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Large deletions in human brca1 gene and use thereof

Large deletions in human brca1 gene and use thereof description/claims

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. § 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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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