U.S. patent application number 10/457030 was filed with the patent office on 2004-02-05 for method of diagnosing pulmonary hypertension.
Invention is credited to Foroud, Tatiana, Lane, Kirk B., Loyd, James E., Machado, Rajiv D., Nichols, William C., Pauciulo, Michael W., Phillips, John A. III, Thomson, Jennifer R., Trembath, Richard C..
Application Number | 20040023280 10/457030 |
Document ID | / |
Family ID | 26913204 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023280 |
Kind Code |
A1 |
Loyd, James E. ; et
al. |
February 5, 2004 |
Method of diagnosing pulmonary hypertension
Abstract
This invention relates generally to a method of identifying an
individual having an increased susceptibility to developing
Familial Primary Pulmonary Hypertension (FPPH), as well as to a
method for diagnosing an individual suffering from FPPH. The
invention also relates to a method of identifying an individual
having an increased susceptibility to developing (non-familial)
Primary Pulmonary Hypertension (PPH), as well as to a method for
diagnosing an individual suffering from PPH.
Inventors: |
Loyd, James E.; (Nashville,
TN) ; Lane, Kirk B.; (Brentwood, TN) ;
Phillips, John A. III; (Brentwood, TN) ; Trembath,
Richard C.; (Oakham, GB) ; Machado, Rajiv D.;
(Leicester, GB) ; Thomson, Jennifer R.; (Leeds,
GB) ; Nichols, William C.; (Loveland, OH) ;
Pauciulo, Michael W.; (Blue Ash, OH) ; Foroud,
Tatiana; (Indianapolis, IN) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
26913204 |
Appl. No.: |
10/457030 |
Filed: |
June 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10457030 |
Jun 6, 2003 |
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09908500 |
Jul 17, 2001 |
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6642002 |
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60218740 |
Jul 17, 2000 |
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60220133 |
Jul 21, 2000 |
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Current U.S.
Class: |
435/6.16 ;
435/6.18; 435/7.1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 ;
435/7.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Goverment Interests
[0002] Some of the research on which the present disclosure is
based was funded by National Institutes of Health Grants HL 48164
and HL 61997.
Claims
We claim:
1. A method of identifying a subject having an increased
susceptibility for developing pulmonary hypertension, comprising
detecting a mutant Bone Morphogenic Protein Receptor II (BMPR-II)
polypeptide or a mutated Bone Morphogenic Protein Receptor 2
(BMPR2) nucleic acid in the subject, thereby identifying a subject
having an increased susceptibility for developing pulmonary
hypertension.
2. The method of claim 1, wherein the subject having an increased
susceptibility for developing pulmonary hypertension is identified
by detecting a mutated BMPR2 nucleic acid in the subject.
3. The method of claim 2, wherein the mutated BMPR2 nucleic acid
encodes a mutant BMPR-II polypeptide.
4. The method of claim 2 wherein the mutated BMPR2 nucleic acid
comprises a missense mutation.
5. The method of claim 2, wherein the mutated BMPR2 nucleic acid
comprises a nonsense mutation.
6. The method of claim 2, wherein the mutated BMPR2 nucleic acid
comprises a deletion mutation.
7. The method of claim 2, wherein the mutated BMPR2 nucleic acid
comprises an insertion mutation.
8. The method of claim 2, wherein the mutated BMPR2 nucleic acid
comprises a truncation mutation.
9. The method of claim 8, wherein the mutated BMPR2 nucleic acid is
truncated at a nucleotide position of the sequence set forth in SEQ
ID NO:1 which is 3' to nucleotide position 2695.
10. The method of claim 2, wherein the mutated BMPR2 nucleic acid
comprises a nucleotide sequence that differs from the sequence set
forth in SEQ ID NO:1.
11. The method of claim 1, wherein the mutated BMPR2 nucleic acid
or mutant BMPR-II polypeptide has a sequence associated with
pulmonary hypertension.
12. The method of claim 11, wherein the mutated BMPR2 nucleic acid
comprises a mutation at a nucleotide position of the sequence set
forth in SEQ ID NO:1 selected from the group consisting of 218,
354, 355, 367, 428, 504, 689, 958, 993, 1042, 1076, 1129, 1191,
1258, 1454, 1535, 1557, 1749, 2292, 2408, 2579, and 2695.
13. The method of claim 12, wherein the mutation in the mutated
BMPR2 nucleic acid results in a non-conservative substitution in
the amino acid sequence encoded by the nucleic acid.
14. The method of claim 13, wherein the mutation in the mutated
BMPR2 nucleic acid results in a non-conservative substitution at a
Cys residue encoded by the nucleotide sequence set forth in SEQ ID
NO:1.
15. The method of claim 13, wherein the mutation is selected from
the group consisting of C218G, T354G, T367C, T367A, C428T, C993T,
G1042A, T 1258 C, A 1454 G, A 1535 C, T 1557 A, C 2695 T.
16. The method of claim 1, wherein the subject having an increased
susceptibility for developing pulmonary hypertension is identified
by detecting a BMPR2 nucleic acid having a sequence associated with
pulmonary hypertension.
17. The method of claim 16, wherein the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension comprises a
truncation mutation.
18. The method of claim 16, wherein the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension comprises a
missense mutation.
19. The method of claim 16 wherein the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension comprises a
nonsense mutation.
20. The method of claim 16, wherein the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension comprises a
deletion mutation.
21. The method of claim 16, wherein the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension comprises a nucleic
acid sequence having an insertion mutation.
22. The method of claim 16, wherein the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension encodes a mutant
BMPR-II polypeptide.
23. The method of claim 22, wherein the mutant BMPR-II polypeptide
comprises at least one mutation at an amino acid position of the
sequence set forth in SEQ ID NO:2.
24. The method of claim 22, wherein the BMPR-II polypeptide
comprises at least one mutation at an amino acid position of the
sequence set forth in SEQ ID NO:2 selected from the group
consisting of 73, 118, 123, 143, 332, 348, 420, 485, 512, 519, and
899.
25. The method of claim 22, wherein the BMPR-II polypeptide
comprises at least one mutation at an amino acid position of the
sequence set forth in SEQ ID NO:2 selected from the group
consisting of a Trp residue at amino acid position 118, an Arg
residue at amino acid position 123, a Ser residue at amino acid
position 123, a Leu residue at amino acid position 143, an Ile
residue at amino acid position 348, an Arg residue at amino acid
position 420, an Ala residue at amino acid position 485, a Gln
residue at amino acid position Gln, and a Lys residue at amino acid
position 519.
26. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension terminates
prematurely.
27. The method of claim 26, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension terminates at an
amino acid position of the sequence set forth in SEQ ID NO:2 which
is N-terminal to amino acid position 73, 332, or 899.
28. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one amino acid
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
29. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Cys
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
30. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Pro
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
31. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Lys
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
32. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Arg
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
33. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Asp
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
34. The method of claim 22, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Glu
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
35. The method of claim 1, wherein the subject having an increased
susceptibility for developing pulmonary hypertension is identified
by detecting a mutant BMPR-II polypeptide in the subject.
36. The method of claim 35, wherein the mutant BMPR-II polypeptide
terminates prematurely.
37. The method of claim 36, wherein the mutant BMPR-II polypeptide
terminates at an amino acid position of the sequence set forth in
SEQ ID NO:2 which is N-terminal to amino acid position 73, 332, or
899.
38. The method of claim 35, wherein the mutant BMPR-II polypeptide
comprises a non-conservative substitution at an amino acid
position.
39. The method of claim 38, wherein the mutant BMPR-II polypeptide
comprises a non-conservative substitution for a Cys residue.
40. The method of claim 38, wherein the non-conservative
substitution is selected from the group consisting of a Trp residue
at amino acid position 118, an Arg residue at amino acid position
123, a Ser residue at amino acid position 123, a Leu residue at
amino acid position 143, an Ile residue at amino acid position 348,
an Arg residue at amino acid position 420, an Ala residue at amino
acid position 485, a Gln residue at amino acid position Gln, and a
Lys residue at amino acid position 519.
41. The method of claim 35, wherein the mutant BMPR-II polypeptide
comprises an amino acid sequence that differs from the sequence set
forth in SEQ ID NO:2.
42. The method of claim 41, wherein the mutant BMPR-II polypeptide
comprises a mutation at an amino acid position of the sequence set
forth in SEQ ID NO:2 selected from the group consisting of 73, 118,
123, 143, 332, 348, 420, 485, 512, 519, and 899.
43. The method of claim 35, wherein the BMPR-II polypeptide
comprises at least one mutation at an amino acid position of the
sequence set forth in SEQ ID NO:2 selected from the group
consisting of 73, 118, 123, 143, 332, 348, 420, 485, 512, 519, and
899.
44. The method of claim 35, wherein the BMPR-II polypeptide
comprises at least one mutation at an amino acid position of the
sequence set forth in SEQ ID NO:2 selected from the group
consisting of a Trp residue at amino acid position 118, an Arg
residue at amino acid position 123, a Ser residue at amino acid
position 123, a Leu residue at amino acid position 143, an Ile
residue at amino acid position 348, an Arg residue at amino acid
position 420, an Ala residue at amino acid position 485, a Gln
residue at amino acid position Gln, and a Lys residue at amino acid
position 519.
45. The method of claim 1, wherein the subject having an increased
susceptibility for developing pulmonary hypertension is identified
by detecting a BMPR-II polypeptide having a sequence associated
with pulmonary hypertension in the subject.
46. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension terminates
prematurely.
47. The method of claim 46, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension terminates at an
amino acid position of the sequence set forth in SEQ ID NO:2 which
is N-terminal to amino acid position 73, 332, or 899.
48. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one amino acid
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
49. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Cys
residue of a BMPR-II encoded by the nucleotide sequence set forth
in SEQ ID NO:1.
50. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Pro
residue of the wild-type BMPR-II encoded by the nucleotide sequence
set forth in SEQ ID NO:1.
51. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Lys
residue of the wild-type BMPR-II encoded by the nucleotide sequence
set forth in SEQ ID NO:1.
52. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Arg
residue of the wild-type BMPR-II encoded by the nucleotide sequence
set forth in SEQ ID NO:1.
53. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Asp
residue of the wild-type BMPR-II encoded by the nucleotide sequence
set forth in SEQ ID NO:1.
54. The method of claim 45, wherein the BMPR-II polypeptide having
a sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one Glu
residue of the wild-type BMPR-II encoded by the nucleotide sequence
set forth in SEQ ID NO:1.
55. The method of claim 1, wherein the mutant BMPR-II polypeptide
or mutated BMPR2 nucleic acid is due to a familial mutation.
56. The method of claim 1, wherein the mutant BMPR-II polypeptide
or mutated BMPR2 nucleic acid is due to a sporadic mutation.
57. The method of claim 1, wherein the mutated BMPR2 nucleic acid
has a sequence associated with pulmonary hypertension, wherein the
mutated BMPR2 nucleic acid results in altered BMPR2 RNA
function.
58. The method of claim 57, wherein the altered BMPR2 RNA function
is reduced BMPR2 mRNA production, altered processing of BMPR2 RNA,
or increased BMPR2 RNA instability.
59. The method of claim 58, wherein the altered BMPR2 RNA function
is altered splicing of BMPR2 RNA.
60. A kit for identifying a subject having an increased
susceptibility for developing pulmonary hypertension, comprising
reagents for detecting a mutant Bone Morphogenic Protein Receptor
II (BMPR-II) polypeptide or a mutated BMPR2 nucleic acid in the
subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/908,500, filed Jul. 17, 2001, which claims benefit of U.S.
Provisional Application No. 60/218,740, filed Jul. 17, 2000, and
U.S. Provisional Application No. 60/220,133, filed Jul. 21, 2000,
application Ser. No. 09/908,500, filed Jul. 17, 2001, Application
Serial No. 60/218,740, filed Jul. 17, 2000, and U.S. Provisional
Application No. 60/220,133, filed Jul. 21, 2000, are hereby
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to a method of identifying
an individual having an increased susceptibility to developing
Familial Primary Pulmonary Hypertension (FPPH), as well as to a
method for diagnosing an individual suffering from FPPH. The
invention also relates to a method of identifying an individual
having an increased susceptibility to developing non-familial, or
sporadic, Primary Pulmonary Hypertension (PPH), as well as to a
method for diagnosing an individual suffering from sporadic PPH.
The invention also relates to a method of identifying an agent
capable of altering the symptoms of PPH in an individual suffering
from familial or sporadic PPH, comprising contacting a test agent
with Bone Morphogenic Protein Receptor II (BMPR-II) and determining
whether the test agent alters BMPR-II activity, wherein an
alteration in BMPR-II activity in the presence of the test agent as
compared with BMPR-II activity in the absence of the test agent
indicates that the test agent is capable of altering the symptoms
of PPH in an individual suffering from familial or sporadic
PPH.
BACKGROUND OF THE INVENTION
[0004] Primary pulmonary hypertension (PPH) is characterized by
sustained elevation of pulmonary artery pressure (greater than 25
mmHg at rest and greater than 30 mmHg during exercise) and with no
identifiable cause, such as recurrent thromboembolism, chronic
hypoxic lung disease or left-sided cardiac disease. PPH is twice as
common in females than males and symptoms develop typically in the
3.sup.rd and 4.sup.th decades of life, although the disease may
occur at any age. Despite advances in therapy, mortality in PPH
remains high with mean survival from onset of disease only 2.5
year.
[0005] At least 6% of individuals diagnosed with PPH have a known
family history of the disorder. The disease can be classified as
being either familial (more than one affected relative has been
identified in at least 6% of cases (familial PPH; MIM 178600) (ref.
3)) or sporadic. Familial PPH (FPPH) segregates as an autosomal
dominant disorder, with markedly reduced penetrance.
[0006] There is a need to identify the genetic basis for this
devastating disease in order to better diagnose and treat patients
suffering from PPH.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention relates to a method of identifying a subject
having an increased susceptibility for developing pulmonary
hypertension, comprising detecting a mutant Bone Morphogenic
Protein Receptor II (BMPR-II) polypeptide or a mutated Bone
Morphogenic Protein Receptor 2 (BMPR2) nucleic acid in the subject,
thereby identifying a subject having an increased susceptibility
for developing pulmonary hypertension. Wild-type BMPR2 nucleotide
sequence is SEQ ID NO:1. Wild-type BMPR-II amino acid sequence is
SEQ ID NO:2.
[0008] In one aspect, the mutated BMPR2 nucleic acid or mutant
BMPR-II polypeptide has a sequence associated with pulmonary
hypertension.
[0009] In another aspect, the mutated BMPR2 nucleic acid comprises
a missense mutation.
[0010] In yet another aspect, the mutated BMPR2 nucleic acid
comprises a nonsense mutation.
[0011] In another aspect, the mutated BMPR2 nucleic acid comprises
a deletion mutation.
[0012] In another aspect, the mutated BMPR2 nucleic acid comprises
an insertion mutation.
[0013] In another aspect, the mutated BMPR2 nucleic acid comprises
a truncation mutation. Preferably, the mutated BMPR2 nucleic acid
is truncated at a nucleotide position of the sequence set forth in
SEQ ID NO:1 which is 3' to nucleotide position 2695.
[0014] In another aspect, the subject having an increased
susceptibility for developing pulmonary hypertension is identified
by detecting a BMPR2 nucleic acid having a sequence associated with
pulmonary hypertension.
[0015] In a preferred aspect, the pulmonary hypertension is primary
pulmonary hypertension. In another aspect, the pulmonary
hypertension is secondary pulmonary hypertension.
[0016] In various preferred embodiments, the mutated BMPR2 nucleic
acid can include a missense mutation or a nonsense mutation.
[0017] In another aspect, the invention features a method of
identifying a mutant BMPR-II polypeptide or a mutated BMPR2 nucleic
acid, including detecting, in a patient with PPH, a BMPR-II
polypeptide that is not present in normal subjects or a BMPR2
nucleic acid that is not present in normal subjects, thereby
identifying a mutant BMPR-II polypeptide or a mutated BMPR2 nucleic
acid.
[0018] In another aspect, the invention features a method of
increasing BMPR-II biological activity.
[0019] In another aspect, the invention features a method of
decreasing BMPR-II biological activity.
[0020] In another aspect, the invention features a method of
identifying a compound that modulates the biological activity of a
BMPR-II polypeptide, including: a) contacting a sample including a
BMPR-II polypeptide or a BMPR2 nucleic acid with the compound; and
b) measuring BMPR-II biological activity in the sample, whereby an
increase or decrease in BMPR-II biological activity, compared to
BMPR-II biological activity in an identical sample not contacted
with the compound, identifies a compound that modulates the
biological activity of the BMPR-II polypeptide.
[0021] In various embodiments of this aspect of the invention,
BMPR-II biological activity is increased or decreased; the BMPR-II
polypeptide is a wild-type BMPR-II polypeptide or the BMPR2 nucleic
acid is a wild-type BMPR2 nucleic acid; the BMPR-II polypeptide is
a polymorphic variant of a BMPR-II polypeptide or the BMPR2 nucleic
acid is a polymorphic variant of a BMPR2 nucleic acid; or the
BMPR-II polypeptide is a mutant BMPR-II polypeptide or the BMPR2
nucleic acid is a mutated BMPR2 nucleic acid.
[0022] In another aspect, the invention features a non-human mammal
having a deleted, mutated, or polymorphic variant BMPR2 gene. In
various aspects of the twelfth aspect of the invention, the
non-human mammal is a mouse; and/or the non-human mammal is
homozygous for the deleted, mutated, or polymorphic variant BMPR2
gene.
[0023] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIGS. 1A-1C are diagrams showing the physical map of the
PPH1 interval at 2q33. FIG. 1A shows the position of microsatellite
markers. The markers are, from left to right, D2S115, D2S348,
D2S2392, D2S2396, D2S 1367, D2S 116, D2S309, D2S2309, D2S2214,
D2S2217, D2S346, D2S2289, 19E07, D2S307, CTLA4, D2S72, D2S105,
D2S2189, D2S1384. FIG. 1B shows the physical map contig (BAC/PAC)
of the region surrounding BMPR2 including other genes analysed in
the examples (12). FIG. 1C shows the BMPR2 genomic structure,
determined by analysis of available sequence data for BAC clone
RP11-345N12 as well as sequence analysis of additional BAC clones
identified by library screening as shown (not to scale).
[0025] FIG. 2A is a diagram of the structure of BMPR2 cDNA. The
location of the exons are indicated by the nucleotide start
position in the cDNA. The cysteine residues within the
extracellular domain are each denoted by *. The filled in box
represents the transmembrane domain and the stippled area
identifies the region encoding the receptor kinase domain.
[0026] FIG. 2B is a diagram of BLAST homology results showing
protein similarity of human BMPR-II with receptors in other species
and human TGF-.beta. receptor type II (TGFBR-II). Amino acid
positions are shown together with the codon substitutions of
conserved amino acids (boxed).
[0027] FIG. 3 is a diagram of a basic function of BMPR-II. Two BMP
type I receptors (BMPRIA and BMPRIB) and a single BMP type II
receptor have been identified in mammals as serine/threonine kinase
receptors. Following ligand binding to BMPR-II, this receptor forms
a heteromeric complex with a type I receptor, resulting in
activation of the type I receptor kinase domain which intiates
phosphorylation of cytoplasmic signalling proteins, termed Smads,
responsible for signal transduction.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The invention relates to the surprising discovery that FPPH
is caused by mutations in the gene encoding a TGF-.beta. type II
receptor, BMPR-II. The invention provides a method of identifying a
subject having an increased susceptibility for developing pulmonary
hypertension, comprising detecting a mutant Bone Morphogenic
Protein Receptor II (BMPR-II) polypeptide or a mutated BMPR2
nucleic acid in the subject, thereby identifying a subject having
an increased susceptibility for developing pulmonary hypertension.
BMPR2 refers to the gene (or other nucleic acid) encoding a BMPR-II
polypeptide. BMPR-II refers to the polypeptide encoded by a BMPR2
gene. Both of these terms are used herein as general identifiers.
Thus, for example, a BMPR2 gene or nucleic acid refers to any gene
or nucleic acid identified with or derived from a wild-type BMPR2
gene. For example, a mutant BMPR2 gene is a form of BMPR2 gene.
[0029] In a preferred embodiment, the pulmonary hypertension is
primary pulmonary hypertension. In another embodiment, the
pulmonary hypertension is secondary pulmonary hypertension.
[0030] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example, "a
molecule" can mean a single molecule or more than one molecule.
[0031] By "about" is meant .+-.10% of a recited value.
[0032] By "BMPR-II biological activity" is meant any physiological
function attributable to a BMPR-II polypeptide molecule, including
signal transduction. BMPR-II biological activity, as referred to
herein, is relative to that of the normal BMPR-II polypeptide
molecule. It may be desirable to increase or decrease BMPR-II
biological activity.
[0033] Mechanisms by which a compound may increase BMPR-II
biological activity include, but are not limited to, mimicry of
endogenous BMPR-II polypeptide activity; stimulation of the
activity of a less active or inactive version (for example, a
mutant) of the BMPR-II polypeptide; or increasing the amount of
BMPR-II polypeptide in a cell (for example, by stimulating BMPR2
transcription and/or translation or by inhibiting BMPR2 mRNA or
polypeptide degradation).
[0034] BMPR-II biological activity in a sample, such as a cell,
tissue, or animal, may be indirectly measured by measuring the
relative amount of BMPR2 mRNA (for example, by reverse
transcription-polymerase chain reaction (RT-PCR) amplification,
ribonuclease protection assay or Northern hybridization); the level
of BMPR-II polypeptide (for example, by ELISA or Western blotting);
or the activity of a reporter gene under the transcriptional
regulation of a BMPR2 transcriptional regulatory region (by
reporter gene assay, for example, employing beta-galactosidase,
chloramphenicol acetyltransferase (CAT), luciferase, or green
fluorescent protein, as is well known in the art). For example, a
compound that increases the amount of wild-type BMPR-II polypeptide
(or any other version of the polypeptide that maintains at least
some activity) in a cell is a compound that increases biological
activity of BMPR-II.
[0035] By "BMPR-II polypeptide" is meant a polypeptide that has, or
is related to, the amino acid sequence of SEQ ID NO:2. A BMPR-II
polypeptide contains an amino acid sequence that bears at least 80%
sequence identity, preferably at least 85% sequence identity, more
preferably at least 90% sequence identity, and most preferably at
least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:2.
[0036] By "wild-type BMPR-II polypeptide" is meant a BMPR-II
polypeptide that has the amino acid sequence of SEQ ID NO:2.
[0037] By "wild-type BMPR2 nucleic acid" is meant a nucleic acid
that encodes a wild-type BMPR-II polypeptide. An example of a
wild-type BMPR2 nucleic acid is SEQ ID NO:1. Other wild-type BMPR2
nucleic acids include those containing introns, such as genomic
BMPR2 nucleic acid.
[0038] By "polymorphic variant of a BMPR-II polypeptide" is meant a
BMPR-II polypeptide containing an amino acid change, relative to
wild-type, that does not result in an increase susceptibility to
PPH. Such polymorphic amino acid variations in BMPR-II are seen in
both PPH patients and in normal individuals.
[0039] By "mutant BMPR-II polypeptide" is meant a BMPR-II
polypeptide having an amino acid sequence that differs from the
sequence of a wild-type BMPR-II polypeptide. One example of a
wild-type BMPR-II polypeptide is a polypeptide having the amino
acid sequence set forth in SEQ ID NO:2.
[0040] By "mutated BMPR2 nucleic acid" is meant a nucleic acid
having a nucleotide sequence that differs from the sequence of the
wild-type BMPR2 nucleic acid. One example of a wild-type BMPR2
nucleic acid is a nucleic acid having the nucleotide sequence set
forth in SEQ ID NO:1. A "mutated nucleic acid" is also a nucleic
acid that encodes a BMPR-II polypeptide having an amino acid
sequence that differs from the sequence of a wild-type BMPR2
polypeptide. One example of a wild-type BMPR-II polypeptide is a
polypeptide having the amino acid sequence set forth in SEQ ID
NO:2. A mutated nucleic acid also includes a nucleic acid having a
mutation (relative to the wild-type nucleic acid) in noncoding
sequences, such as 5' or 3' sequences or intronic sequences.
[0041] By "increased susceptibility for developing pulmonary
hypertension" is meant a subject who has a greater than normal
chance of developing pulmonary hypertension, compared to the
general population. Such subjects include, for example, a subject
that harbors a mutation in a BMPR2 gene such that biological
activity of BMPR-II is altered.
[0042] By "test compound" is meant a molecule, be it naturally
occurring or artificially derived, that is surveyed for its ability
to modulate BMPR-II activity. Test compounds may include, for
example, peptides, polypeptides, synthesized organic molecules,
naturally occurring organic molecules, nucleic acid molecules, and
components thereof.
[0043] By "sample" is meant an animal; a tissue or organ from an
animal; a cell (either within a subject, taken directly from a
subject, or a cell maintained in culture or from a cultured cell
line); a cell lysate (or lysate fraction) or cell extract; or a
solution containing one or more molecules derived from a cell or
cellular material (e.g. a polypeptide or nucleic acid), which is
assayed as described herein. A sample may also be any body fluid or
excretion (for example, but not limited to, blood, urine, stool,
saliva, tears, bile) that contains cells or cell components.
[0044] By "modulate" is meant to alter, by increase or
decrease.
[0045] By "normal subject" is meant an individual who does not have
an increased susceptibility for developing pulmonary
hypertension.
[0046] By an "effective amount" of a compound as provided herein is
meant a sufficient amount of the compound to provide the desired
effect. The exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of disease (or underlying genetic defect)
that is being treated, the particular compound used, its mode of
administration, and the like. Thus, it is not possible to specify
an exact "effective amount." However, an appropriate "effective
amount" may be determined by one of ordinary skill in the art using
only routine experimentation.
[0047] By "isolated polypeptide" or "purified polypeptide" is meant
a polypeptide (or a fragment thereof) that is substantially free
from the materials with which the polypeptide is normally
associated in nature. The polypeptides of the invention, or
fragments thereof, can be obtained, for example, by extraction from
a natural source (for example, a mammalian cell), by expression of
a recombinant nucleic acid encoding the polypeptide (for example,
in a cell or in a cell-free translation system), or by chemically
synthesizing the polypeptide. In addition, polypeptide fragments
may be obtained by any of these methods, or by cleaving full length
polypeptides.
[0048] By "isolated nucleic acid" or "purified nucleic acid" is
meant DNA that is free of the genes that, in the
naturally-occurring genome of the organism from which the DNA of
the invention is derived, flank the gene. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector, such as an autonomously replicating plasmid or virus; or
incorporated into the genomic DNA of a prokaryote or eukaryote
(e.g., a transgene); or which exists as a separate molecule (for
example, a cDNA or a genomic or cDNA fragment produced by PCR,
restriction endonuclease digestion, or chemical or in vitro
synthesis). It also includes a recombinant DNA which is part of a
hybrid gene encoding additional polypeptide sequence. The term
"isolated nucleic acid" also refers to RNA, e.g., an mRNA molecule
that is encoded by an isolated DNA molecule, or that is chemically
synthesized, or that is separated or substantially free from at
least some cellular components, for example, other types of RNA
molecules or polypeptide molecules.
[0049] By a "transgene" is meant a nucleic acid sequence that is
inserted by artifice into a cell and becomes a part of the genome
of that cell and its progeny. Such a transgene may be (but is not
necessarily) partly or entirely heterologous (for example, derived
from a different species) to the cell.
[0050] By "transgenic animal" an animal comprising a transgene as
described above. Transgenic animals are made by techniques that are
well known in the art.
[0051] By "knockout mutation" is meant an alteration in the nucleic
acid sequence that reduces the biological activity of the
polypeptide normally encoded therefrom by at least 80% relative to
the unmutated gene. The mutation may, without limitation, be an
insertion, deletion, frameshift, or missense mutation. A "knockout
animal," for example, a knockout mouse, is an animal containing a
knockout mutation. The knockout animal may be heterozygous or
homozygous for the knockout mutation. Such knockout animals are
generated by techniques that are well known in the art. A preferred
form of knockout mutation is one where the biological activity of
the BMPR-II polypeptide is not completely eliminated.
[0052] By "treat" is meant to administer a compound or molecule of
the invention to a subject, such as a human or other mammal (for
example, an animal model), that has an increased susceptibility for
developing pulmonary hypertension, or that has pulmonary
hypertension, in order to prevent or delay a worsening of the
effects of the disease or condition, or to partially or fully
reverse the effects of the disease.
[0053] By "prevent" is meant to minimize the chance that a subject
who has an increased susceptibility for developing pulmonary
hypertension will develop pulmonary hypertension.
[0054] By "specifically binds" is meant that an antibody recognizes
and physically interacts with its cognate antigen (for example, a
BMPR-II polypeptide) and does not significantly recognize and
interact with other antigens; such an antibody may be a polyclonal
antibody or a monoclonal antibody, which are generated by
techniques that are well known in the art.
[0055] By "probe," "primer," or oligonucleotide is meant a
single-stranded DNA or RNA molecule of defined sequence that can
base-pair to a second DNA or RNA molecule that contains a
complementary sequence (the "target"). The stability of the
resulting hybrid depends upon the extent of the base-pairing that
occurs. The extent of base-pairing is affected by parameters such
as the degree of complementarity between the probe and target
molecules and the degree of stringency of the hybridization
conditions. The degree of hybridization stringency is affected by
parameters such as temperature, salt concentration, and the
concentration of organic molecules such as formamide, and is
determined by methods known to one skilled in the art. Probes or
primers specific for BMPR2 nucleic acids (for example, genes and/or
mRNAs) have at least 80%-90% sequence complementarity, preferably
at least 91%-95% sequence complementarity, more preferably at least
96%-99% sequence complementarity, and most preferably 100% sequence
complementarity to the region of the BMPR2 nucleic acid to which
they hybridize. Probes, primers, and oligonucleotides may be
detectably-labeled, either radioactively, or non-radioactively, by
methods well-known to those skilled in the art. Probes, primers,
and oligonucleotides are used for methods involving nucleic acid
hybridization, such as: nucleic acid sequencing, reverse
transcription and/or nucleic acid amplification by the polymerase
chain reaction, single stranded conformational polymorphism (SSCP)
analysis, restriction fragment polymorphism (RFLP) analysis,
Southern hybridization, Northern hybridization, in situ
hybridization, electrophoretic mobility shift assay (EMSA).
[0056] By "specifically hybridizes" is meant that a probe, primer,
or oligonucleotide recognizes and physically interacts (that is,
base-pairs) with a substantially complementary nucleic acid (for
example, a BMPR2 nucleic acid) under high stringency conditions,
and does not substantially base pair with other nucleic acids.
[0057] By "high stringency conditions" is meant conditions that
allow hybridization comparable with that resulting from the use of
a DNA probe of at least 40 nucleotides in length, in a buffer
containing 0.5 M NaHPO.sub.4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA
(Fraction V), at a temperature of 65.degree. C., or a buffer
containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH 7.6, 1.times.
Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a
temperature of 42.degree. C. Other conditions for high stringency
hybridization, such as for PCR, Northern, Southern, or in situ
hybridization, DNA sequencing, etc., are well-known by those
skilled in the art of molecular biology. See, for example, F.
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York, N.Y., 1998.
[0058] By "familial mutation" or "inherited mutation" is meant a
mutation in an individual that was inherited from a parent and that
was present in somatic cells of the parent. By "sporadic mutation"
or "spontaneous mutation" is meant a mutation in an individual that
arose in the individual and was not present in a parent of the
individual.
[0059] By "BMPR2 RNA function" is meant a unction of the RNA other
than the state of coding for an amino acid sequence. For example,
BMPR2 RNA production, stability, processing (including splicing),
transport, and the ability to be translated are BMPR2 RNA
functions. By "altered BMPR2 RNA function" is meant an alteration
of BMPR2 RNA function relative to the function of wild-type BMPR2
RNA.
[0060] As set forth herein, nucleotides are numbered according to
the cDNA sequence for BMPR2 (SEQ ID NO:1), with the adenosine of
the initiation codon assigned position 1. (Kawabata, M., Chytil, A.
& Moses, H. L. Cloning of a novel type II serine/threonine
kinase receptor through interaction with the type I transforming
growth factor-beta receptor. J. Biol. Chem. 270, 5625-5630 (1995);
Liu, F., Ventura, F., Doody, J. & Massagu, J. Human type II
receptor for bone morphogenic proteins (BMPs): extension of the
two-kinase receptor model to the BMPs. Mol. C ell. Biol. 15,
3479-3486 (1995); Rosenzweig, B. L. et al. Cloning and
characterization of a human type II receptor for bone morphogenetic
proteins. Proc. Natl. Acad. Sci. U.S.A. 92, 7632-7636 (1995).
[0061] The nucleotide and amino acid sequence of BMPR2 are shown in
SEQ ID NO:1 and SEQ ID NO:2, respectively, starting at nucleotide 1
and amino acid 1, respectively. However, the wild-type cDNA
sequence for BMPR2 which is set forth in Genbank Accession No.
NM.sub.--001204, assigns the adenosine of the initiation codon to
position 409. Therefore, nucleotide position 1 used herein
corresponds to nucleotide number 409 of the BMPR2 cDNA sequence set
forth in Genbank Accession No. NM.sub.--001204. Thus, where a
mutation is noted as being at, for example, nucleotide residue
1454, this corresponds to nucleotide residue 1862 of the sequence
set forth in Genbank Accession No. NM.sub.--001204 (that is,
1454+408).
[0062] As used herein, a specific notation will be used to denote
certain types of mutations. All notations referencing a nucleotide
or amino acid residue will be understood to correspond to the
residue number of the wild-type BMPR2 nucleic acid sequence set
forth at SEQ ID NO:1, or of the wild-type BMPR-II polypeptide
sequence set forth at SEQ ID NO:2. Thus, for example, the notation
"T 367 C" will be used to indicate that the nucleotide T at
position 367 of the sequence set forth at SEQ ID NO:1 has been
replaced with a C. Similarly, the notation "355 del A" will be used
to indicate that the nucleotide A at position 355 has been deleted.
Furthermore, the notation 2408 ins TG" will be used to indicate
that the nucleotides T and G, in that order, have been inserted
following the nucleotide at position 2408.
[0063] In the method of the invention, the mutant BMPR-II
polypeptide or mutated BMPR2 nucleic acid identified is associated
with pulmonary hypertension.
[0064] In one embodiment, the subject having an increased
susceptibility for developing pulmonary hypertension is identified
by detecting a mutated BMPR2 nucleic acid in the subject. The
mutated BMPR2 nucleic acid may comprise a missense mutation, that
is, a mutation that changes a codon specific for one amino acid to
a codon specific for another amino acid. As is noted below in the
Examples and in Tables 1, 2, and 4, examples of mutated BMPR2
nucleic acids having a missense mutation which are associated with
pulmonary hypertension include C 218 G, T 354 G, T 367 C, T 367 A,
C 428 T, C 993 T, G 1042 A, T1258 C, A 1454 G, A 1535 C, T 1557 A,
and C 2695 T.
[0065] In another embodiment, the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension comprises a nucleic
acid sequence having an insertion mutation, where one or more
nucleotides are inserted into the wild-type sequence. The mutated
BMPR2 nucleic acid may also comprise a deletion mutation, where one
or more nucleotides are deleted from the wild-type sequence. Such a
deletion or insertion mutation may, for example, result in a
frameshift mutation, altering the reading frame. Frameshift
mutations typically result in truncated (that is, prematurely
terminated) BMPR-II polypeptide. As is noted below in the Examples
and in Tables 1, 2, and 4, examples of BMPR2 nucleic acids having
an insertion mutation which are associated with pulmonary
hypertension include 504 ins T, 2292 ins A, and 2408 ins TG.
Examples of BMPR2 nucleic acids having a deletion mutation which
are associated with pulmonary hypertension include 355 del A, 689
del A, 958 del T, 1076 del C, 1191/1192 del TG, and 2579 del T.
[0066] The mutated BMPR2 nucleic acid may also comprise a nonsense
mutation, that is, a mutation that changes a codon specific for an
amino acid to a chain termination codon. Nonsense mutations result
in truncated (that is, prematurely terminated) BMPR-II polypeptide.
As is set forth below in the Examples and in Table 1, examples of
BMPR2 nucleic acids having a nonsense mutation which are associated
with pulmonary hypertension include C 218 G, C 428 T, C 993 T, and
C 2695 T.
[0067] The mutated BMPR2 nucleic acid may also comprise a
truncation mutation, that is, a mutated BMPR2 nucleic acid which
encodes a truncated BMPR-II polypeptide. This may occur where, for
example, the BMPR2 nucleic acid has a nonsense mutation.
[0068] In another embodiment, the mutated BMPR2 nucleic acid can be
truncated at a nucleotide position of the sequence set forth in SEQ
ID NO:1 which is 3' to nucleotide position 2695 of the sequence set
forth at SEQ ID NO:1. As is set forth below in the Examples, it has
been determined that a mutation at nucleotide 2695, which truncates
the BMPR-II polypeptide at amino acid residue 899, is correlated to
pulmonary hypertension.
[0069] In another embodiment, the mutated BMPR2 nucleic acid
comprises a mutation at a nucleotide position of the sequence set
forth in SEQ ID NO:1 selected from the group consisting of
nucleotide 218, 354, 355, 367, 428, 504, 689, 958, 993, 1042, 1076,
1129, 1191, 1258, 1454, 1535, 1557, 1749, 2292, 2408, 2579, and
2695. The mutation can result in a change in a codon such that the
mutated codon now encodes a different amino acid. The mutation can
result in a polypeptide having a non-conservative substitution at
the relevant amino acid residue. One of ordinary skill will readily
understand the concept of a "non-conservative substitution."
Substitutions such as a charged amino acid for an uncharged amino
acid, or an uncharged amino acid for a charged amino acid, or any
amino acid in place of a Cys, or visa versa, or any amino acid in
place of a Pro, or visa versa, are well known in the art to alter
the structure and often the function of a protein. The mutation can
also result in reduction or elimination of BMPR2 mRNA production,
incorrect or altered processing of BMPR2 RNA, increased BMPR2 RNA
instability, or other effects on expression of BMPR2 prior to
translation. For example, the mutation 1129 CG (Table 1) alters a
splice junction and results in incorrect splicing of BMPR2 RNA. The
mutation C 1749 T, which does not alter the encoded amino acid,
likely affects RNA production, processing, or function.
[0070] In the embodiment wherein the mutation in the mutated BMPR2
nucleic acid results in a non-conservative substitution in the
amino acid sequence encoded by the nucleic acid, the mutation in
the mutated BMPR2 nucleic acid can be selected from the group
consisting of C 218 G, T 354 G, T 367 C, T 367 A, C 428 T, C 993 T,
G 1042 A, T1258 C, A 1454 G, A 1535 C, T 1557 A, C 2695 T. The
non-conservative substitution may comprise at least one
substitution at an amino acid position of the sequence set forth in
SEQ ID NO:2 selected from the group consisting of: a Trp residue at
amino acid position 118, an Arg residue at amino acid position 123,
a Ser residue at amino acid position 123, a Leu residue at amino
acid position 143, an lie residue at amino acid position 348, an
Arg residue at amino acid position 420, an Ala residue at amino
acid position 485, a Gln residue at amino acid position Gln, and a
Lys residue at amino acid position 519.
[0071] In yet another embodiment, the BMPR2 nucleic acid having a
sequence associated with pulmonary hypertension encodes a mutant
BMPR-II polypeptide.
[0072] For example, the mutant BMPR-II polypeptide having a
sequence associated with pulmonary hypertension can comprise at
least one mutation at an amino acid position of the sequence set
forth in SEQ ID NO:2. Preferably, the BMPR-II polypeptide comprises
at least one mutation at an amino acid position of the sequence set
forth in SEQ ID NO:2 selected from the group consisting of 73, 118,
123, 143, 332, 348, 420, 485, 512, 519, and 899.
[0073] For example, the BMPR-II polypeptide acid having a sequence
associated with pulmonary hypertension may comprise at least one
mutation at an amino acid position of the sequence set forth in SEQ
ID NO:2 selected from the group consisting of: a Trp residue at
amino acid position 118, an Arg residue at amino acid position 123,
a Ser residue at amino acid position 123, a Leu residue at amino
acid position 143, an Ile residue at amino acid position 348, an
Arg residue at amino acid position 420, an Ala residue at amino
acid position 485, a Gln residue at amino acid position Gln, and a
Lys residue at amino acid position 519.
[0074] In another embodiment, the BMPR-II polypeptide having a
sequence associated with pulmonary hypertension terminates
prematurely. In a preferred embodiment, the BMPR-II polypeptide
having a sequence associated with pulmonary hypertension terminates
at an amino acid position of the sequence set forth in SEQ ID NO:2
which is at or N-terminal to amino acid position 899, including
amino acid positions 73, 332, and 899.
[0075] In another embodiment, the BMPR-II polypeptide having a
sequence associated with pulmonary hypertension has a
non-conservative amino acid substitution of at least one amino acid
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
[0076] In one embodiment, the non-conservative amino acid
substitution comprises a non-conservative amino acid substitution
of any of the following: at least one Cys residue of a BMPR-II
having the amino acid sequence set forth in SEQ ID NO:2. In another
embodiment, the non-conservative amino acid substitution comprises
a non-conservative amino acid substitution of at least one Pro
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2. In another embodiment, the non-conservative amino acid
substitution comprises a non-conservative amino acid substitution
of at least one Lys residue of a BMPR-II having the amino acid
sequence set forth in SEQ ID NO:2. In another embodiment, the
non-conservative amino acid substitution comprises a
non-conservative amino acid substitution of at least one Arg
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2. In another embodiment, the non-conservative amino acid
substitution comprises a non-conservative amino acid substitution
of at least one Asp residue of a BMPR-II having the amino acid
sequence set forth in SEQ ID NO:2. In another embodiment, the
non-conservative amino acid substitution comprises a
non-conservative amino acid substitution of at least one Glu
residue of a BMPR-II having the amino acid sequence set forth in
SEQ ID NO:2.
[0077] The mutated BMPR2 nucleic acid and mutant BMPR-II
polypeptide that is detected can be from any cause. For example,
mutated BMPR2 nucleic acid can be the result of a familial mutation
or a sporadic mutation.
[0078] Kits
[0079] The disclosed method is preferably carried out using a kit
designed or adapted to detect one or more mutant BMPR-II
polypeptides and/or one or more mutated BMPR2 nucleic acids. An
example would be a kit for detecting a variety of mutated BMPR2
nucleic acids. Many such kits, and methods for using them are
known.
[0080] Nucleic Acid Delivery
[0081] BMPR-II biological activity can be stimulated (or correct
activity provided) in a subject by administering to the subject a
nucleic acid encoding BMPR-II, using any method known for nucleic
acid delivery into the cells of a subject. The BMPR2 nucleic acid
is taken up by the cells of the subject and directs expression of
the encoded BMPR-II in those cells that have taken up the nucleic
acid. The BMPR2 nucleic acids of the present invention can be in
the form of naked DNA or RNA, or the nucleic acids can be within a
vector for delivering the nucleic acids to the cells. The vector
can be a commercially available preparation, such as an adenovirus
vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada).
Delivery of the nucleic acid or vector to cells can be via a
variety of mechanisms. As one example, delivery can be via a
liposome, using commercially available liposome preparations such
as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),
SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega
Biotec, Inc., Madison, Wis.), as well as other liposomes developed
according to procedures standard in the art. In addition, the
nucleic acid or vector of this invention can be delivered in vivo
by electroporation, the technology for which is available from
Genetronics, Inc. (San Diego, Calif.) as well as by means of a
SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson,
Ariz.).
[0082] As one example, vector delivery can be via a viral system,
such as a retroviral vector system which can package a recombinant
retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci.
U.S.A. 85:4486, 1988; Miller et al., Mol. Cell. Biol. 6:2895,
1986). The recombinant retrovirus can then be used to infect and
thereby deliver to the infected cells a nucleic acid that encodes a
BMPR-II polypeptide. The exact method of introducing the altered
nucleic acid into mammalian cells is, of course, not limited to the
use of retroviral vectors. Other techniques are widely available
for this procedure including the use of adenoviral vectors (Mitani
et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral
(AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994),
lentiviral vectors (Naidini et al., Science 272:263-267, 1996),
pseudo-typed retroviral vectors (Agrawal et al., Exper. Hematol.
24:738-747, 1996). Physical transduction techniques can also be
used, such as liposome delivery and receptor-mediated and other
endocytosis mechanisms (see, for example, Schwartzenberger et al.,
Blood 87:472-478, 1996). The present invention can be used in
conjunction with any of these or other commonly used gene transfer
methods.
[0083] In a particular example, to deliver a BMPR2 nucleic acid to
the cells of a human subject in an adenovirus vector, the dosage
can range from about 10.sup.7 to 10.sup.9 plaque forming unit (pfu)
per injection but can be as high as 10.sup.12 pfu per injection
(Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel,
Hum. Gene Ther. 8:597-613, 1997). Ideally, a subject will receive a
single injection. If additional injections are necessary, they can
be repeated at six month intervals for an indefinite period and/or
until the efficacy of the treatment has been established.
[0084] Parenteral administration of the nucleic acid or vector of
the present invention, if used, is generally characterized by
injection. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution of suspension in liquid prior to injection, or as
emulsions. A more recently revised approach for parenteral
administration involves use of a slow release or sustained release
system such that a constant dosage is maintained. See, e.g., U.S.
Pat. No. 3,610,795. For additional discussion of suitable
formulations and various routes of administration of therapeutic
compounds, see, e.g., Remington: The Science and Practice of
Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,
Easton, Pa. 1995.
[0085] Animal Models
[0086] Compounds identified as modulating BMPR2 or BMPR-II
expression or proposed to affect PPH may be subsequently screened
in any available animal model system, including, but not limited
to, mice, rats, pigs, rabbits, and chickens (Smith, J D, Lab. Anim.
Sci. 48:573-579, 1998; Narayanaswamy et al., J. Vasc. Interv.
Radiol. 11:517, 2000; Poemama et al., Aterioscler. Thromb.
12:601-607, 1992; and Schreyer et al., Aterioscler. Thromb.
14:2053-2059, 1994). Test compounds are administered to these
animals according to standard methods. Known animal models include
monocrotalin injection, continuous air embolism, and fawn hooded
rat. Some useful models are described by Johnson et al., Pulmonary
veins and bronchial vessels undergo remodeling in sustained
pulmonary hypertension induced by continuous air embolization into
sheep, Experimental Lung Research. 23(5):459-73 (1997); Perkett et
al., Expression of transforming growth factor-beta mRNAs and
proteins in pulmonary vascular remodeling in the sheep air
embolization model of pulmonary hypertension, American Journal of
Respiratory Cell & Molecular Biology. 11 (1): 16-24 (1994);
Perkett et al., Insulin-like growth factor I and pulmonary
hypertension induced by continuous air embolization in sheep,
American Journal of Respiratory Cell & Molecular Biology.
6(1):82-7 (1992); Perkett et al., Sequence of structural changes
and elastin peptide release during vascular remodelling in sheep
with chronic pulmonary hypertension induced by air embolization,
American Journal of Pathology. 139(6):1319-32 (1991); Perkett et
al., Continuous air embolization into sheep causes sustained
pulmonary hypertension and increased pulmonary vasoreactivity,
American Journal of Pathology. 132(3):444-54 (1988); Morio et al.,
Distal airspace enlargement in the fawn-hooded rat: influences of
aging and alveolar wall destruction, Respiration. 68(1):78-86
(2001); Le Cras et al., Early abnormalities of pulmonary vascular
development in the Fawn-Hooded rat raised at Denver's altitude,
American Journal of Physiology--Lung Cellular & Molecular
Physiology. 279(2):L283-91 (2000); Le Cras et al., Abnormal lung
growth and the development of pulmonary hypertension in the
Fawn-Hooded rat, American Journal of Physiology. 277(4 Pt
1):L709-18 (1999); Gonzalez et al., The pulmonary hypertensive
fawn-hooded rat has a normal serotonin transporter coding sequence,
American Journal of Respiratory Cell & Molecular Biology.
19(2):245-9 (1998); Gonzalez et al., Pulmonary hypertension, family
and environment, Journal of Human Hypertension. 11(9):559-61
(1997); Provoost, Spontaneous glomerulosclerosis: insights from the
fawn-hooded rat, Kidney International--Supplement. 45:S2-5 (1994);
Sato et al., Factors influencing the idiopathic development of
pulmonary hypertension in the fawn hooded rat, American Review of
Respiratory Disease. 145(4 Pt 1):793-7 (1992); Ashmore et al.,
Paradoxical constriction to platelets by arteries from rats with
pulmonary hypertension, American Journal of Physiology. 260(6 Pt
2):H1929-34 (1991); Nagaya et al., Gene transfer of human
prostacyclin synthase ameliorates monocrotaline-induced pulmonary
hypertension in rats, Circulation. 102(16):2005-10 (2000); Shubat
et al., Pulmonary vascular responses induced by the pyrrolizidine
alkaloid, monocrotaline, in rats, Toxicon. 25(9):995-1002 (1987);
Gust and Schuster, Vascular remodeling in experimentally induced
subacute canine pulmonary hypertension, Experimental Lung Research.
27(1):1-12 (2001); Ito et al., Alterations of endothelium and
smooth muscle function in monocrotaline-induced pulmonary
hypertensive arteries, American Journal of Physiology--Heart &
Circulatory Physiology. 279(4):H1786-95 (2000); Tanabe et al.,
Experimental study on monocrotaline induced pulmonary hypertensive
rats. (1) Effect of long-term injection of immunosuppressant, Tokai
Journal of Experimental & Clinical Medicine. 6(1):41-8 (1981);
and Kameji et al., Increase of collagen synthesis in pulmonary
arteries of monocrotaline-treated rats, Experientia. 36(4):441-2
(1980).
[0087] Animal models that mimic PPH can be developed using
conventional molecular biology methods. For example, a transgenic
animal (for example, a mouse) that overexpresses BMPR2 can be
generated by inserting a BMPR2-encoding nucleic acid under the
transcriptional regulation of the appropriate tissue-specific
promoter into the genome of the animal.
[0088] Test Compounds
[0089] In general, novel drugs that modulate BMPR-II biological
activity may be identified from large libraries of natural products
or synthetic (or semi-synthetic) extracts or chemical libraries
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of test extracts or compounds is not critical to the
screening procedure(s) of the invention. Accordingly, virtually any
number of chemical extracts or compounds can be screened using the
exemplary methods described herein. Examples of such extracts or
compounds include, but are not limited to, plant-, fungal-,
prokaryotic- or animal-based extracts, fermentation broths, and
synthetic compounds, as well as modification of existing compounds.
Numerous methods are also available for generating random or
directed synthesis (for example, semi-synthesis or total synthesis)
of any number of chemical compounds, including, but not limited to,
saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
Synthetic compound libraries are commercially available, e.g., from
Brandon Associates (Merrimack, N.H.) and Aldrich Chemical
(Milwaukee, Wis.). Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant, and animal extracts are
commercially available from a number of sources, including Biotics
(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics
Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge,
Mass.). In addition, natural and synthetically produced libraries
are generated, if desired, according to methods known in the art,
for example, by standard extraction and fractionation methods.
Furthermore, if desired, any library or compound is readily
modified using standard chemical, physical, or biochemical
methods.
[0090] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication (for
example, taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their BMPR-II-modulatory activities should be employed whenever
possible.
[0091] When a crude extract is found to modulate BMPR-II activity,
further fractionation of the positive lead extract is necessary to
isolate chemical constituents responsible for the observed effect.
Thus, the goal of the extraction, fractionation, and purification
process is the careful characterization and identification of a
chemical entity within the crude extract having an activity that
mimics, stimulates, or antagonizes BMPR-II, depending upon the
effect desired. The same assays described herein for the detection
of activities in mixtures of compounds can be used to purify the
active component and to test derivatives thereof. Methods of
fractionation and purification of such heterogenous extracts are
known in the art. If desired, compounds shown to be useful agents
for treatment are chemically modified according to methods known in
the art. Compounds identified as being of therapeutic value can be
subsequently analyzed using any animal models for PPH.
[0092] Administration of Compounds that Modulate BMPR-II Biological
Activity
[0093] The compositions and methods described herein can be used
therapeutically in combination with a pharmaceutically acceptable
carrier. By "pharmaceutically acceptable carrier" is meant a
material that is not biologically or otherwise undesirable, that
is, the material may be administered to an individual along with a
polypeptide, nucleic acid, or other compound of the invention
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the components of the
pharmaceutical composition in which it is contained. Pharmaceutical
carriers are well-known in the art. These most typically are
standard carriers for administration of vaccines or pharmaceuticals
to humans, including solutions such as sterile water, saline, and
buffered solutions at physiological pH.
[0094] Molecules intended for pharmaceutical delivery may be
formulated in a pharmaceutical composition. Pharmaceutical
compositions may include carriers, thickeners, diluents, buffers,
preservatives, surface active agents and the like in addition to
the molecule of choice. Pharmaceutical compositions may also
include one or more active ingredients such as antimicrobial
agents, anti-inflammatory agents, anesthetics, and the like.
Methods for making such formulations are well known in the art, and
are described, for example, in: Remington: The Science and Practice
of Pharmacy (19.sup.th ed.), ed. A. R. Gennaro, E. W. Martin Mack
Publishing Co., Easton, Pa., 1995.
[0095] The pharmaceutical compositions may be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration may be
topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection. The compounds and compositions of the present invention
can be administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity, or transdermally.
[0096] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0097] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0098] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
Formulations for parenteral administration may include sterile
aqueous solutions which may also contain buffers, diluents and
other suitable additives.
[0099] The compounds of the invention are administered in an
effective amount, using standard approaches. Effective dosages and
schedules for administering the compounds may be determined
empirically, and making such determinations is routine to one of
ordinary skill in the art. The skilled artisan will understand that
the dosage will vary, depending upon, for example, the species of
the subject the route of administration, the particular compound to
be used, other drugs being administered, and the age, condition,
sex and extent of the disease in the subject. The dosage can be
adjusted by the individual physician in the event of any
counterindications. A dose of a compound of the invention generally
will range between about 1 .mu.g/kg of body weight and 1 g/kg of
body weight. Examples of such dosage ranges are, e.g., about 1
.mu.g-100 .mu.g/kg, 100 .mu.g/kg-10 mg/kg, or 10 mg-1 g/kg, once a
week, bi-weekly, daily, or two to four times daily. Compounds of
the invention include BMPR-II polypeptides, BMPR2 nucleic acids,
and molecules that regulate expression and/or biological activity
of endogenous wild-type, polymorphic, and/or mutant BMPR-II
polypeptides and/or nucleic acids (for example, DNA or RNA
molecules) encoding such BMPR-II polypeptides.
[0100] The following Examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention.
EXAMPLES
Example 1
[0101] This example describes identification of BMPR2 mutations
associated with susceptibility to PPH. To enable positional cloning
of the FPPH gene (PPH1) a published YAC map was verified and
extended at 2q33 to anchor a BAC/PAC contig including genomic
sequences available at GenBank (Hadano, S. et al. A yeast
artificial chromosome-based physical map of the juvenile
amyotrophic lateral sclerosis (ALS2) critical region on human
chromosome 2q33-q34. Genomics 55, 106-112 (1999)). This contig
covers the entire 5.8 Mb PPH1 region defined by recently detected
recombination events limited by the polymorphic STS markers D2S115
and D2S1384 (ref. 12), and includes the nucleotide sequence
encoding bone morphogenetic protein receptor type II (BMPR-II).
[0102] PCR primers were designed for amplification of patient
genomic DNA after determination of the intron/exon boundaries of
BMPR2. Sequence variants were identified from the panel of kindreds
studied. Heterogeneous mutations, including frameshift, nonsense
and missense mutations, were identified. These mutations were
distributed across the gene.
[0103] Either restriction enzyme or sequence analysis of DNA from
affected and unaffected first-degree relatives was used to show
co-segregation of the mutations with the disease phenotype
(including obligate gene carriers) in all but one of the pedigrees.
150 normal chromosomes derived from the same population as the
affected families were screened together with a panel of 64
chromosomes from normal, but ethnically diverse, controls subjects.
None of the mutations were detected in either panel.
[0104] Materials and Methods
[0105] Patients
[0106] We ascertained families in which at least two members had
the typical manifestations of PPH after exclusion of known
associated disorders, as previously described (5). We collected
venous blood samples and extracted genomic DNA following informed
consent. Obligate gene carriers are defined as those individuals
who inherit and transmit the disease gene to an affected offspring
but who themselves show no clinical manifestations of the
disease.
[0107] Determination of the Genomic Structure of BMPR2
[0108] Available genomic sequence of BAC RP11-354N12
(http://www.ncbi.nlm.nih.gov/ Genbank) was compared to the
published cDNA sequence (Genbank Z48923) and the intron/exon
boundaries for the 3' portion of the gene (exons 8-13) determined
(14). To determine the intron/exon boundaries for exons 1-7,
additional BAC clones were isolated by PCR screening of a human
genomic BAC library (CITB B&C, Research Genetics) using both an
exon 1 and exon 3 STS designed from the BMPR2 cDNA. Direct sequence
analysis of the BAC clones with primer predicted to be near
intron/exon junctions, based on the mouse Bmpr2 genomic structure,
generated flanking intronic sequence for the remaining introns
(18). Comparison between the human and published mouse genomic
organization shows strong conservation of the intron/exon
boundaries (18).
[0109] Mutational Analysis
[0110] We screened the entire coding and intron/exon boundaries by
direct sequencing of both forward and reverse strands on either an
ABI 377 sequencer or an ABI 3700 DNA analyzer, using the Applied
Biosystems DyeDeoxy or BigDye terminator kit and analysed the data
using Sequence Analysis v3.2 or v3.6NT software (Perkin Elmer). The
PCR primers for each exon were:
1 Exon1: 5'-AGCTAGGTCCTCTCATCAGC-3' (SEQ ID NO:3)
5'-CAGCCGCAGTGCTGACCAGC-3'; (SEQ ID NO:4) Exon2:
5'-GTCATTCGGATAAGACAAAG-3' (SEQ ID NO:5)
5'-TTTAACATACTCCCATGTCC-3'; (SEQ ID NO:6) Exon3:
5'-TAGCTTACACGTACTCTCAC-3' (SEQ ID NO:7)
5'-CCTGGCTTCAACCTTGAATG-3'; (SEQ ID NO:8) Exon4:
5'-GGGTACAGCCTTTCTAAAGG-3' (SEQ ID NO:9)
5'-GATACTATTGAGGCTGGGTG-3'; (SEQ ID NO:10) Exon5:
5'-GCTGCTAATCTTTCTGCAGC-3' (SEQ ID NO:11)
5'-GAATGAAGTCACTGTTCCAG-3'; (SEQ ID NO:12) Exon6:
5'-CAGAGAGCTGTAGCATTCTG-3' (SEQ ID NO:13)
5'-AAGTGATCCACCTGCCTTAG-3'; (SEQ ID NO:14) Exon7:
5'-ACTCTTCATGTTAAAGTGAG-3' (SEQ ID NO:15)
5'-CTTTGAAGATATAATTAAAATTTCC-3'; (SEQ ID NO:16) Exon8:
5'-CACCTGGCCAGTAGATGTTT-3' (SEQ ID NO:17)
5'-TGTTCAATAGTCCCTTTTATTCATTG-3'; (SEQ ID NO:18) Exon9:
5'-CTAATTTGCATCCTGCTGCT-3' (SEQ ID NO:19) 5'-TGTTCTTCAGAATATGCTACG
TTCTC-3'; (SEQ ID NO:20) Exon10: 5'-TTGTGGCATTAGGCAACTCC-3' (SEQ ID
NO:21) 5'-GCCTGAAGGGGATGAA AAA-3'; (SEQ ID NO:22) Exon11:
5'-CCACACCCCTTAGGGTCTTA-3' (SEQ ID NO:23) 5'-CACATGGTTTGACATGTAC
TTTG-3'; (SEQ ID NO:24) Exon12A: 5'-CATCAGAGCTTTCCTTGAGGTT-3' (SEQ
ID NO:25) 5'-CAGAGGTGTTAAATTT GGAG-3'; (SEQ ID NO:26) Exon12B:
5'-TCTACCTGCCACACCATTCA-3' (SEQ ID NO:27)
5'-TGGAAACCAACAAGCTAGACC-3'; (SEQ ID NO:28) Exon12C:
5'-CCCCAAAAGACACACAGGAG-3' (SEQ ID NO:29)
5'-TGAATGGTGTGGCAGGTAGA-3'; (SEQ ID NO:30) Exon13:
5'-GCTGACAGGAGGATAAAGCA-3' (SEQ ID NO:31)
5'-CACCCTCCTGAGACATTGGT-3'. (SEQ ID NO:32)
[0111] Restriction Endonuclease Digestion
[0112] We confirmed segregation of the mutations within families
and excluded the presence of the mutations in controls, including a
panel from the DNA Polymorphism Discovery Resource, Coriell Cell
Repositories, by PCR amplification of the relevant exon. This was
followed by either mutation specific restriction fragment length
polymorphism (RFLP) analysis or direct sequencing as previously
described (23). Exon 12 (2579-2580delT) was PCR amplified using the
following primers as a nested PCR reaction: 5'
ACCCAATATGCCAATGGGAC-3' (SEQ ID NO:33), 5'TTCGCCACCTTCTAGTGGCT-3'
(SEQ ID NO:34) followed by 5'CATGTGGTAAACTGAAAAGCTCA-3' (SEQ ID
NO:35), 5'TTGAGACCACTTTGATACACACA-3' (SEQ ID NO:36). We digested an
aliquot (10 .mu.l) overnight at 37.degree. C. with the appropriate
enzyme (10U; Gibco) and separated the fragments on a 4% agarose
gel.
[0113] In keeping with the recognized reduced penetrance of FPPH,
some individuals over 40 years of age exhibited the restriction
fragments of a mutant but were not affected (individuals 6 and 10
in NLO I). Nucleic acid mutations included A 1454 G (exon 11,
AvaII, pedigree NL01), T 354 G (exon 3, Bsp12861, pedigree US 14),
25792580 delT (exon 12, Asel, pedigree US55), C 2695 T (exon 12,
HaeIII, pedigree US33), 355 del A (exon 3, Bsp12861, pedigree
UK13), G 1042 A (exon 8, pedigree UK06), and C 218 G (exon 2,
pedigree US35). Polypeptide mutations included D 485 G (pedigree
NL01), C 118 W (pedigree US14), PTC+10aa (pedigree US55), R 899 X
(pedigree US33), PTC+8aa (pedigree UK13), C 347 Y (pedigree UK06),
and S 73 X (pedigree US35). The downstream amino acid position of
the premature termination codon (PTC) is indicated by the
designation "+Xaa" where X is number of amino acids downstream of
the codon that is mutated. Sequence analysis of both forward and
reverse strands was performed for those PPH families in which the
observed mutation did not create or destroy a restriction site: G
1042 A (exon 8, pedigree UK06), and C 218 G (exon 2,
pedigreeUS35).
[0114] Discussion
[0115] Members of the TGF-.beta. superfamily transduce signals by
binding to heteromeric complexes of type I and II receptors,
activating serine/threonine kinases, leading to transcriptional
regulation by phosphorylated Smads (Massagu, J. & Chen, Y-G.
Controlling TGF-.beta. signalling. Genes Dev. 14, 627-644 (2000)).
In FPPH, mutations in the gene encoding BMPR-II lead to alterations
in domains which have been identified in TGF-.beta. type II
receptors as being involved in ligand binding, kinase activity and
heteromeric dimer formation (Wrana, J. L. et al. Two distinct
transmembrane serine/threonine kinases from Drosophila melanogaster
form an activin receptor complex. Mol. Cell. Biol. 14, 944-950
(1994); Carcamo, J., Zentella, A. & Massagu, J. Disruption of
transforming growth factor beta signalling by a mutation that
prevents transphosphorylation within the receptor complex. Mol.
Cell. Biol. 15, 1573-1581 (1995); Gilboa, L. et al. Bone
morphogenetic protein receptor complexes on the surface of live
cells: A new oligomerization mode for serine/ threonine kinase
receptors. Mol. Biol. Cell. 11, 1023-1035 (2000)).
REFERENCES
[0116] 1. Rubin, L. ACCP consensus statement: primary pulmonary
hypertension. Chest 104, 236-250 (1993).
[0117] 2. Gaine, S. P. & Rubin, L. J. Primary pulmonary
hypertension. Lancet 352, 719725 (1998).
[0118] 3. Rich, S. et al. Primary pulmonary hypertension: a
national prospective study. Ann. Intern. Med. 107, 216-223
(1987).
[0119] 4. Loyd, J. E., Primm, R. K. & Newman, J. H. Familial
primary pulmonary hypertension: clinical patterns. Am. Rev. Respir.
Dis.129, 194-197 (1984).
[0120] 5. Nichols, W. C. et al. Localisation of the gene for
familial primary pulmonary hypertension to chromosome 2q31-32.
Nature Genet. 15 , 277-280 (1997). Morse, J. H. et al. Mapping of
familial pulmonary hypertension locus (PPH1) to chromosome 2q31-32.
Circulation 95, 2603-2606 (1997).
[0121] 7. Massagu, J. & Chen, Y-G. Controlling TGF-.beta.
signalling. Genes Dev. 14, 627644 (2000).
[0122] 8. Wrana, J. L. et al. Two distinct transmembrane
serine/threonine kinases from Drosophila melanogaster form an
activin receptor complex. Mol. Cell. Biol. 14, 944950 (1994).
[0123] 9. Carcamo, J., Zentella, A. & Massagu, J. Disruption of
transforming growth factor beta signalling by a mutation that
prevents transphosphorylation within the receptor complex. Mol.
Cell. Biol. 15, 1573-1581 (1995).
[0124] 10. Gilboa, L. et al. Bone morphogenetic protein receptor
complexes on the surface of live cells: A new oligomerization mode
for serine/threonine kinase receptors. Mol. Biol. Cell. 11,
1023-1035 (2000).
[0125] 11. Hadano, S. et al. A yeast artificial chromosome-based
physical map of the juvenile amyotrophic lateral sclerosis (ALS2)
critical region on human chromosome 2q33-q34. Genomics 55, 106-112
(1999).
[0126] 13. Kawabata, M., Chytil, A. & Moses, H. L. Cloning of a
novel type II serine/threonine kinase receptor through interaction
with the type I transforming growth factor-beta receptor. J. Biol.
Chem. 270, 5625-5630 (1995).
[0127] 14. Liu, F., Ventura, F., Doody, J. & Massagu, J. Human
type II receptor for bone morpbogenic proteins (BMPs): extension of
the two-kinase receptor model to the BMPs. Mol. Cell. Biol. 15,
3479-3486 (1995).
[0128] 15. Rosenzweig, B. L. et al. Cloning and characterization of
a human type II receptor for bone morphogenetic proteins. Proc.
Natl. Acad. Sci. U.S.A. 92 , 7632-7636 (1995).
[0129] 16. Botney, M. D., Bahadori, L. & Gold, L. I. Vascular
remodeling in primary pulmonary hypertension. Potential role for
transforming growth factor-beta. Am. J. Pathol. 144, 286-295
(1994).
[0130] 17. Marchuk, D. A. Genetic abnormalities in hereditary
hemorrhagic telangiectasia. Curr. Opin. Hematol. 5, 332-338
(1998).
[0131] 18. Beppu, H., Minowa, O., Miyazono, K. & Kawabata, M.
cDNA cloning and genomic organization of the mouse BMP type II
receptor. Biochem. Biophys. Res. Commun. 235, 499-504 (1997).
[0132] 19. Wilkie, A. O. The molecular basis of genetic dominance.
J. Med. Genet. 31, 8998 (1994).
[0133] 21. Lu, S. L. et al. HNPCC associated with germline mutation
in the TGF-beta type II receptor gene. Nature Genet. 19, 17-18
(1998).
[0134] 22. Lee, S. D. et al. Monoclonal endothelial cell
proliferation is present in primary but not secondary pulmonary
hypertension. J. Clin. Invest. 101, 927-934 (1998). Shackleton, S.
et al. LMNA, encoding lamin A/C, is mutated in partial
lipodystrophy. Nature Genet. 24, 153-156 (2000).
Example 2
[0135] Materials and Methods
[0136] Patients
[0137] Patients (age range 14-55 years) were recruited through
physicians at specialist pulmonary vascular clinics in the UK
(n=35), France (n=13) and the USA (n=2). PPH was defined by
standard clinical methods, including cardiac catheterization
revealing pulmonary hypertension (mean pulmonary artery
pressure>25 mm Hg) and a normal pulmonary artery wedge pressure,
without other abnormalities such as lung disease, heart disease,
pulmonary embolism or systemic disease such as connective tissue
diseases. All studies were performed with consent and approval by
the Leicestershire Health Authority Ethics Committee (England).
[0138] Typical Presentation
[0139] At age 36, patient 10 a previously fit and well nulliparous
white female, developed shortness of breath and reduced exercise
tolerance. She had no previous history of tobacco consumption, nor
use of appetite suppressants. A five generation detailed pedigree
revealed no preceding family history of PPH. The following year,
she was investigated for worsening breathlessness. On clinical
examination, external appearance was normal, cardiac auscultation
revealed a loud second pulmonary heart sound. A radiograph of the
chest showed enlarged pulmonary arteries with `pruning` of
peripheral vessels, electrocardiogram had evidence of right
ventricular strain and an echocardiogram revealed an enlarged right
ventricle and moderate tricuspid regurgitation. An autoantibody
screen and ventilation/perfusion scan were normal. At right heart
catheterisation, pulmonary artery pressure was 97/41 mmHg with a
pulmonary wedge pressure of 8 mmHg, and a diagnosis of primary
pulmonary hypertension was made. The patient was anti-coagulated on
warfarin and commenced on a calcium channel blocking agent,
diltiazam. Her condition deteriorated over the following year with
increasing episodes of retro-sternal chest pain, haemoptysis,
syncope and the development of peripheral oedema. Domicilary oxygen
was provided and she was assessed for heart-lung transplantation,
which she received at age 41. Histology of the explanted lungs
showed marked intimal expansion and atheromatous plaques in the
main branches of pulmonary arteries. Distal arteries showed
hypertrophied muscularised media, intimal expansion and
obliteration, with formation of plexiform lesions. The alveoli and
bronchi appeared normal. A year following transplant she returned
to work and remains under follow-up on a regime of
immunosuppression.
[0140] DNA Sequence Analysis of BMPR2 Gene
[0141] We obtained 10 to 20 ml of peripheral blood from each family
member studied. DNA was isolated from whole blood as described
elsewhere. Parental relationships were confirmed through the
segregation analysis of 10 independent highly polymorphic markers.
Protein coding sequences from exons 1 to 13 were amplified from
genomic DNA using primers derived from intron sequence as described
in Example 1. Genomic fragments amplified by the polymerase chain
reaction (PCR) were sequenced with a dye-terminator cycle-sequence
system (ABI 3700, Perkin-Elmer Applied Biosystems, Foster City,
Calif.).
[0142] Confirmation of Genotypes and Detection of Spontaneous (De
Novo) Mutations
[0143] Variants of the BMPR2 gene were identified by sequence
analysis and, when possible, were independently confirmed by
restriction endonuclease digestion. Relevant exons were PCR
amplified using primers as described, digested with restriction
enzymes (Hae III, Taq I, Mse I, Fnu4H I, New England Biolabs)
according to the manufacturer's instructions, and size-separated on
a 4% composite agarose gel (FMC BioProducts, Gibco BRL). The
presence or absence of the sequence variants from available family
members and at least 150 normal control chromosomes was determined
by analysis of the restriction digest or direct sequencing
results.
[0144] Results
[0145] Analysis of the BMPR2 Gene
[0146] Sequencing of genomic DNA of the panel of sporadic PPH
subjects demonstrated a variety of novel heterozygous mutations of
the BMPR2 gene (Table 4). In patients 1 and 2, the nucleotide
sequence revealed substitutions of guanine for adenine in exons 2
and 3 respectively. These change the sequences of codon 60 from TGC
to TAC (patient 1) and codon 117 from TGT to TAT (patient 2); both
changing a highly conserved encoded amino acid from cysteine to
tyrosine (Table 4). As these mutations do not result in either gain
or loss of a restriction site, genomic sequencing of parental
samples demonstrated the presence of the mutation in the fathers of
both sporadic patients.
[0147] In patient 8, genomic sequencing identified a substitution
of thymine for cytosine in exon 11, changing the sequence of codon
483 from TGT to CGT and the encoded amino acid from cysteine to
arginine (Table 2). No additional family members were available for
study. In patients 6 and 10, deletion of an adenine in exon 9 and a
guanine in exon 12 both lead to a change in the coding reading
frame, and predict premature truncation of the 1038 amino-acid
protein at codon positions 423 and 803 respectively. Analysis of
samples from unaffected parents, either by direct sequencing of
genomic DNA (patient 6) or restriction enzyme analysis with Fnu4HI
(patient 10), demonstrated the absence of the mutation, confirming
these patients had spontaneous mutations of the BMPR2 gene (Table
4).
[0148] The possibility of incorrect paternity was excluded by the
analysis of informative markers. The mutation observed in patient
10, was also detected in two further sporadic patients, 11 and 12
ascertained independently. No parental samples were available and
the possibility of the patients having inherited the mutation from
a common ancestor was excluded through the examination of genotypes
from microsatellite markers from within and surrounding the BMPR2
gene on chromosome 2.
[0149] In three additional patients, insertions of residues in the
genomic sequence occurred. In patient 4, an additional thymine was
detected in exon 6 at position 787 (Table 4). In patient 7 both a
guanine and adenine were inserted at nucleotide position 1247-8 of
exon 9, while in patient 9 an adenine is inserted in exon 12 at
position 1969 and confirmed through restriction digest analysis
with Mse I (Table 4). Each of the mutations predicts premature
truncation of the BMPR-II protein through shifts of the reading
frame (Table 4). Parental material was not available for analysis
for these subjects.
[0150] In patients 3 and 5, the substitution of cytosine for
thymine occurred in exons 6 and 8 respectively (Table 4). In both
patients the mutations result in the change of the encoded
amino-acid arginine CGA to the stop signal TGA. These sequence
changes were confirmed by restriction digest analysis of genomic
DNA with HaeIII and TaqI respectively; however samples from other
family members were not available.
[0151] None of these sequence changes were detected in the analysis
of a large panel of chromosomes from unrelated normal individuals,
indicating that these mutations are not polymorphisms. Mutations of
the entire coding sequence of the BMPR2 gene were also excluded in
the remaining 38 patients diagnosed with sporadic PPH.
[0152] Pulmonary Artery Myocytes from Patients with Familial and
Sporadic PPH Exhibit Abnormal Responses to TGF-.beta. Family
Ligands
[0153] PPH myocytes exhibited specifically heightened
.sup.3H-thymidine incorporation to BMP2, a known BMPR-II ligand,
and TGF-.beta.. In contrast, no increased DNA synthesis was
observed in response to these peptides in cells from control
subjects or patients with secondary pulmonary hypertension. Indeed,
TGF-.beta. suppressed basal .sup.3H-thymidine incorporation in
pulmonary artery myocytes from controls. PDGF-.beta. stimulated
.sup.3H-thymidine incorporation by an equal amount in all cells,
with no significant difference between patient groups. Growth
arrested pulmonary artery myocytes from patients with PPH were
used. Incubations were for 48 hours with [methyl]-.sup.3H-thymidine
added for final 24 hours. All subjects were age-matched and
patients had a comparable degree of pulmonary hypertension: PPH
(mean pulmonary artery pressure 604 mmHg); SPH (655 mmHg).
*p<0.05, **p<0.01 compared with corresponding 0.1% FBS.
REFERENCES
[0154] 1. Rubin L J. Primary Pulmonary Hypertension. The New
England Journal of Medicine 1997; 336:111-117.
[0155] 2. Game S P, Rubin L J. Primary pulmonary hypertension.
Lancet 1998; 352:719-725.
[0156] 3. Rich S, Dantzker D R, Ayres S M, Bergofsky E H, Brundage
B H, Detre K M, et al. Primary pulmonary hypertension. A national
prospective study. Ann.Intern.Med. 1987; 107:216-223.
[0157] 4. Loyd J E, Primm R K, Newman J H. Familial primary
pulmonary hypertension: clinical patterns. Am.Rev.Respir.Dis. 1984;
129:194-197.
[0158] 5. Machado R D, Pauciulo M W, Fretwell N, Veal C, Thomson J
R, Guell C V, et al. A physical and transcript map based upon
refinement of the critical interval for PPH1, a gene for familial
primary pulmonary hypertension. Genomics 2000; In press.
[0159] 6. Nichols W C, Koller D L, Slovis B, Foroud T M, Terry V H,
Arnold N D, et al. Localization of the gene for familial primary
pulmonary hypertension to chromosome 2q31-32. Nat.Genet. 1997;
15:277-280.
[0160] 7. Morse J H, Jones A C, Barst R J, Hodge S E, Wilhelmsen K
C, Nygaard T G. Mapping of familial primary pulmonary hypertension
locus (PPH1) to chromosome 2q31-q32. Circulation 1997;
95:2603-2606.
[0161] 8. Deng Z, Haghighi F, Helleby L, Vanterpool K, Horn E M,
Barst R J, et al. Fine mapping of PPH1, a gene for familial primary
pulmonary hypertension, to a 3 cM region on chromosome 2q33.
American Journal of Respiratory & Critical Care Medicine 2000;
161:1055-1059.
[0162] 9. The International PPH Consortium, Lane K B, Machado R D,
Pauciulo M W, Thomson J R, Phillips III J A, et al. Heterozygous
germline mutations in a TGF-.beta. receptor, BMPR2, are the cause
of familial primary pulmonary hypertension. Nat.Genet. 2000; In
press.
[0163] 10. Massagu J, Chen Y G. Controlling TGF-.beta. signaling.
Genes & Development 2000; 14:627-644.
[0164] 11. Loyd J E, Atkinson J B, Pietra G G, Virmani R, Newman J
H. Heterogeneity of Pathologic Lesions in Familial Primary
Pulmonary Hypertension. Am.Rev.Respir.Dis. 1988; 138:952-957.
[0165] 12. Pietra G G, Edwards W D, Kay J M, Rich S, Kernis J,
Schloo B, et al. Histopathology of primary pulmonary hypertension.
A qualitative and quantitative study o f pulmonary blood vessels
from 58 patients in the National Heart, Lung, and Blood Institute,
Primary Pulmonary Hypertension Registry. Circulation 1989;
80:1198-1206.
[0166] 13. Elliott G, Alexander G, Leppert M, Yeates S, Kerber R.
Coancestry in apparently sporadic primary pulmonary hypertension.
Chest 1995; 108:973-977.
[0167] 14. Morrell N W, Upton P D, Kotecha S, Huntley A, Yacoub M
H, Polak J M, et al. Angiotensin II activates MAPK and stimulates
growth of human pulmonary artery smooth muscle via ATI receptors.
American Journal of Physiology 1999; 277:L440-L448
[0168] 15. Rich S. Primary pulmonary hypertension. Executive
summary from the world symposium. Primary Pulmonary Hypertension.
World Health Organisation Publications 1998;
[0169] 16. Marchuk DA. Genetic abnormalities in hereditary
hemorrhagic telangiectasia. Current Opinion in Hematology 1998;
5:332-338.
[0170] 17. Blobe G C, Schiemann W P, Lodish H F. Role of
transforming growth factor b in human disease. N Engl J Med 2000;
342:1350-1358.
[0171] 18. Mesa R A, Edell E S, Dunn W F, Edwards W D. Human
immunodeficiency virus infection and pulmonary hypertension: two
new cases and a review of 86 reported cases. Mayo Clinic
Proceedings 1998; 73:37-45.
[0172] 19. Abenhaim L, Moride Y, Brenot F, Rich S, Benichou J, Kurz
X, et al. Appetite-suppressant drugs and the risk of primary
pulmonary hypertension. International Primary Pulmonary
Hypertension Study Group [see comments]. N.Engl.J.Med. 1996;
335:609-616.
[0173] 20. Gomez-Sanchez M A, Saenz d I C, Gomez-Pajuelo C,
Martinez-Tello F J, Mestre d J M, James T N. Clinical and
pathologic manifestations of pulmonary vascular disease in the
toxic oil syndrome. Journal of the American College of Cardiology
1991; 18:1539-1545.
[0174] 21. Lee S D, Shroyer K R, Markham N E, Cool C D, Voelkel N
F, Tuder R M. Monoclonal endothelial cell proliferation is present
in primary but not secondary pulmonary hypertension. Journal of
Clinical Investigation 1998; 101:927-934.
[0175] 22. Lu S L, Kawabata M, Imamura T, Akiyama Y, Nomizu T,
Miyazono K, et al. HNPCC associated with germline mutation in the
TGF-.beta. type II receptor gene. Nat.Genet. 1998; 19:17-18.
2TABLE 1 Family/Pt Location Mutation Nucleotide change Consequence
Segregation +UK13 Exon 3 Deletion 355delA GCTGTTGTA frameshift
Bsp1286I UK09 Exon 3 C123R 367 TC missense MseI UK21 Exon 4
Insertion 504insT GTTGCCTTT frameshift FRA Exon 6 Deletion
689/90delAA TGCTGTAAA frameshift DS +UK06 Exon 8 C347T G1042A
missense DS GER01 Exon 9 C420R T1258C missense
AfaI/Bsp1407I/BsrGl/SspBI/MaeI +NL01 Exon 11 D485G A1454G missense
AvaII GRE01 Exon 11 L512T A1535 C missense SWE01 Exon 12 R584X
C1749T nonsense UK22 Exon 12 Insertion 2292insA ACCAAAAAA
frameshift DS UK11 Exon 12 Deletion 2579-2580delT ATTAATT
frameshift (PTC + 10 aa) AseI UK04 Exon 7 Deletion 958 + 3 delT
AGGAGGTA inactivates exon 7 donor splice site +US35 Exon 2 S73X
C218G nonsense DS +US14 Exon 3 C118W T354G missense Bsp1286I +US33
Exon 12 R899X C2695T nonsense HaeIII +US55 Exon 12 Deletion
2579-2580delT frameshift (PTC +10 aa) AseI ATTAATT US94 Exon 8
Deletion 1076delC GGTGAC frameshift (PTC + 15 aa) US89 Exon 9
Deletion 1191/1192delTG GGACTGTG frameshift (PTC + 48 aa) US80 Exon
9 Splice 1129-3CG inactivates exon 9 acceptor splice site Defect
US37 Exon 4 R147X C428T nonsense US49 Exon 11 N519K T1557A missense
DS US79 Exon 12 Insertion 2408insTG TGGTGTG frameshift (PTC + 3 aa)
NOR01 Exon 3 C123S T367A missense MseI US50 Exon 8 R332X C993T
nonsense
[0176]
3TABLE 2 Patient Location Mutation Nucleotide change Consequence
Segregation Mat Pat Centre 5226 Exon 2 C60Y C179A missense DS WT
C196Y Manchester 3576 Exon 6 Insertion TTTATAGTTT frameshift DS
Declined Declined Newcastle 5949 Exon 6 R211X C631T nonsense HaeIII
Alive, sample Alive, sample being France being arranged arranged
5591 Exon 8 R332X C994T nonsense TaqI Alive, to arrange Alive, to
arrange CXH sample sample 5508 Exon 9 Insertion GGGAGAGA frameshift
DS Alive, to arrange ? Glasgow 1247/48insGA sample 5927 Exon 11
C483R T1447C missense DS Blood sample ? France taken 5545 Exon 12
Insertion TCTGCTAA frameshift MseI WT RIP Newcastle 1969insA 5163
Exon 12 Deletion CAATCAATG frameshift Fnu4HI WT WT Harefield
2386delG 5937 Exon 12 Deletion CAATCAATG frameshift Fnu4HI To
request To request France 2386delG 5943 Exon 12 Deletion CAATCAATG
frameshift Fnu4HI To request To request France 2386delG Ut. 01 Exon
9 Deletion GGGAGATA frameshift DS WT WT Utah 1248delA Ut. 11 Exon 3
C117Y 350G--A missense DS WT C117Y Utah
[0177]
4TABLE 3 Clinical features of PPH. Characteristics 1 2 3 4 5 6 7 8
9 10 11 12 Sex F M F F F M M F F F F M Age (years) 23 31 27 35 29
42 34 43 Age of onset 17 25 22 29 22 30 36 (years) Family History -
- - - - - - - - - - - Therapy + + + + + - + + + - + +
(vasodilators) HLT - - - - - + - - - + - - Mutation C60Y C117Y
R211X 787insT R332X 1248-delA 1247/48-insGA C483R 1969-insA
2386-delG 2386-delG 2386-delG BMPR2
[0178]
5TABLE 4 Patient Patient Mutation Nucleotide Codon Amino Acid
Restriction No. Identifier Exon Type Change Position Change
Inheritance Enzyme 1 5226 2 Missense G(179) A 60 Cys to Tyr
Paternal -- 2 Ut.11 3 Missense G(350) A 117 Cys to Tyr Paternal 3
3576 6 Frameshift 787insT 263 PTC.sup.+3 4 5949 6 Nonsense C(631) T
211 Arg to Stop -- HaeIII 5 5591 8 Nonsense C(994) T 332 Arg to
Stop -- TaqI 6 Ut.01 9 Frameshift 1248delA 416 PTC.sup.+7 de novo
-- 7 5508 9 Frameshift 1247/8ins GA 416 PTC.sup.+4 -- -- 8 5927 11
Missense T(1447) C 483 Cys to Arg -- -- 9 5545 12 Frameshift
1969insA 657 PTC.sup.+18 -- MseI 10 5163 12 Frameshift 2386delG 796
PTC.sup.+7 de novo Fnu4HI 11 5937 12 Frameshift 2386delG 796
PTC.sup.+7 -- Fnu4HI 12 5943 12 Frameshift 2386delG 796 PTC.sup.+7
-- Fnu4HI 13 5597 1-6 Deletion
[0179] It is understood that the disclosed invention is not limited
to the particular methodology, protocols, and reagents described as
these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
[0180] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to "the antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0181] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are as
described. Publications cited herein and the material for which
they are cited are specifically incorporated by reference. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0182] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
36 1 3122 DNA Homo Sapiens CDS (1)...(3115) 1 atg act tcc tcg ctg
cag cgg ccc tgg cgg gtg ccc tgg cta cca tgg 48 Met Thr Ser Ser Leu
Gln Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15 acc atc ctg
ctg gtc agc act gcg gct gct tcg cag aat caa gaa cgg 96 Thr Ile Leu
Leu Val Ser Thr Ala Ala Ala Ser Gln Asn Gln Glu Arg 20 25 30 cta
tgt gcg ttt aaa gat ccg tat cag caa gac ctt ggg ata ggt gag 144 Leu
Cys Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu 35 40
45 agt aga atc tct cat gaa aat ggg aca ata tta tgc tcg aaa ggt agc
192 Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser
50 55 60 acc tgc tat ggc ctt tgg gag aaa tca aaa ggg gac ata aat
ctt gta 240 Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn
Leu Val 65 70 75 80 aaa caa gga tgt tgg tct cac att gga gat ccc caa
gag tgt cac tat 288 Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln
Glu Cys His Tyr 85 90 95 gaa gaa tgt gta gta act acc act cct ccc
tca att cag aat gga aca 336 Glu Glu Cys Val Val Thr Thr Thr Pro Pro
Ser Ile Gln Asn Gly Thr 100 105 110 tac cgt ttc tgc tgt tgt agc aca
gat tta tgt aat gtc aac ttt act 384 Tyr Arg Phe Cys Cys Cys Ser Thr
Asp Leu Cys Asn Val Asn Phe Thr 115 120 125 gag aat ttt cca cct cct
gac aca aca cca ctc agt cca cct cat tca 432 Glu Asn Phe Pro Pro Pro
Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140 ttt aac cga gat
gag aca ata atc att gct ttg gca tca gtc tct gta 480 Phe Asn Arg Asp
Glu Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val 145 150 155 160 tta
gct gtt ttg ata gtt gcc tta tgc ttt gga tac aga atg ttg aca 528 Leu
Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170
175 gga gac cgt aaa caa ggt ctt cac agt atg aac atg atg gag gca gca
576 Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala
180 185 190 gca tcc gaa ccc tct ctt gat cta gat aat ctg aaa ctg ttg
gag ctg 624 Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu
Glu Leu 195 200 205 att ggc cga ggt cga tat gga gca gta tat aaa ggc
tcc ttg gat gag 672 Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly
Ser Leu Asp Glu 210 215 220 cgt cca gtt gct gta aaa gtg ttt tcc ttt
gca aac cgt cag aat ttt 720 Arg Pro Val Ala Val Lys Val Phe Ser Phe
Ala Asn Arg Gln Asn Phe 225 230 235 240 atc aac gaa aag aac att tac
aga gtg cct ttg atg gaa cat gac aac 768 Ile Asn Glu Lys Asn Ile Tyr
Arg Val Pro Leu Met Glu His Asp Asn 245 250 255 att gcc cgc ttt ata
gtt gga gat gag aga gtc act gca gat gga cgc 816 Ile Ala Arg Phe Ile
Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270 atg gaa tat
ttg ctt gtg atg gag tac tat ccc aat gga tct tta tgc 864 Met Glu Tyr
Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285 aag
tat tta agt ctc cac aca agt gac tgg gta agc tct tgc cgt ctt 912 Lys
Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295
300 gct cat tct gtt act aga gga ctg gct tat ctt cac aca gaa tta cca
960 Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro
305 310 315 320 cga gga gat cat tat aaa cct gca att tcc cat cga gat
tta aac agc 1008 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg
Asp Leu Asn Ser 325 330 335 aga aat gtc cta gtg aaa aat gat gga acc
tgt gtt att agt gac ttt 1056 Arg Asn Val Leu Val Lys Asn Asp Gly
Thr Cys Val Ile Ser Asp Phe 340 345 350 gga ctg tcc atg agg ctg act
gga aat aga ctg gtg cgc cca ggg gag 1104 Gly Leu Ser Met Arg Leu
Thr Gly Asn Arg Leu Val Arg Pro Gly Glu 355 360 365 gaa gat aat gca
gcc ata agc gag gtt ggc act atc aga tat atg gca 1152 Glu Asp Asn
Ala Ala Ile Ser Glu Val Gly Thr Ile Arg Tyr Met Ala 370 375 380 cca
gaa gtg cta gaa gga gct gtg aac ttg agg gac tgt gaa tca gct 1200
Pro Glu Val Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala 385
390 395 400 ttg aaa caa gta gac atg tat gct ctt gga cta atc tat tgg
gag ata 1248 Leu Lys Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr
Trp Glu Ile 405 410 415 ttt atg aga tgt aca gac ctc ttc cca ggg gaa
tcc gta cca gag tac 1296 Phe Met Arg Cys Thr Asp Leu Phe Pro Gly
Glu Ser Val Pro Glu Tyr 420 425 430 cag atg gct ttt cag aca gag gtt
gga aac cat ccc act ttt gag gat 1344 Gln Met Ala Phe Gln Thr Glu
Val Gly Asn His Pro Thr Phe Glu Asp 435 440 445 atg cag gtt ctc gtg
tct agg gaa aaa cag aga ccc aag ttc cca gaa 1392 Met Gln Val Leu
Val Ser Arg Glu Lys Gln Arg Pro Lys Phe Pro Glu 450 455 460 gcc tgg
aaa gaa aat agc ctg gca gtg agg tca ctc aag gag aca atc 1440 Ala
Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr Ile 465 470
475 480 gaa gac tgt tgg gac cag gat gca gag gct cgg ctt act gca cag
tgt 1488 Glu Asp Cys Trp Asp Gln Asp Ala Glu Ala Arg Leu Thr Ala
Gln Cys 485 490 495 gct gag gaa agg atg gct gaa ctt atg atg att tgg
gaa aga aac aaa 1536 Ala Glu Glu Arg Met Ala Glu Leu Met Met Ile
Trp Glu Arg Asn Lys 500 505 510 tct gtg agc cca aca gtc aat cca atg
tct act gct atg cag aat gaa 1584 Ser Val Ser Pro Thr Val Asn Pro
Met Ser Thr Ala Met Gln Asn Glu 515 520 525 cgc aac ctg tca cat aat
agg cgt gtg cca aaa att ggt cct tat cca 1632 Arg Asn Leu Ser His
Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro 530 535 540 gat tat tct
tcc tcc tca tac att gaa gac tct atc cat cat act gac 1680 Asp Tyr
Ser Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp 545 550 555
560 agc atc gtg aag aat att tcc tct gag cat tct atg tcc agc aca cct
1728 Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr
Pro 565 570 575 ttg act ata ggg gaa aaa aac cga aat tca att aac tat
gaa cga cag 1776 Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn
Tyr Glu Arg Gln 580 585 590 caa gca caa gct cga atc ccc agc cct gaa
aca agt gtc acc agc ctc 1824 Gln Ala Gln Ala Arg Ile Pro Ser Pro
Glu Thr Ser Val Thr Ser Leu 595 600 605 tcc acc aac aca aca acc aca
aac acc aca gga ctc acg cca agt act 1872 Ser Thr Asn Thr Thr Thr
Thr Asn Thr Thr Gly Leu Thr Pro Ser Thr 610 615 620 ggc atg act act
ata tct gag atg cca tac cca gat gaa aca aat ctg 1920 Gly Met Thr
Thr Ile Ser Glu Met Pro Tyr Pro Asp Glu Thr Asn Leu 625 630 635 640
cat acc aca aat gtt gca cag tca att ggg cca acc cct gtc tgc tta
1968 His Thr Thr Asn Val Ala Gln Ser Ile Gly Pro Thr Pro Val Cys
Leu 645 650 655 cag ctg aca gaa gaa gac ttg gaa acc aac aag cta gac
cca aaa gaa 2016 Gln Leu Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu
Asp Pro Lys Glu 660 665 670 gtt gat aag aac ctc aag gaa agc tct gat
gag aat ctc atg gag cac 2064 Val Asp Lys Asn Leu Lys Glu Ser Ser
Asp Glu Asn Leu Met Glu His 675 680 685 tct ctt aaa cag ttc agt ggc
cca gac cca ctg agc agt act agt tct 2112 Ser Leu Lys Gln Phe Ser
Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser 690 695 700 agc ttg ctt tac
cca ctc ata aaa ctt gca gta gaa gca act gga cag 2160 Ser Leu Leu
Tyr Pro Leu Ile Lys Leu Ala Val Glu Ala Thr Gly Gln 705 710 715 720
cag gac ttc aca cag act gca aat ggc caa gca tgt ttg att cct gat
2208 Gln Asp Phe Thr Gln Thr Ala Asn Gly Gln Ala Cys Leu Ile Pro
Asp 725 730 735 gtt ctg cct act cag atc tat cct ctc ccc aag cag cag
aac ctt ccc 2256 Val Leu Pro Thr Gln Ile Tyr Pro Leu Pro Lys Gln
Gln Asn Leu Pro 740 745 750 aag aga cct act agt ttg cct ttg aac acc
aaa aat tca aca aaa gag 2304 Lys Arg Pro Thr Ser Leu Pro Leu Asn
Thr Lys Asn Ser Thr Lys Glu 755 760 765 ccc cgg cta aaa ttt ggc agc
aag cac aaa tca aac ttg aaa caa gtc 2352 Pro Arg Leu Lys Phe Gly
Ser Lys His Lys Ser Asn Leu Lys Gln Val 770 775 780 gaa act gga gtt
gcc aag atg aat aca atc aat gca gca gaa cct cat 2400 Glu Thr Gly
Val Ala Lys Met Asn Thr Ile Asn Ala Ala Glu Pro His 785 790 795 800
gtg gtg aca gtc acc atg aat ggt gtg gca ggt aga aac cac agt gtt
2448 Val Val Thr Val Thr Met Asn Gly Val Ala Gly Arg Asn His Ser
Val 805 810 815 aac tcc cat gct gcc aca acc caa tat gcc aat agg aca
gta cta tct 2496 Asn Ser His Ala Ala Thr Thr Gln Tyr Ala Asn Arg
Thr Val Leu Ser 820 825 830 ggc caa aca acc aac ata gtg aca cat agg
gcc caa gaa atg ttg cag 2544 Gly Gln Thr Thr Asn Ile Val Thr His
Arg Ala Gln Glu Met Leu Gln 835 840 845 aat cag ttt att ggt gag gac
acc cgg ctg aat att aat tcc agt cct 2592 Asn Gln Phe Ile Gly Glu
Asp Thr Arg Leu Asn Ile Asn Ser Ser Pro 850 855 860 gat gag cat gag
cct tta ctg aga cga gag caa caa gct ggc cat gat 2640 Asp Glu His
Glu Pro Leu Leu Arg Arg Glu Gln Gln Ala Gly His Asp 865 870 875 880
gaa ggt gtt ctg gat cgt ctt gtg gac agg agg gaa cgg cca cta gaa
2688 Glu Gly Val Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu
Glu 885 890 895 ggt ggc cga act aat tcc aat aac aac aac agc aat cca
tgt tca gaa 2736 Gly Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser Asn
Pro Cys Ser Glu 900 905 910 caa gat gtt ctt gca cag ggt gtt cca agc
aca gca gca gat cct ggg 2784 Gln Asp Val Leu Ala Gln Gly Val Pro
Ser Thr Ala Ala Asp Pro Gly 915 920 925 cca tca aag ccc aga aga gca
cag agg cct aat tct ctg gat ctt tca 2832 Pro Ser Lys Pro Arg Arg
Ala Gln Arg Pro Asn Ser Leu Asp Leu Ser 930 935 940 gcc aca aat gtc
ctg gat ggc agc agt ata cag ata ggt gag tca aca 2880 Ala Thr Asn
Val Leu Asp Gly Ser Ser Ile Gln Ile Gly Glu Ser Thr 945 950 955 960
caa gat ggc aaa tca gga tca ggt gaa aag atc aag aaa cgt gtg aaa
2928 Gln Asp Gly Lys Ser Gly Ser Gly Glu Lys Ile Lys Lys Arg Val
Lys 965 970 975 act ccc tat tct ctt aag cgg tgg cgc ccc tcc acc tgg
gtc atc tcc 2976 Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser Thr
Trp Val Ile Ser 980 985 990 act gaa tcg ctg gac tgt gaa gtc aac aat
aat ggc agt aac agg gca 3024 Thr Glu Ser Leu Asp Cys Glu Val Asn
Asn Asn Gly Ser Asn Arg Ala 995 1000 1005 gtt cat tcc aaa tcc agc
act gct gtt tac ctt gca gaa gga ggc act 3072 Val His Ser Lys Ser
Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr 1010 1015 1020 gct aca
acc atg gtg tct aaa gat ata gga atg aac tgt ctg t 3115 Ala Thr Thr
Met Val Ser Lys Asp Ile Gly Met Asn Cys Leu 1025 1030 1035 gaaatgt
3122 2 1038 PRT Homo Sapiens 2 Met Thr Ser Ser Leu Gln Arg Pro Trp
Arg Val Pro Trp Leu Pro Trp 1 5 10 15 Thr Ile Leu Leu Val Ser Thr
Ala Ala Ala Ser Gln Asn Gln Glu Arg 20 25 30 Leu Cys Ala Phe Lys
Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu 35 40 45 Ser Arg Ile
Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser 50 55 60 Thr
Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn Leu Val 65 70
75 80 Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln Glu Cys His
Tyr 85 90 95 Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser Ile Gln
Asn Gly Thr 100 105 110 Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys
Asn Val Asn Phe Thr 115 120 125 Glu Asn Phe Pro Pro Pro Asp Thr Thr
Pro Leu Ser Pro Pro His Ser 130 135 140 Phe Asn Arg Asp Glu Thr Ile
Ile Ile Ala Leu Ala Ser Val Ser Val 145 150 155 160 Leu Ala Val Leu
Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170 175 Gly Asp
Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala 180 185 190
Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu 195
200 205 Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp
Glu 210 215 220 Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala Asn Arg
Gln Asn Phe 225 230 235 240 Ile Asn Glu Lys Asn Ile Tyr Arg Val Pro
Leu Met Glu His Asp Asn 245 250 255 Ile Ala Arg Phe Ile Val Gly Asp
Glu Arg Val Thr Ala Asp Gly Arg 260 265 270 Met Glu Tyr Leu Leu Val
Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285 Lys Tyr Leu Ser
Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295 300 Ala His
Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro 305 310 315
320 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg Asp Leu Asn Ser
325 330 335 Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys Val Ile Ser
Asp Phe 340 345 350 Gly Leu Ser Met Arg Leu Thr Gly Asn Arg Leu Val
Arg Pro Gly Glu 355 360 365 Glu Asp Asn Ala Ala Ile Ser Glu Val Gly
Thr Ile Arg Tyr Met Ala 370 375 380 Pro Glu Val Leu Glu Gly Ala Val
Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395 400 Leu Lys Gln Val Asp
Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile 405 410 415 Phe Met Arg
Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr 420 425 430 Gln
Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp 435 440
445 Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg Pro Lys Phe Pro Glu
450 455 460 Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu
Thr Ile 465 470 475 480 Glu Asp Cys Trp Asp Gln Asp Ala Glu Ala Arg
Leu Thr Ala Gln Cys 485 490 495 Ala Glu Glu Arg Met Ala Glu Leu Met
Met Ile Trp Glu Arg Asn Lys 500 505 510 Ser Val Ser Pro Thr Val Asn
Pro Met Ser Thr Ala Met Gln Asn Glu 515 520 525 Arg Asn Leu Ser His
Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro 530 535 540 Asp Tyr Ser
Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp 545 550 555 560
Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr Pro 565
570 575 Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr Glu Arg
Gln 580 585 590 Gln Ala Gln Ala Arg Ile Pro Ser Pro Glu Thr Ser Val
Thr Ser Leu 595 600 605 Ser Thr Asn Thr Thr Thr Thr Asn Thr Thr Gly
Leu Thr Pro Ser Thr 610 615 620 Gly Met Thr Thr Ile Ser Glu Met Pro
Tyr Pro Asp Glu Thr Asn Leu 625 630 635 640 His Thr Thr Asn Val Ala
Gln Ser Ile Gly Pro Thr Pro Val Cys Leu 645 650 655 Gln Leu Thr Glu
Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu 660 665 670 Val Asp
Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Met Glu His 675 680 685
Ser Leu Lys Gln Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser 690
695 700 Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val Glu Ala Thr Gly
Gln 705 710 715 720 Gln Asp Phe Thr Gln Thr Ala Asn Gly Gln Ala Cys
Leu Ile Pro Asp 725 730 735 Val Leu Pro Thr Gln Ile Tyr Pro Leu Pro
Lys Gln Gln Asn Leu Pro 740 745 750 Lys Arg Pro Thr Ser Leu Pro Leu
Asn Thr Lys
Asn Ser Thr Lys Glu 755 760 765 Pro Arg Leu Lys Phe Gly Ser Lys His
Lys Ser Asn Leu Lys Gln Val 770 775 780 Glu Thr Gly Val Ala Lys Met
Asn Thr Ile Asn Ala Ala Glu Pro His 785 790 795 800 Val Val Thr Val
Thr Met Asn Gly Val Ala Gly Arg Asn His Ser Val 805 810 815 Asn Ser
His Ala Ala Thr Thr Gln Tyr Ala Asn Arg Thr Val Leu Ser 820 825 830
Gly Gln Thr Thr Asn Ile Val Thr His Arg Ala Gln Glu Met Leu Gln 835
840 845 Asn Gln Phe Ile Gly Glu Asp Thr Arg Leu Asn Ile Asn Ser Ser
Pro 850 855 860 Asp Glu His Glu Pro Leu Leu Arg Arg Glu Gln Gln Ala
Gly His Asp 865 870 875 880 Glu Gly Val Leu Asp Arg Leu Val Asp Arg
Arg Glu Arg Pro Leu Glu 885 890 895 Gly Gly Arg Thr Asn Ser Asn Asn
Asn Asn Ser Asn Pro Cys Ser Glu 900 905 910 Gln Asp Val Leu Ala Gln
Gly Val Pro Ser Thr Ala Ala Asp Pro Gly 915 920 925 Pro Ser Lys Pro
Arg Arg Ala Gln Arg Pro Asn Ser Leu Asp Leu Ser 930 935 940 Ala Thr
Asn Val Leu Asp Gly Ser Ser Ile Gln Ile Gly Glu Ser Thr 945 950 955
960 Gln Asp Gly Lys Ser Gly Ser Gly Glu Lys Ile Lys Lys Arg Val Lys
965 970 975 Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser Thr Trp Val
Ile Ser 980 985 990 Thr Glu Ser Leu Asp Cys Glu Val Asn Asn Asn Gly
Ser Asn Arg Ala 995 1000 1005 Val His Ser Lys Ser Ser Thr Ala Val
Tyr Leu Ala Glu Gly Gly Thr 1010 1015 1020 Ala Thr Thr Met Val Ser
Lys Asp Ile Gly Met Asn Cys Leu 1025 1030 1035 3 20 DNA Artificial
Sequence Description of Artificial Sequence; Note = Syntheic
Construct 3 agctaggtcc tctcatcagc 20 4 20 DNA Artificial Sequence
Description of Artificial Sequence; Note = Syntheic Construct 4
cagccgcagt gctgaccagc 20 5 20 DNA Artificial Sequence Description
of Artificial Sequence; Note = Syntheic Construct 5 gtcattcgga
taagacaaag 20 6 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 6 tttaacatac
tcccatgtcc 20 7 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 7 tagcttacac
gtactctcac 20 8 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 8 cctggcttca
accttgaatg 20 9 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 9 gggtacagcc
tttctaaagg 20 10 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 10 gatactattg
aggctgggtg 20 11 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 11 gctgctaatc
tttctgcagc 20 12 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 12 gaatgaagtc
actgttccag 20 13 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 13 cagagagctg
tagcattctg 20 14 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 14 aagtgatcca
cctgccttag 20 15 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 15 actcttcatg
ttaaagtgag 20 16 25 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 16 ctttgaagat
ataattaaaa tttcc 25 17 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 17 cacctggcca
gtagatgttt 20 18 26 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 18 tgttcaatag
tcccttttat tcattg 26 19 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 19 ctaatttgca
tcctgctgct 20 20 26 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 20 tgttcttcag
aatatgctac gttctc 26 21 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 21 ttgtggcatt
aggcaactcc 20 22 19 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 22 gcctgaaggg
gatgaaaaa 19 23 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 23 ccacacccct
tagggtctta 20 24 23 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 24 cacatggttt
gacatgtact ttg 23 25 22 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 25 catcagagct
ttccttgagg tt 22 26 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 26 cagaggtgtt
aaatttggag 20 27 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 27 tctacctgcc
acaccattca 20 28 21 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 28 tggaaaccaa
caagctagac c 21 29 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 29 ccccaaaaga
cacacaggag 20 30 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 30 tgaatggtgt
ggcaggtaga 20 31 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 31 gctgacagga
ggataaagca 20 32 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 32 caccctcctg
agacattggt 20 33 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 33 acccaatatg
ccaatgggac 20 34 20 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 34 ttcgccacct
tctagtggct 20 35 23 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 35 catgtggtaa
actgaaaagc tca 23 36 23 DNA Artificial Sequence Description of
Artificial Sequence; Note = Syntheic Construct 36 ttgagaccac
tttgatacac aca 23
* * * * *
References