Mutations in the BRCA2 interacting DSS1 are not a risk factor for male breast cancer (2006)

Male breast cancer (MBC) is a rare disease and, because of its rarity, its etiology is poorly understood. However, germ-line mutations in breast cancer susceptibility gene BRCA2 are known to predispose to MBC.1 Male carriers of BRCA2 mutations have an ∼80–100 times high risk for the development of breast cancer than the general male population thus making BRCA2 the strongest presently known MBC linked gene.2 In addition to BRCA2, germ-line mutations in BRCA1, CHEK2, AR and PTEN have also been linked to increased MBC risk,1 thus further supporting the role of genetic predisposition in the development of MBC. However, only a proportion of MBC cases (∼4–40% depending on the population) can be explained by mutations in BRCA2 and these other susceptibility genes.1, 3 Defects in genes functionally linked to BRCA2 might account for additional MBC cases. One such candidate gene is DSS1 (also known as SHFM1 and SEM1). DSS1 (deleted in split hand/split foot) encodes a 70 amino acids long, structurally and functionally highly conserved protein.4, 5, 6 The gene was originally linked with split hand/split foot malformation (SHFM) on chromosome 7q21.3-q22.14, but no definitive evidence for its role in this syndrome has been found.7 Instead, DSS1 has been shown to interact directly with BRCA2.6 Interactions of BRCA2 and DSS1, or their orthologues in lower organisms, have been convincingly demonstrated at least in yeast, fungi and mammalian cells.6, 8, 9 The physical binding between these 2 proteins occurs in the most conserved portions of both proteins.6, 8 Interestingly, some of the known cancer predisposing mutations in BRCA2 affect amino acids that mediate the interaction of DSS1 with BRCA2 and, essentially all BRCA2 in mammalian cells is associated with DSS1.8, 10 Most importantly, the strong physical interaction between DSS1 and BRCA2 seems to be crucial for the BRCA2 function in the repair of DNA double-strand breaks.8 Repair of DNA double-strand breaks during the G1/S and S/G2 phases of the mitotic cell cycle occurs through 2 main mechanisms, non-homologous end-joining and homologous recombination.11, 12, 13 Of these, the homologous recombination is an error-free repair mechanism and its proper function is critical for maintaining the chromosomal integrity.12 BRCA2 functions in homologous recombination together with RAD51 recombinase and colocalizes to the sites of DNA damage.14, 15 Interestingly, Yang et al.8 demonstrated that the same ∼90-kDa BRCA2 domain that binds to DSS1 also binds to single-stranded DNA. The function of DSS1 in this interaction remained unclear but DSS1 may regulate the accessibility of the DNA binding sites on BRCA2.8 This model raises both DSS1 and BRCA2 in critical role in homologous recombination. In fact, loss of DSS1 function has been shown to result in highly similar phenotypes in DNA repair and homologous recombination than inactivation of BRCA2 or RAD51.9, 16, 17, 18 Furthermore, DSS1 is needed for DNA damage-induced RAD51 locus formation and is recruited to double stranded DNA breaks in vivo together with proteasome subcomponents with which DSS1 can interact physically.19, 20, 21 Collectively, these results intimately link DSS1 in DNA repair and implicate it as a putative cancer susceptibility gene. Because DSS1 displays strong functional and molecular links to BRCA2 protein, DNA repair, and homologous recombination processes previously implicated in cancer predisposition, we were interested in elucidating whether mutations in DSS1 could be identified in MBC patients and thus explain some of the BRCA2 independent MBC cases. DNA samples from either whole blood (n = 41) or paraffin embedded tissue specimens (n = 104) of 145 Finnish MBC patients were available for mutation analysis of DSS1 gene. These patients were diagnosed between 1967 and 1996 and represent 60% of the MBC cases diagnosed during those 30 yr in Finland. The average age at MBC diagnosed was 65.8 yr (range from 30 to 94 yr). Eleven MBC patients had a first degree relative diagnosed with breast cancer, 1 with ovarian cancer, and 2 patients had both breast and ovarian cancer in family. All of the patients had been screened for Finnish BRCA2 founder mutations by solid-phase minisequencing (single-base extension reaction).22 Eleven BRCA2 mutation carriers were found and included in this study: 999del5 (n = 1), 4075delGT (n = 1), 5080del5 (n = 1), 7708C > T (n = 1), and 9346(−2)A > G (n = 7). Nineteen commercially available breast cancer cell lines (American Type Culture Collection, Manassas, VA) were also analyzed (Table I). Cells were grown under recommended culture conditions. The research protocol was approved by the Ethical Committee of the Tampere University Hospital and the Ministry of Social Affairs and Health in Finland. The entire coding region including exon-intron boundaries of DSS1 was sequenced using ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit and an automated ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequenced products were made as short as possible by primer designing to enable sufficient amplification from paraffin-embedded tissue samples. Variants were identified using Sequencher software version 4.5 (Gene Codes Corporation, Ann Arbor, MI). Our study design relied on the fact that, similarly to BRCA2, DSS1 mutations might be associated with increased risk for MBC. Our cohort of 145 Finnish MBC patients provided an excellent material for investigation of the possible role of DSS1 mutations in MBC predisposition. We also chose to study 19 commercially available breast cancer cell lines to further increase the strength of the analysis. Most of the DNA samples (n = 104) included in this study were derived from paraffin-embedded tissues from which the extraction of high quality DNA can be difficult. DNA sequence analyses were successfully preformed from the majority of the paraffin samples but especially the longer exons 2 and 3 were occasionally problematic and the analysis failed in 27 and 6 cases, respectively (Table I). Somewhat unexpectedly, no DSS1 mutations were found among the 145 MBC patients or among the 19 breast cancer cell lines (Table I). However, it has to be noted that our mutation screen would have missed deletions affecting complete exons or the entire DSS1 gene. Such alterations as well as other changes, such as epigenetic events, might also affect DSS1 function in breast cancer. Nevertheless, 3 silent, synonymous nucleotide substitutions 143G > A, 311G > A, and 341G > A (numbers refer to GenBank sequence NM_006304.1) were observed (Table I). The 143G > A variant was most frequent and occurred in 8 patient samples and in the SK-BR-3 cell line, whereas the other 2 variants were observed in a single patient sample each. None of these variants were detected among the eleven carriers of the BRCA2 mutations. Polymorphisms in the DNA repair genes have previously associated with increased risk of breast cancer.8, 17, 23 More specifically, the genetic variation in DNA repair genes has been suggested to influence their capability to repair DNA damage.23 However, these polymorphisms have been amino acid substitutions whereas the 3 DSS1 variants were synonymous nucleotide substitutions which do not change the amino acid in the polypeptide. Thus, the contribution of these variants to breast cancer susceptibility is unlikely. In summary, we have performed a comprehensive mutation screen of the entire coding region of DSS1 gene in a cohort of Finnish MBC patients and 19 breast cancer cell lines but did not identify any mutations. These results indicate that despite of the critical role of DSS1 in DNA repair and its tight association with BRCA2, mutations in DSS1 does not appear to be involved in increased breast cancer susceptibility, at least in the Finnish population. Yours sincerely, We wish to thank all the patients participated in this study and Kati Rouhento for technical assistance. Kirsi Syrjäkoski, Jussi Jäntti, Anne Kallioniemi, Ritva Karhu.