 485 490 495
Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu Lys Asp 500
505 510 Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu
Met 515 520 525 Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu
Leu Gly Ala 530 535 540 Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val
Glu Tyr Ala Ala Lys 545 550 555 560 Gly Asn Leu Arg Glu Phe Leu Arg
Ala Arg Arg Pro Pro Gly Leu Asp 565 570 575 Tyr Ser Phe Asp Thr Cys
Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys 580 585 590 Asp Leu Val Ser
Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu 595 600 605 Ala Ser
Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu 610 615 620
Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg 625
630 635 640 Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly
Arg Leu 645 650 655 Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp
Arg Val Tyr Thr 660 665 670 His Gln Ser Asp Val Trp Ser Phe Gly Val
Leu Leu Trp Glu Ile Phe 675 680 685 Thr Leu Gly Gly Ser Pro Tyr Pro
Gly Ile Pro Val Glu Glu Leu Phe 690 695 700 Lys Leu Leu Lys Glu Gly
His Arg Met Asp Lys Pro Ala Asn Cys Thr 705 710 715 720 His Asp Leu
Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser 725 730 735 Gln
Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu 740 745
750 Thr Val Thr Ser Thr Asp Val Lys Ala Thr Gln Glu Glu Asn Arg Glu
755 760 765 Leu Arg Ser Arg Cys Glu Glu Leu His Gly Lys Asn Leu Glu
Leu Gly 770 775 780 Lys Ile Met Asp Arg Phe Glu Glu Val Val Tyr Gln
Ala Met Glu Glu 785 790 795 800 Val Gln Lys Gln Lys Glu Leu Ser Lys
Ala Glu Ile Gln Lys Val Leu 805 810 815 Lys Glu Lys Asp Gln Leu Thr
Thr Asp Leu Asn Ser Met Glu Lys Ser 820 825 830 Phe Ser Asp Leu Phe
Lys Arg Phe Glu Lys Gln Lys Glu Val Ile Glu 835 840 845 Gly Tyr Arg
Lys Asn Glu Glu Ser Leu Lys Lys Cys Val Glu Asp Tyr 850 855 860 Leu
Ala Arg Ile Thr Gln Glu Gly Gln Arg Tyr Gln Ala Leu Lys Ala 865 870
875 880 His Ala Glu Glu Lys Leu Gln Leu Ala Asn Glu Glu Ile Ala Gln
Val 885 890 895 Arg Ser Lys Ala Gln Ala Glu Ala Leu Ala Leu Gln Ala
Ser Leu Arg 900 905 910 Lys Glu Gln Met Arg Ile Gln Ser Leu Glu Lys
Thr Val Glu Gln Lys 915 920 925 Thr Lys Glu Asn Glu Glu Leu Thr Arg
Ile Cys Asp Asp Leu Ile Ser 930 935 940 Lys Met Glu Lys Ile 945
16220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 162gtaacctgcg ggagtttctg 2016320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
163acaccaggtc cttgaaggtg 2016420DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 164cctgagggac agtcctggta
2016520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 165agtgctccca agaaatcgaa 2016623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
166cgtgaagatg ctgaaagacg atg 2316721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
167aaacgcttga agaggtcgga g 2116820DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 168atgctagcag gggtctctga
2016920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 169cccttccaga acacctttca 2017018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
170atgatcatgc gggagtgc 1817120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 171gggggtcgaa cttgaggtat
2017220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 172cgcaggcttt ttgtagtgag 2017318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
173tgtaggcgcg aaaggaag 1817420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 174gaactcatcc ggacccctat
2017520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 175gctttcccca ttgcacttta 2017620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
176gaggagagaa gcacgtggag 2017720DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 177ggcagacgtg tgaggtgtaa
2017820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 178gtgatcagca gctggactgt 2017918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
179gagcctgggc atggatct 1818020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 180agaggtgacc accaatcagc
2018120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 181cgtgtcccac acagagacag 2018221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 182tgcgtcgtgg agaacaagtt t 2118321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 183gttccactgc aaggtgtaca g 2118421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 184gcacaacctc gactactaca a 21185822PRTHomo sapiens
185Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala
1 5 10 15 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln
Ala Gln 20 25 30 Pro Trp Gly Ala Pro Val Glu Val Glu Ser Phe Leu
Val His Pro Gly 35 40 45 Asp Leu Leu Gln Leu Arg Cys Arg Leu Arg
Asp Asp Val Gln Ser Ile 50 55 60 Asn Trp Leu Arg Asp Gly Val Gln
Leu Ala Glu Ser Asn Arg Thr Arg 65 70 75 80 Ile Thr Gly Glu Glu Val
Glu Val Gln Asp Ser Val Pro Ala Asp Ser 85 90 95 Gly Leu Tyr Ala
Cys Val Thr Ser Ser Pro Ser Gly Ser Asp Thr Thr 100 105 110 Tyr Phe
Ser Val Asn Val Ser Asp Ala Leu Pro Ser Ser Glu Asp Asp 115 120 125
Asp Asp Asp Asp Asp Ser Ser Ser Glu Glu Lys Glu Thr Asp Asn Thr 130
135 140 Lys Pro Asn Arg Met Pro Val Ala Pro Tyr Trp Thr Ser Pro Glu
Lys 145 150 155 160 Met Glu Lys Lys Leu His Ala Val Pro Ala Ala Lys
Thr Val Lys Phe 165 170 175 Lys Cys Pro Ser Ser Gly Thr Pro Asn Pro
Thr Leu Arg Trp Leu Lys 180 185 190 Asn Gly Lys Glu Phe Lys Pro Asp
His Arg Ile Gly Gly Tyr Lys Val 195 200 205 Arg Tyr Ala Thr Trp Ser
Ile Ile Met Asp Ser Val Val Pro Ser Asp 210 215 220 Lys Gly Asn Tyr
Thr Cys Ile Val Glu Asn Glu Tyr Gly Ser Ile Asn 225 230 235 240 His
Thr Tyr Gln Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile 245 250
255 Leu Gln Ala Gly Leu Pro Ala Asn Lys Thr Val Ala Leu Gly Ser Asn
260 265 270 Val Glu Phe Met Cys Lys Val Tyr Ser Asp Pro Gln Pro His
Ile Gln 275 280 285 Trp Leu Lys His Ile Glu Val Asn Gly Ser Lys Ile
Gly Pro Asp Asn 290 295 300 Leu Pro Tyr Val Gln Ile Leu Lys Thr Ala
Gly Val Asn Thr Thr Asp 305 310 315 320 Lys Glu Met Glu Val Leu His
Leu Arg Asn Val Ser Phe Glu Asp Ala 325 330 335 Gly Glu Tyr Thr Cys
Leu Ala Gly Asn Ser Ile Gly Leu Ser His His 340 345 350 Ser Ala Trp
Leu Thr Val Leu Glu Ala Leu Glu Glu Arg Pro Ala Val 355 360 365 Met
Thr Ser Pro Leu Tyr Leu Glu Ile Ile Ile Tyr Cys Thr Gly Ala 370 375
380 Phe Leu Ile Ser Cys Met Val Gly Ser Val Ile Val Tyr Lys Met Lys
385 390 395 400 Ser Gly Thr Lys Lys Ser Asp Phe His Ser Gln Met Ala
Val His Lys 405 410 415 Leu Ala Lys Ser Ile Pro Leu Arg Arg Gln Val
Thr Val Ser Ala Asp 420 425 430 Ser Ser Ala Ser Met Asn Ser Gly Val
Leu Leu Val Arg Pro Ser Arg 435 440 445 Leu Ser Ser Ser Gly Thr Pro
Met Leu Ala Gly Val Ser Glu Tyr Glu 450 455 460 Leu Pro Glu Asp Pro
Arg Trp Glu Leu Pro Arg Asp Arg Leu Val Leu 465 470 475 480 Gly Lys
Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Leu Ala Glu 485 490 495
Ala Ile Gly Leu Asp Lys Asp Lys Pro Asn Arg Val Thr Lys Val Ala 500
505 510 Val Lys Met Leu Lys Ser Asp Ala Thr Glu Lys Asp Leu Ser Asp
Leu 515 520 525 Ile Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His
Lys Asn Ile 530 535 540 Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly
Pro Leu Tyr Val Ile 545 550 555 560 Val Glu Tyr Ala Ser Lys Gly Asn
Leu Arg Glu Tyr Leu Gln Ala Arg 565 570 575 Arg Pro Pro Gly Leu Glu
Tyr Cys Tyr Asn Pro Ser His Asn
Pro Glu 580 585 590 Glu Gln Leu Ser Ser Lys Asp Leu Val Ser Cys Ala
Tyr Gln Val Ala 595 600 605 Arg Gly Met Glu Tyr Leu Ala Ser Lys Lys
Cys Ile His Arg Asp Leu 610 615 620 Ala Ala Arg Asn Val Leu Val Thr
Glu Asp Asn Val Met Lys Ile Ala 625 630 635 640 Asp Phe Gly Leu Ala
Arg Asp Ile His His Ile Asp Tyr Tyr Lys Lys 645 650 655 Thr Thr Asn
Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu 660 665 670 Phe
Asp Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val 675 680
685 Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Val
690 695 700 Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg
Met Asp 705 710 715 720 Lys Pro Ser Asn Cys Thr Asn Glu Leu Tyr Met
Met Met Arg Asp Cys 725 730 735 Trp His Ala Val Pro Ser Gln Arg Pro
Thr Phe Lys Gln Leu Val Glu 740 745 750 Asp Leu Asp Arg Ile Val Ala
Leu Thr Ser Asn Gln Glu Tyr Leu Asp 755 760 765 Leu Ser Met Pro Leu
Asp Gln Tyr Ser Pro Ser Phe Pro Asp Thr Arg 770 775 780 Ser Ser Thr
Cys Ser Ser Gly Glu Asp Ser Val Phe Ser His Glu Pro 785 790 795 800
Leu Pro Glu Glu Pro Cys Leu Pro Arg His Pro Ala Gln Leu Ala Asn 805
810 815 Gly Gly Leu Lys Arg Arg 820 1865917DNAHomo sapiens
186agatgcaggg gcgcaaacgc caaaggagac caggctgtag gaagagaagg
gcagagcgcc 60ggacagctcg gcccgctccc cgtcctttgg ggccgcggct ggggaactac
aaggcccagc 120aggcagctgc agggggcgga ggcggaggag ggaccagcgc
gggtgggagt gagagagcga 180gccctcgcgc cccgccggcg catagcgctc
ggagcgctct tgcggccaca ggcgcggcgt 240cctcggcggc gggcggcagc
tagcgggagc cgggacgccg gtgcagccgc agcgcgcgga 300ggaacccggg
tgtgccggga gctgggcggc cacgtccgga cgggaccgag acccctcgta
360gcgcattgcg gcgacctcgc cttccccggc cgcgagcgcg ccgctgcttg
aaaagccgcg 420gaacccaagg acttttctcc ggtccgagct cggggcgccc
cgcagggcgc acggtacccg 480tgctgcagtc gggcacgccg cggcgccggg
gcctccgcag ggcgatggag cccggtctgc 540aaggaaagtg aggcgccgcc
gctgcgttct ggaggagggg ggcacaaggt ctggagaccc 600cgggtggcgg
acgggagccc tccccccgcc ccgcctccgg ggcaccagct ccggctccat
660tgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg
agcgccggcc 720gcggagctct tgcgaccccg ccaggacccg aacagagccc
gggggcggcg ggccggagcc 780ggggacgcgg gcacacgccc gctcgcacaa
gccacggcgg actctcccga ggcggaacct 840ccacgccgag cgagggtcag
tttgaaaagg aggatcgagc tcactgtgga gtatccatgg 900agatgtggag
ccttgtcacc aacctctaac tgcagaactg ggatgtggag ctggaagtgc
960ctcctcttct gggctgtgct ggtcacagcc acactctgca ccgctaggcc
gtccccgacc 1020ttgcctgaac aagcccagcc ctggggagcc cctgtggaag
tggagtcctt cctggtccac 1080cccggtgacc tgctgcagct tcgctgtcgg
ctgcgggacg atgtgcagag catcaactgg 1140ctgcgggacg gggtgcagct
ggcggaaagc aaccgcaccc gcatcacagg ggaggaggtg 1200gaggtgcagg
actccgtgcc cgcagactcc ggcctctatg cttgcgtaac cagcagcccc
1260tcgggcagtg acaccaccta cttctccgtc aatgtttcag atgctctccc
ctcctcggag 1320gatgatgatg atgatgatga ctcctcttca gaggagaaag
aaacagataa caccaaacca 1380aaccgtatgc ccgtagctcc atattggaca
tccccagaaa agatggaaaa gaaattgcat 1440gcagtgccgg ctgccaagac
agtgaagttc aaatgccctt ccagtgggac cccaaacccc 1500acactgcgct
ggttgaaaaa tggcaaagaa ttcaaacctg accacagaat tggaggctac
1560aaggtccgtt atgccacctg gagcatcata atggactctg tggtgccctc
tgacaagggc 1620aactacacct gcattgtgga gaatgagtac ggcagcatca
accacacata ccagctggat 1680gtcgtggagc ggtcccctca ccggcccatc
ctgcaagcag ggttgcccgc caacaaaaca 1740gtggccctgg gtagcaacgt
ggagttcatg tgtaaggtgt acagtgaccc gcagccgcac 1800atccagtggc
taaagcacat cgaggtgaat gggagcaaga ttggcccaga caacctgcct
1860tatgtccaga tcttgaagac tgctggagtt aataccaccg acaaagagat
ggaggtgctt 1920cacttaagaa atgtctcctt tgaggacgca ggggagtata
cgtgcttggc gggtaactct 1980atcggactct cccatcactc tgcatggttg
accgttctgg aagccctgga agagaggccg 2040gcagtgatga cctcgcccct
gtacctggag atcatcatct attgcacagg ggccttcctc 2100atctcctgca
tggtggggtc ggtcatcgtc tacaagatga agagtggtac caagaagagt
2160gacttccaca gccagatggc tgtgcacaag ctggccaaga gcatccctct
gcgcagacag 2220gtaacagtgt ctgctgactc cagtgcatcc atgaactctg
gggttcttct ggttcggcca 2280tcacggctct cctccagtgg gactcccatg
ctagcagggg tctctgagta tgagcttccc 2340gaagaccctc gctgggagct
gcctcgggac agactggtct taggcaaacc cctgggagag 2400ggctgctttg
ggcaggtggt gttggcagag gctatcgggc tggacaagga caaacccaac
2460cgtgtgacca aagtggctgt gaagatgttg aagtcggacg caacagagaa
agacttgtca 2520gacctgatct cagaaatgga gatgatgaag atgatcggga
agcataagaa tatcatcaac 2580ctgctggggg cctgcacgca ggatggtccc
ttgtatgtca tcgtggagta tgcctccaag 2640ggcaacctgc gggagtacct
gcaggcccgg aggcccccag ggctggaata ctgctacaac 2700cccagccaca
acccagagga gcagctctcc tccaaggacc tggtgtcctg cgcctaccag
2760gtggcccgag gcatggagta tctggcctcc aagaagtgca tacaccgaga
cctggcagcc 2820aggaatgtcc tggtgacaga ggacaatgtg atgaagatag
cagactttgg cctcgcacgg 2880gacattcacc acatcgacta ctataaaaag
acaaccaacg gccgactgcc tgtgaagtgg 2940atggcacccg aggcattatt
tgaccggatc tacacccacc agagtgatgt gtggtctttc 3000ggggtgctcc
tgtgggagat cttcactctg ggcggctccc cataccccgg tgtgcctgtg
3060gaggaacttt tcaagctgct gaaggagggt caccgcatgg acaagcccag
taactgcacc 3120aacgagctgt acatgatgat gcgggactgc tggcatgcag
tgccctcaca gagacccacc 3180ttcaagcagc tggtggaaga cctggaccgc
atcgtggcct tgacctccaa ccaggagtac 3240ctggacctgt ccatgcccct
ggaccagtac tcccccagct ttcccgacac ccggagctct 3300acgtgctcct
caggggagga ttccgtcttc tctcatgagc cgctgcccga ggagccctgc
3360ctgccccgac acccagccca gcttgccaat ggcggactca aacgccgctg
actgccaccc 3420acacgccctc cccagactcc accgtcagct gtaaccctca
cccacagccc ctgctgggcc 3480caccacctgt ccgtccctgt cccctttcct
gctggcagga gccggctgcc taccaggggc 3540cttcctgtgt ggcctgcctt
caccccactc agctcacctc tccctccacc tcctctccac 3600ctgctggtga
gaggtgcaaa gaggcagatc tttgctgcca gccacttcat cccctcccag
3660atgttggacc aacacccctc cctgccacca ggcactgcct ggagggcagg
gagtgggagc 3720caatgaacag gcatgcaagt gagagcttcc tgagctttct
cctgtcggtt tggtctgttt 3780tgccttcacc cataagcccc tcgcactctg
gtggcaggtg ccttgtcctc agggctacag 3840cagtagggag gtcagtgctt
cgtgcctcga ttgaaggtga cctctgcccc agataggtgg 3900tgccagtggc
ttattaattc cgatactagt ttgctttgct gaccaaatgc ctggtaccag
3960aggatggtga ggcgaaggcc aggttggggg cagtgttgtg gccctggggc
ccagccccaa 4020actgggggct ctgtatatag ctatgaagaa aacacaaagt
gtataaatct gagtatatat 4080ttacatgtct ttttaaaagg gtcgttacca
gagatttacc catcgggtaa gatgctcctg 4140gtggctggga ggcatcagtt
gctatatatt aaaaacaaaa aagaaaaaaa aggaaaatgt 4200ttttaaaaag
gtcatatatt ttttgctact tttgctgttt tattttttta aattatgttc
4260taaacctatt ttcagtttag gtccctcaat aaaaattgct gctgcttcat
ttatctatgg 4320gctgtatgaa aagggtggga atgtccactg gaaagaaggg
acacccacgg gccctggggc 4380taggtctgtc ccgagggcac cgcatgctcc
cggcgcaggt tccttgtaac ctcttcttcc 4440taggtcctgc acccagacct
cacgacgcac ctcctgcctc tccgctgctt ttggaaagtc 4500agaaaaagaa
gatgtctgct tcgagggcag gaaccccatc catgcagtag aggcgctggg
4560cagagagtca aggcccagca gccatcgacc atggatggtt tcctccaagg
aaaccggtgg 4620ggttgggctg gggagggggc acctacctag gaatagccac
ggggtagagc tacagtgatt 4680aagaggaaag caagggcgcg gttgctcacg
cctgtaatcc cagcactttg ggacaccgag 4740gtgggcagat cacttcaggt
caggagtttg agaccagcct ggccaactta gtgaaacccc 4800atctctacta
aaaatgcaaa aattatccag gcatggtggc acacgcctgt aatcccagct
4860ccacaggagg ctgaggcaga atcccttgaa gctgggaggc ggaggttgca
gtgagccgag 4920attgcgccat tgcactccag cctgggcaac agagaaaaca
aaaaggaaaa caaatgatga 4980aggtctgcag aaactgaaac ccagacatgt
gtctgccccc tctatgtggg catggttttg 5040ccagtgcttc taagtgcagg
agaacatgtc acctgaggct agttttgcat tcaggtccct 5100ggcttcgttt
cttgttggta tgcctcccca gatcgtcctt cctgtatcca tgtgaccaga
5160ctgtatttgt tgggactgtc gcagatcttg gcttcttaca gttcttcctg
tccaaactcc 5220atcctgtccc tcaggaacgg ggggaaaatt ctccgaatgt
ttttggtttt ttggctgctt 5280ggaatttact tctgccacct gctggtcatc
actgtcctca ctaagtggat tctggctccc 5340ccgtacctca tggctcaaac
taccactcct cagtcgctat attaaagctt atattttgct 5400ggattactgc
taaatacaaa agaaagttca atatgttttc atttctgtag ggaaaatggg
5460attgctgctt taaatttctg agctagggat tttttggcag ctgcagtgtt
ggcgactatt 5520gtaaaattct ctttgtttct ctctgtaaat agcacctgct
aacattacaa tttgtattta 5580tgtttaaaga aggcatcatt tggtgaacag
aactaggaaa tgaattttta gctcttaaaa 5640gcatttgctt tgagaccgca
caggagtgtc tttccttgta aaacagtgat gataatttct 5700gccttggccc
taccttgaag caatgttgtg tgaagggatg aagaatctaa aagtcttcat
5760aagtccttgg gagaggtgct agaaaaatat aaggcactat cataattaca
gtgatgtcct 5820tgctgttact actcaaatca cccacaaatt tccccaaaga
ctgcgctagc tgtcaaataa 5880aagacagtga aattgacctg aaaaaaaaaa aaaaaaa
591718776DNAHomo sapiens 187gtgctggcat gccgcgccct cccagaggcc
caccttcaag cagctggtgg aggacctgga 60ccgtgtcctt accgtg 7618876DNAHomo
sapiens 188atgcgggagt gctggcatga cgcgccctcc cagaggccca ccttcaagca
gctggtggag 60gacctggacc gtgtcc 7618976DNAHomo sapiens 189agctggtgga
ggacctggac cgtgtcctta ccgtgacgtc caccgacgtg agtgctggct 60ctggcctggt
gccacc 7619076DNAHomo sapiens 190cccttaaaac aactcgttcc ctcagaccac
acacaagaca gttcaagagg gactcaagga 60cttacaggaa tgtcca 7619176DNAHomo
sapiens 191aaccaaaggc tcagaccccc aggaatagaa aatataggcc cttaaaacaa
ctcgttccct 60cagaccacac acaaga 7619276DNAHomo sapiens 192tcaaggactt
acaggaatgt ccagtgctcc caagaaatcg aactccacaa gcttggcttc 60ccgcgcacgt
cctgag 7619376DNAHomo sapiens 193ggacttacag gaatgtccag tgctcccaag
aaatcgaact ccacaagctt ggcttcccgc 60ggacgtcctg agggat 7619476DNAHomo
sapiens 194cagaccacac acaagacagt tcaagaggga ctcaaggact tacaggaatg
tccagtgctc 60ccaagaaatc gaactc 7619576DNAHomo sapiens 195cttaccgtga
cgtccaccga cgtgagtgct ggctctggcc tggtgccacc cgcctatgcc 60cctcccctgc
ccttag 7619676DNAHomo sapiens 196aaacttgagg tataaggact gcttcctcaa
ggccgactcc ttaaactggg gacaagaggg 60caagtgatca ggtctg 7619776DNAHomo
sapiens 197aacttgaggt ataaggactg cttcctcaag gccgactcct taaactgggg
acaagagggc 60aagtgatcag gtctga 7619876DNAHomo sapiens 198gcccgcaggt
acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 60ttcaagcagc
tggtgg 7619976DNAHomo sapiens 199accgtgacgt ccaccgacgt gagtgctggc
tctggcctgg tgcgacccgc cgatctctct 60cccctgtcct tttcct 7620076DNAHomo
sapiens 200tgggagggtg cggggggccg ggggggggag tgtgcaggtg agctccctgg
cccttggccc 60cctgccctct gggggg 7620176DNAHomo sapiens 201ctgggaatgg
tggtgtctcg ggcagggttg tgggtgaccg ggggtgggag ggtgcggggg 60accggggggg
ggaggg 7620276DNAHomo sapiens 202agcgccctgc ccgcaggtac atgatcatgc
gggagtgctg gcatgccgcg ccctcccaga 60ggcccacctt caagca 7620376DNAHomo
sapiens 203gccaacgcca tgcccaggcc ggagagtccc ggggaggctg ctggtgggca
gctgactgcg 60gggacactgg gtggaa 7620476DNAHomo sapiens 204aggccaccag
aggccaacgc catgcccagg ccggagagtc ccggggaggc tgctggtggg 60gaggcgaacg
cgggga 7620576DNAHomo sapiens 205tgcccaggcc ggagagtccc ggggcggctg
ctggggggga gctgactggg ggggcactgg 60gggggagacc cgggcc 7620676DNAHomo
sapiens 206taggccctta aaacaactcg ttccctcaga ccacacacaa gacagttcaa
gagggactca 60aggacttaca ggaatg 7620776DNAHomo sapiens 207actcaaggac
ttacaggaat gtccagtgct cccaagaaat cgaactccac aagcttggct 60tcccgcggac
gtcctg 7620876DNAHomo sapiens 208aggcccttaa aacaactcgt tccctcagac
cacacacaag acagttcaag agggactcaa 60ggacttacag gaatgt 7620976DNAHomo
sapiens 209gccctcccag aggcccacct tcaagcagct ggtggaggac ctggaccgtg
tccttaccgt 60gacgtccacc gacgtg 7621076DNAHomo sapiens 210gccgcgccct
cccagaggcc caccttcaag cagctggtgg aggacctgga ccgtgtcctt 60accgtgacgt
ccaccg 7621176DNAHomo sapiens 211ggtggaggac ctggaccgtg accttaccgg
gacgtccacc gacgggagtg ctggctctgg 60cctggtgcca cccgcc 7621276DNAHomo
sapiens 212gacgtccacc gacgtgagtg ctggctctgg cctggtgcca cccgcctatg
cccctccccc 60tgccgtcccc ggccat 7621376DNAHomo sapiens 213tgtccttacc
gtgacgtcca ccgacgtgag tgctggctct ggcctggtgc cacccgccta 60tgcccctccc
cctgcc 7621476DNAHomo sapiens 214tacctgctgg tctcggtggc cacgggcact
ggtctaccag ggctgtccct ccggaggggg 60tcaaacttga gggata 7621576DNAHomo
sapiens 215ctggaccgtg tccttaccgt gacgtccacc gacgtgagtg ctggctctgg
cctggtgcca 60cccgcccatg cccctc 7621676DNAHomo sapiens 216tgtccttacc
gtgacgtcca ccgacgtgag tgctggctct ggcctggtgc cacccgccta 60tgcccctccc
ctgccc 7621776DNAHomo sapiens 217aaaagattta agtttagatc tttaatatac
ctagaacggt ggctgtaacc agcaaggcag 60gagccctttg tgttgg 7621876DNAHomo
sapiens 218tgggtcaaac ttgaggtata aggactgctt cctcaaggcc gactccttat
actggggaca 60agagggcaag tgatca 7621976DNAHomo sapiens 219gagtgctggc
tctggcctgg tgccacccgc ctatgcccct ccccctggcg tccccggcca 60tcctgccccc
cagagt 7622076DNAHomo sapiens 220gagtgctggc tctggcctgg tgccacccgc
ctatgcccct ccccctgccg tccccggcca 60tcctgccccc cagagt 7622176DNAHomo
sapiens 221gccaacgcca tgcccaggcc ggagagtccc ggggaggctg ctggtgggga
gctgacttcg 60gggacactgg ggggaa 7622276DNAHomo sapiens 222catgcgggag
tgctggcatg gcgcgccctc ccagcggccc accttcaagc agctggtggg 60ggacctggac
cgtgtc 7622376DNAHomo sapiens 223acgtgagtgc tggctctggc ctggtgccac
ccgcctatgc ccctccccct gccgtccccg 60gccatcctgc ccccca 7622476DNAHomo
sapiens 224ccctcccaga ggcccacctt caagcagctg gtggaggacc tggaccgtgt
ccttaccgtg 60acgtccaccg acgtga 7622576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 225ggacttacag gaatgtccag tgctcccaag aaatcgaact
ccacaagctt ggcttcccgc 60ggacgtcctg agggat 7622676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 226catccctcag gacgtccgcg ggaagccaag cttgtggagt
tcgatttctt gggagcactg 60gacattcctg taagtc 7622776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 227catccctcag gacgtccgcg ggaagccaag cttgtggagt
tcgatttctt gggagcactg 60gacattcctg taagtc 7622876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 228catccctcag gacgtccgcg ggaagccaag cttgtggagt
tcgatttctt gggagcactg 60gacattcctg taagtc 7622976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 229gccatccctc aggacgtccg cgggaagcca agcttgtgga
gttcgatttc ttgggagcac 60tggacattcc tgtaag 7623076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 230ccggccatcc ctcaggacgt ccgcgggaag ccaagcttgt
ggagttcgat ttcttgggag 60cactggacat tcctgt 7623176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 231caggaatgtc cagtgctacc aagaaatcga actccacaag
cttgggttcc cgcggacgtc 60ctccgggatg gccgtg 7623276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 232caggaatgtc cagtgctccc aagaaatcga actccacaag
cttggcttcc cgcggacgtc 60ctgagggatg gccggg 7623376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 233tccccggcca tccctcagga cgtccgcggg aagccaagct
tgtggagttc gatttcttgg 60gagcactgga cattcc 7623476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 234tccccggcca tccctcagga ngtccgcggg aagccaagct
tgtggagttc gatttcttgg 60gagcactgga cattcc 7623576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 235gtccccggcc atccctcagg acgtccgcgg gaagccaagc
ttgtggagtt cgatttcttg 60ggagcactgg acattc 7623676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 236ccgtccccgg ccatccctca ggacgtccgc gggaagccaa
gcttgtggag ttcgatttct 60tgggagcact ggacat 7623776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 237tgctcccaag aaatcgaact ccacaagctt ggcttcccgc
ggacgtcctg agggatggcc 60ggggacggca ggggga 7623876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 238aagaaatcga actccacaag cttggcttcc cgcggacgtc
ctgagggatg gccggggacg 60gcagggggag gggcat 7623976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 239aaatcgaact ccacaagctt ggcttcccgc ggacgtcctg
agggatggcc gggggcggca 60gggggagggg catagg 7624076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 240ctggcctggt gccacccgcc tatgcccctc cccctgccgt
ccccggccat ccctcaggac 60gtccgcggga agccaa 7624176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 241tcgtcccgcg gacttcctga tggatcgccg gggacggcag
ggggaggggc ataggcgtgt 60ggcaccaggc cagctc 7624276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 242cttcccgcgg acgtcctgag ggatggccgg ggacggnagg
gggaggggca taggcgggtg 60gcaccaggcc agagcc 7624376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 243gtgctggctc tggcctggtg ccacccgcct atgcccctcc
ccctgccgtc cccggccatc 60cctcaggacg tccgcg 7624476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 244gagggatggc cggggacggc agggggaggg gcataggcgg
gtggcaccag gccagagcca 60gcactcacgt cggtgg 7624576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 245ggacaagagg gcaagtgatc aggtctgact gccatcccct
aacacacaca ggggggctaa 60gggcagggga ggggca 7624676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 246ggacaagagg gcaagtgatc aggtctgact gccatcccct
aacacacaca ggggggctaa 60gggcagggga ggggca 7624776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 247atgcccctcc cctgccctta gcccccctgt gtgtgttagg
ggatggcagt cagacctgat 60cacttgccct cttgtc 7624876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 248gcctatgccc ctcccctgcc cttagccccc ctgtgtgtgt
taggggatgg cagtcagacc 60tgatcacttg ccctct 7624976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 249gtgccacccg cctatgcccc tcccctgccc ttagcccccc
tgtgtgtgtt aggggatggc 60agtcagacct gatcac 7625076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 250tgactgccat cccctaacac acacaggggg gctaagggca
ggggaggggc ataggcgggg 60ggcaccaggc cagagc 7625176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 251tgactgccat cccctaacac acacaggggg gctaagggca
ggggaggggc ataggcgggg 60ggcaccaggc cagagc 7625276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 252tgccatcccc taacacacac aggggggcta agggcagggg
aggggcatag gcggggggca 60ccaggacaga ggcagc 7625376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 253cacacagggg ggctaagggc aggggagggg cataggcggg
ggggaccagg cccgagccag 60cactcacgtc gggggg 7625476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 254gacgtccacc gacgtgagtg ctggctctgg cctggtgcca
cccgcctatg cccctcccct 60gcccttagac cccctg 7625576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 255cttaccgtga cgtccaccga cgtgagtgct ggctctggcc
tggtgccacc cgcctatgcc 60cctcccctgc ccttag 7625676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 256tgtccttacc gtgacgtcca ccgacgtgag tgctggctct
ggcctggtgc cacccgccta 60tgcccctccc ctgccc 7625776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 257cgggggtggg agtgtgcggg tgaccggggg tgggagtgtg
caggtgacct ccctggccct 60tagccccctg cactct 7625876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 258cgggtgaccg ggggagggag tgtgcagggg acctccctgg
cccttagccc cctgcactct 60ggggggcagg atggcc 7625976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 259ggagtgtgca ggtgacctcc ctggccctta gccccctgca
ctctgggggg caggatggcc 60ggggacggca ggggga 7626076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 260ggggaggctg ctggtgggca gctgactgcg gggacactgg
gaggaagcct ggaccctcag 60cgaacttcgc ccagcc 7626176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 261acagcctggg cacagaggtg gctgtgcgaa ggtcgctgag
ggtccaggct tccacccagt 60gtccccgcag tcagct 7626276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 262tgactgcggg gacactgggt ggaagcctgg accctcagcg
accttcgcac agccacctct 60gtgcccaggc tgtgcc 7626376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 263cggggacact gggtggaagc ctggaccctc agcgaccttc
gcacagccac ctctgtgccc 60aggctgtgcc ccagaa 7626476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 264ggggacactg ggtggaagcc tggaccctca gcgaccttcg
cacagccacc tctgtggcca 60ggctgtgcca cagaag 7626576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 265ggacactggg tggaagcctg gaccctcagc gaccttcgca
cagccacctc tgtgcccagg 60ctgtgcccca gaaggc 7626676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 266gaagcctgga ccctcagcga ccttcgcaca gccacctctg
tgccccggct gtgccccagc 60cggcccgccc cacacc 7626776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 267gaagcctgga ccctcagcga ccttcgcaca gccacctctg
tgcccaggct gtgccccaga 60aggcccgccc cacacc 7626876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 268tggaccctca gcgaccttcg cacagccacc tctgtgccca
ggctgtgccc cagaaggccc 60gccccacacc tcagca 7626976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 269gaccctcagc gaccttcgca cagccacctc tgtgcccagg
ctgtgcccca gaaggcccgc 60cccacacctc agcact 7627076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 270tcagcgacct tcgcacagcc acctctgtgc ccaggctgtg
ccccagaagg cccgccccac 60acctcagcac tctggg 7627176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 271ccttcgcaca gccacctctg tgcccaggct gtgccccaga
aggcccgccc cacacctcag 60cactctgggg ggcagg 7627276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 272gacgtccacc gacgtgagtg ctggctctgg cctggtgcca
cccgcctatg cccctccccc 60tgccgtcccc ggccat 7627376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 273caagagggac tcaaggactt acaggaatgt ccagtgctcc
caagaaatcg aactccacaa 60gcttggcttc ccgcgg 7627476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 274caagagggac tcaaggactt acaggaatgt ccagtgctcc
caagaaatcg aactccacaa 60gcttggcttc ccgcgg 7627576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 275ataggccctt aaaacaactc gttccctcag accacacaca
agacagttca agagggactc 60aaggacttac aggaat 7627676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 276tcaagaggga ctcaaggact tacaggaatg tccagtgctc
ccaagaaatc gaactccaca 60agcttggctt cccgcg 7627776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 277accacacaca agacagttca agagggactc aaggacttac
aggaatgtcc agtgctccca 60agaaatcgaa ctccac 7627876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 278gagctggcct ggtgccacac gcctatgccc ctccccctgc
cgtccccggc gatccatcag 60gaagtccgcg ggacga 7627976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 279ccaccgacgt gagtgctggc tctggcctgg tgccacccgc
ctatgcccct ccccctgccg 60tccccggcca tccctc 7628076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 280caagagcctc agacagtgca tgagggaccc gagacagtgc
ggcgagggaa cagcacagcg 60gccccatgcc cccaac 7628176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 281caagagcctc agacagtgca tgagggaccc gagacagtgc
ggcgagggaa cagcacaggg 60gccccatgcc cccaac 7628276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 282cgttccctca gaccacacac aagacagttc aagagggact
caaggactta caggaatgtc 60cagtgctccc aagaga 7628376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 283ccaggaatag aaaatatagg cccttaaaac aactcgttcc
ctcagaccac acacaagaca 60gttcaagagg gactca 7628476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 284ggctctggcc tggtgccacc cgcctatgcc cctccccctn
ccgtccccgg ccatccctca 60ggacgtccgc gggaag 7628576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 285gccctgcccg caggtacatg atcatgcggg agtgctggca
tgccgcgccc tcccagaggc 60ccaccttcaa gcagct 7628676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 286ctggcatgcc gcgccctccc agaggcccac ctttaagcag
ctggtagagg gcctggaccg 60tgtccttacc gtgacg 7628776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 287taaaacaact cgttccctca gaccacacac aagacagttc
aagagggact caaggactta 60caggaatgtc cagtgc 7628876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 288cacggccatc ccggaggacg tccgcgggaa cccaagcttg
tggagttcga tttcttggta 60gcactggaca ttcctg 7628976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 289tccccctgcc gtccccggcc atccctcagg acgtccgcgg
gaagccaagc ttgtggagtt 60cgatttcttg ggagca 7629076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 290agaccacaca caagacagtt caagagggac tcaaggactt
acaggaatgt ccagtgctcc 60caagaaatcg aactcc 7629176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 291cccggccatc cctcaggacg tccgcgggaa gccaagcttg
tggagttcga tttcttggga 60gcactggaca ttcctg 7629276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 292ggcatgccgc gccctcccag aggcccacct tcaagcagct
ggtggaggac ctggaccgtg 60tccttaccgt gacgtc 7629376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 293ggcatgccgc gccctcccag aggcccacct tcaagcagct
ggtggaggac ctggaccgtg 60tccttaccgt gacgtc 7629476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 294cggcgcacat acctgctggt ctcggtggcc acgggcactg
gtctaccagg actgtccctc 60aggagggggt caaact 7629576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 295atacctgctg gtctcggtgg ccacgggcac tggtctacca
ggactgtccc tcaggagggg 60gtcaaacttg aggtat 7629676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 296aggtataagg actgcttcct caaggccgac tccttaaact
ggggacaaga gggcaagtga 60tcaggtctga ctgcca 7629776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 297ggaggacctg gactgtgtcc ttaccgtgac gtccaccgac
gtgagtgctg gctctggcct 60ggtgccaccc gcctat 7629876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 298ggaggacctg gaccgtgtcc ttaccgtgac gtccaccgac
gtgagtgctg gctctggcct 60ggtgccaccc gcctat 7629976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 299caagcagctg gtggaggacc tggaccgtgt ccttaccgtg
acgtccaccg acgtgagtgc 60tggctctggc ctggtg 7630076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 300accttcaagc agctggtgga ggacctggac cgtgtcctta
ccgtgacgtc caccgacgtg 60agtgctggct ctggcc 7630176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 301caaacttgag gtataaggac tgcttcctca aggccgactc
cttaaactgg ggacaagagg 60gcaagtgatc aggtct 7630276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 302tacctgctgg tctcggtggc cacgggcact ggtctaccag
ggctgtccct ccggaggggg 60tcaaacttga gggata 7630376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 303aacttgaggt ataaggactg cttcctcaag gccgactcct
taaactgggg acaagagggc 60aagtgatcag gtctga 7630476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 304agctggtgga ggacctggac cgtgtcctta ccgtgacgtc
caccgacgtg agtgctggct 60ctggcctggt gccacc 7630576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 305gcgccctccc agaggcccac cttcaagcag ctggtggagg
acctggaccg tgtccttacc 60gtgacgtcca ccgacg 7630676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 306gcgggagtgc tggcatgccg cgccctccca gaggcccacc
ttcaagcagc tggtggagga 60cctggaccgt gtcctt 7630776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 307cctccactgg gtcctcaggg gtgggggtcc ctccggggct
gggcggggga gggactggca 60ggcctgcagg ggggtt 7630876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 308tcacggcagc aagaaccaca ctcactgctg caaggccacc
agaggccaac gccatgccca 60ggccggagag tcccgg 7630976DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 309tacatgatca tgcgggaggg ctggcatgcc gcgccctccc
agaggcccac cttcaagcag 60ctggtggagg gccggg 7631076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 310ggtgggaagc ggcggggctc actcctgagc gccctgcccg
cagggacatg atcatgcggg 60ggtgctggcc ttgcgg 7631176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 311gcgccctccc agaggcccac cttcaagcag ctggtggagg
acctggaccg tgtccttacc 60gtgacgtcca ccgacg 7631276DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 312cctgcccccc agagtgctga ggtgtggggc gggccttctg
gggcacagcc tgggcacaga 60ggtggctgtg cgaagg 7631376DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 313gcaggtacat gatcatgcgg gagtgccggc atttcgggac
cttccctcgg gccaccctct 60tccggttgtt gtgggc 7631476DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 314gcaggtacat gatcatgcgg gagtgctggc atgccgcgcc
ctccccgagg accaccttcc 60agcagccggg ggaggg 7631576DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 315cccgaataag gtgggaagcg gcggggctca ctcctgagcg
ccctgaccgc aggtacatga 60gcatgcggga gtggcg 7631676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 316cgtgtcctta ccgtgacgtc caccgacgtg agtgctggct
ctggcctggt gccacccgcc 60tatgcccctc cccctg 7631776DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 317acatgatcat gcgggagtgc tggcatgccg cgccccccca
gaggcccacc ttcaagcagc 60tggtggagga cctgga 7631876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 318gccttctggg gcacagcctg ggcacagagg tggctgtgcg
aaggtcgctg agggtccagg 60cttccaccca gtgtcc 7631927DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 319t ccg gat gaa ctg gtc cct gtc ctt cg 27 Pro Asp
Glu Leu Val Pro Val Leu 1 5 3208PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 320Pro Asp Glu Leu Val Pro
Val Leu 1 5 32126DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 321ctg atc gac ctg gat gtc ccc
gtc ga 26Leu Ile Asp Leu Asp Val Pro Val 1 5 3228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 322Leu
Ile Asp Leu Asp Val Pro Val 1 5 32326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 323at gct gtc aga ggg ttt tgt gat gaa 26 Ala Val
Arg Gly Phe Cys Asp Glu 1 5 3248PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 324Ala Val Arg Gly Phe Cys
Asp Glu 1 5 32527DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 325ac tac gat aaa aga cat aac
cag tgc g 27 Tyr Asp Lys Arg His Asn Gln Cys 1 5 3268PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 326Tyr
Asp Lys Arg His Asn Gln Cys 1 5 327176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
327cgggggtggc cccccctcgg gggacagcgc atgcccgctg cgcaccatca
agagagtcca 60gttcggagtc ctgagtccgg atgaactggt ccctgtcctt cgaatggtgg
aaggtgatac 120catctatgat tactgctggt attctctgat gtcctcagcc
cagccagaca cctcct 17632859PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 328Gly Gly Gly Pro Pro
Ser Gly Asp Ser Ala Cys Pro Leu Arg Thr Ile 1 5 10 15 Lys Arg Val
Gln Phe Gly Val Leu Ser Pro Asp Glu Leu Val Pro Val 20 25 30 Leu
Arg Met Val Glu Gly Asp Thr Ile Tyr Asp Tyr Cys Trp Tyr Ser 35 40
45 Leu Met Ser Ser Ala Gln Pro Asp Thr Ser Xaa 50 55
32990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 329tggtccctgt ccttcgaatg gtggaaggtg
ataccatcta tgattactgc tggtattctc 60tgatgtcctc agcccagcca gacacctcct
9033090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 330ctggtccctg tccttcgaat ggtggaaggt
gataccatct atgattactg ctggtattct 60ctgatgtcct cagcccggcc agacacctcc
9033190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 331actggtccct gtccttcgaa tggtggaagg
tgataccatc tatgattact gctggtattc 60tctgatgtcc tcagcccagc cagacaccac
9033290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 332aactggtccc tgtccttcga atggtggaag
gtgataccat ctatgattac tgctggtatt 60ctctgatgtc ctcagcccag ccagacacct
9033390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 333gaactggtcc ctgtccttcg aatggtggaa
ggtgatacca tctatgatta ctgctggtat 60tctctgatgt cctcagccca gccagacacc
9033490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 334tgaactggtc cctgtccttc gaatggtgga
aggtgatacc atctatgatt actgctggta 60ttctctgatg tcctcagccc agccagacac
9033591DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 335atgaactggt ccctgtcctt cgaatggtgg
aaggtgatac catctatgat tactgctgta 60ttctctgatg tcctcagccc agccagacac
c 9133690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 336gatgaactgg tccctgtcct tcgaatggtg
gaaggtgata ccatctatga ttactgctgg 60tattctctga tgtcctcagc ccagccagac
9033790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 337ggatgaactg gtccctgtcc ttcgaatggt
ggaaggtgat accatctatg attactgctg 60gtattctctg atgtcctcag cccagccaga
9033890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 338cggatgaact ggtccctgtc cttcgaatgg
tggaaggtga taccatctat gattactgct 60ggtattctct gatgtcctca gcccagccag
9033990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 339ccggatgaac tggtccctgt ccttcgaatg
gtggaaggtg ataccatcta tgattactgc 60tggtattctc tgatgtcctc agcccagcca
9034090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 340tccggatgaa ctggtccctg tccttcgaat
ggtggaaggt gataccatct atgattactg 60ctggtattct ctgatgtcct cagcccagcc
9034190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 341gtccggagga actggtccct gtccttcgaa
tggtggaagg tgataccatc tatgattgct 60gctggtattc tctgatgtcc tcagcccagc
9034290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 342agtccggatg aactggtccc tgtccttcga
atggtggaag gtgataccat ctatgattac 60tgctggtatt ctctgatgtc ctcagcccag
9034390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 343gagtccggat gaactggtcc ctgtccttcg
aatggtggaa ggtgatacca tctatgatta 60ctgctggtat tctctgatgt cctcagcccg
9034490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 344tgagtccgga tgaactggtc cctgtccttc
gaatggtgga aggtgatacc atctatgatt 60actgctggta ttctctgatg tcctcagccc
9034590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 345ctgagtccgg atgaactggt ccctgtcctt
cgaatggtgg aaggtgatac catctatgat 60tactgctggt attctctgat gtcctcagcc
9034690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 346cctgagtccg gatgaactgg tccctgtcct
tcgaatggtg gaaggtgata ccatctatga 60ttactgctgg tattctctga tgtcctcagc
9034790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 347tcctgagtcc ggatgaactg gtccctgtcc
ttcgaatggt ggaaggtgat accatctatg 60attactgctg gtattctctg atgtcctcag
9034890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 348gtcctgagtc cggatgaact ggtccctgtc
cttcgaatgg tggaaggtga taccatctat 60gattactgct ggtattctct gatgtcctca
9034990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 349agtcctgagt ccggatgaac tggtccctgt
ccttcgaatg gtggaaggtg ataccatcta 60tgattactgc tggtattctc tgatgtcctc
9035090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 350gagtcctgag tccggatgaa ctggtccctg
tccttcgaat ggtggaaggt gataccatct 60atgattactg ctggtattct ctgatgtcct
9035190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 351ggagtcctga gtccggatga actggtccct
gtccttcgaa tggtggaagg tgataccatc 60tatgattact gctggtattc tctgatgtcc
9035290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 352cggagtcctg agtccggatg aactggtccc
tgtccttcga atggtggaag gtgataccat 60ctatgattac tgctggtatt ctctgatgtc
9035390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 353tcggagtcct gagtccggat gaactggtcc
ctgtccttcg aatggtggaa ggtgatacca 60tctatgatta ctgctggtat tctctgatgt
9035490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 354ttcggagtcc tgagtccgga tgaactggtc
cctgtccttc gaatggtgga aggtgatacc 60atctatgatt actgctggta ttctctgatg
9035590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 355gttcggagtc ctgagtccgg atgaactggt
ccctgtcctt cgaatggtgg aaggtgatac 60catctatgat tactgctggt attctctgat
9035690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 356cagttcggag tcctgagtcc ggatgaactg
gtccctgtcc ttcgaatggt ggaaggtgat 60accatctatg attactgctg gtattctctg
9035790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 357ccagttcgga gtcctgagtc cggatgaact
ggtccctgtc cttcgaatgg tggaaggtga 60taccatctat gattactgct ggtattctct
9035890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 358tccagttcgg agtcctgagt ccggatgaac
tggtccctgt ccttcgaatg gtggaaggtg 60ataccatcta tgattactgc tggtattctc
9035990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 359gtccagttcg gagtcctgag tccggatgaa
ctggtccctg tccttcgaat ggtggaaggt 60gataccatct atgattactg ctggtattct
9036090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 360gagtccagtt cggagtcctg agtccggatg
aactggtccc tgtccttcga atggtggaag 60gtgataccat ctatgattac tgctggtatt
9036190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 361agagtccagt tcggagtcct gagtccggat
gaactggtcc ctgtccttcg aatggtggaa 60ggtgatacca tctatgatta ctgctggtat
9036290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 362gagagtccag ttcggagtcc tgagtccgga
tgaactggtc cctgtccttc gaatggtgga 60aggtgatacc atctatgatt actgctggta
9036390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 363agagagtcca gttcggagtc ctgagtccgg
atgaactggt ccctgtcctt cgaatggtgg 60aaggtgatac catctatgat tactgctggt
9036490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 364aagagagtcc agttcggagt cctgagtccg
gatgaactgg tccctgtcct tcgaatggtg 60gaaggggata ccatctatga ttactgccgg
9036590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 365caagagagtc cagttcggag tcctgagtcc
ggatgaactg gtccctgtcc ttcgaatggt 60ggaaggtgat accatctatg attactgctg
9036690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 366tcaagagagt ccagttcgga gtcctgagtc
cggatgaact ggtccctgtc cttcgaatgg 60tggaaggtga taccatctat gattactgct
9036790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 367atcaagagag tccagttcgg agtcctgagt
ccggatgaac tggtccctgt ccttcgaatg 60gtggaaggtg ataccatcta tgattactgc
9036890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 368caccatcaag agagtccagt tcggagtcct
gagtccggat gaactggtcc ctgtccttcg 60aatggtggaa ggtgatacca tctatgatta
9036990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 369cgcaccatca agagagtcca gttcggagtc
ctgagtccgg atgaactggt ccctgtcctt 60cgaatggtgg aaggtgatac catctatgat
9037090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 370gcgcaccatc aagagagtcc agttcggagt
cctgagtccg gatgaactgg tccctgtcct 60tcgaatggtg gaaggtgata ccatctatga
9037190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 371gcccgctgcg caccatcaag agagtccagt
tcggagtcct gagtccggat gaactggtcc 60ctgtccttcg aatggtggaa ggtgatacca
9037290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 372cgcatgcccg ctgcgcacca tcaagagagt
ccagttcgga gtcctgagtc cggatgaact 60ggtccctgtc cttcgaatgg tggaaggtga
9037390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 373gcgcatgccc gctgcgcacc atcaagagag
tccagttcgg agtcctgagt ccggatgaac 60tggtccctgt ccttcgaatg gtggaaggtg
9037490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 374agcgcatgcc cgctgcgcac catcaagaga
gtccagttcg gagtcctgag tccggatgaa 60ctggtccctg tccttcgaat ggtggaaggt
9037590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 375cagcgcatgc ccgctgcgca ccatcaagag
agtccagttc ggagtcctga gtccggatga 60actggtccct gtccttcgaa tggtggaagg
9037690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 376gggacagcgc atgcccgctg cgcaccatca
agagagtcca gttcggagtc ctgagtccgg 60atgaactggt ccctgtcctt cgaatggtgg
9037790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 377cgggggacag cgcatgcccg ctgcgcacca
tcaagagagt ccagttcgga gtcctgagtc 60cggatgaact ggtccctgtc cttcgaatgg
9037890DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 378cccccctcgg
gggacagcgc atgcccgctg cgcaccatca agagagtcca gttcggagtc 60ctgagtccgg
atgaactggt ccctgtcctt 9037990DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 379ccccccctcg
ggggacagcg catgcccgct gcgcaccatc aagagagtcc agttcggagt 60cctgagtccg
gatgaactgg tccctgtcct 9038090DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 380ggccccccct
cgggggacag cgcatgcccg ctgcgcacca tcaagagagt ccagttcgga 60gtcctgagtc
cggatgaact ggtccctgtc 9038190DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 381ggggtggccc
cccctcgggg gacagcgcat gcccgctgcg caccatcaag agagtccagt 60tcggagtcct
gagtccggat gaactggtcc 9038290DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 382cgggggtggc
cccccctcgg gggacagcgc atgcccgctg cgcaccatca agagagtcca 60gttcggagtc
ctgagtccgg atgaactggt 90383170DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 383g cag ctg gac tgt
gcc ttg gac cta atg agg cgc ctg cct ccc cag caa 49 Gln Leu Asp Cys
Ala Leu Asp Leu Met Arg Arg Leu Pro Pro Gln Gln 1 5 10 15 atc gag
aaa aac ctc agc gac ctg atc gac ctg gat gtc ccc gtt gag 97Ile Glu
Lys Asn Leu Ser Asp Leu Ile Asp Leu Asp Val Pro Val Glu 20 25 30
gcc ctc acc acg gtg aag cca tac tgc aat gag atc cat gcc cag gct
145Ala Leu Thr Thr Val Lys Pro Tyr Cys Asn Glu Ile His Ala Gln Ala
35 40 45 caa ctg tgg ctc aag aga gac ccc a 170Gln Leu Trp Leu Lys
Arg Asp Pro 50 55 38456PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 384Gln Leu Asp Cys Ala
Leu Asp Leu Met Arg Arg Leu Pro Pro Gln Gln 1 5 10 15 Ile Glu Lys
Asn Leu Ser Asp Leu Ile Asp Leu Asp Val Pro Val Glu 20 25 30 Ala
Leu Thr Thr Val Lys Pro Tyr Cys Asn Glu Ile His Ala Gln Ala 35 40
45 Gln Leu Trp Leu Lys Arg Asp Pro 50 55 38590DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 385tggatgtccc cgtgggggcc ctcaccacgg tgaagccata
ctgcaatgag atccatgccc 60aggctcaact gtggctcaag agagacccca
9038690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 386ctggatctcc ccgtcgagcc cctccccacg
gtgaagccat actgcaatga gatccatgcc 60caggctcaac tgtggctcaa gagagacccc
9038790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 387caaggatgtc cccgttgagg ccctcaccac
ggtgaagcca tactgcaatg agatccatgc 60ccaggctcaa ctgtggctca agagagacct
9038890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 388acctggatgt ccccgtcgag gccctcccca
cggtgaagcc atactgcaat gagatccatg 60cccaggctca actgtggctc aagagagacc
9038990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 389gccctggatg tccccgtcga ggccctcacc
acggtgaagc catactgcaa tgagatccat 60gcccaggctc aactgtggct caagagagac
9039090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 390ccaccttgat gtccccgtcg aggccctcac
cacggtgaag ccatactgca atgagatcca 60tgcccaggct caactgtggc tcaagagaga
9039190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 391gcgacctgga tgtccccgtc gaggccctcc
ccacggtgaa gccatactgc aatgagatcc 60atgcccaggc tcaactgtgg ctcaagagag
9039290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 392gatcgacctg gatgtccccg tcgaggccct
caccacggtg aagccatact gcaatgagat 60ccatgcccag gctcaactgt ggctcaagag
9039390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 393acctgatcga cctggatgtc cccgtcgagg
ccctcaccac ggtgaagcca tactgcaatg 60agatccatgc ccaggctcaa ctgtggctca
9039490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 394cgacctgatc gacctggatg tccccgtcga
ggccctcacc acggtgaagc catactgcaa 60tgagatccat gcccaggctc aactgtggct
9039590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 395ctcagcgacc tgatcgacct ggatgtcccc
gtcgaggccc tcaccacggt gaagccatac 60tgcaatgaga tccatgccca ggctcaactg
9039690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 396aaaacctcag cgacctgatc gacctggatg
tccccgtcga ggccctcacc acggtgaagc 60catactgcaa tgagatccat gcccaggctc
9039790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 397gaaaaacctc agcgacctga tcgacctgga
tgtccccgtc gaggccctca ccacggtgaa 60gccatactgc aatgagatcc atgcccaggc
9039890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 398agaaaaacct cagcgacctg atcgacctgg
atgtccccgt cgaggccctc accacggtga 60agccatactg caatgagatc catgcccagg
9039990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 399cgagaaaaac ctcagcgacc tgatcgacct
ggatgtcccc gtcgaggccc tcaccacggt 60gaagccatac tgcaatgaga tccatgccca
9040090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 400tcgagaaaaa cctcagcgac ctgatcgacc
tggatgtccc cgtcgaggcc ctcaccacgg 60tgaagccata ctgcaatgag atccatgccc
9040190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 401atcgagaaaa acctcagcga cctgatcgac
ctggatgtcc ccgtcgaggc cctcaccacg 60gtgaagccat actgcaatga gatccatgcc
9040290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 402aatcgagaaa aacctcagcg acctgatcga
cctggatgtc cccgtcgagg ccctcaccac 60ggtgaagcca tactgcaatg agatccatgc
9040390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 403aaatcgaaaa aaacctcagc gacctgatcg
acctggatgg ccccggcgag gccctcacca 60cggtgaagcc atactgcaat gagatccatg
9040490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 404caaatcgaga aaaacctcag cgacctgatc
gacctggatg tccccgtcga ggccctcacc 60acggtgaagc catactgcaa tgagatccat
9040590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 405gcaaatcgag aaaaacctca gcgacctgat
cgacctggat gtccccgtcg aggccctcac 60cacggtgaag ccatactgca atgagatcca
9040690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 406agcaaatcga gaaaaacctc agcgacctga
tcgacctgga tgtccccgtc gaggccctca 60ccacggtgaa gccatactgc aatgagatcc
9040790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 407cagcaaatcg agaaaaacct cagcgacctg
atcgacctgg atgtccccgt cgaggccctc 60accacggtga agccatactg caatgagatc
9040890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 408ccccagcaaa tcgagaaaaa cctcagcgac
ctgatcgacc tggatgtccc cgtcgaggcc 60ctcaccacgg tgaagccata ctgcaatgag
9040990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 409ctccccagca aatcgagaaa aacctcagcg
acctgatcga cctggatgtc cccgtcgagg 60ccctcaccac ggtgaagcca tactgcaatg
9041090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 410cctccccagc aaatcgagaa aaacctcagc
gacctgatcg acctggatgt ccccgtcgag 60gccctcacca cggtgaagcc atactgcaat
9041190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 411gcctccccag caaatcgaga aaaacctcag
cgacctgatc gacctggatg tccccgtcga 60ggccctcacc acggtgaagc catactgcaa
9041290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 412tgcctcccca gcaaatcgag aaaaacctca
gcgacccgat cgacctggat gtccccgtcg 60aggccctcac cacggtgaag ccatactgca
9041390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 413ctgcctcccc agcaaatcga gaaaaacctc
agcgacctga tcgacctgga tgtccccgtc 60gaggccctca ccacggtgaa gccatactgc
9041490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 414cctgcctccc cagcaaatcg agaaaaacct
cagcgacctg atcgacctgg atgtccccgt 60cgaggccctc accacggtga agccatactg
9041590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 415cgcctgcctc cccagcaaat cgagaaaaac
ctcagcgacc tgatcgacct ggatgtcccc 60gtcgaggccc tcaccacggt gaagccatac
9041690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 416ggcgcctgcc tccccagcaa atcgagaaaa
acctcagcga cctgatcgac ctggatgtcc 60ccgtcgaggc cctcaccacg gtgaagccat
9041790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 417gcggtgcctg cctccccagc aaatcgagaa
aaacctcagc gacctgatcg acctggatgt 60ccccgtcgag gccctcacca cggtgaagcc
9041890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 418tgaggcgcct gcctccccag caaatcgaga
aaaacctcag cgacctgatc gacctggatg 60tccccgtcga ggccctcacc acggtgaagc
9041990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 419atgaggcgcc tgcctcccca gcaaatcgag
aaaaacctca gcgacctgat cgacctggat 60gtccccgtcg aggccctcac cacggtgaag
9042090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 420aatgaggcgc ctgcctcccc agcaaatcga
gaaaaacctc agcgacctga tcgacctgga 60tgtccccgtc gaggccctca ccacggtgaa
9042190DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 421taatgaggcg cctgcctccc cagcaaatcg
agaaaaacct cagcgacctg atcgacctgg 60atgtccccgt cgaggccctc accacggtga
9042290DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 422gacctaatga ggcgcctgcc tccccagcaa
atcgagaaaa acctcagcga cctgatcgac 60ctggatgtcc ccgtcgaggc cctcaccacg
9042390DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 423ttggacctaa tgaggcgcct gcctccccag
caaatcgaga aaaacctcag cgacctgatc 60gacctggatg tccccgtcga ggccctcacc
9042490DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 424ctgggaccta aggaggcgcc tgcctcccca
gcaaatcgag aaaaacctca gcgacctgat 60cgacctggat gtccccgtcg aggccctcac
9042590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 425gccttggacc taatgaggcg cctgcctccc
cagcaaatcg agaaaaacct cagcgacctg 60atcgacctgg atgtccccgt cgaggccctc
9042690DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 426gtgccttgga cctaatgagg cgcctgcctc
cccagcaaat cgagaaaaac ctcagcgacc 60tgatcgacct ggatgtcccc gtcgaggccc
9042790DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 427actgggcctg gggcctaatg aggcgcctgc
ctccccagca aatcgagaaa aacctcagcg 60acctgatcga cctggatgtc cccgtcgagg
9042890DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 428gactgtgcct gggccctaag gaggcgcctg
cctccccagc aaatcgagaa aaacctcagc 60gacctgatcg acctggatgt ccccgtcgag
9042990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 429catggactgt gccttggacc taatgaggcg
cctgcctccc cagcaaatcg agaaaaacct 60cagcgacctg atcgacctgg atgtccccga
9043090DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 430gctggactgt gccttggacc taatgaggcc
ccttcctccc cagcaaatcg agaaaaacct 60cagcgacctg atcgacctgg atgtcccccg
9043191DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 431agctggactg tgccttggac ctaatgaggc
gcctgcctcc ccagcaaatc gagaaaaacc 60ccagcgacct gatcgacctg gatgtccccg
g 9143291DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 432gcagctggac tgtgccttgg acctaatgag
gcgcctgcct ccccagcaaa tcgagaaaaa 60cctcagcgac ctgatcgacc tggatgtccc
c 91433176DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 433ggagtgggtt aatgcattaa tccttaagaa
taaactgaaa gtgcgaactg cctatccgtc 60attgagactt attcatgctg tcagagggtt
ttgtgatgaa ggaacctgta cagataaagc 120caatattctg tatgcctggg
cgagaaatgc tccccctacc cggctcccca aaggtg 17643459PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
434Glu Trp Val Asn Ala Leu Ile Leu Lys Asn Lys Leu Lys Val Arg Thr
1 5 10 15 Ala Tyr Pro Ser Leu Arg Leu Ile His Ala Val Arg Gly Phe
Cys Asp 20 25 30 Glu Gly Thr Cys Thr Asp Lys Ala Asn Ile Leu Tyr
Ala Trp Ala Arg 35 40 45 Asn Ala Pro Pro Thr Arg Leu Pro Lys Gly
Xaa 50 55 43590DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 435ggttttgtga tgaaggaacc
tgtacagata aagccaatat tctgtatgcc tgggcgagaa 60atgctccccc tacccggctc
cccaaaggtg 9043690DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 436tggttttgtg atgaaggaac
ctgtacagat aaagccaata ttctgtatgc ctgggcgaga 60aatgctcccc ctacccggct
ccccaaaggt 9043790DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 437ctggttttgt gatgaaggaa
cctgtacaga taaagccaat attctgtatg cctgggcgag 60aaatgctccc cctacgcggc
tccccaaagg 9043890DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 438gagggttttg tgatgaagga
acctgtacag ataaagccaa tattctgtat gcctgggcga 60gaaatgctcc ccctacccgg
ctccccaaag 9043991DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 439gttactggtt ttgtgatgaa
ggaacctgta cagataaagc caatattctg tatgcctggg 60cgagaaatgc tccccctacc
cggctcccca a 9144090DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 440gctgtcagag ggttttgtga
tgaaggaacc tgtacagata aagccaatat tctgtatgcc 60tgggcgagaa atgctccccc
tacccggctc 9044191DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 441tgctgtcaga gggttttgtg
atgaaggaac ctgtacagat aaagccaata ttctgtatgc 60ctgggcgaga aatgctcccc
ctacccggcc c 9144290DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 442atgctgtcag agggttttgt
gatgaaggaa cctgtacaga taaagccaat attctgtatg 60cctgggcgag aaatgctccc
cctacccggc 9044390DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 443catgctgtca gagggttttg
tgatgaagga gcctgtacag ataaagccaa tattctgtat 60gcctgggcga gaaatgctcc
ccctacccgg 9044490DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 444tcatgctgtc
agagggtttt gtgatgaagg aacctgtaca gataaagcca atattctgta 60tgcctgggcg
agaaatgctc cccctacccg 9044590DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 445tattcatgct
gtcagagggt tttgtgatga aggaacctgt acagataaag ccaatattct 60gtatgcctgg
gcgagaaatg ctccccctac 9044690DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 446cttattcatg
ctgtcagagg gttttgtgat gaaggaacct gtacagataa agccaatatt 60ctgtatgcct
gggcgagaaa tactccccct 9044790DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 447acttattcat
gctgtcagag ggttttgtga tgaaggaacc tgtacagata aagccaatat 60tctgtatgcc
tgggcgagaa atgctccccc 9044890DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 448gagacttatt
catgctgtca gagggttttg tgatgaagga acctgtacag ataaagccaa 60tattctgtat
gcctgggcga gaaatgctcc 9044990DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 449tgagacttat
tcatgctgtc agagggtttt gtgatgaagg aacctgtaca gataaagcca 60atattctgta
tgcctgggcg agaaatgctc 9045090DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 450ttgagactta
ttcatgctgt cagagggttt tgtgatgaag gaacctgtac agataaagcc 60aatattctgt
atgcctgggc gagaaatgct 9045190DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 451gtcattgaga
cttattcatg ctgtcagagg gttttgtgat gaaggaacct gtacagataa 60agccaatatt
ctgtatgcct gggcgagaaa 9045290DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 452ccgtcattga
gacttattca tgctgtcaga gggttttgtg atgaaggaac ctgtacagat 60aaagccaata
ttctgtatgc ctgggcgaga 9045390DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 453tatccgtcat
tgagacttat tcatgctgtc agagggtttt gtgatgaagg aacctgtaca 60gataaagcca
atattctgta tgcctgggcg 9045490DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 454ctatccgtca
ttgagactta ttcatgctgt cagagggttt tgtgatgaag gaacctgtac 60agataaagcc
aatattctgt atgcctgggc 9045590DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 455cctatccgtc
attgagactt attcatgctg tcagagggtt ttgtgatgaa ggaacctgta 60cagataaagc
caatattctg tatgcctggg 9045690DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 456gcctatccgt
cattgagact tattcatgct gtcagagggt tttgtgatga aggaacctgt 60acagataaag
ccaatattct gtatgcctgg 9045790DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 457tgaaagtgcg
aactgcctat ccgtcattga gacttattca tgctgtcaga gggttttgtg 60atgaaggaac
ctgtacagat aaagccaata 9045890DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 458ctgaaagtgc
gaactgccta tccgtcattg agacttattc atgctgtcag agggttttgt 60gatgaaggaa
cctgtacaga taaagccaat 9045990DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 459aaactgaaag
tgcgaactgc ctatccgtca ttgagactta ttcatgctgt cagagggttt 60tgtgatgaag
gaacctgtac agataaagcc 9046090DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 460taaactgaaa
gtgcgaactg cctatccgtc attgagactt attcatgctg tcagagggtt 60ttgtgatgaa
ggaacctgta cagataaagc 9046190DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 461attaatcctt
aagaataaac tgaaagtgcg aactgcctat ccgtcattga gacttattca 60tgctgtcaga
gggttttgtg atgaaggaac 9046290DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 462gcattaatcc
ttaagaataa actgaaagtg cgaactgcct atccgtcatt gagacttatt 60catgctgtca
gagggttttg tgatgaagga 9046390DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 463ggagtgggtt
aatgcattaa tccttaagaa taaactgaaa gtgcgaactg cctatccgtc 60attgagactt
attcatgctg tcagagggtt 90464167DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 464acacgatttt
gtcttgtgtt gccaagtcta cctgtgccat caacaacacc ctcattgctt 60tcttcatttt
gactacgata aaagacataa ccagtgcggt gcaatccaag cgaagaaaat
120ccaagtaaac aagcaggact gcgacttgat acttggaaat gtgtgtg
16746556PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 465His Asp Phe Val Leu Cys Cys Gln Val Tyr
Leu Cys His Gln Gln His 1 5 10 15 Pro His Cys Phe Leu His Phe Asp
Tyr Asp Lys Arg His Asn Gln Cys 20 25 30 Gly Ala Ile Gln Ala Lys
Lys Ile Gln Val Asn Lys Gln Asp Cys Asp 35 40 45 Leu Ile Leu Gly
Asn Val Cys Xaa 50 55 46690DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 466ataaaagaca
taaccagtgc ggtgcaatcc aagcgaagaa aatccaagta aacaagcagg 60actgcgactt
gatacttgga aatgtgtgtg 9046790DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 467ttgctttctt
cattttgact acgataaaag acataaccag tgcggtgcaa tccaagcgaa 60gaaaatccaa
gtaaacaagc aggactgcga 9046890DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 468cctcattgct
ttcttcattt tgactacgat aaaagacata accagtgcgg tgcaatccaa 60gcgaagaaaa
tccaagtaaa caagcaggac 9046990DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 469ccctcattgc
tttcttcatt ttgactacga taaaagacat aaccagtgcg gtgcaatcca 60agcgaagaaa
atccaagtaa acaagcagga 9047090DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 470caccctcatt
gctttcttca ttttgactac gataaaagac ataaccagtg cggtgcaatc 60caagcgaaga
aaatccaagt aaacaagcag 9047190DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 471caacaccctc
attgctttct tcattttgac tacgataaaa gacataacca gtgcggtgca 60atccaagcga
agaaaatcca agtaaacaag 9047290DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 472caacaacacc
ctcattgctt tcttcatttt gactacgata aaagacataa ccagtgcggt 60gcaatccaag
cgaagaaaat ccaagtaaac 9047390DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 473atcaacaaca
ccctcattgc tttcttcatt ttgactacga taaaagacat aaccagtgcg 60gtgcaatcca
agcgaagaaa atccaagtaa 9047490DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 474catcaacaac
accctcattg ctttcttcat tttgactacg ataaaagaca taaccagtgc 60ggtgcaatcc
aagcgaagaa aatccaagta 9047590DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 475gtgccatcaa
caacaccctc attgctttct tcattttgac tacgataaaa gacataacca 60gtgcggtgca
atccaagcga agaaaatcca 9047690DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 476gtctacctgt
gccatcaaca acaccctcat tgctttcttc attttgacta cgataaaaga 60cataaccagt
gcggtgcaat ccaagcgaag 9047790DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 477caagtctacc
tgtgccatca acaacaccct cattgctttc ttcattttga ctacgataaa 60agacataacc
agtgcggtgc aatccaagcg 9047890DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 478ccaagtctac
ctgtgccatc aacaacaccc tcattgcttt cttcattttg actacgataa 60aagacataac
cagtgcggtg caatccaagc 9047990DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 479gtcttgtgtt
gccaagtcta cctgtgccat caacaacacc ctcattgctt tcttcatttt 60gactacgata
aaagacataa ccagtgcggt 9048090DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 480ttttcttgtg
ttgccaagtc tacctgtgcc atcaacaaca ccctcattgc tttcttcatt 60ttgactacga
taaaagacat aaccagtgcg 9048190DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 481attttgtctt
gtgttgccaa gtctacctgt gccatcaaca acaccctcat tgctttcttc 60attttgacta
cgataaaaga cataaccagt 9048290DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 482acacgatttt
gtcttgtgtt gccaagtcta cctgtgccat caacaacacc ctcattgctt 60tcttcatttt
gactacgata aaagacataa 90483166DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 483atttagagca
cagtgatgag caagcagtaa taaagtctcc cttaaaatgc accctccttc 60cacctggcca
ggaatcagca ttgggaatgg taccacctcc cgaaaatgtc agaatgaatt
120ctgttaattt caagaacatt ctacagtggg agtcacctgc ttttgc
16648455PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 484Leu Glu His Ser Asp Glu Gln Ala Val Ile
Lys Ser Pro Leu Lys Cys 1 5 10 15 Thr Leu Leu Pro Pro Gly Gln Glu
Ser Ala Leu Gly Met Val Pro Pro 20 25 30 Pro Glu Asn Val Arg Met
Asn Ser Val Asn Phe Lys Asn Ile Leu Gln 35 40 45 Trp Glu Ser Pro
Ala Phe Xaa 50 55 48590DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 485agcattggga
atggtaccac ctcccgaaaa tgtcagaatg aattctgtta atttcaagaa 60cattctacag
tgggagtcac ctgcttttgc 9048690DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 486gtcagcattg
ggaatggtac cacctcccga aaatgtcaga atgaattctg ttaatttcaa 60gaacattcta
cagtgggagt cacctgcttt 9048790DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 487cagtaataaa
gtctccctta aaatgcaccc tccttccacc tggccaggaa tcagcattgg 60gaatggtacc
acctcccgaa aatgtcagaa 9048890DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 488gtgatgagca
agcagtaata aagtctccct taaaatgcac cctccttcca cctggccagg 60aatcagcatt
gggaatggta ccacctcccg 9048990DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 489acagtgatga
gcaagcagta ataaagtctc ccttaaaatg caccctcctt ccacctggcc 60aggaatcagc
attgggaatg gtaccaccaa 9049090DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 490atttagagca
cagtgatgag caagcagtaa taaagtctcc cttaaaatgc accctccttc 60cacctggcca
ggaatcagca ttgggaatgg 9049112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 491Leu Gln Leu Ala Asn Glu
Glu Ile Ala Gln Val Arg 1 5 10 49210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 492Asp
Gly Thr Gly Leu Val Pro Ser Glu Arg 1 5 10 49313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 493Val
Gly Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys 1 5 10
4947PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 494Val Leu Val Gly Pro Gln Arg 1 5
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