Genetics of Breast and Ovarian Cancer Predisposition with a Focus on RAD51C and RAD51D Genes

Breast and ovarian cancers are common cancers affecting women. Family history of the disease is a major risk factor for both cancers. Even though several susceptibility alleles have been identified, genetic predisposition to breast and ovarian cancer is still largely unexplained. Most of the known risk-genes play a role in DNA repair and especially in homologous recombination repair. RAD51C, RAD51D, and RAD54L are genes involved in homologous recombination and rare mutations in RAD51C and RAD51D have been identified in breast and ovarian cancer families. With the growing number of identified risk-genes and the development of efficient next-generation sequencing methods, multigene-panels have now largely replaced traditional BRCA1 and BRCA2 testing in clinical genetic testing of breast and ovarian cancer patients. The aim of this study was to identify pathogenic germline mutations in RAD51C, RAD51D, and RAD54L in breast and ovarian cancer families and to evaluate the association of the identified mutations with breast, ovarian, prostate, and colorectal cancer risk in the Finnish population. In addition, we studied the mutation spectrum of 10 known susceptibility genes in 95 high-risk breast or ovarian cancer patients with a gene panel. Sequencing of the RAD51C gene in 277 and the RAD51D and RAD54L genes in 95 familial breast or ovarian cancer patients revealed two protein-truncating mutations in RAD51C, c.93delG and c.837+1G>A, and one in RAD51D, c.576+1G>A, which were subsequently genotyped in breast (n ≈ 2000), ovarian (n ≈ 500), prostate (n ≈ 1000), and colorectal (n ≈ 1000) cancer patients and population controls (n ≈ 2000). Mutations in both genes were enriched in ovarian cancer patients. The mutation frequency was significantly increased among patients with a personal or family history of ovarian cancer, but did not significantly differ between unselected breast cancer patients and controls. No mutations were observed in prostate or colorectal cancer patients. These results suggest that germline mutations in RAD51C and RAD51D increase the risk of ovarian cancer, but not the overall risk of breast cancer nor prostate or colorectal cancer risk. The gene-panel testing identified 12 different pathogenic mutations in 18 patients (19%), including 2 patients with two different protein-truncating mutations. BRCA1 or BRCA2 mutations, including one large duplication in BRCA1, were identified in 8 patients (8.4%) and mutations in other genes in 10 patients (10.5%). In addition, a novel duplication covering the RAD51C exons 1–7 was identified. Together, the BRCA1 and RAD51C duplications accounted for approximately 10% of all the observed pathogenic or potentially pathogenic mutations. Genotyping of the RAD51C duplication in breast (n ≈ 2500) and ovarian (n ≈ 500) cancer patients and population controls (n ≈ 1000) revealed seven carriers among the cases, but none among the controls and a significant association with ovarian cancer risk. This thesis study establishes RAD51C as an ovarian cancer susceptibility gene, presents information that strengthens the role of RAD51D mutations in ovarian cancer predisposition, and provides valuable new knowledge on the associated cancer risks for both genes. The results also highlight the importance of comprehensive mutation testing of all the relevant susceptibility genes in the clinical genetic testing of breast and ovarian cancer patients.Rinta- ja munasarjasyövät ovat yleisiä naisten syöpiä ja sukutausta on molempien syöpien merkittävä riskitekijä. Vaikka monia alttiusgeenejä on tunnistettu, perinnöllisen rinta- ja munasarjasyöpäalttiuden geneettinen tausta on vielä suurelta osin selittämättä. Tehokkaiden uudenpolven sekvensointimenetelmien, kuten geenipaneelien, kehitys on mahdollistanut useiden alttiusgeenien samanaikaisen tutkimisen ja geenipaneelit ovatkin nykyään laajalti käytössä myös potilaiden kliinisessä geenitestauksessa. Tunnistetut mutaatiot voivat ohjata potilaiden syöpähoitojen valintaa ja terveille mutaationkantajille voidaan tarjota ennaltaehkäiseviä toimenpiteitä sekä tarkempaa seurantaa. Tämän väitöskirjatyön tavoitteena oli tunnistaa rinta- ja munasarjasyöpäsuvuista haitallisia mutaatiota RAD51C-, RAD51D- ja RAD54L-geeneistä sekä selvittää löydettyjen mutaatioiden vaikutusta rinta-, munasarja-, eturauhas- ja suolistosyövän riskiin suomalaisessa väestössä. RAD51C- ja RAD51D-geeneistä on aiemmin löydetty harvinaisia mutaatioita rinta-munasarjasyöpäsuvuissa ja RAD54L toimii samalla DNA:n korjausreitillä näiden geenien kanssa. Lisäksi kartoitimme korkean riskin rinta- ja munasarjasyöpäpotilailla esiintyviä mutaatioita geenipaneelin avulla. Sekvensoimalla RAD51C-, RAD51D- ja RAD54L-geenit rinta- tai munasarjasyöpäpotilaiden verestä eristetyissä DNA-näytteissä tunnistimme kaksi erilaista RAD51C-mutaatiota ja yhden RAD51D-mutaation. Näiden mutaatioiden seulonta yli 4000 syöpäpotilaan ja yli 2000 väestökontrollin aineistossa paljasti mutaatioita erityisesti munasarjasyöpäpotilailla sekä niillä rintasyöpäpotilailla, joilla oli munasarjasyövän sukutaustaa. Eturauhas- tai suolistosyöpäpotilailta mutaatioita ei löytynyt eikä rintasyöpäpotilailla esiintynyt mutaatioita useammin kuin väestökontrolleilla. Tulokset viittaavat siihen, että perityt mutaatiot RAD51C- ja RAD51D-geeneissä lisäävät riskiä sairastua munasarjasyöpään, mutta eivät nosta rintasyövän kokonaisriskiä tai riskiä sairastua eturauhas- tai suolistosyöpään. Geenipaneelilla testatuista 95 potilaasta 18:lla (19 %) löytyi vähintään yksi haitallinen mutaatio jossakin tutkituista geeneistä. Tärkeimpien alttiusgeenien BRCA1:n tai BRCA2:n mutaatiota löytyi kahdeksalla potilaalla (8,4 %) ja muiden alttiusgeenien mutaatioita kymmenellä potilaalla (10,5 %). Lisäksi löysimme yhdeltä potilaalta suuren, aiemmin tunnistamattoman, genomisen muutoksen: duplikaation, jossa lähes koko RAD51C-geeni oli kahdentunut. Noin 3000 rinta- tai munasarjasyöpäpotilaan ja yli 1000 väestökontrollin seulonnassa duplikaatioita löytyi merkittävästi enemmän munasarjasyöpäpotilailta kuin kontrolleilta, mikä viittaa siihen, että duplikaatio lisää munasarjasyövän riskiä. Suomalaisista munasarjasyöpäpotilaista arviolta noin 2 % kantaa joko tunnistamaamme RAD51C-duplikaatiota tai RAD51C- tai RAD51D-mutaatiota. Tässä väitöskirjatyössä saatiin uutta tietoa RAD51C- ja RAD51D-mutaatioihin liittyvistä syöpäriskeistä. Tutkimuksen tulokset osoittavat RAD51C:n olevan munasarjasyövän alttiusgeeni ja vahvistavat RAD51D:n roolia munasarjasyöpäalttiudessa. Lisäksi tulokset korostavat laajan ja kattavan mutaatiotestauksen merkitystä rinta- ja munasarjasyöpäpotilaiden kliinisessä diagnostiikassa, jolloin mutaatiot kaikissa tunnetuissa riskigeeneissä voidaan tunnistaa samanaikaisesti ja myös suuret genomiset muutokset testataan

Ovarian and Breast Cancer Risks Associated With Pathogenic Variants in RAD51C and RAD51D

Background: The purpose of this study was to estimate precise age-specific tubo-ovarian carcinoma (TOC) and breast cancer (BC) risks for carriers of pathogenic variants in RAD51C and RAD51D. Methods: We analyzed data from 6178 families, 125 with pathogenic variants in RAD51C, and 6690 families, 60 with pathogenic variants in RAD51D. TOC and BC relative and cumulative risks were estimated using complex segregation analysis to model the cancer inheritance patterns in families while adjusting for the mode of ascertainment of each family. All statistical tests were two-sided. Results: Pathogenic variants in both RAD51C and RAD51D were associated with TOC (RAD51C: relative risk [RR] = 7.55, 95% confidence interval [CI] = 5.60 to 10.19; P = 5 x 10(-40); RAD51D: RR = 7.60, 95% CI = 5.61 to 10.30; P = 5 x 10(-39)) and BC (RAD51C: RR =1.99, 95% CI = 1.39 to 2.85; P = 1.55 x 10(-4); RAD51D: RR = 1.83, 95% CI = 1.24 to 2.72; P = .002). For both RAD51C and RAD51D, there was a suggestion that the TOC relative risks increased with age until around age 60 years and decreased thereafter. The estimated cumulative risks of developing TOC to age 80 years were 11% (95% CI = 6% to 21%) for RAD51C and 13% (95% CI = 7% to 23%) for RAD51D pathogenic variant carriers. The estimated cumulative risks of developing BC to 80 years were 21% (95% CI = 15% to 29%) for RAD51C and 20% (95% CI = 14% to 28%) for RAD51D pathogenic variant carriers. Both TOC and BC risks for RAD51C and RAD51D pathogenic variant carriers varied by cancer family history and could be as high as 32-36% for TOC, for carriers with two first-degree relatives diagnosed with TOC, or 44-46% for BC, for carriers with two first-degree relatives diagnosed with BC. Conclusions: These estimates will facilitate the genetic counseling of RAD51C and RAD51D pathogenic variant carriers and justify the incorporation of RAD51C and RAD51D into cancer risk prediction models.Peer reviewe

Screening of HELQ in breast and ovarian cancer families

Several high and moderate risk alleles have been identified for breast and ovarian cancer predisposition and most of them encode proteins that function in DNA repair. A prospective candidate for breast and ovarian cancer susceptibility is the HELQ helicase that has a role in the resolution of DNA interstrand cross-links. HELQ interacts with the RAD51 paralog complex BCDX2. Two components of the complex, RAD51C and RAD51D, increase the risk of ovarian cancer especially, and the other two, RAD51B and XRCC2 have been associated with breast cancer risk. To investigate the role of HELQ in cancer predisposition, we screened the gene for germline variation in 185 Finnish breast or ovarian cancer families and performed haplotype analyses for 1517 breast cancer cases, 308 ovarian cancer cases, and 1234 population controls using five common polymorphisms at the HELQ gene locus. No truncating mutations were identified among the families. One putatively pathogenic missense mutation c.1309A > G was identified but no additional carriers were observed in the subsequent genotyping of 332 familial breast or ovarian cancer patients. Furthermore, the haplotype distribution did not differ between breast or ovarian cancer cases and population controls. Our results indicate that HELQ is not a major breast and ovarian cancer susceptibility gene in the Finnish population. However, we cannot rule out rare risk-variants in the Finnish or other populations and larger datasets are needed to further assess the role of HELQ especially in ovarian cancer predisposition.Peer reviewe

RAD51B in Familial Breast Cancer

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk.Public Library of Science open acces

Screening of Finnish RAD51C founder mutations in prostate and colorectal cancer patients

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RAD51, XRCC3, and XRCC2 mutation screening in Finnish breast cancer families

Majority of the known breast cancer susceptibility genes have a role in DNA repair and the most important high-risk genes BRCA1 and BRCA2 are specifically involved in the homologous recombination repair (HRR) of DNA double-strand breaks. A central player in HRR is RAD51 that binds DNA at the damage site. The RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 facilitate the binding of RAD51 to DNA. While germline mutations in RAD51C and RAD51D are associated with high ovarian cancer risk and RAD51B polymorphisms with breast cancer, the contribution of RAD51, XRCC3, and XRCC2 is more unclear. To investigate the role of RAD51, XRCC3, and XRCC2 in breast cancer predisposition and to identify putative recurrent founder mutations in the Finnish population where such mutations have been observed in most of the currently known susceptibility genes, we screened 182 familial Finnish breast or ovarian cancer patients for germline variation in the RAD51 and XRCC3 genes and 342 patients for variation in XRCC2, with a subset of the patients selected on the basis of decreased RAD51 protein expression on tumors. We also performed haplotype analyses for 1516 breast cancer cases and 1234 controls to assess the common variation in these genes. No pathogenic mutations were detected in any of the genes and the distribution of haplotypes was similar between cases and controls. Our results suggest that RAD51, XRCC3, and XRCC2 do not substantially contribute to breast cancer predisposition in the Finnish population.Peer reviewe

Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer

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FANCM mutation c.5791C > T is a risk factor for triple-negative breast cancer in the Finnish population

The FANCM c.5101C > T nonsense mutation was previously found to associate with breast cancer in the Finnish population, especially among triple-negative cases. Here, we studied the prevalence of three other FANCM variants: c.5791C > T, which has been reported to predispose to familial breast cancer, and the c.4025_4026delCT and c.5293dupA variants recently identified in Finnish cancer patients. We genotyped the FANCM c.5791C > T mutation in 4806 invasive breast cancer patients, including BRCA1/2 mutation negative familial cases and unselected cases, and in 2734 healthy population controls from four different geographical areas of Finland. The association of the mutation with breast cancer risk among patient subgroups was statistically evaluated. We further analyzed the combined risk associated with c.5101C > T and c.5791C > T mutations. We also genotyped 526 unselected ovarian cancer patients for the c.5791C > T mutation and 862 familial breast cancer patients for the c.4025_4026delCT and c.5293dupA variants. The frequency of the FANCM c.5791C > T mutation was higher among breast cancer cases than in controls (OR 1.94, 95% CI 0.87-4.32, P = 0.11), with a statistically significant association with triple-negative breast cancer (OR 5.14, 95% CI 1.65-16.0, P = 0.005). The combined analysis for c.5101C > T and c.5791C > T carriers confirmed a strong association with breast cancer (OR 1.86, 95% CI 1.32-2.49, P = 0.0002), especially among the triple-negative patients (OR 3.08, 95% CI 1.77-5.35, P = 0.00007). For the other variants, only one additional c.4025_4026delCT carrier and no c.5293dupA carriers were observed. These results support the role of FANCM as a breast cancer susceptibility gene, particularly for triple-negative breast cancer.Peer reviewe

Recurrent moderate-risk mutations in Finnish breast and ovarian cancer patients

Mutations in BRCA1 and BRCA2 genes predispose to breast and ovarian cancer (BC/OC) with a high lifetime risk, whereas mutations in PALB2, CHEK2, ATM, FANCM, RAD51C and RAD51D genes cause a moderately elevated risk. In the Finnish population, recurrent mutations have been identified in all of these genes, the latest being CHEK2 c.319+2T>A and c.444+1G>A. By genotyping 3,156 cases and 2,089 controls, we estimated the frequencies of CHEK2 c.319+2T>A and c.444+1G>A in Finnish BC patients. CHEK2 c.319+2T>A was detected in 0.7% of the patients, and it was associated with a high risk of BC in the unselected patient group (OR = 5.40 [95% CI 1.58–18.45], p = 0.007) and similarly in the familial patient group. CHEK2 c.444+1G>A was identified in 0.1% of all patients. Additionally, we evaluated the combined prevalence of recurrent moderate‐risk gene mutations in 2,487 BC patients, 556 OC patients and 261 BRCA1/2 carriers from 109 families. The overall frequency of the mutations was 13.3% in 1,141 BRCA1/2‐negative familial BC patients, 7.5% in 1,727 unselected BC patients and 7.2% in 556 unselected OC patients. At least one moderate‐risk gene mutation was found in 12.5% of BRCA1 families and 7.1% of BRCA1 index patients, as well as in 17.0% of BRCA2 families and 11.3% of BRCA2 index patients, and the mutations were associated with an additional risk in the BRCA1/2 index patients (OR = 2.63 [1.15–5.48], p = 0.011). These results support gene panel testing of even multiple members of BC families where several mutations may segregate in different individuals.</p

Recurrent moderate-risk mutations in Finnish breast and ovarian cancer patients

Mutations in BRCA1 and BRCA2 genes predispose to breast and ovarian cancer (BC/OC) with a high lifetime risk, whereas mutations in PALB2, CHEK2, ATM, FANCM, RAD51C and RAD51D genes cause a moderately elevated risk. In the Finnish population, recurrent mutations have been identified in all of these genes, the latest being CHEK2 c.319+2T>A and c.444+1G>A. By genotyping 3,156 cases and 2,089 controls, we estimated the frequencies of CHEK2 c.319+2T>A and c.444+1G>A in Finnish BC patients. CHEK2 c.319+2T>A was detected in 0.7% of the patients, and it was associated with a high risk of BC in the unselected patient group (OR = 5.40 [95% CI 1.58-18.45], p = 0.007) and similarly in the familial patient group. CHEK2 c.444+1G>A was identified in 0.1% of all patients. Additionally, we evaluated the combined prevalence of recurrent moderate-risk gene mutations in 2,487 BC patients, 556 OC patients and 261 BRCA1/2 carriers from 109 families. The overall frequency of the mutations was 13.3% in 1,141 BRCA1/2-negative familial BC patients, 7.5% in 1,727 unselected BC patients and 7.2% in 556 unselected OC patients. At least one moderate-risk gene mutation was found in 12.5% of BRCA1 families and 7.1% of BRCA1 index patients, as well as in 17.0% of BRCA2 families and 11.3% of BRCA2 index patients, and the mutations were associated with an additional risk in the BRCA1/2 index patients (OR = 2.63 [1.15-5.48], p = 0.011). These results support gene panel testing of even multiple members of BC families where several mutations may segregate in different individuals.Peer reviewe