U.S. patent application number 11/830625 was filed with the patent office on 2008-12-25 for large deletions in human brca1 gene and use thereof.
This patent application is currently assigned to Myriad Genetics, Incorporated. Invention is credited to Brant C. Hendrickson, Dmitry Pruss, Thomas Scholl, Benjamin Ward.
Application Number | 20080318224 11/830625 |
Document ID | / |
Family ID | 29739931 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080318224 |
Kind Code |
A1 |
Scholl; Thomas ; et
al. |
December 25, 2008 |
LARGE DELETIONS IN HUMAN BRCA1 GENE AND USE THEREOF
Abstract
Large deletions have been identified in the BRCA1 gene in
patients. The large deletions predispose the patients to breast
cancer and ovarian cancer. Thus, methods for detecting the genetic
variants are provided which can be used in detecting a
predisposition to cancer.
Inventors: |
Scholl; Thomas; (Salt Lake
City, UT) ; Hendrickson; Brant C.; (Salt Lake City,
UT) ; Ward; Benjamin; (Park City, UT) ; Pruss;
Dmitry; (Salt Lake City, UT) |
Correspondence
Address: |
MYRIAD GENETICS INC.;INTELLECUTAL PROPERTY DEPARTMENT
320 WAKARA WAY
SALT LAKE CITY
UT
84108
US
|
Assignee: |
Myriad Genetics,
Incorporated
Salt Lake City
UT
|
Family ID: |
29739931 |
Appl. No.: |
11/830625 |
Filed: |
July 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10457839 |
Jun 9, 2003 |
7250497 |
|
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11830625 |
|
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60387132 |
Jun 7, 2002 |
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60402430 |
Aug 9, 2002 |
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Current U.S.
Class: |
435/6.12 ;
435/7.1 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C12Q 1/6886 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/6 ;
435/7.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method, comprising: detecting a deletion in the BRCA1 gene,
said deletion resulting from the unequal crossover between a pair
of repetitive sequences in the BRCA1 gene, where said pair of
repetitive sequences is selected from the group consisting of: (1)
a first Alu sequence comprising basepairs 56,705-57,010, and a
second Alu sequence comprising basepairs 63,044-63,346, (2) a third
Alu sequence comprising basepairs 54,676-54,965, and a fourth Alu
sequence comprising basepairs 62,026-62,323, (3) a fifth Alu
sequence comprising basepairs 55,865-56,164, and said fourth Alu
sequence comprising basepairs 62,026-62,323, (4) said fifth Alu
sequence comprising basepairs 55,865-56,164, and a sixth Alu
sequence comprising basepairs 61,616-61,918, (5) a seventh Alu
sequence comprising basepairs 53,006-53,171, and an eighth Alu
sequence comprising basepairs 58,500-58,798, and (6) a ninth Alu
sequence comprising basepairs 50,347-50,642, and a tenth Alu
sequence comprising basepairs 76,803-77,098; wherein the presence
of the deletion would indicate a predisposition to cancer.
2. The method of claim 1, wherein said method is used to predict a
predisposition to cancer in a patient.
3. The method of claim 1 wherein the detection step comprises
analysis of BRCA1 genomic DNA.
4. The method of claim 3 wherein the analysis of BRCA1 genomic DNA
comprises amplifying a region of genomic DNA in which the deletion
occurs.
5. The method of claim 3 wherein the analysis of BRCA1 genomic DNA
comprises hybridizing a nucleic acid probe to a region of genomic
DNA in which the deletion occurs.
6. The method of claim 1 wherein the detection step comprises
analysis of BRCA1 cDNA.
7. The method of claim 6 wherein the analysis of BRCA1 cDNA
comprises amplifying a region of cDNA in which the deletion
occurs.
8. The method of claim 6 wherein the analysis of BRCA1 cDNA
comprises hybridizing a nucleic acid probe to a region of cDNA in
which the deletion occurs.
9. The method of claim 1 wherein the detection step comprises
analysis of a BRCA1 polypeptide.
10. The method of claim 9 wherein the analysis of a BRCA1
polypeptide comprises determining whether the polypeptide is
truncated.
11. The method of claim 9 wherein the analysis of a BRCA1
polypeptide comprises contacting the polypeptide with an
antibody.
12. The method of claim 1 wherein said pair of repetitive sequences
in the BRCA1 gene is selected from the group consisting of: (1) a
first upstream sequence of basepairs 56,960-56,998, and a first
downstream sequence of basepairs 63,296-63,334, (2) a second
upstream sequence of basepairs 54,960-54,965, and a second
downstream sequence of basepairs 62,143-62,147, (3) a third
upstream sequence of basepairs 55,893-55,932, and a third
downstream sequence of basepairs 62,049-62,088, (4) a fourth
upstream sequence of basepairs 56,090-56,095, and a fourth
downstream sequence of basepairs 61,838-61,843, (5) a fifth
upstream sequence of basepairs 53,030-53,075, and a fifth
downstream sequence of basepairs 58,659-58,704, and (6) a sixth
upstream sequence of basepairs 50,524-50,577, and a sixth
downstream sequence of basepairs 76,977-77,031.
13. The method of claim 12 wherein the detection step comprises
analysis of BRCA1 genomic DNA.
14. The method of claim 13 wherein the analysis of BRCA1 genomic
DNA comprises amplifying a region of genomic DNA in which the
deletion occurs.
15. The method of claim 12 wherein the detection step comprises
analysis of BRCA1 cDNA.
16. The method of claim 12 wherein the detection step comprises
analysis of BRCA1 polypeptides.
17. The method of claim 1, wherein the deletion results in a BRCA1
genomic DNA comprising a nucleotide sequence selected from the
group consisting of: (a) SEQ ID NO:1, (b) SEQ ID NO:2, (c) SEQ ID
NO:3, (d) SEQ ID NO:4, (e) SEQ ID NO:5, and (f) SEQ ID NO:6.
18. The method of claim 17 wherein the detection step comprises
amplifying a region of genomic DNA in which the deletion
occurs.
19. The method of claim 17 wherein the detection step comprises
hybridizing a nucleic acid probe to a region of genomic DNA in
which the deletion occurs.
20. An isolated mutant BRCA1 polypeptide comprising a sequence
selected from the group consisting of: (a) SEQ ID NO:10, (b) SEQ ID
NO:11, (c) SEQ ID NO 12, (d) SEQ ID NO 13, and (e) SEQ ID NO:14.
Description
CROSS-REFERENCE TO RELATED U.S. APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 10/457,839 filed on Jun. 9, 2003, expected to issue on
Jul. 31, 2007 as U.S. Pat. No. 7,250,497; which claims the benefit
(under 35 U.S.C. .sctn. 119(e)) of U.S. Provisional Application
Nos. 60/387,132 filed on Jun. 7, 2002 and 60/402,430 filed on Aug.
9, 2002; all of which are incorporated by reference herein in their
entirety.
SEQUENCE LISTING
[0002] The instant application was filed with a formal Sequence
Listing submitted electronically as a text file. This text file,
which was named "3002-01-12D-2007-07-30-SEQ-LST-JBO_ST25", was
created on Jul. 30, 2007, and is 15,974 bytes in size. Its contents
are incorporated by reference herein in their entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention generally relates to human genetics,
particularly to the identification of genetic polymorphic
variations in the human BRCA1 gene and methods of using the
identified genetic polymorphisms.
TECHNICAL BACKGROUND OF THE INVENTION
[0004] Breast cancer susceptibility gene 1 (BRCA1) is a tumor
suppressor gene identified on the basis of its genetic linkage to
familial breast cancers. It is a 220-kilodalton nuclear
phosphoprotein in normal cells. Mutations of the BRCA1 gene in
humans are associated with predisposition to breast and ovarian
cancers. In fact, BRCA1 and BRCA2 mutations are responsible for the
majority of familial breast cancer. Inherited mutations in the
BRCA1 and BRCA2 genes account for approximately 7-10% of all breast
and ovarian cancers. Women with BRCA mutations have a lifetime risk
of breast cancer between 56-87%, and a lifetime risk of ovarian
cancer between 27-44%. In addition, mutations in BRCA1 gene have
also been linked to various other tumors including, e.g.,
proliferative breast disease (PBD), papillary serous carcinoma of
the peritoneum (PSCP), and prostate cancer. Schorge, et al., J.
Nat. Cancer Inst., 90:841-845 (1998); Arason, Am. J. Hum. Genet.,
52:711-717 (1993); Langston, et al., New Eng. J. Med., 334: 137-142
(1996).
[0005] A large number of deleterious mutations in BRCA1 gene have
been discovered. Genetic testing on patients to determine the
presence or absence of such deleterious mutations has proven to be
an effective approach in detecting predispositions to breast and
ovarian cancers. Genetic testing is now commonly accepted as the
most accurate method for diagnosing hereditary breast cancer and
ovarian risk.
[0006] As deleterious mutations in BRCA1 are associated with
predisposition to cancers, particularly breast cancer and ovarian
cancer, it is desirable to identify additional naturally existing
deleterious mutations in the BRCA1 gene, which may serve as
valuable diagnostic markers. One such class of deleterious
mutations includes large deletions.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the discovery of a number
of large deletions in human BRCA1 gene in patients. A detailed
description of the newly discovered deletion mutations is provided
in Table 1. These large deletions are believed to be deleterious
and cause significant alterations in structure or biochemical
activities in the BRCA1 gene products expressed from mutant BRCA1
genes. Patients with such deletions in one of their BRCA1 genes are
predisposed to, and thus have a significantly increased likelihood
of, breast cancer and/or ovarian cancer. Therefore, these deletion
variants are useful in genetic testing as markers for the
prediction of predisposition to cancers, especially breast cancer
and ovarian cancer, and in therapeutic applications for treating
cancers.
[0008] Accordingly, in a first aspect of the present invention,
isolated BRCA1 nucleic acids (genomic DNAs, corresponding mRNAs and
corresponding cDNAs) are provided comprising one of the newly
discovered genetic variants summarized in Table I below.
[0009] In accordance with another aspect of the invention, isolated
polypeptides are provided which are BRCA1 protein variants
comprising at least a portion of the amino acid sequence of a BRCA1
protein. The BRCA1 protein variants are encoded by an isolated
BRCA1 gene sequence of the present invention.
[0010] The present invention also provides a method for preparing
an antibody to a BRCA1 protein variant according to the present
invention. Preferably, the antibody prepared in this method is
selectively immunoreactive with one or more of the newly discovered
BRCA1 protein variants.
[0011] In accordance with another aspect of the invention, a method
is provided for genotyping BRCA1 to determine whether an individual
has a genetic variant or an amino acid variant identified in the
present invention. The presence of the variants would indicate a
predisposition to cancers including breast cancer and ovarian
cancer. In accordance with this aspect of the invention, a sample
containing genomic DNA, mRNA, or cDNA of the BRCA1 gene is obtained
from the individual to be tested. The genomic DNA, mRNA, or cDNA of
the BRCA1 gene in the sample should include at least the nucleotide
sequence surrounding the locus of one or more of the
above-described genetic variants such that the presence or absence
of a particular genetic variant can be determined. Any suitable
method known in the art for genotyping can be used for determining
the nucleotide(s) at a particular position in the BRCA1 gene.
Alternatively, the presence or absence of one or more of the amino
acid variants disclosed in FIGS. 7, 8 or 9 can also be determined
in the BRCA1 protein in a sample isolated from a patient to be
tested. The presence of the nucleotide and/or amino acid variants
provided in the present invention may be indicative of a likelihood
of a predisposition to cancers, e.g., breast cancer and ovarian
cancer.
[0012] In accordance with another aspect of the present invention,
a variety of methods are provided for predicting a predisposition
to cancer in a patient. In one embodiment these methods comprise
detecting a deletion in the BRCA1 gene that can result from an
unequal crossover event between specific pairs of Alu sequences,
wherein the presence of such a deletion would indicate a
predisposition to cancer. The detection step used in such methods
can involve the analysis of BRCA1 genomic DNA, cDNA or
polypeptides. Analyses of nucleic acids in these instances can
involve amplification-based approaches or hybridization-based
approaches. Analyses of polypeptides can involve determining
whether or not the variant BRCA1 polypeptide is truncated, or
contains characteristic epitopes that can be specifically detected
with an appropriate antibody.
[0013] In another embodiment of this aspect of the present
invention these methods comprise detecting a deletion in the BRCA1
gene that can result from an unequal crossover event between
specific repetitive sequences, commonly referred to as
recombination breakpoints or regions, and presented in Table 1,
wherein the presence of such a deletion would also indicate a
predisposition to cancer. As with deletions resulting from the
unequal crossover between specific Alu repeats, the detection step
used in the methods of this embodiment can involve the analysis of
BRCA1 genomic DNA, cDNA or polypeptides, and anlysis of nucleic
acids can involve amplification-based approaches.
[0014] In yet another embodiment of this aspect of the present
invention these methods involve detecting specific sequences in
BRCA1 genomic DNA or cDNA that are formed by the joining of the
normally-separated sequences that occur on either side of the
deleted region. Detection of these indicative or characteristic
nucleic acids in these instances can involve amplification-based
approaches or hybridization-based approaches.
[0015] In accordance with another aspect of the invention, a
detection kit is also provided for detecting, in an individual, an
elevated risk of cancer. In a specific embodiment, the kit is used
in determining a predisposition to breast cancer and ovarian
cancer. The kit may include, in a partitioned carrier or confined
compartment, any nucleic acid probes or primers, or antibodies
useful for detecting the BRCA1 variants of the present invention as
described above. The kit can also include other reagents such as
reverse transcriptase, DNA polymerase, buffers, nucleotides and
other items that can be used in detecting the genetic variations
and/or amino acid variants according to the method of this
invention. In addition, the kit preferably also contains
instructions for its use.
[0016] The present invention further provides a method for
identifying a compound for treating or preventing cancers
associated with a BRCA1 genetic variant of the present invention.
The method includes screening for a compound capable of selectively
interacting with a BRCA1 protein variant of the present
invention.
[0017] The foregoing and other advantages and features of the
invention, and the manner in which the same are accomplished, will
become more readily apparent upon consideration of the following
detailed description of the invention taken in conjunction with the
accompanying examples and drawings, which illustrate preferred and
exemplary embodiments.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIGS. 1-6 show alignments of the upstream and downstream
BRCA1 sequences involved in the unequal crossover events that
resulted in deletions 1-6, respectively. Recombination likely
occurred between the regions underlined in the upstream and
downstream sequences, to produce the observed recombinant sequences
shown (with their region of recombination underlined). These
observed recombinant sequences were discovered in genomic DNA
isolated from human patients. The nucleotide numbers shown
correspond to the reference sequence provided by GenBank Accession
Number L78833.1 (Smith et al., Genome Res. 6:1029-1049 (1996)).
[0019] FIGS. 7, 8, and 9 depict the effects or consequences of the
newly discovered large deletions on the gene products of BRCA1
genes bearing such mutations. In particular, FIG. 7 illustrates the
effects of newly discovered deletion Nos. 1, 2, 3, and 4, which
effectively remove exons 16 and 17 from the BRCA1 gene transcript
(mRNA), thereby removing the 133 codons encoding amino acid
residues E1559-T1691. Although these mutations all result in a
shortened mRNA transcript lacking exons 16 and 17 and a shortened
mutant BRCA1 protein, they do not disrupt the open reading frame of
the remaining transcript.
[0020] FIG. 8 shows the effects of newly discovered Deletion No. 5,
which effectively removes exons 15 and 16 from the BRCA1 gene
transcript (mRNA), thereby removing the third position nucleotide
from codon R1495 and the following 167 codons encoding amino acid
residues S1496-F1661. Removal of the third position nucleotide of
codon R1495 serves to disrupt the downstream open reading frame,
resulting in a frame shift that is maintained until an ochre stop
codon is encountered fourteen codons into exon 17. As a result of
the frame-shift created by the removal of exons 15 and 16 from the
spliced gene transcript, a novel 13-amino acid sequence encoded by
the frame-shifted exon 17 (SEQ ID NO:13) is appended onto R1495 of
the translated BRCA1 polypeptide, and the overall length of the
resulting BRCA1 protein is shortened from 1863 to 1507 amino acid
residues.
[0021] FIG. 9 depicts the effects of newly discovered deletion No.
6, which effectively removes exons 14 through 20 from the BRCA1
gene transcript (mRNA), thereby removing the second and third
position nucleotides from codon A1452 and the following 306 codons
encoding amino acid residues V1453-K1758. Removal of second and
third position nucleotides from codon A1453 serves to disrupt the
downstream open reading frame, resulting in a translational frame
shift that is maintained through the codons encoded by exons 21,
22, 23 and 24, until a UGA stop codon is encountered 7 codons into
exon 24. As a result of the frame-shift created by the removal of
exons 14 through 20 from the spliced gene transcript, a novel
69-amino acid sequence encoded by the frame-shifted exons 21, 22,
23, and 24 (SEQ ID NO:14) is appended onto K1452 of the translated
BRCA1 polypeptide, and the overall length of the resulting BRCA1
protein is shortened from 1863 to 1521 amino acid residues.
[0022] Note: For FIGS. 1-6, the BRCA1 genomic DNA nucleotide or
basepair numbers correspond to the reference sequence provided by
GenBank Accession Number L78833.1. For FIGS. 7, 8, and 9, the BRCA1
cDNA nucleotide and amino acid numbers correspond to the reference
sequence provided by GenBank Accession No. U14680.1
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0023] The terms "genetic variant," "mutation," and "nucleotide
variant" are used herein interchangeably to refer to changes or
alterations to a reference BRCA1 gene sequence at a particular
locus, including, but not limited to, nucleotide base deletions,
insertions, inversions, and substitutions in the coding and
noncoding regions. Deletions may be of a single nucleotide, a
portion or a region of the nucleotide sequence of the gene, or of
the entire gene sequence. Insertions may be of one or more
nucleotides. The genetic variants may occur in transcriptional
regulatory regions, untranslated regions of mRNA, exons, introns,
or exon/intron junctions. The genetic variants may or may not
result in stop codons, frame shifts, deletion of amino acids,
altered amino acid sequence, or altered protein expression level.
The mutations or genetic variants can be somatic, i.e., occur only
in certain tissues of the body and are not inherited in the
germline, or germline mutations, i.e., inherited mutations found in
all tissues.
[0024] "Genetic polymorphism" as used herein refers to the
phenomena that two or more genetic variants in a particular locus
of a gene are found in a population.
[0025] The term "allele" or "gene allele" is used herein to refer
generally to a naturally occurring gene having the reference
sequence or a gene containing a specific genetic variant.
[0026] As used herein, the term "BRCA1 nucleic acid" means a
nucleic acid molecule the nucleotide sequence of which is found
uniquely in a BRCA1 gene or a substantially equivalent form
thereof. That is, the nucleotide sequence of a "BRCA1 nucleic acid"
can be a full-length sequence of, or a portion found in, either
BRCA1 genomic DNA or mRNA/cDNA, either wild-type or naturally
existing variant BRCA1 gene, or an artificial nucleotide sequence
encoding a wild-type BRCA1 protein or naturally existing
polymorphic variant BRCA1 protein.
[0027] The term "BRCA1 nucleic acid variant" refers to a naturally
existing BRCA1 nucleic acid.
[0028] As used herein, the term "amino acid variant" refers to
amino acid changes to a reference BRCA1 protein sequence resulting
from nucleotide variants or mutations to the reference gene
encoding the reference BRCA1 protein. The term "amino acid variant"
is intended to encompass not only single amino acid substitutions,
but also amino acid deletions, insertions, and other significant
changes of amino acid sequence in a BRCA1 protein.
[0029] The term "BRCA1 protein variant" is used herein relative to
a reference BRCA1 protein to mean a BRCA1 protein found in a
population that is the coding product of a BRCA1 gene allele
containing genetic variants such as single nucleotide
substitutions, insertions, deletions, and DNA rearrangements, which
lead to alterations in the protein sequence of the protein
variant.
[0030] The term "locus" refers to a specific position or site in a
nucleotide sequence of a gene, or amino acid sequence of a protein.
Thus, there may be one or more contiguous nucleotides in a
particular gene locus, or one or more amino acids at a particular
locus in a polypeptide. Moreover, "locus" may also be used to refer
to a particular position in a gene sequence where one or more
nucleotides have been deleted, inserted, or inverted.
[0031] The terms "polypeptide," "protein," and "peptide" are used
herein interchangeably to refer to amino acid chains in which the
amino acid residues are linked by peptide bonds or modified peptide
bonds. The amino acid chains can be of any length of greater than
two amino acids. Unless otherwise specified, the terms
"polypeptide," "protein," and "peptide" also encompass various
modified forms thereof. Such modified forms may be naturally
occurring modified forms or chemically modified forms. Examples of
modified forms include, but are not limited to, glycosylated forms,
phosphorylated forms, myristoylated forms, palmitoylated forms,
ribosylated forms, acetylated forms, etc. Modifications also
include intra-molecular crosslinking and covalent attachment of
various moieties such as lipids, flavin, biotin, polyethylene
glycol or derivatives thereof, etc. In addition, modifications may
also include cyclization, branching and cross-linking. Further,
amino acids other than the conventional twenty amino acids encoded
by genes may also be included in a polypeptide.
[0032] The terms "primer," "probe," and "oligonucleotide" may be
used herein interchangeably to refer to a relatively short nucleic
acid fragment or sequence. They can be DNA, RNA, or a hybrid
thereof, or chemically modified analogs or derivatives thereof.
Typically, they are single-stranded. However, they can also be
double-stranded having two complementing strands that can be
separated apart by denaturation. Normally, they have a length of
from about 8 nucleotides to about 200 nucleotides, preferably from
about 12 nucleotides to about 100 nucleotides, and more preferably
about 18 to about 50 nucleotides. They can be labeled with
detectable markers or modified in any conventional manners for
various molecular biological applications.
[0033] The term "isolated," when used in reference to nucleic acids
(which include gene sequences or fragments) of this invention, is
intended to mean that a nucleic acid molecule is present in a form
other than found in nature in its original environment with respect
to its association with other molecules. For example, since a
naturally existing chromosome includes a long nucleic acid
sequence, an "isolated nucleic acid" as used herein means a nucleic
acid molecule having only a portion of the nucleic acid sequence in
the chromosome but not one or more other portions present on the
same chromosome. Thus, for example, an isolated gene typically
includes no more than 25 kb of naturally occurring nucleic acid
sequence which immediately flanks the gene in the naturally
existing chromosome or genomic DNA. However, it is noted that an
"isolated nucleic acid" as used herein is distinct from a clone in
a conventional library such as genomic DNA library and cDNA library
in that the clones in a library are still in admixture with almost
all the other nucleic acids in a chromosome or a cell. An isolated
nucleic acid can be in a vector.
[0034] The term "isolated nucleic acid" embraces "purified nucleic
acid" which means a specified nucleic acid is in a substantially
homogenous preparation of nucleic acid substantially free of other
cellular components, other nucleic acids, viral materials, or
culture medium, or chemical precursors or by-products associated
with chemical reactions for chemical synthesis of nucleic acids.
Typically, a "purified nucleic acid" can be obtained by standard
nucleic acid purification methods. In a purified nucleic acid,
preferably the specified nucleic acid molecule constitutes at least
15 percent of the total nucleic acids in the preparation. The term
"purified nucleic acid" also means nucleic acids prepared from a
recombinant host cell (in which the nucleic acids have been
recombinantly amplified and/or expressed), or chemically
synthesized nucleic acids.
[0035] The term "isolated nucleic acid" also encompasses a
"recombinant nucleic acid" which is used herein to mean a hybrid
nucleic acid produced by recombinant DNA technology having the
specified nucleic acid molecule covalently linked to one or more
nucleic acid molecules that are not the nucleic acids naturally
flanking the specified nucleic acid. Typically, such nucleic acid
molecules flanking the specified nucleic acid are no more than 50
kb. In addition, the specified nucleic acid may have a nucleotide
sequence that is identical to a naturally occurring nucleic acid,
or a modified form, or mutant form thereof having one or more
mutations such as nucleotide substitution, deletion/insertion,
inversion, and the like.
[0036] In addition, "isolated nucleic acid" further includes a
chemically synthesized nucleic acid having a naturally occurring
nucleotide sequence or an artificially modified form thereof (e.g.,
dideoxy forms).
[0037] The term "isolated polypeptide" as used herein means a
polypeptide molecule is present in a form other than found in
nature in its original environment with respect to its association
with other molecules. The term "isolated polypeptide" encompasses a
"purified polypeptide" which is used herein to mean that a
specified polypeptide is in a substantially homogenous preparation,
substantially free of other cellular components, other
polypeptides, viral materials, or culture medium, or when the
polypeptide is chemically synthesized, substantially free of
chemical precursors or by-products associated with the chemical
synthesis. For a purified polypeptide, preferably the specified
polypeptide molecule constitutes at least 15 percent of the total
polypeptide in the preparation. A "purified polypeptide" can be
obtained from natural or recombinant host cells by standard
purification techniques, or by chemical synthesis.
[0038] The term "isolated polypeptide" also encompasses a
"recombinant polypetide," which is used herein to mean a hybrid
polypeptide produced by recombinant DNA technology or chemical
synthesis having a specified polypeptide molecule covalently linked
to one or more polypeptide molecules which do not naturally link to
the specified polypeptide.
[0039] As used herein, "haplotype" is a combination of genetic
(nucleotide) variants in a region of an mRNA or a genomic DNA on a
chromosome found in an individual. Thus, a haplotype includes a
number of genetically linked polymorphic variants that are
typically inherited together as a unit.
[0040] The term "reference sequence" refers to a polynucleotide or
polypeptide sequence known in the art, including those disclosed in
publicly accessible databases (e.g., GenBank), or a newly
identified gene sequence, used simply as a reference with respect
to the variants provided in the present invention. The nucleotide
or amino acid sequence in a reference sequence is contrasted to the
alleles disclosed in the present invention having newly discovered
nucleotide or amino acid variants.
[0041] The terms "crossing-over" and "crossover," are used
interchangeably herein, to refer to the reciprocal exchange of
material between chromosome homologs--by breakage and reunion--that
occurs during meiosis and is responsible for genetic recombination.
The term "unequal crossover," as used herein, refers to a crossover
event occurring between homologous sequences in paired chromosome
homologs that are not perfectly aligned, or, more generally,
describes a recombination event in which the two recombining sites
lie at nonidentical locations in the two parental DNA molecules.
The products of an unequal crossover are two chromosomes, or more
generally two progeny DNA molecules, one of which bears a deletion,
and the other of which bears a duplication of the nucleotide
sequence residing between the mispaired homologous sequences or
recombining sites.
2. Nucleotide and Amino Acid Variants
[0042] Smith and coworkers described the complete genomic sequence
of a 117 kilobase region of human DNA containing the BRCA1 gene,
and deposited the nucleotide sequence of the genomic DNA in the
GenBank under the Accession Number L78833.1 (Smith et al., Genome
Res., 6:1029-1049 (1996)). This nucleotide sequence (referred to as
L78833. 1) is used herein as a reference sequence for identifying
the polymorphic positions of the large deletions of the present
invention and the upstream and downstream sequences that were
likely involved in the unequal crossover events that yielded
Deletion Nos. 1-6. The complete coding sequence corresponding to
the mRNA transcribed from the BRCA1 gene, and the amino acid
sequence encoded therein, were deposited in the GenBank under
Accession Number U14680.1. These sequences (cDNA and amino acid)
are used as reference sequences for identifying the effects or
consequences of the large deletions at the level of the gene
transcript (mRNA), cDNA and encoded protein.
[0043] In accordance with the present invention, analysis of the
nucleotide sequence of genomic DNA corresponding to the BRCA1 genes
of specific human patients has led to the discovery of a number of
mutant BRCA1 alleles that exhibit large deletions relative to the
reference sequence provided by GenBank Accession No. L78833.1.
Specifically, six different genetic variants exhibiting large
deletions have been discovered. These six different genetic
variants, and the effects or consequences they have on the gene
products expressed from the BRCA1 alleles that bear them, are
summarized in Table 1. Of these six different genetic variants
corresponding to six different large deletions of nucleotide
sequence within the BRCA1 gene, four result in the deletion of
exons 16 and 17, one results in deletion of exons 15 and 16, and
one results in deletion of exons 14 through 20, in the mRNAs
transcribed from the variant alleles.
TABLE-US-00001 TABLE I GENETIC VARIANTS OF THE BRCA1 GENE All
numeric designation of nucleotides conform to the sequence in Smith
et al., Genome Res., 6: 1029-1049 (1996) and GenBank Accession
Number L78833.1 Recombination Breakpoint Size of Deletion 5' Region
3' Region Deletion Exons Consequences No. (nt positions) (nt
positions) (bp) Removed of Deletion 1 56,960-56,998 63,296-63,334
6,337 16 & 17 Removal of residues E1559-T1691 2 54,960-54,965
62,143-62,147 7,183 16 & 17 Removal of residues E1559-T1691 3
55,893-55,932 62,049-62,088 6,157 16 & 17 Removal of residues
E1559-T1691 4 56,090-56,095 61,838-61,843 5,749 16 & 17 Removal
of residues E1559-T1691 5 53,030-53,075 58,659-58,704 5,629 15
& 16 Addition of a novel 13- residue carboxyl- terminus onto
R1495 6 50,524-50,577 76,977-77,031 26,454 14-20 Addition of a
novel 69- residue carboxyl- terminus onto K1452
[0044] In further accordance with the present invention, the large
deletions described in Table 1 were found in patients at high risk
of developing breast cancer. Nucleotide sequences obtained from
these individuals indicate that all six of these large deletions
involved the joining of a particular sequence in a more 5' region
of the BRCA1 gene (an upstream sequence), to a similar sequence in
a more 3' region of the BRCA1 gene (a downstream sequence) to
create a recombined or joined sequence that spans the deletion
locus. Further analysis has shown that all upstream sequences, and
all downstream sequences reside within identified Alu repeats
(Smith et al., Genome Res., 6:1029-1049 (1996)). Consequently, the
observed mutations most likely arose from an unequal crossover
event occurring between misaligned Alu sequences in the BRCA1 genes
of paired homologous chromosomes. The specific sequences of the
upstream and downstream loci involved in these six unequal
crossover events, along with the specific joined or recombined
sequences resulting from these unequal crossover events (the
"deletion loci"), which have been observed in the genomic DNA of
specific individuals, are shown in FIGS. 1-6. The consequences of
each of the large deletions observed in mutant BRCA1 genomic DNAs
(as depicted in FIGS. 1-6) on the nucleotide sequence of the mRNA
transcript transcribed therefrom (or on the corresponding cDNA), as
well as on the amino acid sequence of the encoded protein, are
shown in FIGS. 7-9.
[0045] The breakpoint regions (upstream and downstream loci)
believed to be responsible for the unequal crossover that resulted
in Deletion No. 1, and the resulting recombined nucleotide sequence
discovered in human patients are shown underlined in FIG. 1. As
indicated in Table 1, the 5' recombination breakpoint resides
between nucleotides 56,960 and 56,998 (underlined in the upstream
sequence) and the 3' recombination breakpoint resides between
nucleotides 63,296 and 63,334 (underlined in the downstream
sequence). Recombination between the upstream and downstream
breakpoint regions has resulted in the deletion of 6,337 basepairs
of the BRCA1 gene and has produced a novel BRCA1 gene sequence
comprising the junction sequence provided by SEQ ID NO:1. The
resulting recombined genomic DNA sequence, which when transcribed
directs the expression of mutant mRNAs lacking exons 16 and 17
(FIG. 7), was found in three individuals.
[0046] The loci (breakpoint regions) believed to be responsible for
the unequal crossover that resulted in Deletion No. 2, and the
resulting recombined nucleotide sequence discovered in human
patients are shown underlined in FIG. 2. As indicated in Table 1,
the 5' recombination breakpoint resides between nucleotides 54,960
and 54,965 (underlined in the upstream sequence) and the 3'
recombination breakpoint resides between nucleotides 62,143 and
62,147 (underlined in the downstream sequence). Recombination
between the upstream and downstream breakpoint regions has resulted
in the deletion of 7,183 basepairs of the BRCA1 gene and has
produced a novel BRCA1 gene sequence comprising the junction
sequence provided by SEQ ID NO:2. The resulting recombined genomic
DNA sequence, which when transcribed also directs the expression of
mutant mRNAs lacking exons 16 and 17 (FIG. 7), was identified in
one individual.
[0047] The loci (breakpoint regions) believed to be responsible for
the unequal crossover that resulted in Deletion No. 3, and the
resulting recombined nucleotide sequence discovered in human
patients are shown underlined in FIG. 3. As indicated in Table 1,
the 5' recombination breakpoint resides between nucleotides 55,893
and 55,932 (underlined in the upstream sequence) and the 3'
recombination breakpoint resides between nucleotides 62,048 and
62,087 (underlined in the downstream sequence). Recombination
between the upstream and downstream breakpoint regions has resulted
in the deletion of 6,157 basepairs of the BRCA1 gene and has
produced a novel BRCA1 gene sequence comprising the junction
sequence provided by SEQ ID NO:3. The resulting recombined genomic
DNA sequence, which when transcribed also directs the expression of
mutant mRNAs lacking exons 16 and 17 (FIG. 7), was characterized in
one individual.
[0048] The loci (breakpoint regions) believed to be responsible for
the unequal crossover that resulted in Deletion No. 4, and the
resulting recombined nucleotide sequence discovered in human
patients are shown underlined in FIG. 4. As indicated in Table 1,
the 5' recombination breakpoint resides between nucleotides 56,090
and 56,095 (underlined in the upstream sequence) and the 3'
recombination breakpoint resides between nucleotides 61,838 and
61,843 (underlined in the downstream sequence). Recombination
between the upstream and downstream breakpoint regions has resulted
in the deletion of 5,749 basepairs of the BRCA1 gene and has
produced a novel BRCA1 gene sequence comprising the junction
sequence provided by SEQ ID NO:4. The resulting recombined genomic
DNA sequence, which when transcribed also directs the expression of
mutant mRNAs lacking exons 16 and 17 (FIG. 7), was found in one
individual.
[0049] The loci (breakpoint regions) believed to be responsible for
the unequal crossover that resulted in Deletion No. 5, and the
resulting recombined nucleotide sequence discovered in human
patients are shown underlined in FIG. 5. As indicated in Table 1,
the 5' recombination breakpoint resides between nucleotides 53,030
and 53,075 (underlined in the upstream sequence) and the 3'
recombination breakpoint resides between nucleotides 58,659 and
58,704 (underlined in the downstream sequence). Recombination
between the upstream and downstream breakpoint regions has resulted
in the deletion of 5,629 basepairs of the BRCAI gene and has
produced a novel BRCAI gene sequence comprising the junction
sequence provided by SEQ ID NO:5. The resulting recombined genomic
DNA sequence, which when transcribed also directs the expression of
mutant mRNAs lacking exons 15 and 16 (FIG. 8), was identified in
one individual.
[0050] The loci (breakpoint regions) believed to be responsible for
the unequal crossover that resulted in Deletion No. 6, and the
resulting recombined nucleotide sequence discovered in human
patients are shown underlined in FIG. 6. As indicated in Table 1,
the 5' recombination breakpoint resides between nucleotides 50,524
and 50,577 (underlined in the upstream sequence) and the 3'
recombination breakpoint resides between nucleotides 76,977 and
77,031 (underlined in the downstream sequence). Recombination
between the upstream and downstream breakpoint regions has resulted
in the deletion of 26,454 basepairs of the BRCAI gene and has
produced a novel BRCAI gene sequence comprising the junction
sequence provided by SEQ ID NO:6. The resulting recombined genomic
DNA sequence, which when transcribed also directs the expression of
mutant mRNAs lacking exons 14 through 20 (FIG. 9), has now been
characterized in fourteen individuals.
[0051] The consequences of Deletions 1-6 on the gene products
encoded by the BRCAI alleles bearing these mutations are depicted
in FIGS. 7-9. As mentioned above, Deletion Nos. 1, 2, 3, and 4 all
produce mutant alleles of the BRCA1 gene that, when transcribed,
direct the expression of mRNAs lacking exons 16 and 17 (FIG. 7).
Such mRNAs, and cDNAs prepared from them, lack the codons encoding
amino acid residues E1559-T1691, and are characterized by the novel
junction sequence comprising SEQ ID NO:7, which spans the deleted
codons. Despite the omission of the 133 codons encoded by exons 16
and 17, the open reading frame of the remaining nucleotides is not
disrupted (i.e., ntG4675 carries over to ntA5075 and ntT5076, so
that aaD1692 is conserved). Consequently, the mRNAs transcribed
from the mutant alleles characterized by Deletion Nos. 1-4, direct
the translation of a mutant BRCAI protein comprised of 1,730 amino
acid residues, instead of the normal 1,863. These shorter mutant
BRCAI proteins are characterized by the amino acid sequence created
by the juxtaposition of the codon encoding L1558 with the codon
encoding D1692, and comprising SEQ ID NO:10.
[0052] In contrast, Deletion No. 5 produces a mutant allele of the
BRCA1 gene that, when transcribed, directs the expression of mRNA
lacking exons 15 and 16 (FIG. 8). Such mRNA, and the cDNA prepared
from it, lacks the codons encoding amino acid residues S1496-F1662,
as well as the third position nucleotide from codon R1495, and is
characterized by the novel junction sequence comprising SEQ ID
NO:8, which spans the region of the deleted codons. Unlike with
Deletions Nos. 1-4, mRNA transcribed from mutant BRCA1 alleles
encompassing Deletion No. 5 directs a translational frame shift
downstream of the junction between nucleotides encoded by exons 14
and 17. Translation in the shifted frame is maintained until an
ochre stop codon is encountered fourteen codons into exon 17. As a
result of the frame-shift created by the omission of exons 15 and
16, a novel 13-amino acid sequence (SEQ ID NO:13), encoded by
codons within the frame-shifted exon 17, is appended onto R1495 of
the translated mutant BRCA1 polypeptide, and the overall length of
the BRCA1 protein is shortened from 1863 to 1507 amino acid
residues. Consequently, these mutant BRCA1 proteins are
characterized by their shortened length, their novel
carboxy-termini, and by the unique amino acid sequence created by
the splicing of exons 14 and 17, which comprises SEQ ID NO:11
[0053] Deletion No. 6 produces a mutant allele of the BRCA1 gene
that, when transcribed, directs the expression of mRNA lacking
exons 14 through 20 (FIG. 9). Such mRNA, and the cDNA prepared from
it, lacks the codons encoding amino acid residues V1453-K1758, as
well as the second and third position nucleotides from codon A1453,
and is characterized by the novel junction sequence comprising SEQ
ID NO:9, which spans the deleted codons. Like Deletion No. 5,
Deletion No. 6 directs a translational frame shift downstream of
the junction between nucleotides encoded by exons 13 and 21.
Translation in the shifted frame is maintained through the codons
encoded by exons 21, 22, 23 and 24, until a UGA stop is encountered
7 codons into exon 24. As a result of the frame-shift created by
the omission of exons 14 through 20, a novel 69-amino acid sequence
(SEQ ID NO:14) is appended onto K1452 of the translated mutant
BRCA1 polypeptide, and the overall length of the BRCA1 protein is
shortened from 1863 to 1521 amino acid residues. Consequently,
these mutant BRCA1 proteins are also characterized by their
shortened length, their novel carboxy-termini, and by the unique
amino acid sequence created by the splicing of exons 13 and 21,
which comprises SEQ ID NO:12.
[0054] As shown in the Figures, and described above, the genetic
variants according to the present invention are expected to cause
significant changes in the structure and biological activity of the
BRCA1 protein they encode. Individuals who inherit such genetic
variants (large deletion mutations) are predisposed to cancers,
particularly breast cancer and ovarian cancer.
3. BRCA1 Nucleic Acids
[0055] In a first aspect of the present invention, isolated nucleic
acids are provided comprising a nucleotide sequence of a BRCA1
nucleic acid variant identified in accordance with the present
invention. The nucleotide sequence is at least 12, 13, 14, 15, 17,
18, 19, 20, 25, 30, or 35 contiguous nucleotides spanning the
deletion locus in one of the mutant BRCA1 genomic DNAs having one
of the Deletion Nos. 1-6, or the deletion locus in one of the
mutant BRCA1 mRNAs, or cDNAs prepared therefrom, expressed from the
mutant BRCA1 genomic DNAs having one of the Deletion Nos. 1-6. The
nucleic acid molecules can be in a form of DNA, RNA, or a chimeric
or hybrid thereof, and can be in any physical structures including
a single-strand or double-strand or in the form of a triple
helix.
[0056] In one embodiment, the isolated nucleic acids have a
sequence selected from the group consisting of SEQ ID NOs:1-9 or
15-82, and complements thereof. Specifically, SEQ ID NOs:1 and
15-17 are mutant sequences seen in the BRCA1 genomic DNA variant
that resulted from Deletion No. 1. SEQ ID NOs:2 and 18-23 are
mutant sequences seen in the BRCA1 genomic DNA variant that
resulted from Deletion No. 2. SEQ ID NOs:3 and 24-27 are mutant
sequences seen in the BRCA1 genomic DNA variant that resulted from
Deletion No. 3. SEQ ID NOs:4 and 28-32 are mutant sequences seen in
the BRCA1 genomic DNA variant that resulted from Deletion No. 4.
SEQ ID NOs:5 and 33-36 are mutant BRCA1 genomic sequences that
resulted from Deletion No. 5. And, SEQ ID NOs:6 and 37-41 are
mutant BRCA1 genomic sequences that resulted from Deletion No.
6.
[0057] In addition, SEQ ID NOs:7 and 42-52 are mutant BRCA1 cDNA
sequences that span the cDNA deletion locus that results from
Deletion Nos. 1, 2, 3, and 4. SEQ ID NOs:45-47 are portions of the
antisense strand sequence of the mutant BRCA1 cDNA resulted from
Deletion Nos. 1, 2, 3, and 4, while SEQ ID NOs:7, 42-44, and 48-52
are portions of the sense strand that also span the cDNA deletion
locus resulted from Deletion Nos. 1, 2, 3, and 4.
[0058] SEQ ID NOs:8, 53-57, and 64-66 are portions of the sense
strand of mutant BRCA1 cDNA sequences that span the cDNA deletion
locus resulted from Deletion No. 5, while SEQ ID NOs:58-63 are
portions of the antisense strand sequence of the mutant BRCA1 cDNA
resulted from Deletion No. 5.
[0059] SEQ ID NOs:9, 67-74, and 64-66 are portions of the sense
strand of mutant BRCA1 cDNA sequences that span the cDNA deletion
locus resulted from Deletion No. 6, while SEQ ID NOs:75-79 are
portions of the antisense strand sequence of the mutant BRCA1 cDNA
resulted from Deletion No. 6.
[0060] SEQ ID NOs:80 represents the portion of the cDNA encoding
the novel carboxyl-terminal tail of the mutant BRCA1 polypeptide
resulting from Deletion No. 5. SEQ ID NO:81 represents the cDNA
encompassing the junction of the original reading frame and the
novel frame-shifted reading frame that results from the
juxtaposition of exons 13 and 21 seen in the mutant BRCA1 mRNA
resulting from Deletion No. 6, while SEQ ID NO:82 represents the
portion of the cDNA encoding the novel carboxyl-terminal tail of
the mutant BRCA1 polypeptide resulting from Deletion No. 6.
[0061] In a specific embodiment, the isolated nucleic acids of the
present invention are isolated BRCA1 nucleic acid having a sequence
according to one of SEQ ID NOs:1-9 or 15-82, or complements
thereof. Preferably, the isolated BRCA1 nucleic acids are isolated
BRCA1 nucleic acid variants that are mutant BRCA1 genomic DNAs
having one of the Deletion Nos. 1-6, or those mutant BRCA1 mRNAs
derived from the mutant BRCA1 genomic DNAs, having one of the
Deletion Nos. 1-6, or cDNAs derived from such mRNAs. The BRCA1
genomic DNAs, cDNAs and mRNAs can have a full-length sequence
(i.e., including the entire coding regions and, in the case of
genomic DNAs, optionally introns, promoter, and other regulatory
sequences) or partial sequence (i.e., a portion of the full-length
sequence).
[0062] In one embodiment, an isolated BRCA1 nucleic acid is an
oligonucleotide, primer or probe comprising a contiguous span of
the nucleotide sequence of a mutant BRCA1 sequence (either genomic
DNA or cDNA or mRNA sequence) provided in accordance with the
present invention and spanning a cDNA deletion locus resulted from
Deletion Nos. 1, 2, 3, 4, 5 or 6. The oligonucleotide, primer or
probe contains at least 12, preferably from about 15, 18, 20, 22,
25, 30, 40 to about 50, 60, 70, 80, 90, or 100, and more preferably
from about 30 to about 50 nucleotides. In one embodiment, the
oligonucleotides, primers and probes are specific to a BRCA1
nucleic acid variant of the present invention. That is, they
selectively hybridize, under stringent conditions generally
recognized in the art, to a BRCA1 nucleic acid variant of the
present invention, but do not substantially hybridize to a
reference BRCA1 nucleic acid sequence under stringent conditions.
Such oligonucleotides will be useful in hybridization-based
methods, or alternatively amplification-based methods, for
detecting the nucleotide variants of the present invention as
described in detail below. A skilled artisan would recognize
various stringent conditions that enable the oligonucleotides of
the present invention to differentiate between a reference BRCA1
gene sequence and an isolated BRCA1 nucleic acid variant of the
present invention. For example, the hybridization can be conducted
overnight in a solution containing 50% formamide, 5.times.SSC,
pH7.6, 5.times. Denhardt's solution, 10% dextran sulfate, and 20
microgram/ml denatured, sheared salmon sperm DNA. The hybridization
filters can be washed in 0.1.times.SSC at about 65.degree. C.
[0063] The oligonucleotide primers or probes of the present
invention can have a detectable marker selected from, e.g.,
radioisotopes, fluorescent compounds, enzymes, or enzyme co-factors
operably linked to the oligonucleotide. The primers, probes and
oligonucleotide sequences of the present invention are useful in
genotyping and haplotyping as will be apparent from the description
below.
[0064] In another specific embodiment, BRCA1 nucleic acids are
provided having 100, 200, 300, 400 or 500 nucleotides or basepairs,
which contain the BRCA1 variant nucleotide or basepair sequences
provided by SEQ ID NOs:1-9 or 15-82, and/or the complements
thereof. Such nucleic acids can be DNA or RNA, and single-stranded
or double-stranded.
[0065] It should be understood that any nucleic acid molecules
containing a sequence according to one of SEQ ID NOs: 1-9 or 15-82
fall within the scope of this invention. For example, a hybrid
nucleic acid molecule may be provided having a sequence according
to one of SEQ ID NOs: 1-9 or 15-82 operably linked to a non-BRCA1
sequence such that the hybrid nucleic acid encodes a hybrid protein
having a mutant BRCA1 peptide sequence. In another embodiment, the
present invention provides a vector construct containing one of the
nucleic acid molecules of the present invention. As will be
apparent to skilled artisans, the vector may be employed to amplify
a nucleic acid molecule of the present invention that is contained
in the vector construct. Alternatively, the vector construct may be
used in expressing a polypeptide encoded by a nucleic acid molecule
of the present invention that is contained in the vector construct.
Generally, the vector construct may include a promoter operably
linked to an isolated nucleic acid molecule (including a
full-length sequence or a fragment thereof in the 5' to 3'
direction or in the reverse direction for the purpose of producing
antisense nucleic acids), an origin of DNA replication for the
replication of the vectors in host cells and a replication origin
for the amplification of the vectors in, e.g., E. coli, and
selection marker(s) for selecting and maintaining only those host
cells harboring the vectors. Additionally, the vectors preferably
also contain inducible elements, which function to control the
expression of the isolated gene sequence. Other regulatory
sequences such as transcriptional termination sequences and
translation regulation sequences (e.g., Shine-Dalgarno sequence)
can also be included. An epitope tag coding sequence for detection
and/or purification of the encoded polypeptide can also be
incorporated into the vector construct. Examples of useful epitope
tags include, but are not limited to, influenza virus hemagglutinin
(HA), Simian Virus 5 (V5), polyhistidine (6xHis), c-myc, lacZ, GST,
and the like. Proteins with polyhistidine tags can be easily
detected and/or purified with Ni affinity columns, while specific
antibodies to many epitope tags are generally commercially
available. The vector construct can be introduced into the host
cells or organisms by any techniques known in the art, e.g., by
direct DNA transformation, microinjection, electroporation, viral
infection, lipofection, biolystics (gene gun), and the like. The
vector construct can be maintained in host cells in an
extrachromosomal state, i.e., as self-replicating plasmids or
viruses. Alternatively, the vector construct can be integrated into
chromosomes of the host cells by conventional techniques such as
selection of stable cell lines or site-specific recombination. The
vector construct can be designed to be suitable for expression in
various host cells, including but not limited to bacteria, yeast
cells, plant cells, insect cells, and mammalian and human cells. A
skilled artisan will recognize that the designs of the vectors can
vary with the host used.
[0066] In another embodiment, a BRCA1 nucleic acid of the present
invention is incorporated in a microchip or microarray, or other
similar structures. The microarray will allow rapid genotyping
and/or haplotyping in a large scale. As is known in the art, in
microchips, a large number of different nucleic acids are attached
or immobilized in an array on a solid support, e.g., a silicon chip
or glass slide. Target nucleic acid sequences to be analyzed can be
contacted with the immobilized nucleic acids on the microchip. See
Lipshutz et al., Biotechniques, 19:442-447 (1995); Chee et al., i
Science, 274:610-614 (1996); Kozal et al., i Nat. Med. 2:753-759
(1996); Hacia et al., i Nat. Genet., 14:441-447 (1996); Saiki et
al., i Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeras et
al., i Genome Res., 8:435-448 (1998). The microchip technologies
combined with computerized analysis tools allow large-scale high
throughput screening. See, e.g., U.S. Pat. No. 5,925,525 to Fodor
et al. Wilgenbus et al., J. Mol Med., 77:761-786 (1999); Graber et
al., Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat.
Genet., 14:441-447 (1996); Shoemaker et al., Nat. Genet.,
14:450-456 (1996); DeRisi et al., Nat. Genet., 14:457-460 (1996);
Chee et al., Nat. Genet., 14:610-614 (1996); Lockhart et al., Nat.
Genet., 14:675-680 (1996); Drobyshev et al., Gene, 188:45-52
(1997).
[0067] In a preferred embodiment, a microarray is provided
comprising a plurality of the nucleic acids of the present
invention such that the nucleotide identity at each of the genetic
variant sites disclosed in Table I can be determined in one single
microarray.
4. BRCA1 Polypeptides
[0068] The present invention also provides isolated polypeptides
having a novel amino acid sequence of a BRCA1 protein variant
identified in accordance with the present invention. The amino acid
sequence is a contiguous sequence of at least 3, 4, 5, 6, 7, 8, 9,
10, 12, or 13 amino acids spanning the deletion locus resulted from
Deletion Nos. 1, 2, 3, 4, 5, or 6. In addition, the amino acid
sequence can also be a contiguous sequence of at least 3, 4, 5, 6,
7, 8, 9, 10, 12, or 13 amino acids within the carboxyl-terminal
sequence of 13 amino acids of the BRCA1 protein variant resulting
from Deletion No. 5. Alternatively, the amino acid sequence can be
a contiguous sequence of at least 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 25, 30, 35, 40 45, 50, 55, 60, 65, or 69 amino acids within
the carboxyl-terminal sequence of 69 amino acids of the BRCA1
protein variant resulting from Deletion No. 6.
[0069] In one embodiment, the isolated polypeptides of the present
invention comprise an amino acid sequence according to one of SEQ
ID NOs:10-14 or 83-93. The isolated polypeptide of the present
invention can have at least 7, 8, 9, or more amino acids in length,
preferably 10 or more, more preferably 25 or more, and even more
preferably 50 or more amino acids.
[0070] The isolated polypeptides of the present invention
comprising an amino acid sequence according to one of SEQ ID NOs:10
and 83-87 represent novel amino acid sequence fragments of the
BRCA1 protein variant resulting from Deletion Nos. 1, 2, 3 or 4. As
illustrated in FIG. 7, and described above, these polypeptides
correspond to codon sequences in mutant mRNAs created by the direct
splicing of exon 15 to exon 18--mutant mRNAs that are transcribed
from mutant alleles of the BRCA1 gene bearing either Deletion No.
1, 2, 3, or 4--and all contain certain amino acid residues encoded
by codons representing both exons. Hence, the isolated polypeptides
of the present invention comprising an amino acid sequence
according to one of SEQ ID NOs:10-14 or 83-93 all represent novel
junction polypeptides in which amino acid residues encoded by exon
15 are joined with amino acid residues encoded by exon 18.
[0071] Similarly, the isolated polypeptides of the present
invention comprising an amino acid sequence according to one of SEQ
ID NOs:11, 88, and 89 represent novel amino acid sequence fragments
of the BRCA1 protein variant resulting from Deletion No. 5. As
shown in FIG. 8, and described above, these polypeptides correspond
to codon sequences in mutant mRNAs created by the direct splicing
of exon 14 to exon 17--mutant mRNAs that are transcribed from
mutant alleles of the BRCA1 gene bearing Deletion No. 5--and all
contain certain amino acid residues encoded by codons representing
both exons. Hence, the isolated polypeptides of the present
invention comprising an amino acid sequence according to one of SEQ
ID NOs:11, 88, and 89 all represent novel junction polypeptides in
which amino acid residues encoded by exon 14 are joined with amino
acid residues encoded by exon 17.
[0072] And, the isolated polypeptides of the present invention
comprising an amino acid sequence according to one of SEQ ID NOs:12
and 91-93 represent novel amino acid sequence fragments of the
BRCA1 protein variant resulting from Deletion No. 6. As depicted in
FIG. 9, and described above, these polypeptides correspond to codon
sequences in mutant mRNAs created by the direct splicing of exon 13
to exon 21--mutant mRNAs that are transcribed from mutant alleles
of the BRCA1 gene bearing Deletion No. 6--and all contain certain
amino acid residues encoded by codons representing both exons.
Hence, the isolated polypeptides of the present invention
comprising an amino acid sequence according to one of SEQ ID NOs:12
and 91-93 all represent novel junction polypeptides in which amino
acid residues encoded by exon 13 are joined with amino acid
residues encoded by exon 21.
[0073] Further, the isolated polypeptides of the present invention
comprising an amino acid sequence according to one of SEQ ID NOs:13
and 90 represent novel amino acid sequence fragments of a new
carboxy-terminus added to the BRCA1 protein variant resulting from
Deletion No. 5. As illustrated in FIG. 8, these polypeptides are
encoded by codons in exon 17 which are translated from a shifted
reading frame created by the the splicing of exon 14 to exon
17.
[0074] Similarly, the isolated polypeptide of the present invention
comprising an amino acid sequence according to SEQ ID NO:14
represent a novel amino acid sequence fragments of a new
carboxy-terminus added to the BRCA1 protein variant resulting from
Deletion No. 6. As illustrated in FIG. 9, this polypeptide is
encoded by codons in exons 21, 22, 23 and 24 which are translated
from a shifted reading frame created by the the splicing of exon 13
to exon 21.
[0075] In a specific embodiment, the present invention provides
isolated BRCA1 protein variants having one or more amino acid
sequences according to one of SEQ ID NOs:10-14 or 83-93. For
example, the isolated BRCA1 protein variant can be the protein
variant isolated from a patient having Deletion No. 1, 2, 3 or 4.
Alternatively, the isolated BRCA1 protein variant can be the
protein variant isolated from a patient having Deletion No. 5. Or,
the isolated BRCA1 protein variant can be the protein variant
isolated from a patient having Deletion No. 6. Preferably the
isolated BRCA1 protein variants contain at least 10, 20, 30, 40, 50
or 60 amino acid residues which encompass a BRCA1 variant amino
acid sequences provided by SEQ ID NOs:10-14 and 83-93.
Additionally, the isolated BRCA1 protein variants of the present
invention may also include other amino acid variants, such as those
created as a result of single nucleotide polymorphisms in the
coding sequence of the BRCA1 gene.
[0076] It should be understood that hybrid proteins having one of
the above mutant BRCA1 amino acid sequences and a non-BRCA1 amino
acid sequence also fall within the scope of the present
invention.
[0077] As will be apparent to a skilled artisan, the isolated
nucleic acids and polypeptides of the present invention can be
prepared using techniques generally known in the field of molecular
biology. See generally, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989.
5. Antibodies
[0078] The present invention also provides antibodies selectively
immunoreactive with an isolated BRCA1 protein variant of the
present invention. As used herein, the term "antibody" encompasses
both monoclonal and polyclonal antibodies that fall within any
antibody classes, e.g., IgG, IgM, IgA, etc. The term "antibody"
also includes antibody fragments including, but not limited to, Fab
and F(ab').sub.2, conjugates of such fragments, and single-chain
antibodies that can be made in accordance with U.S. Pat. No.
4,704,692, which is incorporated herein by reference. Specifically,
as used herein, the phrase "selectively immunoreactive with an
isolated BRCA1 protein variant of the present invention" means that
the immunoreactivity of the antibody of the present invention with
a BRCA1 protein variant of the present invention is substantially
higher than that with a BRCA1 protein heretofore known in the art
so that the binding of the antibody to the protein variant of the
present invention is readily distinguishable from the binding of
the antibody to the BRCA1 protein known in the art based on the
strength of the binding affinities. Preferably, the binding
constant differs by a magnitude of at least 2 fold, more preferably
at least 5 fold, even more preferably at least 10 fold, and most
preferably at least 100 fold.
[0079] To make the antibody, a BRCA1 protein variant of the present
invention, or a suitable fragment thereof, can be used to immunize
an animal. The BRCA1 protein variant can be made by any methods
known in the art, e.g., by recombinant expression or chemical
synthesis. Additionally, a mutant BRCA1 protein fragment having an
amino acid sequence selected from SEQ ID NOs:10-14 or 83-93 can
also be used. Preferably, the mutant BRCA1 protein fragment
consists of less than 100 amino acids, more preferably less than 50
amino acids, and even more preferably less than 25 amino acids. As
a result, a greater portion of the total antibodies may be
selectively immunoreactive with a BRCA1 protein variant of the
present invention. Techniques for immunizing animals for the
purpose of making polyclonal antibodies are generally known in the
art. See Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988. A carrier
may be necessary to increase the immunogenicity of the polypetide.
Suitable carriers known in the art include, but are not limited to,
liposome, macromolecular protein or polysaccharide, or combination
thereof. Preferably, the carrier has a molecular weight in the
range of about 10,000 to 1,000,000. The polypeptide may also be
administered along with an adjuvant, e.g., complete Freund's
adjuvant.
[0080] The antibodies of the present invention preferably are
monoclonal. Such monoclonal antibodies may be developed using any
conventional techniques known in the art. For example, the popular
hybridoma method disclosed in Kohler and Milstein, Nature,
256:495-497 (1975) is now a well-developed technique that can be
used in the present invention. See U.S. Pat. No. 4,376,110, which
is incorporated herein by reference. Essentially, B-lymphocytes
producing a polyclonal antibody against a protein variant of the
present invention can be fused with myeloma cells to generate a
library of hybridoma clones. The hybridoma population is then
screened for antigen binding specificity and also for
immunoglobulin class (isotype). In this manner, pure hybridoma
clones producing specific homogenous antibodies can be selected.
See generally, Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Press, 1988. Alternatively, other techniques
known in the art may also be used to prepare monoclonal antibodies,
which include but are not limited to the EBV hybridoma technique,
the human N-cell hybridoma technique, and the trioma technique.
[0081] In addition, antibodies selectively immunoreactive with a
protein variant of the present invention may also be recombinantly
produced. For example, cDNAs prepared by PCR amplification from
activated B-lymphocytes or hybridomas may be cloned into an
expression vector to form a cDNA library, which is then introduced
into a host cell for recombinant expression. The cDNA encoding a
specific desired protein may then be isolated from the library. The
isolated cDNA can be introduced into a suitable host cell for the
expression of the protein. Thus, recombinant techniques can be used
to recombinantly produce specific native antibodies, hybrid
antibodies capable of simultaneous reaction with more than one
antigen, chimeric antibodies (e.g., the constant and variable
regions are derived from different sources), univalent antibodies
which comprise one heavy and light chain pair coupled with the Fc
region of a third (heavy) chain, Fab proteins, and the like. See
U.S. Pat. No. 4,816,567; European Patent PublicationNo. 0088994;
Munro, Nature, 312:597 (1984); Morrison, Science, 229:1202 (1985);
Oi et al., BioTechniques, 4:214 (1986); and Wood et al., Nature,
314:446-449 (1985), all of which are incorporated herein by
reference. Antibody fragments such as Fv fragments, single-chain Fv
fragments (scFv), Fab' fragments, and F(ab').sub.2 fragments can
also be recombinantly produced by methods disclosed in, e.g., U.S.
Pat. No. 4,946,778; Skerra & Pluckthun, Science,
240:1038-1041(1988); Better et al., Science, 240:1041-1043 (1988);
and Bird, et al., Science, 242:423-426 (1988), all of which are
incorporated herein by reference.
[0082] In a preferred embodiment, the antibodies provided in
accordance with the present invention are partially or fully
humanized antibodies. For this purpose, any methods known in the
art may be used. For example, partially humanized chimeric
antibodies having V regions derived from the tumor-specific mouse
monoclonal antibody, but human C regions are disclosed in Morrison
and Oi, Adv. Immunol., 44:65-92 (1989). In addition, fully
humanized antibodies can be made using transgenic non-human
animals. For example, transgenic non-human animals such as
transgenic mice can be produced in which endogenous immunoglobulin
genes are suppressed or deleted, while heterologous antibodies are
encoded entirely by exogenous immunoglobulin genes, preferably
human immunoglobulin genes, recombinantly introduced into the
genome. See e.g., U.S. Pat. Nos. 5,530,101; 5,545,806; 6,075,181;
PCT Publication No. WO 94/02602; Green et. al., Nat. Genetics, 7:
13-21 (1994); and Lonberg et al., Nature 368: 856-859 (1994), all
of which are incorporated herein by reference. The transgenic
non-human host animal may be immunized with suitable antigens such
as a protein variant of the present invention to illicit specific
immune response thus producing humanized antibodies. In addition,
cell lines producing specific humanized antibodies can also be
derived from the immunized transgenic non-human animals. For
example, mature B-lymphocytes obtained from a transgenic animal
producing humanized antibodies can be fused to myeloma cells and
the resulting hybridoma clones may be selected for specific
humanized antibodies with desired binding specificities.
Alternatively, cDNAs may be extracted from mature B-lymphocytes and
used in establishing a library that is subsequently screened for
clones encoding humanized antibodies with desired binding
specificities. In addition, antibodies may also be produced in
transgenic plants containing recombinant nucleic acids encoding
antibodies.
[0083] In accordance with another embodiment of the present
invention, a protein microchip or microarray is provided having (1)
a BRCA1 protein variant of the present invention or a fragment
thereof comprising an amino acid sequence according to SEQ ID NOs:
10-14 or 83-93; and/or (2) an antibody selectively immunoreactive
with a BRCA1 protein variant of the present invention.
[0084] Protein microarrays are becoming increasingly important in
both proteomics research and protein-based detection and diagnosis
of diseases. The protein microarrays in accordance with the present
invention will be useful in a variety of applications including,
e.g., high throughput screening for compounds capable of modulating
the activities of a BRCA1 protein variant of the present invention.
The protein microarrays are also useful in detecting the mutant
BRCA1 proteins, and thus can be used in determining a
predisposition to cancer, particularly breast cancer and ovarian
cancer in patients.
[0085] The protein microarray of the present invention can be
prepared by a number of methods known in the art. An example of a
suitable method is that disclosed in MacBeath and Schreiber,
Science, 289:1760-1763 (2000). Essentially, glass microscope slides
are treated with an aldehyde-containing silane reagent
(SuperAldehyde Substrates purchased from TeleChem International,
Cupertino, Calif.). Nanoliter volumes of protein samples in a
phophate-buffered saline with 40% glycerol are then spotted onto
the treated slides using a high-precision contact-printing robot.
After incubation, the slides are immersed in a bovine serum albumin
(BSA)-containing buffer to quench the unreacted aldehydes and to
form a BSA layer which functions to prevent non-specific protein
binding in subsequent applications of the microchip. Alternatively,
as disclosed in MacBeath and Schreiber, proteins or protein
complexes of the present invention can be attached to a BSA-NHS
slide by covalent linkages. BSA-NHS slides are fabricated by first
attaching a molecular layer of BSA to the surface of glass slides
and then activating the BSA with N,N'-disuccinimidyl carbonate. As
a result, the amino groups of the lysine, asparate, and glutamate
residues on the BSA are activated and can form covalent urea or
amide linkages with protein samples spotted on the slides. See
MacBeath and Schreiber, Science, 289:1760-1763 (2000).
[0086] Another example of useful method for preparing the protein
microchip of the present invention is that disclosed in PCT
Publication Nos. WO 00/4389A2 and WO 00/04382, both of which are
assigned to Zyomyx and are incorporated herein by reference. First,
a substrate or chip base is covered with one or more layers of thin
organic film to eliminate any surface defects, insulate proteins
from the base materials, and to ensure a uniform protein array.
Next, a plurality of protein-capturing agents (e.g., antibodies,
peptides, etc.) are arrayed and attached to the base that is
covered with the thin film. Proteins or protein complexes can then
be bound to the capturing agents forming a protein microarray. The
protein microchips are kept in flow chambers with an aqueous
solution.
[0087] The protein microarray of the present invention can also be
made by the method disclosed in PCT Publication No. WO 99/36576
assigned to Packard Bioscience Company, which is incorporated
herein by reference. For example, a three-dimensional hydrophilic
polymer matrix, i.e., a gel, is first deposited on a solid
substrate such as a glass slide. The polymer matrix gel is capable
of expanding or contracting and contains a coupling reagent that
reacts with amine groups. Thus, proteins and protein complexes can
be contacted with the matrix gel in an expanded aqueous and porous
state to allow reactions between the amine groups on the protein or
protein complexes with the coupling reagents thus immobilizing the
proteins and protein complexes on the substrate. Thereafter, the
gel is contracted to embed the attached proteins and protein
complexes in the matrix gel.
[0088] Alternatively, the proteins and protein complexes of the
present invention can be incorporated into a commercially available
protein microchip, e.g., the Proteinchip System from Ciphergen
Biosystems Inc., Palo Alto, Calif. The Proteinchip System comprises
metal chips having a treated surface that interact with proteins.
Basically, a metal chip surface is coated with a silicon dioxide
film. The molecules of interest such as proteins and protein
complexes can then be attached covalently to the chip surface via a
silane coupling agent.
[0089] The protein microchips of the present invention can also be
prepared with other methods known in the art, e.g., those disclosed
in U.S. Pat. Nos. 6,087,102, 6,139,831, 6,087,103; PCT
PublicationNos. WO 99/60156, WO 99/39210, WO 00/54046, WO 00/53625,
WO 99/51773, WO 99/35289, WO 97/42507, WO 01/01142, WO 00/63694, WO
00/61806, WO 99/61148, WO 99/40434, all of which are incorporated
herein by reference.
6. Genotyping
[0090] In another aspect of the present invention, methods are
provided for predicting, in an individual, the likelihood of
developing cancer. As described above, the large deletions in BRCA1
genes identified in accordance with the present invention are
deleterious and predispose individuals having the deletions to
cancer, particularly breast cancer and ovarian cancer. Thus, by
detecting, in an individual, the presence or absence of one or more
of the BRCA1 variants of the present invention, one can reasonably
predict a predisposition to cancer, e.g., breast cancer and ovarian
cancer.
[0091] Numerous techniques for detecting genetic variants are known
in the art and can all be used for the method of this invention.
The techniques can be nucleic acid-based or protein-based. In
either case, the techniques used must be sufficiently sensitive so
as to accurately detect the nucleotide or amino acid variations.
Very often, a probe is utilized which is labeled with a detectable
marker. Unless otherwise specified in a particular technique
described below, any suitable marker known in the art can be used,
including but not limited to, radioactive isotopes, fluorescent
compounds, biotin which is detectable using strepavidin, enzymes
(e.g., alkaline phosphatase), substrates of an enzyme, ligands and
antibodies, etc. See Jablonski et al., Nucleic Acids Res.,
14:6115-6128 (1986); Nguyen et al., Biotechniques, 13:116-123
(1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977).
[0092] In a DNA-based detection method, a target DNA sample, i.e.,
a sample containing BRCA1 gene sequence should be obtained from the
individual to be tested. Any tissue or cell sample containing the
BRCA1 genomic DNA or mRNA, or a portion thereof, can be used.
Preferably, a tissue sample containing cell nuclei and thus genomic
DNA can be obtained from the individual. Blood samples can also be
useful, except that only white blood cells and other lymphocytes
have cell nuclei, while red blood cells are enucleated and contain
mRNA. Nevertheless, mRNA is also useful as it can be analyzed for
the presence of nucleotide variants in its sequence or serve as
template for cDNA synthesis. The tissue or cell samples can be
analyzed directly without much processing. Alternatively, nucleic
acids including the target BRCA1 nucleic acids can be extracted,
purified, or amplified before they are subject to the various
detecting procedures discussed below. Other than tissue or cell
samples, cDNAs or genomic DNAs from a cDNA or genomic DNA library
constructed using a tissue or cell sample obtained from the
individual to be tested are also useful.
[0093] To determine the presence or absence of the deletion
mutations identified in the present invention, one technique is
simply sequencing the target BRCA1 genomic DNA or cDNA,
particularly the region spanning the deletion locus to be detected.
Various sequencing techniques are generally known and widely used
in the art including the Sanger method and the Gilbert chemical
method. The newly developed pyrosequencing method monitors DNA
synthesis in real time using a luminometric detection system.
Pyrosequencing has been shown to be effective in analyzing genetic
polymorphisms such as single-nucleotide polymorphisms and can also
be used in the present invention. See Nordstrom et al., Biotechnol.
Appl. Biochem., 31(2): 107-112 (2000); Ahmadian et al., Anal.
Biochem., 280:103-110 (2000). For example, sequencing primers can
be designed based on either mutant or wild-type BRCA1 gene intronic
or exonic sequences such that the primers have the nucleotide
sequence adjacent to a deletion locus identified in accordance with
the present invention. In another example, PCR primers are designed
based on either mutant or wild-type BRCA1 gene intronic or exonic
sequences such that PCR amplification generates a BRCA1 DNA
fragment spanning the deletion locus. As the large deletions
identified in accordance with the present invention alter the size
of the BRCA1 genomic DNA or cDNA, the presence or absence of a
deletion mutation according to the present invention can be
determined based on the molecular weight of the PCR amplification
products generated using the PCR primers. Optionally, DNA
sequencing is then performed on the amplified fragment to determine
the nucleotide sequence of the suspect region.
[0094] Alternatively, the restriction fragment length polymorphism
(RFLP) method may also prove to be a useful technique. In
particular, the large deletions identified in accordance with the
present invention result in the elimination and creation of
restriction enzyme recognition sites. Digestion of the mutant BRCA1
genomic DNAs or cDNAs with appropriate restriction enzyme(s) will
generate restriction fragment length patterns distinct from those
generated from wild-type BRCA1 genomic DNA or cDNA. Thus, the large
deletions in BRCA1 of the present invention can be detected by
RFLP. The application of the RFLP techniques known in the art to
the present invention will be apparent to skilled artisans.
[0095] Similarly, genomic DNA can be obtained from a patient sample
and digested by appropriate restriction enzyme(s). Southern blot
can be performed using a probe having a wild-type BRCA1 sequence
that is missing from one or more of the BRCA1 genetic variants of
the present invention. Alternatively, probes specific to the mutant
BRCA1 nucleic acids of the present invention can also be used.
[0096] The presence or absence of a BRCA1 deletion mutation
identified according to the present invention can also be detected
using the amplification refractory mutation system (ARMS)
technique. See e.g., European Patent No. 0,332,435; Newton et al.,
Nucleic Acids Res., 17:2503-2515 (1989); Fox et al., Br. J. Cancer,
77:1267-1274 (1998); Robertson et al., Eur. Respir. J., 12:477-482
(1998). In the ARMS method, a primer is synthesized matching the
nucleotide sequence immediately 5' upstream from the locus being
tested except that the 3'-end nucleotide which corresponds to the
nucleotide at the locus is a predetermined nucleotide. For example,
the 3'-end nucleotide can be the same as that in the mutated locus.
The primer can be of any suitable length so long as it hybridizes
to the target DNA under stringent conditions only when its 3'-end
nucleotide matches the nucleotide at the locus being tested.
Preferably the primer has at least 12 nucleotides, more preferably
from about 18 to 50 nucleotides. If the individual tested has a
mutation at the locus and the nucleotide therein matches the 3'-end
nucleotide of the primer, then the primer can be further extended
upon hybridizing to the target DNA template, and the primer can
initiate a PCR amplification reaction in conjunction with another
suitable PCR primer. In contrast, if the nucleotide at the locus is
of wild type, then primer extension cannot be achieved. Various
forms of ARMS techniques developed in the past few years can be
used. See e.g., Gibson et al., Clin. Chem. 43:1336-1341 (1997).
Thus, for example, primers having a sequence selected from SEQ ID
NOs:42-47, 53-63, and 70-79 can all be useful in this
technique.
[0097] Similar to the ARMS technique is the mini sequencing or
single nucleotide primer extension method, which is based on the
incorporation of a single nucleotide. An oligonucleotide primer
matching the nucleotide sequence immediately 5' to the locus being
tested is hybridized to the target DNA or mRNA in the presence of
labeled dideoxyribonucleotides. A labeled nucleotide is
incorporated or linked to the primer only when the
dideoxyribonucleotides matches the nucleotide at the variant locus
being detected. Thus, the identity of the nucleotide at the variant
locus can be revealed based on the detection label attached to the
incorporated dideoxyribonucleotides. See Syvanen et al., Genomics,
8:684-692 (1990); Shumaker et al., Hum. Mutat., 7:346-354 (1996);
Chen et al., Genome Res., 10:549-547 (2000).
[0098] Another set of techniques useful in the present invention is
the so-called "oligonucleotide ligation assay" (OLA) in which
differentiation between a wild-type locus and a mutation is based
on the ability of two oligonucleotides to anneal adjacent to each
other on the target DNA molecule allowing the two oligonucleotides
joined together by a DNA ligase. See Landergren et al., Science,
241:1077-1080 (1988); Chen et al, Genome Res., 8:549-556 (1998);
lannone et al., Cytometry, 39:131-140 (2000). Thus, for example, to
detect a mutation at a particular locus in the BRCA1 gene, two
oligonucleotides can be synthesized, one having the BRCA1 sequence
just 5' upstream from the locus with its 3' end nucleotide being
identical to the nucleotide in the mutant locus of the BRCA1 gene,
the other having a nucleotide sequence matching the BRCA1 sequence
immediately 3' downstream from the locus in the BRCA1 gene. The
oligonucleotides can be labeled for the purpose of detection. Upon
hybridizing to the target BRCA1 gene under a stringent condition,
the two oligonucleotides are subjected to ligation in the presence
of a suitable ligase. The ligation of the two oligonucleotides
would indicate that the target DNA has a nucleotide variant at the
locus being detected. Thus, for example, oligonucleotides can be
readily designed based on the deletion loci present in mutant BRCA1
genomic DNA or cDNA sequences that result from Deletion Nos. 1,2,
3,4, 5, or6.
[0099] Detection of the genetic variations identified in accordance
with the present invention can also be accomplished by a variety of
hybridization-based approaches. Allele-specific oligonucleotides
are useful. See Conner et al., Proc. Natl. Acad. Sci. USA,
80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA,
86:6230-6234 (1989). Oligonucleotide probes hybridizing
specifically to a BRCA1 gene allele having a particular gene
variant at a particular locus but not to other alleles can be
designed by methods known in the art. The probes can have a length
of, e.g., from 10 to about 50 nucleotide bases. The target BRCA1
genomic DNA or cDNA and the oligonucleotide probe can be contacted
with each other under conditions sufficiently stringent such that
the genetic variant can be distinguished from the wild-type BRCA1
gene based on the presence or absence of hybridization. The probe
can be labeled to provide detection signals. Alternatively, the
allele-specific oligonucleotide probe can be used as a PCR
amplification primer in an "allele-specific PCR" and the presence
or absence of a PCR product of the expected length would indicate
the presence or absence of a particular genetic variant. In this
respect, oligos having a sequence selected from SEQ ID NOs:7-9,
15-79 and 81 can be used.
[0100] Another useful technique that is gaining increased
popularity is mass spectrometry. See Graber et al., Curr. Opin.
Biotechnol., 9:14-18 (1998). For example, in the primer oligo base
extension (PROBE.TM.) method, a target nucleic acid is immobilized
to a solid-phase support. A primer is annealed to the target
immediately 5' upstream from the locus to be analyzed. Primer
extension is carried out in the presence of a selected mixture of
deoxyribonucelotides and dideoxyribonucleotides. The resulting
mixture of newly extended primers is then analyzed by MALDI-TOF.
See e.g., Monforte et al., Nat. Med., 3:360-362 (1997). In another
example, primers can be designed based on either mutant or
wild-type BRCA1 gene intronic or exonic sequences such that the
primers have the nucleotide sequences adjacent to and flanking a
deletion locus identified in accordance with the present invention.
PCR amplification on a patient sample is carried out using the
primers. Mass spectrometry is then performed on the PCR
product.
[0101] In addition, the microchip or microarray technologies are
also applicable to the detection method of the present invention.
Essentially, in microchips, a large number of different
oligonucleotide probes are immobilized in an array on a substrate
or carrier, e.g., a silicon chip or glass slide. Target nucleic
acid sequences to be analyzed can be contacted with the immobilized
oligonucleotide probes on the microchip. See Lipshutz et al.,
Biotechniques, 19:442-447 (1995); Chee et al., Science, 274:610-614
(1996); Kozal et al., Nat. Med. 2:753-759 (1996); Hacia et al.,
Nat. Genet., 14:441-447 (1996); Saiki et al., Proc. Natl. Acad.
Sci. USA, 86:6230-6234 (1989); Gingeras et al., Genome Res.,
8:435-448 (1998). Alternatively, the multiple target nucleic acid
sequences to be studied are fixed onto a substrate and an array of
probes is contacted with the immobilized target sequences. See
Drmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous
microchip technologies have been developed incorporating one or
more of the above described techniques for detecting mutations
particularly SNPs. The microchip technologies combined with
computerized analysis tools allow fast screening in a large scale.
The adaptation of the microchip technologies to the present
invention will be apparent to a person of skill in the art apprised
of the present disclosure. See, e.g., U.S. Pat. No. 5,925,525 to
Fodor et al. Wilgenbus et al., J. Mol. Med., 77:761-786 (1999);
Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et
al., Nat. Genet., 14:441-447 (1996); Shoemaker et al., Nat. Genet.,
14:450-456 (1996); DeRisi et al., Nat. Genet., 14:457-460 (1996);
Chee et al., Nat. Genet., 14:610-614 (1996); Lockhart et al., Nat.
Genet., 14:675-680 (1996); Drobyshev et al., Gene, 188:45-52
(1997).
[0102] As is apparent from the above survey of the suitable
detection techniques, it may or may not be necessary to amplify the
target DNA, i.e., the BRCA1 genomic DNA or cDNA sequence to
increase the number of target DNA molecules, depending on the
detection techniques used. For example, most PCR-based techniques
combine the amplification of a portion of the target and the
detection of mutations. PCR amplification is well known in the art
and is disclosed in U.S. Pat. Nos. 4,683,195 and 4,800,159, both of
which are incorporated herein by reference. For non-PCR-based
detection techniques, if necessary, the amplification can be
achieved by, e.g., in vivo plasmid multiplication, or by purifying
the target DNA from a large amount of tissue or cell samples. See
generally, Sambrook et al., Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., 1989. However, even with scarce samples, many sensitive
techniques have been developed in which genetic variations can be
detected without having to amplify the target DNA in the sample.
For example, techniques have been developed that amplify the signal
as opposed to the target DNA by, e.g., employing branched DNA or
dendrimers that can hybridize to the target DNA. The branched or
dendrimer DNAs provide multiple hybridization sites for
hybridization probes to attach thereto thus amplifying the
detection signals. See Detmer et al., J. Clin. Microbiol.,
34:901-907 (1996); Collins et al., Nucleic Acids Res., 25:2979-2984
(1997); Horn et al., Nucleic Acids Res., 25:4835-4841 (1997); Horn
et al., Nucleic Acids Res., 25:4842-4849 (1997); Nilsen et al., J.
Theor. Biol., 187:273-284 (1997).
[0103] A number of other techniques that avoid amplification all
together include, e.g., surface-enhanced resonance Raman scattering
(SERRS), fluorescence correlation spectroscopy, and single-molecule
electrophoresis. In SERRS, a chromophore-nucleic acid conjugate is
absorbed onto colloidal silver and is irradiated with laser light
at a resonant frequency of the chromophore. See Graham et al.,
Anal. Chem., 69:4703-4707 (1997). The fluorescence correlation
spectroscopy is based on the spatio-temporal correlations between
fluctuating light signals and trapping single molecules in an
electric field. See Eigen et al., Proc. Natl. Acad. Sci. USA,
91:5740-5747 (1994). In single-molecule electrophoresis, the
electrophoretic velocity of a fluorescently tagged nucleic acid is
determined by measuring the time required for the molecule to
travel a predetermined distance between two laser beams. See Castro
et al., Anal. Chem., 67:3181-3186 (1995). Additionally, the Invader
assay and the rolling circle amplification technique may also be
used. See e.g. Lyamichev et al., Nat. BiotechnoL, 17:292-296
(1999); Lizardi et al., Nature Genetics, 19:225-232 (1998).
[0104] In addition, the allele-specific oligonucleotides (ASO) can
also be used in in situ hybridization using tissues or cells as
samples. The oligonucleotide probes which can hybridize
differentially with the wild-type gene sequence or the gene
sequence harboring a mutation may be labeled with radioactive
isotopes, fluorescence, or other detectable markers. In situ
hybridization techniques are well known in the art and their
adaptation to the present invention for detecting the presence or
absence of a genetic variant in the BRCA1 gene of a particular
individual should be apparent to a skilled artisan apprised of this
disclosure.
[0105] Protein-based detection techniques may also prove to be
useful, especially when the genetic variant causes amino acid
substitutions or deletions or insertions that affect the protein
primary, secondary or tertiary structure. To detect the amino acid
variations, protein sequencing techniques may be used. For example,
a BRCA1 protein or fragment thereof can be synthesized by
recombinant expression using a BRCA1 DNA fragment isolated from an
individual to be tested. Preferably, a BRCA1 cDNA fragment of no
more than 100 to 150 base pairs encompassing the polymorphic locus
to be determined is used. The amino acid sequence of the peptide
can then be determined by conventional protein sequencing methods.
Alternatively, the recently developed HPLC-microscopy tandem mass
spectrometry technique can be used for determining the amino acid
sequence variations. In this technique, proteolytic digestion is
performed on a protein, and the resulting peptide mixture is
separated by reversed-phase chromatographic separation. Tandem mass
spectrometry is then performed and the data collected therefrom is
analyzed. See Gatlin et al., Anal. Chem., 72:757-763 (2000).
[0106] Other useful protein-based detection techniques include
immunoaffinity assays based on antibodies selectively
immunoreactive with mutant BRCA1 proteins according to the present
invention. Such antibodies may react specifically with epitopes
comprising the polypeptide fragments spanning the junction regions
of BRCA1 proteins that correspond to deletion loci in the mutant
BRCA1 mRNAs transcribed from the mutant BRCA1 genomic DNAs of the
present invention (i.e., the deletion loci of variant BRCA1
polypeptides produced as a result of Deletion Nos. 1-6.
Alternatively, such antibodies may react specifically with epitopes
present on the novel carboxyl-terminal polypeptides of the BRCA1
protein variants resulting from Deletion Nos. 5 and 6. Methods for
producing such antibodies are described above in detail. Antibodies
can be used to immunoprecipitate specific proteins from solution
samples or to immunoblot proteins separated by, e.g.,
polyacrylamide gels. Immunocytochemical methods can also be used in
detecting specific protein polymorphisms in tissues or cells. Other
well known antibody-based techniques can also be used including,
e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays
(IEMA), including sandwich assays using monoclonal or polyclonal
antibodies. See e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530, both
of which are incorporated herein by reference.
[0107] It is noted that heterozygotes of the BRCA1 genetic variants
of the present invention are predisposed to cancer such as breast
cancer and ovarian cancer. That is, as long as an individual has
one chromosome containing a BRCA1 genetic variant of the present
invention, there is an increased likelihood of breast cancer and/or
ovarian cancer in the individual.
[0108] Thus, various techniques can be used in genotyping a BRCA1
gene of an individual to determine, in the individual, the presence
or absence of a BRCA1 genetic variant selected from the group
consisting of Deletion Nos. 1 to 6. Typically, once the presence or
absence of a BRCA1 genetic variant of the present invention is
determined, the result can be cast in a communicable form that can
be communicated to the individual patient. Such a form can vary and
can be tangible or intangible. The result with regard to the
presence or absence of a BRCA1 genetic variant of the present
invention in the individual tested can be embodied in descriptive
statements, diagrams, photographs, charts, images or any other
visual forms. For example, images of gel electrophoresis of PCR
products can be used in explaining the results. Diagrams showing
where a deletion occurs in an individual's BRCA1 gene are also
useful in communicating the test results. The statements and visual
forms can be recorded on a tangible media such as papers, computer
readable media such as floppy disks, compact disks, etc., or on an
intangible media, e.g., an electronic media in the form of e-mail,
or on a preferably secured website on the internet or an intranet.
In addition, the result with regard to the presence or absence of a
BRCA1 genetic variant of the present invention in the individual
tested can also be recorded in a sound form and transmitted through
any suitable media, e.g., analog or digital cable lines, fiber
optic cables, etc., via telephone, facsimile, wireless mobile
phone, internet phone and the like.
[0109] The present invention also provides kits for practicing the
genotyping methods described above. The kits may include a carrier
for the various components of the kit. The carrier can be a
container or support, in the form of, e.g., bag, box, tube, rack,
and is optionally compartmentalized. The carrier may define an
enclosed confinement for safety purposes during shipment and
storage. The kit also includes various components useful in
detecting nucleotide or amino acid variants discovered in
accordance with the present invention using the above-discussed
detection techniques.
[0110] In one embodiment, the detection kit includes one or more
oligonucleotides useful in detecting the genetic variants in BRCA1
gene sequence in accordance with the present invention. Preferably,
the oligonucleotides are designed such that they are specific to a
BRCA1 nucleic acid variant of the present invention under stringent
conditions. That is, the oligonucleotides should be designed such
that it can be used in distinguishing one genetic variant from
another at a particular locus under predetermined stringent
hybridization conditions. Examples of such oligonucleotides include
nucleic acids having a sequence selected from SEQ ID NOs:7-9, 15-79
and 81. Thus, the oligonucleotides can be used in
mutation-detecting techniques such as allele-specific
oligonucleotides (ASO), allele-specific PCR, TaqMan-based
quantitative PCR, chemiluminescence-based techniques, molecular
beacons, and improvements or derivatives thereof, e.g., microchip
technologies.
[0111] In another embodiment of this invention, the kit includes
one or more oligonucleotides suitable for use in detecting
techniques such as ARMS, oligonucleotide ligation assay (OLA), and
the like. For example, the oligonucleotides in this embodiment
include a BRCA1 gene sequence immediately 5' upstream from a
deletion locus to be analyzed. The 3' end nucleotide of the oligo
is the first nucleotide on the 3' side of the deletion locus.
Examples of suitable oligos include, but are not limited to, those
consisting of a sequence selected from SEQ ID NOs:1, 3, 5, 6,
42-47, 53-63, and 73-79.
[0112] The oligonucleotides in the detection kit can be labeled
with any suitable detection marker including but not limited to,
radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline
phosphatase), enzyme substrates, ligands and antibodies, etc. See
Jablonski et al., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen
et al., Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol.
BioL, 113:237-251 (1977). Alternatively, the oligonucleotides
included in the kit are not labeled, and instead, one or more
markers are provided in the kit so that users may label the
oligonucleotides at the time of use.
[0113] In another embodiment of the invention, the detection kit
contains one or more antibodies selectively immunoreactive with a
BRCA1 protein variant of the present invention. Methods for
producing and using such antibodies have been described above in
detail.
[0114] Various other components useful in the detection techniques
may also be included in the detection kit of this invention.
Examples of such components include, but are not limited to, DNA
polymerase, reverse transcriptase, deoxyribonucleotides,
dideoxyribonucleotides other primers suitable for the amplification
of a target DNA or mRNA sequence, RNase A, mutS protein, and the
like. In addition, the detection kit preferably includes
instructions on using the kit for detecting genetic variants in
BRCA1 gene sequences, particularly the genetic variants of the
present invention.
7. Screening Assays
[0115] The present invention further provides a method for
identifying compounds capable of modulating, preferably enhancing
the activities of a BRCA1 protein variant of the present invention.
Such compounds may prove to be useful in treating or preventing
symptoms associated with decreased BRCA1 protein activities, e.g.,
cancer. For this purpose, a mutant BRCA1 protein or fragment
thereof containing a particular deletion in accordance with the
present invention can be used in any of a variety of drug screening
techniques. Drug screening can be performed as described herein or
using well known techniques, such as those described in U.S. Pat.
Nos. 5,800,998 and 5,891,628, both of which are incorporated herein
by reference. The candidate therapeutic compounds may include, but
are not limited to proteins, small peptides, nucleic acids, and
analogs thereof. Preferably, the compounds are small organic
molecules having a molecular weight of no greater than 10,000
dalton, more preferably less than 5,000 dalton.
[0116] In one embodiment of the present invention, the method is
primarily based on binding affinities to screen for compounds
capable of interacting with or binding to a BRCA1 protein variant.
Compounds to be screened may be peptides or derivatives or mimetics
thereof, or non-peptide small molecules. Conveniently, commercially
available combinatorial libraries of compounds or phage display
libraries displaying random peptides are used.
[0117] Various screening techniques known in the art may be used in
the present invention. The BRCA1 protein variants (drug target) can
be prepared by any suitable methods, e.g., by recombinant
expression and purification. The polypeptide or fragment thereof
may be free in solution but preferably is immobilized on a solid
support, e.g., in a protein microchip, or on a cell surface.
Various techniques for immobilizing proteins on a solid support are
known in the art. For example, PCT Publication WO 84/03564
discloses synthesizing a large numbers of small peptide test
compounds on a solid substrate, such as plastic pins or other
surfaces. Alternatively, purified mutant BRCA1 protein, or
fragments thereof, can be coated directly onto plates such as
multi-well plates. Non-neutralizing antibodies, i.e., antibodies
capable binding to the BRCA1 protein, or fragments thereof, that do
not substantially affect its biological activities may also be used
for immobilizing the BRCA1 protein, or fragments thereof, on a
solid support.
[0118] To affect the screening, test compounds can be contacted
with the immobilized BRCA1 protein, or fragments thereof, to allow
binding to occur and complexes to form under standard binding
conditions. Either the drug target or test compounds are labeled
with a detectable marker using well known labeling techniques. To
identify binding compounds, one may measure the steady state or
end-point formation of the drug target-test compound complexes, or
kinetics for the formation thereof.
[0119] Alternatively, a known ligand capable of binding to the drug
target can be used in competitive binding assays. Complexes between
the known ligand and the drug target can be formed and then
contacted with test compounds. The ability of a test compound to
interfere with the interaction between the drug target and the
known ligand is measured using known techniques. One exemplary
ligand is an antibody capable of specifically binding the drug
target. Particularly, such an antibody is especially useful for
identifying peptides that share one or more antigenic determinants
of the BRCA1 protein, or fragments thereof, and preferably
antigenic determinants specific to the BRCA1 protein variants of
the present invention.
[0120] In another embodiment, a yeast two-hybrid system may be
employed to screen for proteins or small peptides capable of
interacting with a BRCA1 protein variant. For example, a battery of
fusion proteins each containing a random small peptide fused to
e.g., Gal 4 activation domain, can be co-expressed in yeast cells
with a fusion protein having the Gal 4 binding domain fused to a
BRCA1 protein variant. In this manner, small peptides capable of
interacting with the BRCA1 protein variant can be identified.
Alternatively, compounds can also be tested in a yeast two-hybrid
system to determine their ability to inhibit the interaction
between the BRCA1 protein variant and a known protein, which is
known to interact with the BRCA1 protein or polypeptide or fragment
thereof. Again, one example of such proteins is an antibody
specifically against the BRCA1 protein variant. Yeast two-hybrid
systems and use thereof are generally known in the art and are
disclosed in, e.g., Bartel et al., in: Cellular Interactions in
Development: A Practical Approach, Oxford University Press, pp.
153-179 (1993); Fields and Song, Nature, 340:245-246 (1989);
Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793
(1992); Lee et al., Science, 268:836-844 (1995); and U.S. Pat. Nos.
6,057,101, 6,051,381, and 5,525,490, all of which are incorporated
herein by reference.
[0121] The compounds thus identified can be further tested for
activities, e.g., in stimulating the mutant BRCA1's biological
activities, e.g., in DNA repair and in interacting with its known
interacting partner proteins.
[0122] Once an effective compound is identified, structural analogs
or mimetics thereof can be produced based on rational drug design
with the aim of improving drug efficacy and stability, and reducing
side effects. Methods known in the art for rational drug design can
be used in the present invention. See, e.g., Hodgson et al.,
Bio/Technology, 9:19-21 (1991); U.S. Pat. Nos. 5,800,998 and
5,891,628, all of which are incorporated herein by reference. An
example of rational drug design is the development of HIV protease
inhibitors. See Erickson et al., Science, 249:527-533 (1990).
Preferably, rational drug design is based on one or more compounds
selectively binding to a mutant BRCA1 protein or a fragment
thereof.
[0123] In one embodiment, the three-dimensional structure of, e.g.,
a BRCA1 protein variant, is determined by biophysical techniques
such as X-ray crystallography, computer modeling, or both.
Desirably, the structure of the complex between an effective
compound and the mutant BRCA1 protein is determined, and the
structural relationship between the compound and the protein is
elucidated. In this manner, the moieties and the three-dimensional
structure of the selected compound, i.e., lead compound, critical
to the its binding to the mutant BRCA1 protein are revealed.
Medicinal chemists can then design analog compounds having similar
moieties and structures. In addition, the three-dimensional
structure of wild-type BRCA1 protein is also desirably deciphered
and compared to that of a mutant BRCA1 protein. This will aid in
designing compounds selectively interacting with the mutant BRCA1
protein.
[0124] In another approach, a selected peptide compound capable of
binding the BRCA1 protein variant can be analyzed by alanine
scanning mutagenesis. See Wells, et al., Methods Enzymol.,
202:301-306 (1991). In this technique, an amino acid residue of the
peptide is replaced by Alanine, and its effect on the peptide's
binding affinity to the mutant BRCA1 protein is tested. Amino acid
residues of the selected peptide are analyzed in this manner to
determine the domains or residues of the peptide important to its
binding to mutant BRCA1 protein. These residues or domains
constituting the active region of the compound are known as its
"pharmacophore". This information can be very helpful in rationally
designing improved compounds.
[0125] Once the pharmacophore has been elucidated, a structural
model can be established by a modeling process which may include
analyzing the physical properties of the pharmacophore such as
stereochemistry, charge, bonding, and size using data from a range
of sources, e.g., NMR analysis, x-ray diffraction data, alanine
scanning, and spectroscopic techniques and the like. Various
techniques including computational analysis, similarity mapping and
the like can all be used in this modeling process. See e.g., Perry
et al., in OSAR: Quantitative Structure-Activity Relationships in
Drug Design, pp.189-193, Alan R. Liss, Inc., 1989; Rotivinen et
al., Acta Pharmaceutical Fennica, 97:159-166 (1988); Lewis et al.,
Proc. R. Soc. Lond., 236:125-140 (1989); McKinaly et al., Annu.
Rev. Pharmacol. Toxiciol., 29:111-122 (1989). Commercial molecular
modeling systems available from Polygen Corporation, Waltham,
Mass., include the CHARMm program, which performs the energy
minimization and molecular dynamics functions, and QUANTA program
which performs the construction, graphic modeling and analysis of
molecular structure. Such programs allow interactive construction,
visualization and modification of molecules. Other computer
modeling programs are also available from BioDesign, Inc.
(Pasadena, Calif.), Hypercube, Inc. (Cambridge, Ontario), and
Allelix, Inc. (Mississauga, Ontario, Canada).
[0126] A template can be formed based on the established model.
Various compounds can then be designed by linking various chemical
groups or moieties to the template. Various moieties of the
template can also be replaced. In addition, in the case of a
peptide lead compound, the peptide or mimetics thereof can be
cyclized, e.g., by linking the N-terminus and C-terminus together,
to increase its stability. These rationally designed compounds are
further tested. In this manner, pharmacologically acceptable and
stable compounds with improved efficacy and reduced side effect can
be developed.
8. Cell and Animal Models
[0127] In yet another aspect of the present invention, a cell line
and a transgenic animal carrying a BRCA1 nucleic acid variant in
accordance with the present invention are provided. The cell line
and transgenic animal can be used as model systems for studying
cancers and testing various therapeutic approaches in treating
cancers, e.g., breast cancer and ovarian cancer.
[0128] To establish the cell line, cells expressing the mutant
BRCA1 protein can be isolated from an individual carrying the
genetic variants. The primary cells can be transformed or
immortalized using techniques known in the art. Alternatively,
normal cells expressing a wild-type BRCA1 protein or other type of
genetic variants can be manipulated to replace the entire
endogenous BRCA1 gene with a BRCA1 nucleic acid variant of the
present invention, or simply to introduce mutations into the
endogenous BRCA1 gene. The genetically engineered cells can further
be immortalized.
[0129] A more valuable model system is a transgenic animal. A
transgenic animal can be made by replacing its endogenous BRCA1
gene ortholog with a human BRCA1 nucleic acid variant of the
present invention. Alternatively, deletions can be introduced into
the endogenous animal BRCA1 gene ortholog to simulate the BRCA1
alleles discovered in accordance with the present invention.
Techniques for making such transgenic animals are well known and
are described in, e.g., Capecchi, et al., Science, 244:1288 (1989);
Hasty et al., Nature, 350:243 (1991); Shinkai et al., Cell, 68:855
(1992); Mombaerts et al., Cell, 68:869 (1992); Philpott et al.,
Science, 256:1448 (1992); Snouwaert et al., Science, 257:1083
(1992); Donehower et al., Nature, 356:215 (1992); Hogan et al.,
Manipulating the Mouse Embryo; A Laboratory Manual, 2.sup.nd
edition, Cold Spring Harbor Laboratory Press, 1994; and U.S. Pat.
Nos. 5,800,998, 5,891,628, and 4,873,191, all of which are
incorporated herein by reference.
[0130] The cell line and transgenic animal are valuable tools for
studying the mutant BRCA1 genes, and in particular for testing in
vivo the compounds identified in the screening method of this
invention and other therapeutic approaches as discussed above. As
is well known in the art, studying drug candidates in a suitable
animal model before advancing them into human clinical trials is
particularly important because not only can efficacy of the drug
candidates can be confirmed in the model animal, but the toxicology
profiles, side effects, and dosage ranges can also be determined.
Such information is then used to guide human clinical trials.
[0131] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
[0132] All publications mentioned in the specification are
indicative of the level of those skilled in the art to which this
invention pertains. All publications and patent applications are
herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
93142DNAHomo sapiens 1tctcggcctc ccaaagtgct gggattacag gtgtgagcca
tc 42213DNAHomo sapiens 2tcaaaaaaaa ata 13342DNAHomo sapiens
3tgtaatccca gcactttggg aggccgaggc gggcagatca ca 42410DNAHomo
sapiens 4ttcagtgacc 10548DNAHomo sapiens 5gtaatcccag ctactcagga
ggctgaggca ggagaattgc ttgaaccc 48656DNAHomo sapiens 6gggaggctaa
ggcaggagaa tcgcttgaac ctggaggcag aggttgcagt gagccc 5678DNAHomo
sapiens 7ctagatgc 888DNAHomo sapiens 8aaagatgc 898DNAHomo sapiens
9aaagatct 8107PRTHomo sapiens 10Gln Asp Leu Asp Ala Glu Phe1
5117PRTHomo sapiens 11Gly Val Glu Arg Cys Ser Cys1 5127PRTHomo
sapiens 12Ser Glu Lys Asp Leu Gln Gly1 51313PRTHomo sapiens 13Cys
Ser Cys Thr Ser Gly Pro Glu Asn Thr Thr Ser Leu1 5 101469PRTHomo
sapiens 14Asp Leu Gln Gly Ala Arg Asn Leu Leu Leu Trp Ala Leu His
Gln His1 5 10 15Ala His Arg Ser Thr Gly Met Asp Gly Thr Ala Val Trp
Cys Phe Cys 20 25 30Gly Glu Gly Ala Phe Ile Ile His Pro Trp His Arg
Cys Pro Pro Asn 35 40 45Cys Gly Cys Ala Ala Arg Cys Leu Asp Arg Gly
Gln Trp Leu Pro Cys 50 55 60Asn Trp Ala Asp Val651549DNAHomo
sapiens 15cccgtctcgg cctcccaaag tgctgggatt acaggtgtga gccatcgcg
491660DNAHomo sapiens 16atccacccgt ctcggcctcc caaagtgctg ggattacagg
tgtgagccat cgcgcctagc 601779DNAHomo sapiens 17tgacctcgtg atccacccgt
ctcggcctcc caaagtgctg ggattacagg tgtgagccat 60cgcgcctagc ctatgatga
791815DNAHomo sapiens 18ctcaaaaaaa aatac 151920DNAHomo sapiens
19catctcaaaa aaaaatacat 202026DNAHomo sapiens 20actccatctc
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acataaattg 302239DNAHomo sapiens 22actccatctc aaaaaaaaat acataaattg
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gaggccgagg cgggcagatc acaa 442549DNAHomo sapiens 25gcctgtaatc
ccagcacttt gggaggccga ggcgggcaga tcacaaggt 492660DNAHomo sapiens
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602780DNAHomo sapiens 27gtggtgggtg gcttacgcct gtaatcccag cactttggga
ggccgaggcg ggcagatcac 60aaggtcagga gttcgagacc 802817DNAHomo sapiens
28gaggtttcag tgaccca 172919DNAHomo sapiens 29cagaggtttc agtgaccca
193026DNAHomo sapiens 30gcagaggttt cagtgaccca agatcg 263130DNAHomo
sapiens 31gcagaggttt cagtgaccca agatcgcacc 303245DNAHomo sapiens
32aacctgggag gcagaggttt cagtgaccca agatcgcacc attgc 453350DNAHomo
sapiens 33tgtaatccca gctactcagg aggctgaggc aggagaattg cttgaacccg
503456DNAHomo sapiens 34tgcctgtaat cccagctact caggaggctg aggcaggaga
attgcttgaa cccggg 563566DNAHomo sapiens 35caggtgcctg taatcccagc
tactcaggag gctgaggcag gagaattgct tgaacccggg 60aggcgg 663686DNAHomo
sapiens 36ggcgtggtgg caggtgcctg taatcccagc tactcaggag gctgaggcag
gagaattgct 60tgaacccggg aggcggaggt tgcggt 863758DNAHomo sapiens
37tgggaggcta aggcaggaga atcgcttgaa cctggaggca gaggttgcag tgagccca
583864DNAHomo sapiens 38tcttgggagg ctaaggcagg agaatcgctt gaacctggag
gcagaggttg cagtgagccc 60agat 643974DNAHomo sapiens 39catcttcttg
ggaggctaag gcaggagaat cgcttgaacc tggaggcaga ggttgcagtg 60agcccagatc
gcac 744094DNAHomo sapiens 40cctataatcc catcttcttg ggaggctaag
gcaggagaat cgcttgaacc tggaggcaga 60ggttgcagtg agcccagatc gcaccactac
gctc 9441154DNAHomo sapiens 41caaaaattag ccaaggggtg gtggtgggca
cctataatcc catcttcttg ggaggctaag 60gcaggagaat cgcttgaacc tggaggcaga
ggttgcagtg agcccagatc gcaccactac 120gctccagcct aggtgacaga
gagagactcc gtct 1544215DNAHomo sapiens 42aggcaagatc tagat
154318DNAHomo sapiens 43ccaaggcaag atctagat 184421DNAHomo sapiens
44ttgccaaggc aagatctaga t 214514DNAHomo sapiens 45aaactcagca tcta
144617DNAHomo sapiens 46cacaaactca gcatcta 174720DNAHomo sapiens
47acacacaaac tcagcatcta 204812DNAHomo sapiens 48gatctagatg ct
124915DNAHomo sapiens 49caagatctag atgct 155015DNAHomo sapiens
50gatctagatg ctgag 155118DNAHomo sapiens 51caagatctag atgctgag
185224DNAHomo sapiens 52aggcaagatc tagatgctga gttt 245312DNAHomo
sapiens 53ggagtggaaa ga 125415DNAHomo sapiens 54ccaggagtgg aaaga
155517DNAHomo sapiens 55aaccaggagt ggaaaga 175618DNAHomo sapiens
56gaaccaggag tggaaaga 185721DNAHomo sapiens 57aaagaaccag gagtggaaag
a 215812DNAHomo sapiens 58tacacgagca tc 125914DNAHomo sapiens
59tgtacacgag catc 146016DNAHomo sapiens 60cttgtacacg agcatc
166118DNAHomo sapiens 61aacttgtaca cgagcatc 186220DNAHomo sapiens
62caaacttgta cacgagcatc 206322DNAHomo sapiens 63ggcaaacttg
tacacgagca tc 226415DNAHomo sapiens 64gtggaaagat gctcg
156521DNAHomo sapiens 65ggagtggaaa gatgctcgtg t 216627DNAHomo
sapiens 66ccaggagtgg aaagatgctc gtgtaca 276715DNAHomo sapiens
67gaaaaagatc ttcag 156821DNAHomo sapiens 68tcagaaaaag atcttcaggg g
216927DNAHomo sapiens 69acatcagaaa aagatcttca gggggct 277014DNAHomo
sapiens 70acatcagaaa aaga 147117DNAHomo sapiens 71agcacatcag
aaaaaga 177220DNAHomo sapiens 72caaagcacat cagaaaaaga 207323DNAHomo
sapiens 73gaacaaagca catcagaaaa aga 237426DNAHomo sapiens
74ccagaacaaa gcacatcaga aaaaga 267515DNAHomo sapiens 75agccccctga
agatc 157618DNAHomo sapiens 76tctagccccc tgaagatc 187721DNAHomo
sapiens 77atttctagcc ccctgaagat c 217824DNAHomo sapiens
78cagatttcta gccccctgaa gatc 247927DNAHomo sapiens 79caacagattt
ctagccccct gaagatc 278039DNAHomo sapiens 80tgctcgtgta caagtttgcc
agaaaacacc acatcactt 3981120DNAHomo sapiens 81ataagtgact cttctgccct
tgaggacctg cgaaatccag aacaaagcac atcagaaaaa 60gatcttcagg gggctagaaa
tctgttgcta tgggcccttc accaacatgc ccacagatca 12082210DNAHomo sapiens
82gatcttcagg gggctagaaa tctgttgcta tgggcccttc accaacatgc ccacagatca
60actggaatgg atggtacagc tgtgtggtgc ttctgtggtg aaggagcttt catcattcac
120ccttggcaca ggtgtccacc caattgtggt tgtgcagcca gatgcctgga
cagaggacaa 180tggcttccat gcaattgggc agatgtgtga 210838PRTHomo
sapiens 83Arg Gln Asp Leu Asp Ala Glu Phe1 58410PRTHomo sapiens
84Pro Arg Gln Asp Leu Asp Ala Glu Phe Val1 5 108512PRTHomo sapiens
85Leu Pro Arg Gln Asp Leu Asp Ala Glu Phe Val Cys1 5 108614PRTHomo
sapiens 86Tyr Leu Pro Arg Gln Asp Leu Asp Ala Glu Phe Val Cys Glu1
5 108716PRTHomo sapiens 87Ser Tyr Leu Pro Arg Gln Asp Leu Asp Ala
Glu Phe Val Cys Glu Arg1 5 10 15889PRTHomo sapiens 88Pro Gly Val
Glu Arg Cys Ser Cys Thr1 58911PRTHomo sapiens 89Glu Pro Gly Val Glu
Arg Cys Ser Cys Thr Ser1 5 10907PRTHomo sapiens 90Pro Glu Asn Thr
Thr Ser Leu1 5919PRTHomo sapiens 91Thr Ser Glu Lys Asp Leu Gln Gly
Ala1 59211PRTHomo sapiens 92Ser Thr Ser Glu Lys Asp Leu Gln Gly Ala
Arg1 5 109340PRTHomo sapiens 93Ile Ser Asp Ser Ser Ala Leu Glu Asp
Leu Arg Asn Pro Glu Gln Ser1 5 10 15Thr Ser Glu Lys Asp Leu Gln Gly
Ala Arg Asn Leu Leu Leu Trp Ala 20 25 30Leu His Gln His Ala His Arg
Ser 35 40
* * * * *