The Genetic and Molecular Analyses of RAD51C and RAD51D Identifies Rare Variants Implicated in Hereditary Ovarian Cancer from a Genetically Unique Population

Abstract

To identify candidate variants in RAD51C and RAD51D ovarian cancer (OC) predisposing genes by investigating French Canadians (FC) exhibiting unique genetic architecture. Candidates were identified by whole exome sequencing analysis of 17 OC families and 53 early-onset OC cases. Carrier frequencies were determined by the genetic analysis of 100 OC or HBOC families, 438 sporadic OC cases and 1025 controls. Variants of unknown function were assayed for their biological impact and/or cellular sensitivity to olaparib. RAD51C c.414G>C;p.Leu138Phe and c.705G>T;p.Lys235Asn and RAD51D c.137C>G;p.Ser46Cys, c.620C>T;p.Ser207Leu and c.694C>T;p.Arg232Ter were identified in 17.6% of families and 11.3% of early-onset cases. The highest carrier frequency was observed in OC families (1/44, 2.3%) and sporadic cases (15/438, 3.4%) harbouring RAD51D c.620C>T versus controls (1/1025, 0.1%). Carriers of c.620C>T (n = 7), c.705G>T (n = 2) and c.137C>G (n = 1) were identified in another 538 FC OC cases. RAD51C c.705G>T affected splicing by skipping exon four, while RAD51D p.Ser46Cys affected protein stability and conferred olaparib sensitivity. Genetic and functional assays implicate RAD51C c.705G>T and RAD51D c.137C>G as likely pathogenic variants in OC. The high carrier frequency of RAD51D c.620C>T in FC OC cases validates previous findings. Our findings further support the role of RAD51C and RAD51D in hereditary OC.

Genetic analyses of DNA repair pathway associated genes implicate new candidate cancer predisposing genes in ancestrally defined ovarian cancer cases

“…For the discovery of new candidate OC risk variants (study phase I; Figure 1A ), WES data from peripheral blood lymphocytes (PBL) DNA was available from 15 OC cases from 13 families, each family having at least two first-, second- or third-degree relatives with OC. These cases were confirmed negative for PVs in the known OC risk genes: BRCA1 , BRCA2, BRIP1, RAD51C or RAD51D as previously reported ( 47 , 43 ). This group includes three index cases harbouring a LPV in FANCI c.1813C>T; p.Leu605Phe ( 24 ).…”

“…Targeted analyses of the candidate variants was performed to determine their carrier frequencies (study phase II; Figure 1B ) on the PBL DNA from FC OC cases, regardless of their carrier status for BRCA1 and BRCA2 PVs, from 42 hereditary breast and ovarian cancer (HBOC) syndrome families having one OC and at least two BC cases in the same familial branch and 435 sporadic OC cases not selected for age at diagnosis with the disease or for family history of any cancers. Genetic data was available from 1025 population-matched controls provided by three independent biobanks as previously described ( 43 ). As known OC predisposing genes are also involved in BC risk (nccn.org/guidelines/category_2), targeted analyses of the candidate variants was also performed on the PBL DNA from FC BC cases, regardless of their BRCA1 and BRCA2 PV carrier status, from 33 HBOC families, 139 hereditary breast cancer (HBC) syndrome families having at least three close relatives with BC within first-, second- or third-degree of relationship from the same familial branch and 563 sporadic BC cases not selected for age at diagnosis with the disease or for family history of any cancers.…”

“…Investigating these populations facilitates the characterization of deleterious variants in known or candidate cancer predisposing genes as all types of variants could be investigated and not only LoF variants ( 33 ). A small number of PVs in BRCA1 and BRCA2 ( 40 , 41 ) and one in each of PALB2 ( 42 ), RAD51C ( 43 ) and RAD51D ( 43 , 44 ) have been shown to be frequently occurring in FC OC and/or BC cases versus population-matched controls. Specific PVs in MLH1 ( 45 ) , MSH2 ( 45 ) and MSH6 ( 46 ) have also been reported in FCs in the context of hereditary non-polyposis colorectal cancer.…”

“…We recently reported that not all remaining BRCA1 and BRCA2 negative families with at least two close relatives with OC from the FC population of Quebec by WES analysis were due to PVs in RAD51C , RAD51D or BRIP1 approximating that 40% of such cases are unaccounted for by known or emerging OC predisposing genes ( 33 , 43 , 47 ). Also, we reported that likely pathogenic variants (LPV) in FANCI , a proposed new OC predisposing gene from the Fanconi anemia (FA) pathway, were rarely implicated in familial and sporadic OC cases in this population ( 24 ).…”

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C

“…The reclassification of VUS is essential to ensure appropriate patient care, and functional assays provide critical information for their interpretation [ 8 , 9 , 10 , 11 ]. RNA splicing is one of the gene expression steps that may be impaired by genetic variants [ 12 , 13 ].…”