De Novo BRCA1 Mutation in a Patient with Breast Cancer and an Inherited BRCA2 Mutation

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Abstract

To the Editor: Individuals with an inherited mutation in both of the breast/ovarian cancer–susceptibility genes, BRCA1 and BRCA2, are rarely described (Ramus et al. Ramus et al., 1997Ramus SJ Friedman LS Gayther SA Ponder BA Bobrow L van der Looji M Papp J et al.A breast/ovarian cancer patient with germline mutations in both BRCA1 and BRCA2.Nat Genet. 1997; 15: 14-15Crossref PubMed Scopus (54) Google Scholar; Liede et al. Liede et al., 1998Liede A Rehal P Vesprini D Jack E Abrahamson J Narod SA A breast cancer patient of Scottish descent with germ-line mutations in BRCA1 and BRCA2.Am J Hum Genet. 1998; 62: 1543-1544Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Despite the large number of variants identified in these genes, there are, in the Breast Cancer Information Core, no published reports of de novo mutations. During the course of the Australian Breast Cancer Family Study, a population-based study of breast cancer occurring in women at age <40 years (McCredie et al. McCredie et al., 1998McCredie MRE Dite G Giles GG Hopper JL Breast cancer in Australian women under the age of 40.Cancer Causes Control. 1998; 9: 189-198Crossref PubMed Scopus (104) Google Scholar; Southey et al. Southey et al., 1998Southey MC Batten LE McCredie MRE Giles G Dite G Hopper JL Vente DJ Estrogen receptor polymorphism at codon 325 and risk of breast cancer in women before age forty.J Natl Cancer Inst. 1998; 90: 532-536Crossref PubMed Scopus (51) Google Scholar, Southey et al., 1999Southey MC Tesoriero AA Andersen CR Jennings KM Brown SM Dite GS Jenkins MA et al.BRCA1 mutations and other sequence variants in a population-based sample of Australian women with breast cancer.Br J Cancer. 1999; 79: 34-39Crossref PubMed Scopus (68) Google Scholar), we identified a proband who developed high-grade breast cancer with axillary nodal metastases. Her father developed prostate carcinoma during his early 50s. Her mother had no history of cancer. The studies outlined below were performed with informed consent from the individuals and were approved by the Peter MacCallum Cancer Institute's review board. The proband's leukocyte-derived DNA revealed two germline protein-truncating mutations: one in exon 11 of BRCA2 (6174delT) and a second in exon 11 of BRCA1 (3888delGA). These mutations were identified by the protein-truncation test and by manual DNA sequencing in our laboratory (fig. 1A), and were confirmed, after collection of a further blood sample, by heteroduplex analysis in an outside laboratory (data not shown). The 6174delT BRCA2 mutation has been reported frequently in individuals of Jewish descent (Neuhausen et al. Neuhausen et al., 1996Neuhausen S Gilewski T Norton L Tran T Mc Guire P Swensen J Hampel H et al.Recurrent BRCA2 6174delT mutations in Ashkenazi Jewish woman affected by breast cancer.Nat Genet. 1996; 13: 126-128Crossref PubMed Scopus (244) Google Scholar); however, there was no known Jewish ancestry in this family. The 3888delGA BRCA1 mutation has not been reported before by the Breast Cancer Information Core. The father of the proband carried only the 6174delT BRCA2 mutation (fig. 1A), and neither parent carried the 3888delGA BRCA1 mutation. The proband's brother was unavailable for testing. DNA fingerprinting at five informative microsatellite loci supported the genetic relatedness of the parents' and proband's DNA samples (data not shown). The absence of a germline 3888delGA BRCA1 mutation in either parent indicated that this variant could represent a de novo alteration that occurred during parental germ-cell gametogenesis. We evaluated this possibility by utilizing a heterozygous polymorphism at nucleotide 1186 (1186 A→G) in the proband's BRCA1 exon 11. The proband's BRCA1 exon 11 alleles were separated by cloning, and then were sequenced. The 1186 A→G polymorphism was present on her wild type 3888 allele and in her mother's germline but was not present in her father's germline (fig. 1A). These data indicate that the BRCA1 3888delGA mutation was on the father's allele and further suggest that this mutation arose as a de novo event in a testicular germ cell. The presence, in the proband, of heterozygous germline mutations in BRCA1 and BRCA2 raised the question of whether dysfunction of both these genes was involved in the development of her breast carcinoma. Loss of heterozygosity (LOH) studies performed on microdissected archival breast-cancer tissue demonstrated consistent LOH of the maternal (wild type) allele at the BRCA2-flanking markers D13S260 and D13S290, in multiple independently dissected areas of invasive ductal carcinoma. Loss of the maternal allele was also seen in three adjacent axillary-lymph-node metastases and in multiple apparently separate foci of high-grade ductal carcinoma in situ, a preinvasive lesion (fig. 2). These data are consistent with clonal expansion and dominance of cells that harbor dysfunction of both alleles of BRCA2, through multiple phases of malignant progression. In contrast, evaluation of identical microdissected foci at the intragenic BRCA1 marker D17S855 showed that both alleles were retained, suggesting that LOH-induced inactivation of the second BRCA1 allele was not selected for during carcinogenesis (fig. 2). Interestingly, the sequence adjacent to the BRCA1 mutation—AGAGGAGAAT (where GA constitutes the deleted doublet) is similar to that adjacent to the BRCA2 6174delT mutation—AGTGGAAAAT (where T denotes the deleted nucleotide). Although this similarity could be due to chance, it is also possible that these sequences are problematic for the germ-cell DNA-replication machinery and that they are thus prone to mutation. In support of this, haplotype studies suggest that the 6174delT BRCA2 mutation has arisen on multiple separate occasions in individuals of varying ethnic origins (Berman et al. Berman et al., 1996Berman DB Costalas J Schultz DC Grana G Daly M Godwin AK A common mutation in BRCA2 that predisposes to a variety of cancers is found in both Jewish Ashkenazi and non-Jewish individuals.Cancer Res. 1996; 56: 3409-3414PubMed Google Scholar). In an attempt to define a possible common mechanism underlying the development of these two mutations, we used the mfold software of M. Zuker to evaluate the areas of sequence similarity, together with 100 bp of their respective wild-type BRCA1- and BRCA2-flanking regions, for their ability to form secondary structures. The segments of both genes preferentially formed consistent hairpin loops arising from unpaired nucleotides at a wide range of bond energies under physiological conditions. Moreover, in each case, the deleted bases lay <2 bp from an unpaired sequence. It is therefore possible that these frameshift mutations occurred because of deletion of nucleotides adjacent to unpaired DNA sequences arising in these quasipalindromic regions. Frameshift mutations occurring in quasipalindromic sequences have been well characterized in Escherichia coli (De Boer and Ripley De Boer and Ripley, 1984De Boer JG Ripley LS Demonstration of the production of frameshift and base-substitution mutations by quasipalindromic DNA sequences.Proc Natl Acad Sci USA. 1984; 81: 5528-5531Crossref PubMed Scopus (80) Google Scholar; Rosche et al. Rosche et al., 1998Rosche WA Ripley LS Sinden RR Primer-template misalignment during leading strand DNA synthesis account for the most frequent spontaneous mutations in a quasipalindromic region in Escherichia coli.J Mol Biol. 1998; 284: 633-646Crossref PubMed Scopus (26) Google Scholar) and have been suggested as a basis of the deletion mutations seen in several human genes (Cooper and Krawczak Cooper and Krawczak, 1993Cooper DN Krawczak M Human gene mutation. Bios Scientific, Oxford1993Google Scholar, pp. 185–188). There is a further potential explanation for the occurrence of the two mutations occurring in this case: BRCA1 and BRCA2 have been implicated in the maintenance of genomic integrity (Rajan et al. Rajan et al., 1996Rajan JV Wang M Marquis ST Chodosh LA BRCA2 is coordinately regulated with BRCA1 during proliferation and differentiation in mammary epithelial cells.Proc Natl Acad Sci USA. 1996; 93: 13078-13083Crossref PubMed Scopus (197) Google Scholar; Connor et al. Connor et al., 1997Connor F Bertwistle D Mee PJ Ross GM Swift S Grigorieva E Tybulewicz VL et al.Tumorigenesis and a DNA repair defect in mice with a truncating BRCA2 mutation.Nat Genet. 1997; 17: 423-430Crossref PubMed Scopus (359) Google Scholar; Scully et al. Scully et al., 1997Scully R Chen J Plug A Xiao Y Weaver D Feunteun J Ashley T et al.Association of BRCA1 with Rad51 in mitotic and meiotic cells.Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar; Bertwistle and Ashworth Bertwistle and Ashworth, 1998Bertwistle D Ashworth A Functions of BRCA1 and BRCA2 genes.Curr Opin Genet Dev. 1998; 8: 14-20Crossref PubMed Scopus (88) Google Scholar; Chen et al. Chen et al., 1998Chen J Silver DP Walpita D Cantor SB Gazdar AF Tomilinson G Couch FJ et al.Stable interaction between the products of the BRCA 1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells.Mol Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar; Marmorstein et al. Marmorstein et al., 1998Marmorstein LY Ouchi T Aaronson SA The BRCA2 gene product functionally interacts with p53 and RAD51.Proc Natl Acad Sci USA. 1998; 95: 13869-13874Crossref PubMed Scopus (232) Google Scholar; Patel et al. Patel et al., 1998Patel KJ Yu VPCC Lee H Corcoran A Thistlethwaite FC Evans MJ Colledge WH et al.Involvement of BRCA2 in DNA repair.Mol Cell. 1998; 1: 347-357Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Moreover, in the mouse testes, Brca1 and Brca2 are expressed in the mitotic spermatogonia as well as in meiotic spermatocytes (Blackshear et al. Blackshear et al., 1998Blackshear PE Goldsworthy SM Foley JF McAllister KA Bennett LM Collins NK Bunch DO et al.BRCA1 and BRCA2 expression patterns in mitotic and meiotic cells of mice.Oncogene. 1998; 16: 61-68Crossref PubMed Scopus (76) Google Scholar). Therefore, the presence, in the father, of one mutated BRCA2 allele in developing germ cells could confer a dosage insufficiency and thus result in a generally increased genomic mutation rate. Such an abnormality could theoretically affect any gene and would thus have a minimal likelihood of being detected, thus explaining the absence of reports of supporting evidence. However, the serendipitous coexistent BRCA1 variant described here may indicate the need for more-rigorous searches for de novo germline mutations in other genes arising in the background of inherited BRCA1/2 mutations. Further studies using in vitro methods can address whether sequences such as those described above are indeed more susceptible to mutation, whether BRCA2 haploinsufficiency results in an increased mutation rate, and whether these two mutation mechanisms could display synergistic effects. This case is, to our knowledge, the first reported example of a de novo mutation arising in BRCA1, and it suggests a potential mechanism underlying the development of recurrent germline mutations occurring in association with specific sequences. We thank P. van der Spek, J. Hoeijmakers, S. Venitt, M. Stratton, S. Easteal, R. Sinden and T. Kunkel for discussions; L. Trute for technical assistance; M. Gardner for genetic counseling of patients; and E. Edkins for heteroduplex analysis. This work was funded by the National Health and Medical Research Council of Australia and by the Victorian Health Promotion Foundation.