Mutation analysis of BRCA1 and BRCA2 in a male breast cancer population.

A population-based series of 54 male breast cancer cases from Southern California were analyzed for germ-line mutations in the inherited breast/ovarian cancer genes, BRCA1 and BRCA2. Nine (17%) of the patients had a family history of breast and/or ovarian cancer in at least one first-degree relative. A further seven (13%) of the patients reported breast/ovarian cancer in at least one second-degree relative and in no first-degree relatives. No germ-line BRCA1 mutations were found. Two male breast cancer patients (4% of the total) were found to carry novel truncating mutations in the BRCA2 gene. Only one of the two male breast cancer patients carrying a BRCA2 mutation had a family history of cancer, with one case of ovarian cancer in a first-degree relative. The remaining eight cases (89%) of male breast cancer with a family history of breast/ovarian cancer in first-degree relatives remain unaccounted for by mutations in either the BRCA1 gene or the BRCA2 gene.     

Founder BRCA1 and BRCA2 mutations in Ashkenazi Jews in Israel: frequency and differential penetrance in ovarian cancer and in breast-ovarian cancer families.

Germ-line BRCA1 and BRCA2 mutations account for most of familial breast-ovarian cancer. In Ashkenazi Jews, there is a high population frequency (approximately 2%) of three founder mutations: BRCA1 185delAG, BRCA1 5382insC, and BRCA2 6174delT. This study examined the frequency of these mutations in a series of Ashkenazi women with ovarian cancer unselected for family history, compared with the frequency of these mutations in families ascertained on the basis of family history of at least two affected women. Penetrance was compared, both according to the method of family ascertainment (i.e., on the basis of an unselected ovarian cancer proband vs. on the basis of family history) and for the BRCA1 founder mutations compared with the BRCA2 6174delT mutation. There was a high frequency (10/22; [45%]) of germ-line mutations in Ashkenazi women with ovarian cancer, even in those with minimal or no family history (7/18 [39%]). In high-risk Ashkenazi families, a founder mutation was found in 59% (25/42). Families with any case of ovarian cancer were significantly more likely to segregate a founder mutation than were families with site-specific breast cancer. Penetrance was higher in families ascertained on the basis of family history than in families ascertained on the basis of an unselected proband, but this difference was not significant. Penetrance of BRCA1 185delAG and BRCA1 5382insC was significantly higher than penetrance of BRCA2 6174delT (hazard ratio 2.1 [95% CI 1.2-3.8]; two-tailed P = .01). Thus, the high rate of germ-line BRCA1/BRCA2 mutations in Ashkenazi women and families with ovarian cancer is coupled with penetrance that is lower than previously estimated. This has been shown specifically for the BRCA2 6174delT mutation, but, because of ascertainment bias, it also may be true for BRCA1 mutations.     

Prevalence of<Emphasis Type="Italic"> BRCA1</Emphasis> and<Emphasis Type="Italic"> BRCA2</Emphasis> mutations among clinic-based African American families with breast cancer

To define the prevalence and relative contributions of BRCA1 and BRCA2 mutations among African American families with breast cancer, we analyzed 28 DNA samples from patients identified through two oncology clinics. The entire coding regions of BRCA1 and BRCA2 were screened by protein truncation test, heteroduplex analysis, or single-stranded conformation polymorphism followed by DNA sequencing of variant bands. Deleterious protein-truncating BRCA1 and BRCA2 mutations were identified in five patients or 18% of the entire cohort. Only 8% (1 of 13) of women with a family history of breast cancer, but no ovarian cancer, had mutations. The mutation rates were higher for women from families with a history of breast cancer and at least one ovarian cancer (three of six, 50%). One woman with a family history of undocumented cancers was also found to carry a deleterious mutation in BRCA2. The spectrum of mutations was unique in that one novel BRCA1 mutation (1625del5) and three novel BRCA2 mutations (1536del4, 6696delTC, and 7795delCT) were identified. No recurrent mutations were identified in this cohort, although one BRCA2 (2816insA) mutation had been previously reported. In addition, two BRCA1 and four BRCA2 missense mutations of unknown significance were identified, one of which was novel. Taken together with our previous report on recurrent mutations seen in unrelated families, we conclude that African Americans have a unique mutation spectrum in BRCA1 and BRCA2 genes, but recurrent mutations are likely to be more widely dispersed and therefore not readily identifiable in this population.     

BRCA1 and BRCA2 mutation analysis of 208 Ashkenazi Jewish women with ovarian cancer

Ovarian cancer is a component of the autosomal-dominant hereditary breast-ovarian cancer syndrome and may be due to a mutation in either the BRCA1 or BRCA2 genes. Two mutations in BRCA1 (185delAG and 5382insC) and one mutation in BRCA2 (6174delT) are common in the Ashkenazi Jewish population. One of these three mutations is present in approximately 2% of the Jewish population. Each mutation is associated with an increased risk of ovarian cancer, and it is expected that a significant proportion of Jewish women with ovarian cancer will carry one of these mutations. To estimate the proportion of ovarian cancers attributable to founding mutations in BRCA1 and BRCA2 in the Jewish population and the familial cancer risks associated with each, we interviewed 213 Jewish women with ovarian cancer at 11 medical centers in North America and Israel and offered these women genetic testing for the three founder mutations. To establish the presence of nonfounder mutations in this population, we also completed the protein-truncation test on exon 11 of BRCA1 and exons 10 and 11 of BRCA2. We obtained a detailed family history on all women we studied who had cancer and on a control population of 386 Ashkenazi Jewish women without ovarian or breast cancer. A founder mutation was present in 41.3% of the women we studied. The cumulative incidence of ovarian cancer to age 75 years was found to be 6.3% for female first-degree relatives of the patients with ovarian cancer, compared with 2.0% for the female relatives of healthy controls (relative risk 3.2; 95% CI 1.5-6.8; P=.002). The relative risk to age 75 years for breast cancer among the female first-degree relatives was 2.0 (95% CI 1.4-3.0; P=.0001). Only one nonfounder mutation was identified (in this instance, in a woman of mixed ancestry), and the three founding mutations accounted for most of the observed excess risk of ovarian and breast cancer in relatives.     

Founding BRCA1 mutations in hereditary breast and ovarian cancer in southern Sweden.

Nine different germ-line mutations in the BRCA1 breast and ovarian cancer susceptibility gene were identified in 15 of 47 kindreds from southern Sweden, by use of SSCP and heteroduplex analysis of all exons and flanking intron region and by a protein-truncation test for exon 11, followed by direct sequencing. All but one of the mutations are predicted to give rise to premature translation termination and include seven frameshift insertions or deletions, a nonsense mutation, and a splice acceptor site mutation. The remaining mutation is a missense mutation (Cys61Gly) in the zinc-binding motif. Four novel Swedish founding mutations were identified: the nucleotide 2595 deletion A was found in five families, the C 1806 T nonsense mutation in three families, the 3166 insertion TGAGA in three families, and the nucleotide 1201 deletion 11 in two families. Analysis of the intragenic polymorphism D17S855 supports common origins of the mutations. Eleven of the 15 kindreds manifesting BRCA1 mutations were breast-ovarian cancer families, several of them with a predominant ovarian cancer phenotype. The set of 32 families in which no BRCA1 alterations were detected included 1 breast-ovarian cancer kindred manifesting clear linkage to the BRCA1 region and loss of the wild-type chromosome in associated tumors. Other tumor types found in BRCA1 mutation/haplotype carriers included prostatic, pancreas, skin, and lung cancer, a malignant melanoma, an oligodendroglioma, and a carcinosarcoma. In all, 12 of 16 kindreds manifesting BRCA1 mutation or linkage contained ovarian cancer, as compared with only 6 of the remaining 31 families (P<.001). The present study confirms the involvement of BRCA1 in disease predisposition for a subset of hereditary breast cancer families often characterized by ovarian cancers.     

Founder mutations in the BRCA1 gene in Polish families with breast-ovarian cancer

We have undertaken a hospital-based study, to identify possible BRCA1 and BRCA2 founder mutations in the Polish population. The study group consisted of 66 Polish families with cancer who have at least three related females affected with breast or ovarian cancer and who had cancer diagnosed, in at least one of the three affected females, at age <50 years. A total of 26 families had both breast and ovarian cancers, 4 families had ovarian cancers only, and 36 families had breast cancers only. Genomic DNA was prepared from the peripheral blood leukocytes of at least one affected woman from each family. The entire coding region of BRCA1 and BRCA2 was screened for the presence of germline mutations, by use of SSCP followed by direct sequencing of observed variants. Mutations were found in 35 (53%) of the 66 families studied. All but one of the mutations were detected within the BRCA1 gene. BRCA1 abnormalities were identified in all four families with ovarian cancer only, in 67% of 27 families with both breast and ovarian cancer, and in 34% of 35 families with breast cancer only. The single family with a BRCA2 mutation had the breast-ovarian cancer syndrome. Seven distinct mutations were identified; five of these occurred in two or more families. In total, recurrent mutations were found in 33 (94%) of the 35 families with detected mutations. Three BRCA1 abnormalities-5382insC, C61G, and 4153delA-accounted for 51%, 20%, and 11% of the identified mutations, respectively.     

Two distinct origins of a common BRCA1 mutation in breast-ovarian cancer families: a genetic study of 15 185delAG-mutation kindreds.

We screened 163 women from breast-ovarian cancer-prone families, as well as 178 individuals affected with breast and/or ovarian cancer but unselected for family history, for germ-line mutations in exon 2 of BRCA1, by SSCP analysis and direct sequencing. A total of 25 mutations were detected. Thirteen of 64 Jewish Ashkenazi women and 2 non-Jewish individuals were found to possess the 185delAG mutation. Haplotype data for all 15 individuals, with markers intragenic to BRCA1, suggest that the Jewish Ashkenazi individuals share a common ancestry that is distinct from the lineage shared by the other two women. These data provide the first evidence of two distinct lines of transmission for the 185delAG mutation, only one of which has its origins in the Jewish Ashkenazi population. Our screening also uncovered 10 affected individuals with an 11-bp deletion at nucleotide 188 of BRCA1 (188del11), 4 of whom are Ashkenazi Jews. This is only the third reported mutation detected within the Jewish Ashkenazi population and may represent the second most common alteration in BRCA1 found in Ashkenazi Jews in the United States. The observed overrepresentation of specific mutations within a subgroup of the general population may eventually contribute to the development of inexpensive and routine tests for BRCA1 mutations, as well as to the elucidation of other contributory factors (e.g., diet, environment, and chemical exposures) that may play a key role in cancer initiation and development. The implications of the mutational data, as well as the role that founder effect, demographic history, and penetrance play in the resulting observed phenomena, are discussed.     

Moderate frequency of BRCA1 and BRCA2 germ-line mutations in Scandinavian familial breast cancer.

Previous studies of high-risk breast cancer families have proposed that two major breast cancer-susceptibility genes, BRCA1 and BRCA2, may account for at least two-thirds of all hereditary breast cancer. We have screened index cases from 106 Scandinavian (mainly southern Swedish) breast cancer and breast-ovarian cancer families for germ-line mutations in all coding exons of the BRCA1 and BRCA2 genes, using the protein-truncation test, SSCP analysis, or direct sequencing. A total of 24 families exhibited 11 different BRCA1 mutations, whereas 11 different BRCA2 mutations were detected in 12 families, of which 3 contained cases of male breast cancer. One BRCA2 mutation, 4486delG, was found in two families of the present study and, in a separate study, also in breast tumors from three unrelated males with unknown family history, suggesting that at least one BRCA2 founder mutation exists in the Scandinavian population. We report 1 novel BRCA1 mutation, eight additional cases of 4 BRCA1 mutations described elsewhere, and 11 novel BRCA2 mutations (9 frameshift deletions and 2 nonsense mutations), of which all are predicted to cause premature truncation of the translated products. The relatively low frequency of BRCA1 and BRCA2 mutations in the present study could be explained by insufficient screening sensitivity to the location of mutations in uncharacterized regulatory regions, the analysis of phenocopies, or, most likely, within predisposed families, additional uncharacterized BRCA genes.     

Analysis of PALB2 Gene in BRCA1/BRCA2 Negative Spanish Hereditary Breast/Ovarian Cancer Families with Pancreatic Cancer Cases

Background

The PALB2 gene, also known as FANCN, forms a bond and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1% of familial breast cancer and 3–4% of familial pancreatic cancer. The goal of this study was to determine the prevalence of PALB2 mutations in a population of BRCA1/BRCA2 negative breast cancer patients selected from either a personal or family history of pancreatic cancer.

Methods

132 non-BRCA1/BRCA2 breast/ovarian cancer families with at least one pancreatic cancer case were included in the study. PALB2 mutational analysis was performed by direct sequencing of all coding exons and intron/exon boundaries, as well as multiplex ligation-dependent probe amplification.

Results

Two PALB2 truncating mutations, the c.1653T>A (p.Tyr551Stop) previously reported, and c.3362del (p.Gly1121ValfsX3) which is a novel frameshift mutation, were identified. Moreover, several PALB2 variants were detected; some of them were predicted as pathological by bioinformatic analysis. Considering truncating mutations, the prevalence rate of our population of BRCA1/2-negative breast cancer patients with pancreatic cancer is 1.5%.

Conclusions

The prevalence rate of PALB2 mutations in non-BRCA1/BRCA2 breast/ovarian cancer families, selected from either a personal or family pancreatic cancer history, is similar to that previously described for unselected breast/ovarian cancer families. Future research directed towards identifying other gene(s) involved in the development of breast/pancreatic cancer families is required.     

<Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> germline mutation analysis among Indian women from south India: identification of four novel mutations and high-frequency occurrence of 185delAG mutation

Mutations in the BRCA1 and BRCA2 genes profoundly increase the risk of developing breast and/or ovarian cancer among women. To explore the contribution of BRCA1 and BRCA2 mutations in the development of hereditary breast cancer among Indian women, we carried out mutation analysis of the BRCA1 and BRCA2 genes in 61 breast or ovarian cancer patients from south India with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation-sensitive gel electrophoresis (CSGE) followed by sequencing. Mutations were identified in 17 patients (28.0%); 15 (24.6%) had BRCA1 mutations and two (3.28%) had BRCA2 mutations. While no specific association between BRCA1 or BRCA2 mutations with cancer type was seen, mutations were more often seen in families with ovarian cancer. While 40% (4/10) and 30.8% (4/12) of families with ovarian or breast and ovarian cancer had mutations, only 23.1% (9/39) of families with breast cancer carried mutations in the BRCA1 and BRCA2 genes. In addition, while BRCA1 mutations were found in all age groups, BRCA2 mutations were found only in the age group of ≤40 years. Of the BRCA1 mutations, there were three novel mutations (295delCA; 4213T→A; 5267T→G) and three mutations that have been reported earlier. Interestingly, 185delAG, a BRCA1 mutation which occurs at a very high frequency in Ashkenazi Jews, was found at a frequency of 16.4% (10/61). There was one novel mutation (4866insT) and one reported mutation in BRCA2. Thus, our study emphasizes the importance of mutation screening in familial breast and/or ovarian cancers, and the potential implications of these findings in genetic counselling and preventive therapy.     

Penetrances of BRCA1 1675delA and 1135insA with respect to breast cancer and ovarian cancer.

For genetic counseling and predictive testing in families with inherited breast-ovarian cancer, penetrances and expressions of the underlying mutations should be known. We have previously reported two BRCA1 founder mutations in the Norwegian population. Index cases for the present study were found two different ways: through a series of consecutive ovarian cancers (n=16) and through our family cancer clinic (n=14). Altogether, 20 of the patients had BRCA1 1675delA, and 10 had 1135insA. Their relatives were described with respect to absence/presence of breast and/or ovarian cancer. Of 133 living female relatives, 83 (62%) were tested for the presence of a mutation. No difference, in penetrance and expression, between the two mutations were found, whereas differences according to method of ascertainment were seen. The overall findings were that disease started to occur at age 30 years and that by age 50 years 48% of the mutation-carrying women had experienced breast and/or ovarian cancer. More ovarian cancers than breast cancers were recorded. Both penetrance and expression (breast cancer vs. ovarian cancer) were different from those in reports of the Ashkenazi founder mutations. Whether the reported differences reflect true differences and/or methodological problems is discussed. An observed excess of mutation carriers could not be accounted for by methodological problems; possible explanations were a "true" low penetrance or preferential segregation.     

BRCA1 mutations in a population-based study of breast cancer in Stockholm County

The mutation frequency of BRCA1 and BRCA2 in women with breast cancer varies according to family history, age at diagnosis and ethnicity. The contribution of BRCA1 and BRCA2 mutations in breast cancer populations, unselected for age and family history, has been examined in several studies reporting mutation frequencies between 1% and 12% by screening methods, population sizes, and to what extent the gene/s were screened differed in the studies. We wanted to clarify the proportion of breast cancer attributable to mutations in BRCA1 in an unselected breast cancer population from the Stockholm region. All incident breast cancer patients treated surgically in a 19-month period were eligible for the study and 70% (489/696) participated. Exon 11 of BRCA1 was screened for mutations using the protein truncation test, and the mutation frequency was estimated from that. In previous studies on high-risk families from Stockholm, more than 70% of the mutations were detected in exon 11. Two mutations were found, both in patients with a family history or their own medical history of ovarian cancer, giving a mutation frequency in exon 11 of 0.4% and an estimated BRCA1 mutation frequency of <1%. Mutations in BRCA1 in unselected breast cancer cases in our region are rare and likely to be found only in high-risk families. Our BRCA1 prevalence is the lowest of all studies on unselected breast cancer patients, probably reflecting the comparatively low rates detected also in high-risk breast cancer families from the region.     

The prevalence of common BRCA1 and BRCA2 mutations among Ashkenazi Jews

Three founder mutations in the cancer-associated genes BRCA1 and BRCA2 occur frequently enough among Ashkenazi Jews to warrant consideration of genetic testing outside the setting of high-risk families with multiple cases of breast or ovarian cancer. We estimated the prevalence of these founder mutations in BRCA1 and BRCA2 in the general population of Ashkenazi Jews according to age at testing, personal cancer history, and family cancer history. We compared the results of anonymous genetic testing of blood samples obtained in a survey of >5,000 Jewish participants from the Washington, DC, area with personal and family cancer histories obtained from questionnaires completed by the participants. In all subgroups defined by age and cancer history, fewer mutations were found in this community sample than in clinical series studied to date. For example, 11 (10%) of 109 Jewish women who had been given a diagnosis of breast cancer in their forties carried one of the mutations. The most important predictor of mutation status was a previous diagnosis of breast or ovarian cancer. In men and in women never given a diagnosis of cancer, family history of breast cancer before age 50 years was the strongest predictor. As interest in genetic testing for BRCA1 and BRCA2 in the Jewish community broadens, community-based estimates such as these help guide those seeking and those offering such testing. Even with accurate estimates of the likelihood of carrying a mutation and the likelihood of developing cancer if a mutation is detected, the most vexing clinical problems remain.     

The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women.

The mutations 185delAG, 188del11, and 5382insC in the BRCA1 gene and 6174delT in the BRCA2 gene were analyzed in 199 Ashkenazi and 44 non-Ashkenazi Jewish unrelated patients with breast and/or ovarian cancer. Of the Jewish Ashkenazi women with ovarian cancer, 62% (13/21) had one of the target mutations, as did 30% (13/43) of women with breast cancer alone diagnosed before the age 40 years and 10% (15/141) of those with breast cancer diagnosed after the age 40 years. Age at ovarian cancer diagnosis was not associated with carrier status. Of 99 Ashkenazi patients with no family history of breast and/or ovarian cancer, 10% carried one of the mutations; in two of them the mutation was proved to be paternally transmitted. One non-Ashkenazi Jewish ovarian cancer patient from Iraq carried the 185delAG mutation. Individual mutation frequencies among breast cancer Ashkenazi patients were 6.7% for 185delAG, 2.2% for 5382insC, and 4.5% for 6174delT, among ovarian cancer patients; 185delAG and 6174delT were about equally common (33% and 29%, respectively), but no ovarian cancer patient carried the 5382insC. More mutations responsible for inherited breast and ovarian cancer probably remain to be found in this population, since 79% of high-incidence breast cancer families and 35% of high-incidence breast/ovarian cancer families had none of the three known founder mutations.     

BRCA2 mutations and triple-negative breast cancer

Recently, BRCA1 germline mutations were found in a high proportion (14-34%) of patients with triple-negative breast cancer (TNBC). BRCA2 was either not analyzed or showed much lower mutation frequencies. Therefore, we screened a group of TNBC patients (n = 30) of white European descent for mutations in BRCA2 as well as in BRCA1. Cases were unselected for age of disease-onset (median age at breast cancer diagnosis was 58 years, ranging from 37 to 74 years), family history of cancer and BRCA1 and BRCA2 mutation status. Half of the patients (15/30) showed a family history of breast and/or ovarian cancer. A high frequency of deleterious germline mutations was observed in BRCA2 (5/30; 16.7%), and only one case showed a BRCA1 mutation (3.3%). Although the study group was small, these results point to BRCA2 mutations being important in TNBC.     

Prevalence of PALB2 Mutations in Breast Cancer Patients in Multi-Ethnic Asian Population in Malaysia and Singapore

Background

The partner and localizer of breast cancer 2 (PALB2) is responsible for facilitating BRCA2-mediated DNA repair by serving as a bridging molecule, acting as the physical and functional link between the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins. Truncating mutations in the PALB2 gene are rare but are thought to be associated with increased risks of developing breast cancer in various populations.

Methods

We evaluated the contribution of PALB2 germline mutations in 122 Asian women with breast cancer, all of whom had significant family history of breast and other cancers. Further screening for nine PALB2 mutations was conducted in 874 Malaysian and 532 Singaporean breast cancer patients, and in 1342 unaffected Malaysian and 541 unaffected Singaporean women.

Results

By analyzing the entire coding region of PALB2, we found two novel truncating mutations and ten missense mutations in families tested negative for BRCA1/2-mutations. One additional novel truncating PALB2 mutation was identified in one patient through genotyping analysis. Our results indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the Malaysian and Singaporean populations.     

Missense mutations (p.H371Y, p.D438Y) in gene CHEK2 are associated with breast cancer risk in women of Balochistan origin

CHEK2 encodes a serine/threonine-protein kinase which plays a critical role in DNA damage signaling pathways. CHEK2 directly phosphorylates and regulates the functions of p53 and BRCA1. Most women with breast and/or ovarian cancer are not carriers of mutant BRCA1 or BRCA2. Multiple studies have shown that a CHEK2*1100delC confers about a two-fold increased risk of breast cancer in unselected females and a tenfold increase in males. Moreover, studies have shown that first-degree relatives of bilateral breast cancer cases who carried the CHEK2*1100delC allele had an eight-fold increased risk of breast cancer. It has been suggested that CHEK2 functions as a low-penetrance susceptibility gene for cancers and multiplies the risks associated with other gene(s) to increase cancer risk. The main goal of this study was to evaluate and to compare the role of truncating mutations, splice junction mutations and rare missense substitutions in breast cancer susceptibility gene CHEK2. Present study was performed on 140 individuals including 70 breast cancer patients both with and without family history and 70 normal individuals. Written consent was obtained and 3 ml intravenous blood was drawn from all the subjects. DNA was extracted from all the samples through inorganic method published already. Primers were synthesized for all the 14 exons of CHEK2 gene. Coding and adjacent intronic sequences of CHEK2 gene were amplified and sequenced. Two genetic variants (p.H371Y, p.D438Y) were found in exon 10 and exon 11 of gene CHEK2 which were not found in any of the 70 control individuals from same geographical area and ethnic group. The genetic variant c.1312G>T (p.D438Y) identified in a patient with a family history of breast cancer. To our knowledge, this is first mutation scanning study of gene CHEK2 from Balochistan population.     

Establishing a control population to screen for the occurrence of nineteen unclassified variants in the BRCA1 gene by denaturing high-performance liquid chromatography

Numerous missense mutations in BRCA1 and BRCA2 are detected during clinical screening of breast and ovarian cancer patients. Because of the lack of a functional protein assay to determine the functional consequence of these mutations, patients are often frustrated by inconclusive results due to unclassified variants (UV). To determine whether a reported UV is also present in a control collective and therefore more likely be a rare polymorphism than a deleterious mutation, we collected a control population consisting of 95 females and 25 males aged over 60 years (mean 73 years) without a family history of BRCA associated cancers. The age of the control group is beyond the median onset of breast and ovarian cancer with a hereditary background. These controls were analysed for the presence of 19 known UVs in BRCA1 with the DHPLC technique. Only four of the 19 variants (R496H, R866C, S1040N and M1652I) were detected and can be considered polymorphims. However, no firm conclusion can be drawn about the functional relevance of the other 15 variants.     

RAD51C germline mutations in breast and ovarian cancer cases from high-risk families

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.