Coding sequence haplotype of the human BRCA1 gene

Details Coding sequence haplotype of the human BRCA1 gene Coding sequence haplotype of the human BRCA1 gene

2001-12-20

2004-02-03

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S320100, C435S069100, C435S325000, C536S023100, C536S024300, C536S024320, C536S023500, C530S350000

Reexamination Certificate

active

06686163

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a gene which has been associated with breast and ovarian cancer where the gene is found to be mutated. More specifically, this invention relates to a coding sequence of the BRCA1 gene, BRCA1(omi4), isolated from human subjects which defines a new haplotype.
BACKGROUND OF THE INVENTION
It has been estimated that about 5-10% of breast cancer is inherited Rowell, et al.,
American Journal of Human Genetics,
55:861-865 (1994). Located on chromosome 17, BRCA1 is the first gene identified to be conferring increased risk for breast and ovarian cancer. Miki et al.,
Science,
266:66-71 (1994). Mutations in this “tumor suppressor” gene are thought to account for roughly 45% of inherited breast cancer and 80-90% of families with increased risk of early onset breast and ovarian cancer. Easton et al.,
American Journal of Human Genetics,
52:678-701 (1993).
Locating one or more mutations in the BRCA1 region of chromosome 17 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 DNA sequencing technology, genomic DNA is extracted from whole blood and the coding sequences of the BRCA1 gene are amplified. The coding sequences might be sequenced completely and the results are compared to the DNA sequence of the gene. Alternatively, the coding sequence of the sample gene may be compared to a panel of known mutations or other screening procedure before completely sequencing the gene and comparing it to a normal sequence of the gene.
If a mutation in the BRCA1 coding sequence is found, it may be possible to provide the individual with increased expression of the gene through gene transfer therapy. It has been demonstrated that the gene transfer of the BRCA1 coding sequence into cancer cells inhibits their growth and reduces tumorigenesis of human cancer cells in nude mice. Jeffrey Holt and his colleagues conclude that the product of BRCA1 expression is a secreted tumor growth inhibitor, making BRCA1 an ideal gene for gene therapy studies. Transduction of only a moderate percentage of tumor cells apparently produces enough growth inhibitor to inhibit all tumor cells. Arteaga and Holt,
Cancer Research,
56: 1098-1103 (1996), Holt, et al.,
Nature Genetics,
12: 298-302 (1996). The observation of Holt et al, that the BRCA1 growth inhibitor is a secreted protein leads to the possible use of infection of the BRCA1 growth inhibitor into the area of the tumor for tumor suppression.
The BRCA1 gene is divided into 24 separate exons. Exons 1 and 4 are noncoding, in that they are not part of the final functional BRCA1 protein product. The BRCA1 coding sequence spans roughly 5600 base pairs (bp). Each exon consists of 200-400 bp, except for exon 11 which contains about 3600 bp. To sequence the coding sequence of the BRCA1 gene, each exon is amplified separately and the resulting PCR products are sequenced in the forward and reverse directions. Because exon 11 is so large, we have divided it into twelve overlapping PCR fragments of roughly 350 bp each (segments “A” through “L” of BRCA1 exon 11).
Many mutations and polymorphisms have already been reported in the BRCA1 gene. A world wide web site has been built to facilitate the detection and characterization of alterations in breast cancer susceptibility genes. Such mutations in BRCA1 can be accessed through the Breast Cancer Information Core (BIC) at: www.nchgr.nih.gov/dir/lab_transfer/bic. This data site became publicly available on Nov. 1, 1995. Friend, et al.
Nature Genetics,
11:238, (1995).
The genetics of Breast/Ovarian Cancer Syndrome is autosomal dominant with reduced penetrance. In simple terms, this means that the syndrome runs through families: (1) both sexes can be carriers (mostly women get the disease but men can both pass it on and occasionally get breast cancer); (2) most generations will likely have breast cancer; (3) occasionally women carriers either die young before they have the time to manifest disease (and yet have offspring who get it) or they never develop breast or ovarian cancer and die of old age (the latter people are said to have “reduced penetrance” because they never develop cancer). Pedigree analysis and genetic counseling is absolutely essential to the proper workup of a family prior to any lab work.
Until recently, only a single coding sequence for the BRCA1 gene has been available for comparison to patient samples. That sequence is available as GENBANK Accession Number U14680. Recently another sequence has been made known in U.S. Pat. No. 5,654,155. There is a need in the art, to have available a coding sequence which is the BRCA1 coding sequence found in a portion of the normal population in order to determine whether or not a patient's BRCA1 gene sequence is mutant with increased succeptibility for cancer or a different normal sequence with no such succeptability. This will make it possible for true mutations to be easily identified or differentiated from naturally occurring sequence variations such as polymorphisms. Identification of mutations of the BRCA1 gene and protein allow more widespread diagnostic screening for hereditary breast and ovarian cancer than is currently possible and also have utility in gene therapy, protein replacement therapy and diagnosis.
Knowing the coding sequences which do not represent a higher susceptibility to an individual for cancer will reduce the likelihood of misinterpreting a “sequence variation” found in the population (i.e. polymorphism) with a pathologic “mutation” (i.e. causes disease in the individual or puts the individual at a high risk of developing the disease). With large interest in breast cancer predisposition testing, misinterpretation is particularly worrisome. People who already have breast cancer are asking the clinical question: “is my disease caused by a heritable genetic mutation?” The relatives of the those with breast cancer are asking the question: “Am I also a carrier of the mutation my relative has? Thus, is my risk increased, and should I undergo a more aggressive surveillance program.”
SUMMARY OF THE INVENTION
The present invention is based on the isolation of a coding sequence of the BRCA1 gene found in human individuals.
It is an object of the invention to define a haplotype of a BRCA1 gene, BRCA1(omi4), in normal individuals which does not correspond to increased cancer susceptibility.
It is another object of this invention to sequence a BRCA1 gene or portion thereof and compare it to the newly discovered haplotype, BRCA1(omi4), to determine whether a sequence variation noted represents a polymorphism or a potentially harmful mutation.
It is another object of the invention to provide a protein sequence coded for by the coding sequence of the BRCA1 gene.
It is another object of the invention to provide a list of the codon pairs which occur at each of seven polymorphic points on the BRCA1 gene.
It is another object of the invention to provide a method wherein the BRCA1 gene, or parts thereof, are amplified with one or more oligonucleotide primers.
It is another object of this invention to provide a method of identifying individuals who carry no mutation(s) of the BRCA1 coding sequence and therefore have no increased genetic susceptibility to breast or ovarian cancer based on their BRCA1 genes.
It is another object of this invention to provide a method of identifying a mutation leading to an increased genetic susceptibility to breast or ovarian cancer.
It is another object of the invention to encompass prokaryotic or eukaryotic host cells comprising an expression vector having a DNA sequence that encodes for all or a fragment of the BRCA1(omi4) protein, a BRCA1(omi4) polypeptide, or a functional equivalent thereof.
It is another object of the invention to encompass all or fragments o

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