Evaluation of RAD51C as cancer susceptibility gene in a large breast-ovarian cancer patient population referred for genetic testing

Despite extensive analysis of the BRCA1 and BRCA2 genes, germline mutations are detected in <20% of families with a presumed genetic predisposition for breast and ovarian cancer. Recent literature reported RAD51C as a new breast cancer susceptibility gene. In this study, we report the analysis of 410 patients from 351 unrelated pedigrees. All were referred for genetic testing and we selected families with at least one reported case of ovarian cancer in which BRCA1&2 mutations were previously ruled out. We analyzed the coding exons, intron-exons boundaries, and UTRs of RAD51C. Our mutation analysis did not reveal any unequivocal deleterious mutation. In total 12 unique sequence variations were identified of which two were novel. Our study and others suggest a low prevalence of RAD51C mutations with an exception for some founder populations. This observation is in favor of the rare allele hypothesis in the debate over the nature of the genetic contribution to individual susceptibility to breast and ovarian cancer and further genome-wide studies in high risk families are warranted.     

RAD51C germline mutations found in Spanish site-specific breast cancer and breast-ovarian cancer families

BRCA1 and BRCA2 are the most well-known breast and ovarian cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. Recently, RAD51C, a new Fanconi Anemia gene, essential for homologous recombination repair, has been reported to be a rare hereditary breast and ovarian cancer susceptibility gene. Indeed, several pathogenic mutations have been identified in BRCA1/BRCA2-negative hereditary breast and ovarian cancer families. Here, we present the results of the screening of RAD51C mutations in a large series of 516 BRCA1/BRCA2-negative Spanish patients from breast and/or ovarian cancer families, and the evaluation of these results in the context of all RAD51C carriers. RAD51C mutation screening was performed by DNA analysis for all index cases. All the genetic variants identified were analyzed in silico for splicing and protein predictions. cDNA analysis was performed for three selected variants. All previous RAD51C mutation studies on breast and/or ovarian cancer were reviewed. We identified three inactivating RAD51C mutations. Two mutations were found in breast and ovarian cancer families and one mutation in a site-specific breast cancer family. Based on the mean age of ovarian cancer diagnosis in RAD51C carriers, we would recommend prophylactic bilateral salpingo-ophorectomy in premenopausal RAD51C mutation carriers. Our results support that RAD51C is a rare breast and ovarian cancer susceptibility gene and may contribute to a small fraction of families including breast and ovarian cancer cases and families with only breast cancer. Thus, RAD51C testing should be offered to hereditary breast and/or ovarian cancer families without selecting for specific cancer origin.     

Screening for RAD51 and BRCA2 BRC repeat mutations in breast and ovarian cancer families

Together, germline mutations in the two major susceptibility genes BRCA1 and BRCA2 account for approximately 20-30% and 70-80% of the familial breast and ovarian cancer cases, respectively. This indicates involvement of additional susceptibility genes, perhaps in combination with a polygenic effect. However, it is also possible that part of the mutations disrupting BRCA1 and BRCA2 function still remains to be discovered. In response to double-strand DNA damage the co-operation between RAD51 and BRCA2 is of great importance, and the conserved BRC repeat motifs in BRCA2 are crucial for this interaction. In the current study, patients belonging to 126 breast and/or ovarian cancer families were screened for RAD51 and BRCA2 BRC repeat mutations in order to uncover aberrations that may contribute to hereditary cancer susceptibility. The performed study revealed several novel alterations, however, none of them appeared to be disease-related. Thus, it seems likely that germline mutations in the highly conserved RAD51 gene are extremely rare and generally poorly tolerated.     

The variant E233G of the RAD51D gene could be a low-penetrance allele in high-risk breast cancer families without BRCA1/2 mutations

Six SNPs have been detected in the DNA repair genes RAD51C and RAD51D, not previously characterized. The novel variant E233G in RAD51D is more highly represented in high-risk, site-specific, familial breast cancer cases that are not associated with the BRCA1/2 genes, with a frequency of 5.74% (n = 174) compared to a control population (n = 567) and another subset of breast cancer patients (n = 765) with a prevalence of around 2% only (comparison to controls, OR = 2.6, 95% CI 1.12-6.03; p < 0.021). We found that the immunohistochemical profile detected in available tumors from these patients differs slightly from those described in non-BRCA1/2 tumors. Finally, the structural prediction of the putative functional consequence of this change indicates that it can diminish protein stability and structure. This suggests a role for E233G as a low-penetrance susceptibility gene in the specific subgroup of high-risk familial breast cancer cases that are not related to BRCA1/2.     

Common nonsense mutations in RAD52.

RAD51, RAD52, and RAD54 encode proteins that are critical to the repair of double-strand DNA breaks by homologous recombination. The physical interactions among the products of RAD51, BRCA1, and BRCA2 have suggested that the BRCA1 and BRCA2 breast cancer susceptibility genes may function, at least in part, in this DNA damage repair pathway. Given the observation that different genes within a common functional pathway may be targeted by mutations in human cancers, we analyzed RAD51, RAD52, and RAD54 for the presence of germ-line mutations in 100 cases with early-onset breast cancer and for somatic mutations in 15 human breast cancer cell lines. Two premature stop codons, Ser346ter and Tyr415ter, were identified in germ-line RAD52 alleles from 5% of early-onset breast cancer cases. Together, these two heterozygous mutations were also found in 8% of a healthy control population, indicating that they do not confer an increased risk for breast cancer. A rare germ-line missense mutation was identified in RAD54, whereas no sequence variants were found in RAD51. None of the three RAD genes demonstrated somatic mutations in breast cancer cell lines. We conclude that, despite their potential functional association with the BRCA gene products, RAD51, RAD52, and RAD54 are not themselves targeted by mutations in human breast cancer. The presence of common nonsense mutations in RAD52 within the population may have significance for other conditions associated with potential alterations in DNA damage repair pathways.     

Mutation screening of RAD51C in high-risk breast and ovarian cancer families

Deleterious mutations in the RAD51C gene, which encodes a DNA double-strand break repair protein, have been reported to confer high-penetrance susceptibility to both breast and ovarian cancer. To confirm this we conducted a mutation screen of the RAD51C gene in 192 probands from high-risk breast and/or ovarian cancer families that do not carry BRCA1 or BRCA2 mutations. The nine exons of the RAD51C gene containing protein coding sequence were screened for mutations in genomic DNA from family probands by high-resolution melting analysis and direct DNA sequencing. Four missense variants, p.Ser364Gly, p.Ala126Thr, p.Val169Ala, and p.Thr287Ala were detected in six patients. The p.Ser364Gly variant is a novel variant predicted to have little influence on RAD51C activity. The p.Ala126Thr and p.Val169Ala variants have been reported to have no association with risk of breast cancer in a case–control study. However, p.Thr287Ala disrupts the DNA repair activity of RAD51C, suggesting some influence on risk. Consistent with published results from similar follow-up studies, we suggest that RAD51C mutations are rare events among high-risk breast cancer and breast/ovarian cancer families. Large population-based studies will be needed to reliably assess the prevalence and penetrance of inactivating mutations in the RAD51C susceptibility gene.     

BRIP1, PALB2, and RAD51C mutation analysis reveals their relative importance as genetic susceptibility factors for breast cancer

Mutations in the recognized breast cancer susceptibility genes BRCA1, BRCA2, TP53, ATM, and CHEK2 account for approximately 20% of hereditary breast cancer. This raises the possibility that mutations in other biologically relevant genes may be involved in genetic predisposition to breast cancer. In this study, BRIP1, PALB2, and RAD51C were sequenced for mutations as a result of previously being associated with breast cancer risk due to their role in the double-strand break repair pathway and their close association with BRCA1 and BRCA2. Two truncating mutations in PALB2 (Q66X and W1038X), one of which is has not been reported before, were detected in an independent Australian cohort of 70 individuals with breast or ovarian cancer, and have strong family histories of breast or breast/ovarian cancer. In addition, six missense variants predicted to be causative were detected, one in BRIP1 and five in PALB2. No causative variants were identified in RAD51C. This study supports recent observations that although rare, PALB2 mutations are present in a small but substantial proportion of inherited breast cancer cases, and indicates that RAD51C at a population level does not account for a substantial number of familial breast cancer cases.     

Mutation analysis of RAD51D in non-BRCA1/2 ovarian and breast cancer families

Background:

Recent data show that mutations in RAD51D have an aetiological role in ovarian carcinoma, yet mutations do not appear to be associated with an increased risk for breast cancer. We studied ovarian and breast cancer families having at least one woman affected by ovarian carcinoma, to assess the importance of RAD51D mutations in such families.

Methods:

The coding region of the RAD51D gene was analysed in 175 BRCA1/2-negative families with family histories of both ovarian and breast cancer ascertained from two Canadian and two Belgian institutions.

Results:

We identified one previously reported deleterious mutation, p.Arg186^* (c.556C>T), and two novel variants; missense substitution p.Cys119Arg and an intronic variant c.83-26A>G. p.Arg186^* segregated with the disease in the family and two ovarian carcinomas available for analysis showed loss of the wild-type allele, but the novel variants are likely neutral.

Conclusion:

RAD51D should be included in genetic screening of ovarian cancer families that do not have BRCA1/BRCA2 mutations. We show that mutations are more likely to be found in families with two or more ovarian cancers, or in probands with first-degree relatives with ovarian cancer, and we feel testing should be preferentially offered to affected women from such families.     

A single nucleotide polymorphism in the 5' untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers.

RAD51 colocalizes with both BRCA1 and BRCA2, and genetic variants in RAD51 would be candidate BRCA1/2 modifiers. We searched for RAD51 polymorphisms by sequencing 20 individuals. We compared the polymorphism allele frequencies between female BRCA1/2 mutation carriers with and without breast or ovarian cancer and between population-based ovarian cancer cases with BRCA1/2 mutations to cases and controls without mutations. We discovered two single nucleotide polymorphisms (SNPs) at positions 135 g-->c and 172 g-->t of the 5' untranslated region. In an initial group of BRCA1/2 mutation carriers, 14 (21%) of 67 breast cancer cases carried a "c" allele at RAD51:135 g-->c, whereas 8 (7%) of 119 women without breast cancer carried this allele. In a second set of 466 mutation carriers from three centers, the association of RAD51:135 g-->c with breast cancer risk was not confirmed. Analyses restricted to the 216 BRCA2 mutation carriers, however, showed a statistically significant association of the 135 "c" allele with the risk of breast cancer (adjusted odds ratio, 3.2; 95% confidence limit, 1.4-40). BRCA1/2 mutation carriers with ovarian cancer were only about one half as likely to carry the RAD51:135 g-->c SNP. Analysis of the RAD51:135 g-->c SNP in 738 subjects from an Israeli ovarian cancer case-control study was consistent with a lower risk of ovarian cancer among BRCA1/2 mutation carriers with the "c" allele. We have identified a RAD51 5' untranslated region SNP that may be associated with an increased risk of breast cancer and a lower risk of ovarian cancer among BRCA2 mutation carriers. The biochemical basis of this risk modifier is currently unknown.     

RAD51 up-regulation bypasses BRCA1 function and is a common feature of BRCA1-deficient breast tumors

The breast cancer susceptibility gene BRCA1 encodes a large protein thought to contribute to a variety of cellular processes, although the critical determinants of BRCA1-deficient tumorigenesis remain unclear. Given that BRCA1 is required for cell proliferation, suppressor mutations are believed to modify BRCA1 phenotypes and contribute to the etiology of BRCA1-deficient tumors. Here, we show that overexpression of the homologous recombinase RAD51 in a DT40 BRCA1Delta/Delta mutant rescues defects in proliferation, DNA damage survival, and homologous recombination (HR). In addition, epistasis analysis with BRCA1 and the DNA end-joining factor KU70 indicates that these factors operate independently of one another to repair double-strand breaks. Consistent with this genetic finding, cell synchronization studies show that the ability of BRCA1 to promote radioresistance is restricted to the late S and G2 phases of the cell cycle, as predicted for genes whose function is specific to homology-mediated repair rather than nonhomologous end-joining. Notably, retrospective analyses of microarray expression data reveal elevated expression of RAD51 and two of its late-acting cofactors, RAD54 and RAD51AP1, in BRCA1-deficient versus sporadic breast tumors. Taken together, our results indicate that up-regulation of HR provides a permissive genetic context for cells lacking BRCA1 function by circumventing its requirement in RAD51 subnuclear assembly. Furthermore, the data support a model in which enhanced HR activity contributes to the etiology of BRCA1-deficient tumors.     

Germline mutations in RAD51C in Jewish high cancer risk families

Germline mutations in BRCA1 and BRCA2 account for ~30 % of inherited breast cancer. RAD51C was reported as an additional breast/ovarian cancer susceptibility gene in some populations. There is a paucity of data on the putative contribution of this gene to inherited breast/ovarian cancer in Jewish high risk families. High risk Jewish women, none of whom was a carrier of the predominant Jewish mutations in BRCA1 or BRCA2, were screened for RAD51C germline mutations by direct sequencing of exons and flanking intronic sequences. Overall, 206 high risk women, 79 (38.3 %) of Ashkenazi origin, were genotyped for RAD51C mutations: 190 (92.3 %) with uni- or bilateral breast cancer (mean age at diagnosis 51.3 ± 11.1 years), 14 with ovarian cancer (mean age at diagnosis 55.6 ± 8.7 years), and two with both breast and ovarian cancer. No truncating mutations were noted, and two previously described missense mutations were detected: p.Ile144Thr and p.Thr287Ala in Iraqi and mixed ethnicity Balkan-North African participants, respectively. These missense mutations were evolutionarily conserved, possibly pathogenic, based on some prediction algorithms, and were not detected in any of healthy Iraqi (n = 60) and mixed ethnicity (n = 140), cancer free controls, respectively. Germline mutations in RAD51C contribute marginally to breast and ovarian cancer susceptibility in ethnically diverse, Jewish high risk families. The p.Thr287Ala missense mutation may be a recurring, pathogenic RAD51C mutation in ethnically diverse populations.     

RAD51 135G>C does not modify breast cancer risk in non-BRCA1/2 mutation carriers: evidence from a meta-analysis of 12 studies

A single-nucleotide polymorphism (SNP) in the 5′-untranslated region (UTR) of RAD51, 135G>C (rs1801320), was reported to be associated with an increased risk of breast cancer among BRCA2 as well as BRCA1 carriers. A few studies have also investigated the genetic contribution of RAD51 135G>C to the risk of sporadic breast cancers or breast cancer in non-BRCA1/2 carriers, though the results are yet controversial and inconclusive. We, in this study, performed a more precise estimation of the relationship between 135G>C and breast cancer among non-BRCA1/2 mutation carriers by meta-analyzing the currently available evidence from the literature. A total of 12 studies involving 7,065 cases and 6,981 controls were identified. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. When all the studies were pooled into the meta-analysis, there was no evidence for a significant association between 135G>C and breast cancer risk in non-BRCA1/2 mutation carriers (for CC vs. GG: OR = 0.995, 95%CI: 0.741–1.336; for GC vs. GG: OR = 0.959, 95%CI: 0.869–1.057; for dominant model: OR = 0.988, 95%CI: 0.902–1.082; and for recessive model: OR = 1.037, 95%CI: 0.782–1.376). We also performed subgroup analysis by ethnicity (Caucasian) as well as did analysis using the studies fulfilling Hardy–Weinberg equilibrium, and the results did not change. In summary, the present meta-analysis suggests that the RAD51 135G>C does not modify breast cancer risk in non-BRCA1/2 mutation carriers.     

RAD51C: a novel cancer susceptibility gene is linked to Fanconi anemia and breast cancer

Germline mutations in many of the genes that are involved in homologous recombination (HR)-mediated DNA double-strand break repair (DSBR) are associated with various human genetic disorders and cancer. RAD51 and RAD51 paralogs are important for HR and in the maintenance of genome stability. Despite the identification of five RAD51 paralogs over a decade ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remains obscure. In addition to the known roles of RAD51C in early and late stages of HR, it also contributes to activation of the checkpoint kinase CHK2. One recent study identifies biallelic mutation in RAD51C leading to Fanconi anemia-like disorder. Whereas a second study reports monoallelic mutation in RAD51C associated with increased risk of breast and ovarian cancer. These reports show RAD51C is a cancer susceptibility gene. In this review, we focus on describing the functions of RAD51C in HR, DNA damage signaling and as a tumor suppressor with an emphasis on the new roles of RAD51C unveiled by these reports.     

About 1% of the breast and ovarian Spanish families testing negative for BRCA1 and BRCA2 are carriers of RAD51D pathogenic variants

RAD51D mutations have been recently identified in breast (BC) and ovarian cancer (OC) families. Although an etiological role in OC appears to be present, the association of RAD51D mutations and BC risk is more unclear. We aimed to determine the prevalence of germline RAD51D mutations in Spanish BC/OC families negative for BRCA1/BRCA2 mutations. We analyzed 842 index patients: 491 from BC/OC families, 171 BC families, 51 OC families and 129 patients without family history but with early‐onset BC or OC or metachronous BC and OC. Mutation detection was performed with high‐resolution melting, denaturing high‐performance liquid chromatography or Sanger sequencing. Three mutations were found in four families with BC and OC cases (0.82%). Two were novel: c.1A>T (p.Met1?) and c.667+2_667+23del, leading to the exon 7 skipping and one previously described: c.674C>T (p.Arg232*). All were present in BC/OC families with only one OC. The c.667+2_667+23del cosegregated in the family with one early‐onset BC and two bilateral BC cases. We also identified the c.629C>T (p.Ala210Val) variant, which was predicted in silico to be potentially pathogenic. About 1% of the BC and OC Spanish families negative for BRCA1/BRCA2 are carriers of RAD51D mutations. The presence of several BC mutation carriers, albeit in the context of familial OC, suggests an increased risk for BC, which should be taken into account in the follow‐up and early detection measures. RAD51D testing should be considered in clinical setting for families with BC and OC, irrespective of the number of OC cases in the family.     

Deleterious RAD51C germline mutations rarely predispose to breast and ovarian cancer in Pakistan

RAD51C plays a key role in homologous recombination-mediated DNA repair and maintenance of genomic stability. Biallelic RAD51C mutations cause Fanconi anemia, and monoallelic mutations predispose women to breast and ovarian cancer. Genetic variability of RAD51C and its impact in Asian populations have been poorly studied. Here, we report the results of comprehensive mutational screening of the RAD51C gene in 348 BRCA1/2-negative breast and/or ovarian cancer patients from Pakistan. Mutation analysis of the complete RAD51C-coding region was performed using denaturing high-performance liquid chromatography analysis, followed by DNA sequencing of variant fragments. Three novel protein-truncating mutations, c.204T>A, c.225T>G, and c.701C>G, were identified. c.204T>A was found in one out of 22 (4.5 %) early-onset (≤45 years of age) ovarian cancer patients and c.225T>G in one out of 119 (0.8 %) patients from breast cancer only families. c.701C>G was found in a 60-year-old control with no family history of breast/ovarian cancer. Furthermore, three novel in silico-predicted potentially functional mutations, a missense mutation, c.873T>G, a variant in 5′UTR, c.1-34T>G, and a recurrent intronic variant, c.965+21A>G, were identified. The missense mutation was observed in a patient with bilateral breast cancer from a breast and ovarian cancer family (HBOC), the 5′UTR variant was noted in an early-onset breast cancer patient, and the intronic variant in one early-onset breast cancer patient and one ovarian cancer patient from a HBOC family. Five of the six mutations described were not detected in 400 healthy controls. These findings suggest that RAD51C plays a marginal role in breast and ovarian cancer predisposition in Pakistan. Reliable estimation of the clinical implications of carrying a deleterious RAD51C mutation will require identification of additional mutation-positive patients/families.     

A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2

Genes mutated in patients with Fanconi anemia (FA) interact with the DNA repair genes BRCA1 and BRCA2/FANCD1 to suppress tumorigenesis, but the molecular functions ascribed to them cannot fully explain all of their cellular roles. Here, we show a repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation. Fork protection is surprisingly rescued in FANCD2-deficient cells by elevated RAD51 levels or stabilized RAD51 filaments. Moreover, FANCD2-mediated fork protection is epistatic with RAD51 functions, revealing an unanticipated fork protection pathway that connects FA genes to RAD51 and the BRCA1/2 breast cancer suppressors. Collective results imply a unified molecular mechanism for repair-independent functions of FA, RAD51, and BRCA1/2 proteins in preventing genomic instability and suppressing tumorigenesis.     

The CHEK2*1100delC variant acts as a breast cancer risk modifier in non-BRCA1/BRCA2 multiple-case families

The frame-shifting mutation 1100delC in the cell-cycle-checkpoint kinase 2 gene (CHEK2) has been reported to be associated with familial breast cancer in families in which mutations in BRCA1 and BRCA2 were excluded. To investigate the role of this variant as a candidate breast cancer susceptibility allele, we determined its prevalence in 237 breast cancer patients and 331 healthy relatives derived from 71 non-BRCA1/BRCA2 multiple-case early onset breast cancer families. Twenty-seven patients (11.4%) were carrying the CHEK2*1100delC variant. At least one carrier was found in 15 of the 71 families (21.1%). There was no evidence of cosegregation between the variant and breast cancer, but carrier patients developed breast cancer earlier than did noncarriers. We studied CHEK2 protein expression in 111, and loss of heterozygosity at CHEK2 in 88 breast tumors from these patients. Twelve of 15 tumors from carriers showed absent protein expression as opposed to 3 of 76 tumors from noncarriers (P < 0.001). CHEK2 loss of heterozygosity was associated with absence of protein expression but not with 1100delC carrier status. Thus, selecting for breast cancer cases with a strong familial background not accounted for by BRCA1 or BRCA2 strongly enriches for carriers of CHEK2*1100delC. Our results support a model in which CHEK2*1100delC interacts with an as yet unknown gene (or genes) to increase breast cancer risk.     

Variation in the RAD51 gene and familial breast cancer

Introduction  

Human RAD51 is a homologue of the Escherichia coli RecA protein and is known to function in recombinational repair of double-stranded DNA breaks. Mutations in the lower eukaryotic homologues of RAD51 result in a deficiency in the repair of double-stranded DNA breaks. Loss of RAD51 function would therefore be expected to result in an elevated mutation rate, leading to accumulation of DNA damage and, hence, to increased cancer risk. RAD51 interacts directly or indirectly with a number of proteins implicated in breast cancer, such as BRCA1 and BRCA2. Similar to BRCA1 mice, RAD51 ^-/- mice are embryonic lethal. The RAD51 gene region has been shown to exhibit loss of heterozygosity in breast tumours, and deregulated RAD51 expression in breast cancer patients has also been reported. Few studies have investigated the role of coding region variation in the RAD51 gene in familial breast cancer, with only one coding region variant – exon 6 c.449G>A (p.R150Q) – reported to date.     

Immunohistochemical expression of DNA repair proteins in familial breast cancer differentiate BRCA2-associated tumors.

PURPOSE: Morphologic and immunohistochemical studies of familial breast cancers have identified specific characteristics associated with BRCA1 mutation-associated tumors when compared with BRCA2 and non-BRCA1/2 tumors, but have not identified differences between BRCA2 and non-BRCA1/2 tumors. Because BRCA1 and BRCA2 genes participate in the DNA repair pathway, we have performed an immunohistochemical study with markers related to this pathway to establish the profile of the three groups. MATERIALS AND METHODS: We have studied two tissue microarrays that include 103 familial and 104 sporadic breast tumors, with a panel of DNA repair markers including ATM, CHEK2, RAD51, RAD50, XRCC3, and proliferating cell nuclear antigen. RESULTS: We found more frequent expression of CHEK2 in BRCA1 and BRCA2 tumors than in non-BRCA1/2 and sporadic tumors. We found absence of nuclear expression and presence of cytoplasmic expression of RAD51 in BRCA2 tumors that differentiate them from other familial tumors. We validated these results with a new series of patient cases. The final study with 253 familial patient cases (74 BRCA1, 71 BRCA2, 108 non-BRCA1/2), and 288 sporadic patient cases, has allowed us to confirm our preliminary results. Because BRCA2 tumors present a specific immunohistochemical profile for RAD51 and CHEK2 markers that is different from non-BRCA1/2 tumors, we have built a multivariate model with these markers that distinguish both tumors with an estimated probability of at least 76%. CONCLUSION: Our results suggest that BRCA2 tumors demonstrate more cytoplasmic and less nuclear RAD51 staining, and increased CHEK2 staining. This pattern may distinguish BRCA2 from familial non-BRCA1/2 tumors.     

A HRM-based screening method detects RAD51C germ-line deleterious mutations in Spanish breast and ovarian cancer families

The RAD51C gene has been recently proposed as a high-penetrance breast and ovarian cancer gene. However, early replication studies have failed to confirm the finding. Thus, further studies in larger cohorts should be conducted in order to clarify the role of RAD51C as a cancer susceptibility gene. Here, we describe a high-resolution melting analysis (HRMA)-based method developed for presequence screening of RAD51C sequence variants. We have screened RAD51C sequence variants by HRMA in 492 breast cancer patients with family history of breast and/or ovarian cancer that were previously tested negative for BRCA1/2. All variants were confirmed by direct sequencing. We have detected 12 different RAD51C germ-line sequence variants, including eight transitions, two transversion, and two indels (insA, and delT). All these variants generated melting profiles which differ from wild type homozygous controls. Interestingly, we have identified one clearly pathogenic mutation (c.774delT) in the subset of 101 breast and ovarian cancer families, supporting that RAD51C is a human breast and ovarian cancer susceptibility gene.