Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-07
2002-03-12
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06355427
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention relates to a diagnostic assay for determining susceptibility to breast cancer based on the sequence of the 3′ untranslated region of the prohibitin gene.
BACKGROUND OF THE INVENTION
Breast cancer is the second leading cause of cancer-related deaths of women in North America. A distinction must be drawn, however, between sporadic and familial or inherited breast cancer. Approximately 10% of all breast cancers are currently classified as strongly familial with many of these appearing to be caused by mutations in the hereditary breast cancer genes BRCA1 or BRCA2. However, at least one-third of breast cancers which seem to run in families are not linked to BRCA1 or BRCA2, suggesting the existence of an additional hereditary breast cancer gene or genes. Recently, studies have suggested the possibility that additional genes important for breast cancer development are located on chromosome 17, based on the observation of tumor suppression in breast cancer cells following the introduction of normal human chromosome 17 without the inclusion of active BRCA1 or p53 (Theile, et al., “Suppression of tumorigenicity of breast cancer cells by transfer of human chromosome 17 does not require transferred BRCA1 and p53 genes,”
Oncogene
10:439-443 (1995)).
The antiproliferative gene prohibitin was discovered using a subtraction hybridization to enrich for mRNAs preferentially expressed in normally proliferating cells compared to regenerating rat liver cells (McClung, et al., “Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver,”
Biochem Biophys Res Comm
164:1316-1322 (1989); Nuell, et al., “Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells,”
Mol Cell Biol
11:1372-1381 (1991)). The human prohibitin gene, which maps to chromosome 17 at q21 (White, et al., “Assignment of the human prohibitin gene (PHB) to chromosome 17 and identification of a DNA polymorphism,”
Genomics
11:228-230 (1991)), was an initial candidate gene for the familial breast and ovarian tumor suppressor locus based on a frequent loss of heterozygosity in this region in familial and sporadic breast cancers (Black, et al., “A somatic cell hybrid map of the long arm of human chromosome 17, containing the familial breast cancer locus (BRCA1),”
Am J Hum Genet
52:702-710 (1993); Nagai, et al., “Detailed deletion mapping of chromosome segment 17q12-21 in sporadic breast tumors,”
Genes, Chromosome and Cancer
11:58-62 (1994)). Furthermore, Sato, et al., “The human prohibitin gene located on chromosome 17q21 is mutated in sporadic breast cancer,”
Cancer Res
52:1643-1646 (1992), reported four mutations in a highly conserved region of prohibitin exon 4 in an analysis of 23 sporadic human breast cancers. However, positional cloning studies resulted in the identification of BRCA1 rather than prohibitin (Miki, et al., “A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1,”
Science
266:66-71 (1994)) as a familial breast cancer gene on chromosome 17.
Previous studies of the prohibitin gene from DNA purified from familial breast cancer patients provided no evidence that any of the patients carried a germline change of the protein coding region of prohibitin genomic DNA. It was therefore concluded that there was no relationship between familial/hereditary breast cancer and mutations in the prohibitin protein coding region. Tokino, et al.,
Internat'l J Oncol
3:769-772 (1993). Additional studies did not identify any somatic mutations in the prohibitin protein coding region in familial/hereditary breast cancers suggesting that the protein coding region is not frequently mutated in breast cancers. Sato et al.,
Genomics
17:762-764 (1993).
Previous work by Jupe et al. disclosed a diagnostic test for increased susceptibility to sporadic breast cancer. It was reported that individuals who are heterozygous for the two prohibitin alleles (designated as “non-B” and “B” based on sequence variations found in intron 2 and 5) or homozygous for non-B allele would have low risk for developing sporadic cancer. The probability of developing cancer would increase for those who are homozygous for the B-type allele and again for those who have a mutation in the 3′ untranslated region (“3′UTR”) of at least one of the B-type alleles. Analyses were reported of breast-cancer derived cell lines and primary breast tumors showing homozygosity for the B-type allele and somatic mutations in the 3′UTR.
Full length prohibitin cDNAs for the BT-20, MCF7 and SK-BR-3 breast cancer cell lines were sequenced, and mutations restricted to the 3′UTR were identified. These three cell lines were arrested in cell cycle progression when full length prohibitin transcript was introduced by microinjection. All of them were also homozygous for the B-allele. Compared to the sequence of the wild type prohibitin 3′UTR, two point mutations were identified for BT-20: G (guanine) to A (adenine) at position 758 and T (thymine) to C (cytosine) at position 814. MCF7 also had two point mutations: G to A at position 236 and C to T at position 729. SK-BR-3 showed 26 base changes including a change of C to T at position 729. Thus, MCF7 and SK-BR-3 both had a change of C to T at position 729. Jupe, et al., “Prohibitin in breast cancer cell lines: Loss of antiproliferative activity is linked to 3′ untranslated region mutations,”
Cell Growth and Differentiation
7:871-878 (1996).
It has now been found, contrary to the teachings of the prior prohibitin work, that this change from C to T at position 729 is the result not of a somatic mutation, but rather the result of a natural allelic variation at this point, i.e., it is a germline polymorphism. Furthermore, it is a germline polymorphism that can be used as a susceptibility marker for breast cancer. Data indicate that the frequency of homozygosity for 729-T appears to be approximately 4-5-fold higher in breast cancer patients than in unaffected females, that 4% of all breast cancers develop in women who are homozygous T/T (which likely make up less than 1% of unaffected women), and that their lifetime risk of developing breast cancer is approximately 50%.
Thus, it has now been found that the prohibitin gene, located on chromosome 17q21 near the BRCA1 locus, exhibits a germline polymorphism in the 3′UTR that can be used as a susceptibility marker for breast cancer.
REFERENCES:
patent: 5401635 (1995-03-01), Nakamura et al.
patent: 5776738 (1998-07-01), Dell'Orco et al.
patent: WO9640919 (1996-12-01), None
Jupe, et al. 1996. “The 3′ untranslated region of prohibitin and cellular immortalization,”Experimental Cell Research224: 128-135.
Jupe, et al. 1996. “Prohibitin in breast cancer cell lines: loss of antiproliferative activity is linked to 3′ untranslated region mutations,”Cell Growth&Differentiationvol. 7, 871-878.
Jupe, et al. 1995. “Prohibitin antiproliferative activity and lack of heterozygosity in immortalized cell lines,”Experimental Cell Research218: 577-580.
Sato, et al. 1992. “The human prohibitin gene located on chromosome 17q21 is mutated in sporadic breast cancer,”Cancer Research52 :1643-1646.
Bentley, et al 1991. “Rapid methods for detection of polymorphic markers in genomic DNA,”Methods in Molecular Biologyvol. 9: Protocols in Human Molecular Genetics pp. 51-68.
Liu, et al. 1995. “Restriction endonuclease fingerprinting (REF): a sensitive method for screening mutations in long, contiguous segments of DNA,”BioTechniquesvol. 18: 470-477.
Dell'Orco Robert T.
Jupe Eldon R.
Resta Regina
Thompson Linda F.
Campbell Eggerton A.
Fitch Even Tabin & Flannery
Oklahoma Medical Research Foundation
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