Functional analysis of germline RAD51C missense variants highlight the role of RAD51C in replication fork protection.

Monoallelic or biallelic RAD51C germline mutations results in chromosome instability disorders such as Fanconi anemia and cancers. The bona fide function of RAD51C is to assist RAD51 nucleoprotein filament onto single-strand DNA to complete homologous recombination (HR) repair. In addition to HR repair, the role of RAD51C in DNA replication is emerging when replication forks are transiently or irreversibly stalled. We identified novel RAD51C variants of uncertain significance (VUS) from breast, ovarian, pancreatic and gastric cancer patients and functionally characterized the effect of these variants in replication fork protection and double-strand breaks (DSB's) repair. In RAD51C-deficient Chinese hamster CL-V4B cells, expression of RAD51C F164S, A87E, L134S and E49K variants heightened sensitivity to mitomycin C (MMC), etoposide and PARP inhibition. Differently, expression of subset of RAD51C variants R24L, R24W and R212H displayed mild sensitivity to MMC, etoposide and PARP inhibition. Further functional characterization of a subset of variants revealed that Rad51C F164S, A87E, L134S and E49K variants displayed reduced RAD51 foci formation and increased overall nuclear single strand DNA levels in the presence of replication stress. Additionally, DNA fiber assay revealed that RAD51C F164S, A87E, L134S and E49K variants displayed defective replication fork protection upon prolonged fork stalling. Investigations using patient-derived lymphoblastoid cell line carrying heterozygous RAD51C L134S variant showed an impairment in RAD51 chromatin association and replication fork protection, suggestive of deleteriousness of this VUS variant. Overall, our findings provide more insights into molecular roles of RAD51C in replication fork integrity maintenance and in DSB repair.

Homologous recombination-deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants.

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Primary fallopian tube cancer followed by primary breast cancer in RAD51C mutation carrier treated with niraparib as first line maintenance therapy: a case report.

Given the rarity of RAD51C mutations, the risk and treatment of metachronous breast cancer after the diagnosis of ovarian cancer in RAD51C mutation carriers is not clear, especially for those who have received PARPi treatment. We report the case of a 65-year-old woman diagnosed with stage IIIC high-grade serous primary fallopian tube cancer. The patient had no family history of breast or ovarian cancer. The patient received three cycles of neoadjuvant chemotherapy with paclitaxel and carboplatin and achieved a complete response. After interval debulking surgery, the patient received three cycles of adjuvant chemotherapy. Collection and extraction of saliva DNA for next-generation sequencing identified a RAD51C mutation c.838-2 A > G. The patient received niraparib as front-line maintenance treatment. After 36 months of niraparib treatment, the patient had grade II invasive ductal carcinoma of the left breast that was positive for estrogen receptor (90%) and Ki-67 (30%) and negative for progesterone receptor and human epidermal growth factor receptor 2. Computed tomography revealed the absence of distant metastases. Modified radical mastectomy and axillary lymph node dissection were then performed. The final pathological report of the breast showed a 1.8 cm Bloom-Richardson grade II invasive ductal carcinoma in the left breast with axillary lymph node metastasis (1/21). Finally, the breast cancer was stage IIA, pT1cN1M0. The metachronous breast cancer in this case may be the first report of second primary cancer in fallopian tube cancer patient harboring a RAD51C mutation during niraparib treatment. Further studies are required to determine optimal treatment.

RAD51C-RAD51D interplays with MSH5 and regulates crossover maturation in rice meiosis.

Meiotic crossovers ensure accurate chromosome segregation and increase genetic diversity. RAD51C and RAD51D play an early role in facilitating RAD51 during homologous recombination. However, their later function in meiosis is largely unknown in plants. Here, through targeted disruption of RAD51C and RAD51D, we generated three new mutants and revealed their later meiotic role in crossover maturation. The rad51c-3 and rad51d-4 mutants showed a mixture of bivalents and univalents and no chromosomal entanglements, whereas rad51d-5 exhibited an intermediate phenotype with reduced chromosomal entanglements and increased bivalent formation compared with knockout alleles. Comparisons of RAD51 loadings and chromosomal entanglements in these single mutants, rad51c-3 rad51d-4, rad51c-3 dmc1a dmc1b, and rad51d-4 dmc1a dmc1b suggest that the retained level of RAD51 in mutants is required for uncovering their function in crossover formation. Reductions in chiasma frequency and later HEI10 foci in these mutants support that crossover maturation requires RAD51C and RAD51D. Moreover, interaction between RAD51D and MSH5 indicates that RAD51 paralogs may cooperate with MSH5 to ensure accurate Holliday junction processing into crossover products. This finding of the role of RAD51 paralogs in crossover control may be conserved from mammals to plants and advances our current understanding of these proteins.

Hereditary cancer predispositions: Comparison of multigene panel sequencing on fresh-frozen breast/ovarian tumor versus blood.

In breast or ovarian cancer (BC/OC) patients with evocative personal and/or family history, multigene panel sequencing is performed on blood to diagnose hereditary predispositions. Additionally, BRCA1/BRCA2 testing can be performed on tumor sample for therapeutic purpose. The accuracy of multigene panel tumor analysis on BC/OC to detect predisposing germline pathogenic variants (gPV) has not been precisely assessed. By comparing sequencing data from blood and fresh-frozen tumor we show that tumor genomic instability causes pitfalls to consider when performing tumor testing to detect gPV. Even if loss of heterozygosity increases germline signal in most cases, somatic copy number variants (CNV) can mask germline CNV and collapse point gPV variant allele frequency (VAF). Moreover, VAF does not allow an accurate distinction between germline and somatic pathogenic variants.

Patterns of care and outcomes of risk reducing surgery in women with pathogenic variants in non-BRCA and Lynch syndrome ovarian cancer susceptibility genes.

OBJECTIVES: Guidelines recommend risk-reducing bilateral salpingo-oophorectomy (RRSO) for women with pathogenic variants of non-BRCA and Lynch syndrome-associated ovarian cancer susceptibility genes. Optimal timing and findings at the time of RRSO for these women remains unclear. We sought to characterize practice patterns and frequency of occult gynecologic cancers for these women at our two institutions. METHODS: Women with germline ovarian cancer susceptibility gene pathogenic variants who underwent RRSO between 1/2000-9/2019 were reviewed in an IRB-approved study. All patients were asymptomatic with no suspicion for malignancy at time of RRSO. Clinico-pathologic characteristics were extracted from the medical records. RESULTS: 26 Non-BRCA (9 BRIP1, 9 RAD51C, and 8 RAD51D) and 75 Lynch (36 MLH1, 18 MSH2, 21 MSH6) pathogenic variants carriers were identified. Median age at time of RRSO was 47. There were no occurrences of occult ovarian or fallopian tube cancer in either group. Two patients (3%) in the Lynch group had occult endometrial cancer. Median follow up was 18 and 35 months for non-BRCA and Lynch patients, respectively. No patient developed primary peritoneal cancer upon follow up. Post-surgical complications occurred in 9/101 (9%) of patients. Hormone replacement therapy (HRT) was rarely used despite reported post-menopausal symptoms in 6/25 (23%) and 7/75 (37%) patients, respectively. CONCLUSIONS: No occult ovarian or tubal cancers were observed in either group. No recurrent or primary gynecologic-related cancers occurred upon follow-up. Despite frequent menopausal symptoms, HRT use was rare. Both groups experienced surgical complications when hysterectomy and/or concurrent colon surgery was performed suggesting concurrent surgeries should only be performed when indicated.

Radiation-Induced Lymphopoenia and Treatment Outcome in Hereditary Breast Cancer Patients.

Many breast cancer (BC) predisposition genes encode proteins involved in DNA damage repair (DDR). Identification of germline pathogenic va-riants (PV) in DDR genes raises the question whether their presence can influence the treatment outcomes and potential radiation-induced toxicity in their carriers treated by adjuvant radiotherapy, which has not yet been answered conclusively. We retrospectively examined records of 213 BC patients treated by adjuvant radiotherapy, including 39 (18.3 %) BRCA1/2 PV carriers, 25 carriers (11.7 %) of PV in other breast cancer-predisposing genes, and 149 (70 %) non-carriers. Our goal was to examine 5-year disease-free survival (5y DFS) rates among the study groups and determine the impact of radiotherapy-induced lymphopoenia (RIL) on this outcome. While we found no significant difference in 5y DFS between non-carriers and carriers of BRCA mutations (86.4 % vs 78.4 % P = 0.24) or between non-carriers and other studied mutations (86.4 % vs 93.3 %; P = 0.27), respectively, we observed that the entire group of PV carriers had a significantly lower proportion of patients without RIL (P = 0.04) than the non-carriers. In contrast, subsequent analyses indicated a non-significant trend toward an increased 5y DFS in PV carriers with RIL. Our single-centre study indicated that the presence of PV in BC patients has an insignificant impact on DFS but can reduce the risk of RIL associated with adjuvant radiotherapy. It remains unclear whether this may result from the paradoxical activation of anti-tumour immunity in PV carriers with higher lymphocyte consumption resulting from higher immune effectiveness.

A decade of RAD51C and RAD51D germline variants in cancer.

Defects in DNA repair genes have been extensively associated with cancer susceptibility. Germline pathogenic variants (GPV) in genes involved in homologous recombination repair pathways predispose to cancers arising mainly in the breast and ovary, but also other tissues. The RAD51 paralogs RAD51C and RAD51D were included in this group 10 years ago when germline variants were associated with non-BRCA1/2 familial ovarian cancer. Here, we have reviewed the landscape of RAD51C and RAD51D germline variants in cancer reported in the literature during the last decade, integrating this list with variants identified by in-house patient screening. A comprehensive catalog of 341 variants that have been classified applying ACMG/AMP criteria has been generated pinpointing the existence of recurrent variants in both genes. Recurrent variants have been extensively discussed compiling data on population frequencies and functional characterization if available, highlighting variants that have not been fully characterized yet to properly establish their pathogenicity. Finally, we have complemented this data with relevant information regarding the conservation of mutated residues among RAD51 paralogs and modeling of putative hotspot areas, which contributes to generating an exhaustive update on these two cancer predisposition genes.

Association of recurrent mutations in BRCA1, BRCA2, RAD51C, PALB2, and CHEK2 with the risk of borderline ovarian tumor.

BACKGROUND: There are several genes associated with ovarian cancer risk. Molecular changes in borderline ovarian tumor (BOT) indicate linkage of this disease to type I ovarian tumors (low-grade ovarian carcinomas). This study determined the prevalence and association of mutations in BRCA1, BRCA2, PALB2, RAD51C, and CHEK2 with the risk of BOTs. METHODS: The study group consisted of 102 patients with histologically confirmed BOT and 1743 healthy controls. In addition, 167 cases with ovarian cancer G1 were analyzed. The analyses included genotyping of 21 founder and recurrent mutations localized in 5 genes (BRCA1, BRCA2, PALB2, RAD51C, and CHEK2). The risk for developing BOT and low-grade ovarian cancer, as well as the association of tested mutations with survival, was estimated. RESULTS: The CHEK2 missense mutation (c.470T>C) was associated with 2-times increased risk of BOT (OR=2.05, p=0.03), at an earlier age at diagnosis and about 10% worse rate of a 10-year survival. Mutations in BRCA1 and PALB2 were associated with a high risk of ovarian cancer G1 (OR=8.53, p=0.005 and OR=7.03, p=0.03, respectively) and were related to worse all-cause survival for BRCA1 carriers (HR=4.73, 95%CI 1.45-15.43, p=0.01). CONCLUSIONS: Results suggest that CHEK2 (c.470T>C) may possibly play a role in the pathogenesis of BOT, but due to the low number of BOT patients, obtained results should be considered as preliminary. Larger more in-depth studies are required.

TAZ maintains telomere length in TNBC cells by mediating Rad51C expression.

BACKGROUND: Telomere maintenance is crucial for the unlimited proliferation of cancer cells and essential for the "stemness" of multiple cancer cells. TAZ is more extensively expressed in triple negative breast cancers (TNBC) than in other types of breast cancers, and promotes proliferation, transformation and EMT of cancer cells. It was reported that TAZ renders breast cancer cells with cancer stem cell features. However, whether TAZ regulates telomeres is still unclear. In this study, we explored the roles of TAZ in the regulation of telomere maintenance in TNBC cells. METHODS: siRNA and shRNA was used to generate TAZ-depleted TNBC cell lines. qPCR and Southern analysis of terminal restriction fragments techniques were used to test telomere length. Co-immunoprecipitation, Western blotting, immunofluorescence, Luciferase reporter assay and Chromatin-IP were conducted to investigate the underlying mechanism. RESULTS: By knocking down the expression of TAZ in TNBC cells, we found, for the first time, that TAZ is essential for the maintenance of telomeres in TNBC cells. Moreover, loss of TAZ causes senescence phenotype of TNBC cells. The observed extremely shortened telomeres in late passages of TAZ knocked down cells correlate with an elevated hTERT expression, reductions of shelterin proteins, and an activated DNA damage response pathway. Our data also showed that depletion of TAZ results in overexpression of TERRAs, which are a group of telomeric repeat-containing RNAs and regulate telomere length and integrity. Furthermore, we discovered that TAZ maintains telomere length of TNBC cells likely by facilitating the expression of Rad51C, a crucial element of homologous recombination pathway that promotes telomere replication. CONCLUSIONS: This study supports the notion that TAZ is an oncogenic factor in TNBC, and further reveals a novel telomere-related pathway that is employed by TAZ to regulate TNBC.

Association of RAD51C germline mutations with breast cancer among Bahamians.

PURPOSE: RAD51C is known as an ovarian cancer gene; however, its role in breast cancer susceptibility is less clear. As part of a larger study, we assessed the role of germline RAD51C mutations in breast cancer development. METHODS: We studied 387 unselected, BRCA1- and BRCA2-negative, Bahamian breast cancer cases and 653 controls to search for novel genetic associations with breast cancer development. During the first phase of the study, whole exome sequencing was utilized in 96 cases to identify an association between novel genes and breast cancer susceptibility. In the second phase of the study, targeted gene sequencing was utilized in the entirety of the cases and controls to identify an association between novel genetic mutations and breast cancer development. RESULTS: A RAD51C mutation was found in five breast cancer cases and in no control (5/387 versus 0/653; p = 0.007). None of the mutation-positive cases reported a family history of ovarian cancer. CONCLUSIONS: These data support increasing evidence that RAD51C mutations contribute to breast cancer susceptibility, although the impact may vary substantially from country to country.

BRCA1 promoter methylation in breast cancer patients is associated with response to olaparib/eribulin combination therapy.

BACKGROUND: A PARP inhibitor is effective in breast cancer patients with BRCA1/2 germline mutations, and in cell lines with BRCA1 promoter methylation. However, its efficacy in breast cancer patients with BRCA1 promoter methylation is still unknown. METHODS: Biopsy samples were obtained from 32 triple-negative breast cancer (TNBC) patients treated with eribulin/olaparib combination therapy in a clinical trial (UMINID: 000009498) and analyzed for their mutations by FoundationOne CDx. DNA methylation was evaluated by quantitative methylation-specific PCR and bisulfite sequencing, and its level was adjusted for tumor cell fraction. RESULTS: Among 20 TNBC patients evaluable for both methylation and mutations, one (5%) and five (25%) patients had a high (> 80%) and low (30-80%) BRCA1 promoter methylation levels, respectively. One patient with a high methylation level, also having a BRCA2 mutation of unknown significance, displayed complete response. Among the 5 patients with low methylation levels, only one patient with a BRCA2 mutation of unknown significance displayed long-lasting disease control (24 weeks). Patients with a BRCA1 or BRCA2 mutation, or high BRCA1 promoter methylation showed better 6-month progression-free survival (PFS) compared with the other patients (P = 0.009). CONCLUSION: Quantitative methylation analysis suggested that addition of homozygous BRCA1 promoter methylation to mutations may more accurately identify TNBC patients who would benefit from olaparib/eribulin combination therapy. (209 words).

Patients with pathogenic variants for breast cancer other than BRCA1 and BRCA2: qualitative interviews about health care experiences.

BACKGROUND: Genetic testing for hereditary cancer syndromes has been revolutionized by next-generation sequencing, which allows for simultaneous review of numerous genes. Multigene panels are regularly offered to patients because of their scope and decreased cost and turnaround time. However, many genes included on larger panels have not been studied as extensively as BRCA1 and BRCA2 (BRCA1/2), and their clinical effects are often not as well established. METHODS: We identified patients who received positive test results for pathogenic variants of breast cancer genes from January 2012 through May 2018. We mailed a survey and conducted qualitative interviews to explore the personal and health care experiences of patients with pathogenic variants of BRCA1/2 and patients with "other" (ie, non-BRCA1/2 or PALB2; PTEN; ATM; TP53; NBM, RAD51C; MSH6) variants. We compared the experiences of these patients. RESULTS: Fifty-nine out of 128 individuals responded to the survey (46%). Thirty-two patients had BRCA1/2 variants, and 27 had other variants. (49 women and 10 men; median [range] age, 63 [34-87] years). We interviewed 21 patients (17 women and 4 men; median [range] age, 59.6 [34-82] years). Of the interview participants, ten patients had BRCA1/2 variants, and 11 had non-BRCA1/2 variants. Patients reported receiving poor information about their genetic test results, and they often educated their physicians about their results. Some patients believed that they had been ignored or "brushed off" by health care professionals because non-BRCA1/2 genes are less understood outside the genetics research community. Patients with BRCA1/2 variants had similar problems with health care providers, despite increased awareness and established guidelines about BRCA1/2. CONCLUSIONS: Research is required to understand the clinical significance and proper management of diseases attributable to newly characterized hereditary cancer genes. Additional evaluation of patient and provider education should be at the forefront of efforts to improve patient care.

ZmRAD51C is Essential for Double-Strand Break Repair and Homologous Recombination in Maize Meiosis.

Radiation sensitive 51 (RAD51) recombinases play crucial roles in meiotic double-strand break (DSB) repair mediated by homologous recombination (HR) to ensure the correct segregation of homologous chromosomes. In this study, we identified the meiotic functions of ZmRAD51C, the maize homolog of Arabidopsis and rice RAD51C. The Zmrad51c mutants exhibited regular vegetative growth but complete sterility for both male and female inflorescence. However, the mutants showed hypersensitivity to DNA damage by mitomycin C. Cytological analysis indicated that homologous chromosome pairing and synapsis were rigorously inhibited, and meiotic chromosomes were often entangled from diplotene to metaphase I, leading to chromosome fragmentation at anaphase I. Immunofluorescence analysis showed that although the signals of the axial element absence of first division (AFD1) and asynaptic1 (ASY1) were normal, the assembly of the central element zipper1 (ZYP1) was severely disrupted. The DSB formation was normal in Zmrad51c meiocytes, symbolized by the regular occurrence of γH2AX signals. However, RAD51 and disrupted meiotic cDNA 1 (DMC1) signals were never detected at the early stage of prophase I in the mutant. Taken together, our results indicate that ZmRAD51C functions crucially for both meiotic DSB repair and homologous recombination in maize.

Gene-panel testing of breast and ovarian cancer patients identifies a recurrent RAD51C duplication.

Gene-panel sequencing allows comprehensive analysis of multiple genes simultaneously and is now routinely used in clinical mutation testing of high-risk breast and ovarian cancer patients. However, only BRCA1 and BRCA2 are often analyzed also for large genomic changes. Here, we have analyzed 10 clinically relevant susceptibility genes in 95 breast or ovarian cancer patients with gene-panel sequencing including also copy number variants (CNV) analysis for genomic changes. We identified 12 different pathogenic BRCA1, BRCA2, TP53, PTEN, CHEK2, or RAD51C mutations in 18 of 95 patients (19%). BRCA1/2 mutations were observed in 8 patients (8.4%) and CHEK2 protein-truncating mutations in 7 patients (7.4%). In addition, we identified a novel duplication encompassing most of the RAD51C gene. We further genotyped the duplication in breast or ovarian cancer families (n = 1149), in unselected breast (n = 1729) and ovarian cancer cohorts (n = 553), and in population controls (n = 1273). Seven additional duplication carries were observed among cases but none among controls. The duplication associated with ovarian cancer risk (3/590 of all ovarian cancer patients, 0.5%, P = .032 compared with controls) and was found to represent a large fraction of all identified RAD51C mutations in the Finnish population. Our data emphasizes the importance of comprehensive mutation analysis including CNV detection in all the relevant genes.

Routine germline BRCA1 and BRCA2 testing in patients with ovarian carcinoma: analysis of the Scottish real-life experience.

OBJECTIVE: To determine the rates of germline BRCA1 and BRCA2 mutations in Scottish patients with ovarian cancer, before and after a change in testing policy. DESIGN: Retrospective cohort study. SETTING: Four cancer/genetics centres in Scotland. POPULATION: Patients with ovarian cancer undergoing germline BRCA1 and BRCA2 (gBRCA1/2) sequencing before 2013 (under the 'old criteria', with selection based solely on family history), after 2013 (under the 'new criteria', with sequencing offered to newly presenting patients with non-mucinous ovarian cancer), and in the 'prevalent population' (who presented before 2013, but were not eligible for sequencing under the old criteria but were sequenced under the new criteria). METHODS: Clinicopathological and sequence data were collected before and for 18 months after this change in selection criteria. MAIN OUTCOME MEASURES: Frequency of germline BRCA1, BRCA2, RAD51C, and RAD51D mutations. RESULTS: Of 599 patients sequenced, 205, 236, and 158 were in the 'old criteria', 'new criteria', and 'prevalent' populations, respectively. The frequency of gBRCA1/2 mutations was 30.7, 13.1, and 12.7%, respectively. The annual rate of gBRCA1/2 mutation detection was 4.2 before and 20.7 after the policy change. A total of 48% (15/31) 'new criteria' patients with gBRCA1/2 mutations had a Manchester score of <15 and would not have been offered sequencing based on family history criteria. In addition, 20 patients with gBRCA1/2 were identified in the prevalent population. The prevalence of gBRCA1/2 mutations in patients aged >70 years was 8.2%. CONCLUSIONS: Sequencing all patients with non-mucinous ovarian cancer gives a much higher annual gBRCA1/2 mutation detection rate, with the frequency of positive tests still exceeding the 10% threshold upon which many family history-based models operate. TWEETABLE ABSTRACT: BRCA sequencing all non-mucinous cancer patients increases mutation detection five fold.

Metastatic Prostate Cancer in a RAD51C Mutation Carrier.

A man, aged 61 years, with a history of hypogonadism and family history of cancer experienced persistent urinary difficulties with no visible prostate abnormalities. Laboratory testing and diagnostic imaging revealed a primary lesion in the prostate with lymph node involvement and multiple bone metastases. Treatment with androgen-deprivation therapy, 17,20-lyase inhibition, and bisphosphonates for 7 months was unsuccessful in preventing disease progression, but second-line chemotherapy and continued androgen-deprivation therapy improved prostate specific antigen levels. During the patient's second treatment regimen, his daughter received a diagnosis of breast cancer. The patient's daughter underwent genetic testing for oncogenic mutations, and it was discovered that she carried a mutation in RAD51C, a gene encoding a protein involved in DNA repair and genomic maintenance. Subsequent genetic testing of the patient revealed mutation in RAD51C as well. For patients with metastatic prostate cancer who are unresponsive to standard treatment and who have a positive family history of cancer, genetic testing may be warranted to develop alternative treatment regimens for the patient and guide family discussions regarding cancer risk. Targeted agents like poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors may be a consideration in prostate cancer patients with DNA repair mutations and with refractory disease.

Mutation status of RAD51C, PALB2 and BRIP1 in 100 Japanese familial breast cancer cases without BRCA1 and BRCA2 mutations.

In addition to BRCA1 and BRCA2, RAD51C, PALB2 and BRIP1 are known as breast cancer susceptibility genes. However, the mutation status of these genes in Japanese familial breast cancer cases has not yet been evaluated. To this end, we analyzed the exon sequence and genomic rearrangement of RAD51C, PALB2 and BRIP1 in 100 Japanese patients diagnosed with familial breast and ovarian cancer and without BRCA1 and BRCA2 mutations. We detected a large deletion from exons 6 to 9 in RAD51C, 4 novel BRIP1 missense variants containing 3 novel non-synonymous variants, c.89A>C, c.736A>G and c.2131A>G, and a splice donor site variant c.918+2T>C. No deleterious variant of PALB2 was detected. The results of pedigree analysis showed that the proband with a large deletion on RAD51C had a family history of both breast and ovarian cancer, and the families of probands with novel BRIP1 missense variants included a male patient with breast cancer or many patients with breast cancer within the second-degree relatives. We showed that the mutation frequency of RAD51C in Japanese familial breast cancer cases was similar to that in Western countries and that the prevalence of deleterious mutation of PALB2 was possibly lower. Furthermore, our results suggested that BRIP1 mutation frequency in Japan might differ from that in Western countries.

Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function.

Rad51C-ATXN7 fusion gene expression in colorectal tumors.

BACKGROUND: Fusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown. METHODS: We evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques. RESULTS: We identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1-7 and ATXN7 exons 6-13), and a Variant 2 (between Rad51C exons 1-6 and ATXN7 exons 6-13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR. CONCLUSION: In conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.