Susceptibility mutation 6495delGC of BRCA2

Details Susceptibility mutation 6495delGC of BRCA2 Susceptibility mutation 6495delGC of BRCA2

1998-09-21

2002-12-10

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S024310, C536S024330, C536S023100

Reexamination Certificate

active

06492109

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a gene which predisposes individuals to breast and ovarian cancer. More specifically, this invention relates to a specific mutation in the BRCA2 gene. In addition, it also relates to a method for detecting the presence of the mutation.
BACKGROUND OF THE INVENTION
BRCA2, located on chromosome 13q 12-q13, consists of over 70 kb of genomic DNA. The coding sequence produces a protein of 3,418 amino acids. Although most of the exons are small, exons 10 and 11 represent approximately 60% of the entire coding region. Germline mutations of BRCA2 are predicted to account for approximately 35% of families with multiple case, early onset female breast cancer, and they are also associated with an increased risk of male breast cancer, ovarian cancer, prostate cancer and pancreatic cancer.
The location of one or more mutations in the BRCA2 gene provides a promising approach to reducing the high incidence and mortality associated with breast and ovarian cancer through the early detection of women at high risk. These women, once identified, can be targeted for more aggressive prevention programs. Screening is carried out by a variety of methods which include karyotyping, probe binding and DNA sequencing. In such cases where one or only a few known mutations are responsible for the disease, methods for detecting the mutations are targeted to the site within the gene at which they are known to occur.
There is a need in the art to identify mutations in the BRCA2 gene. Identification of mutations of the BRCA2 gene and protein would allow more widespread diagnostic screening for hereditary breast and ovarian cancer than is currently possible.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a two base pair deletion of nucleotide 6495 of the published BRCA2 cDNA sequence which is associated with susceptibility to and development of breast and ovarian cancer.
It is an object of the invention to provide a method for determining a predisposition or higher susceptibility to breast and ovarian cancer.
It is another object of the invention to provide primers for detecting and amplifying a region of DNA which contains the 6495delGC mutation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based on the discovery of a two base pair deletion of nucleotide 6495 of the published BRCA2 cDNA sequence. This deletion mutation is referred to as 6495delGC. The BRCA2 gene is a tumor suppressor gene associated with breast and ovarian cancer.
The 6495delGC interrupts the normal reading frame of the BRCA2 transcript, resulting in the appearance of an in-frame terminator TAG at codon position 2090. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein.
Useful DNA molecules according to the invention are those which will specifically hybridize to BRCA2 sequences in the region of the 6495delGC mutation. Typically these are 17 to 20 nucleotides in length and have the nucleotide sequence corresponding to the region of the 6495delGC mutation at nucleotides 6495 of the BRCA2 cDNA sequence. Such molecules can be labeled, according to any technique known in the art, such as with radiolabels, fluorescent labels, enzymatic labels, sequence tags, etc. According to another aspect of the invention, the DNA molecules contain the 6495delGC mutation. Such molecules can be used as allele-specific oligonucleotide probes to track a particular mutation through a family.
Blood samples can be tested to determine whether the BRCA2 gene contains the 6495delGC mutation. In one embodiment of the invention a pair of isolated oligonucleotide primers are provided.
BRCA2-11F: 5′-TAC AGC AAG TGG AAA GC-3′(SEQ ID NO: 1), and BRCA2-11-R: 5′-AAG TTT CAG TTT TAC CAA T-3′(SEQ ID NO:2). The designation BRCA2-11 refers to a sequence in exon 11 of the BRCA2 gene. F and R refer to forward and reverse. The oligonucleotide primers are useful in direct amplification of a target polynucleotide prior to sequencing. These unique BRCA2 exon 11 oligonucleotide primers were designed and produced at Oncormed based upon identification of the 6495delGC mutation.
In another embodiment of the invention a pair of isolated allele specific oligonucleotides are provided.
5′-GAA CTG AGC ATA GTC TT-3′(SEQ ID NO:3), and
5′-GAA CTG AAT AGT CTT CA-3′(SEQ ID NO:4).
The allele specific oligonucleotides are useful in diagnosis of a subject at risk of having breast or ovarian cancer. The allele specific oligonucleotides hybridize with a target polynucleotide sequence containing the 6495delGC mutation. 5′-GAA CTG AGC ATA GTC TT-3′(SEQ ID NO:3) hybridizes preferentially to the wildtype sequence and is useful as a control sequence. 5′-GAA CTG AAT AGT CTT CA-3′(SEQ ID NO:4) is designed to hybridize preferentially to the mutant sequence.
The term “substantially complementary to” or “substantially the sequence” refers to (e.g., SEQ ID NO:3 and SEQ ID NO:4) sequences which hybridize to the sequences provided under stringent conditions and/or sequences having sufficient homology with SEQ ID NO:3 and SEQ ID NO:4, such that the allele specific oligonucleotides of the invention hybridize to the sequence. The term “isolated” as used herein includes oligonucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they may be associated. Such association being either in cellular material or in a synthesis medium. A “target polynucleotide” refers to the nucleic acid sequence of interest e.g., the BRCA2 encoding polynucleotide. Other primers which can be used for primer hybridization will be known or readily ascertainable to those of skill in the art.
The primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a significant number of nucleic acids in the polymorphic locus. Specifically, the term “primer” as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and more preferably more than eight and most preferably at least 20 nucleotides of the BRCA2 gene wherein said DNA sequence contains the 6495delGC mutation relative to BRCA2 contained in SEQ ID NO's:3 and 4. Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of the primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains 12-20 or more nucleotides, although it may contain fewer nucleotides.
Primers of the invention are designed to be “substantially” complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5′ and 3′ sequences flanking the mutation to hybridize therewith and permit amplification of the genomic locus.
Oligonucleotide primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved. Typically, one primer is complementary to the negative (−) strand of the polymorphic locus and the other is complementary to the positive (+) strand. Annealing the primers to denatured nucleic

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