ClinVar Genomic variation as it relates to human health

This sequence change affects an acceptor splice site in intron 4 of the RAD51C gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs587780259, gnomAD 0.005%). Disruption of this splice site has been observed in individual(s) with breast cancer and ovarian cancer (PMID: 22006311, 22538716, 24549055, 25452441, 26261251, 26681312, 29255180). ClinVar contains an entry for this variant (Variation ID: 128209). Studies have shown that disruption of this splice site results in skipping of exon 5, but is expected to preserve the integrity of the reading-frame (PMID: 22006311, 24139550; Invitae). This variant disrupts a region of the RAD51C protein in which other variant(s) (p.Arg258His) have been determined to be pathogenic (PMID: 20400963, 22167183, 26354865). This suggests that this is a clinically significant region of the protein, and that variants that disrupt it are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The c.706-2A>G intronic pathogenic mutation results from an A to G substitution two nucleotides upstream from coding exon 5 of the RAD51C gene. This alteration has been reported in multiple individuals with personal and/or family history of breast and/or ovarian cancer (Meindl A et al. Nat. Genet. 2010 May;42:410-4; Walsh T et al. Proc. Natl. Acad. Sci. USA. 2011 Nov;108:18032-7; Loveday C et al. Nat. Genet. 2012 May;44:475-6; Golmard L et al. BMC Cancer. 2013 Oct;13:484; Castéra L et al. Eur. J. Hum. Genet. 2014 Nov;22:1305-13; Song H et al. J. Clin. Oncol. 2015 Sep;33:2901-7; Couch FJ et al. J. Clin. Oncol. 2015 Feb;33:304-11; Susswein LR et al. Genet. Med. 2016 Aug;18(8):823-32; Kotoula V et al. Am J Cancer Res. 2017 Jan;7:98-114; Harter P et al. PLoS ONE. 2017 Oct;12:e0186043; Golmard L et al. Eur. J. Hum. Genet. 2017 12;25:1345-1353). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and will result in the creation or strengthening of a novel splice acceptor site. RNA analyses have shown that this mutation leads to skipping of exon 5, resulting in an in-frame deletion of 44 amino acid residues including the Walker B box in the functionally important RecA domain (Walsh T et al. Proc. Natl. Acad. Sci. USA. 2011 Nov;108:18032-7; Golmard L et al. BMC Cancer. 2013 Oct;13:484; Davy G et al. Eur. J. Hum. Genet. 2017 10;25:1147-1154; Ambry internal data). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

This variant was determined to be pathogenic according to ACMG Guidelines, 2015 [PMID:25741868].

Variant summary: RAD51C c.706-2A>G is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Five predict the variant abolishes the canonical 3' splice acceptor site. Experimental evidence demonstrated that this variant affects mRNA splicing leading to the loss of 44 amino acids in a functional domain of the protein by an in-frame exon 5 skipping (Golmard_2013). The variant allele was found at a frequency of 2e-05 in 251368 control chromosomes. c.706-2A>G has been reported in the literature in multiple individuals affected with Breast and Ovarian Cancer (example, Couch_2015, Golmard_2013). These data indicate that the variant is very likely to be associated with disease. Multiple clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 with complete concordance as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

The RAD51C c.706-2A>G variant is predicted to disrupt the AG splice acceptor site and interfere with normal splicing. This variant was reported in an individual with peritoneal carcinoma, an individual with squamous cell carcinoma of the head and neck, an individual with pancreatic cancer, individuals with breast and/or ovarian cancer, individuals undergoing hereditary cancer genetic testing, (Table S2, Walsh et al. 2011. PubMed ID: 22006311; Table 1, Loveday et al. 2012. PubMed ID: 22538716; Table 2, Scheckenbach et al. 2013. PubMed ID: 24315737; Table S1, Susswein et al. 2015. PubMed ID: 26681312; Table 2, Yurgelun et al. 2018. PubMed ID: 29961768; Table 1, Li et al. 2019. PubMed ID: 30949688;). RT-PCR analysis and minigene assays suggest this variant results in the in-frame skipping of exon 5 (Table 1, Davy et al. 2017. PubMed ID: 28905878; Sanoguera-Miralles et al. 2020. PubMed ID: 33333735). This variant is reported in 0.0046% of alleles in individuals of European (Non-Finnish) descent in gnomAD (http://gnomad.broadinstitute.org/variant/17-56787218-A-G). It is interpreted as pathogenic and likely pathogenic in ClinVar (https://preview.ncbi.nlm.nih.gov/clinvar/variation/128209/). Variants that disrupt the consensus splice acceptor site in RAD51C are expected to be pathogenic. This variant is interpreted as pathogenic.

Canonical splice site variant demonstrated to result in an in-frame loss of the adjacent exon (PMID: 35740625) in a gene for which loss of function is a known mechanism of disease; Observed in individuals with breast and ovarian cancer as well as an individual with a chordoma (PMID: 22006311, 22538716, 24139550, 25452441, 26261251, 29255180, 29053726, 28123851, 28888541, 30441849, 30949688, 34326862, 29522266, 36495689, 38700789); Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 25470109, 24240112, 28123851, 29053726, 29143133, 30949688, 24315737, 22006311, 22538716, 24139550, 24549055, 25452441, 26261251, 26681312, 26720728, 29255180, 28152038, 28776603, 28905878, 31173646, 30441849, 30322717, 31589614, 33333735, 29961768, 32107557, 32359370, 20400964, 22167183, 26354865, 21990120, 20400963, 35740625, 14704354, 34326862, 29922827, 28888541, 29522266, 36495689, 38700789)

RAD51C: PVS1, PS3:Supporting, PS4:Supporting

PVS1, PS3, PS4_STR, PP1

This variant is considered likely pathogenic. This variant occurs within a consensus splice junction and is predicted to result in abnormal mRNA splicing of either an out-of-frame exon or an in-frame exon necessary for protein stability and/or normal function.

This variant causes an A to G nucleotide substitution at the -2 position of intron 4 of the RAD51C gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. RNA studies have shown that this variant causes the skipping of exon 5, resulting in an in-frame deletion of 44 amino acids including the Walker B motif in the ATP-binding domain of the RAD51C protein (PMID: 22006311, 24139550, 28905878, 33333735). This variant is expected to cause a defect in RAD51C protein function. This variant has been reported in many individuals affected with breast or ovarian cancer (PMID: 22006311, 22538716, 24139550, 25452441, 26261251, 26681312, 29053726, 29255180). This variant has been identified in 6/282746 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Loss of RAD51C function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic.

The RAD51C c.706-2A>G variant was identified in 12 of 15014 proband chromosomes (frequency: 0.0008) from individuals or families with breast and ovarian cancer (Castera 2014, Couch 2015, Golmard 2013, Loveday 2012, Song 2015). The variant was also identified in the following databases: dbSNP (ID: rs587780259) as "With Pathogenic allele", ClinVar (5x pathogenic, 2x likely pathogenic), Clinvitae, and LOVD 3.0 (1x). The variant was not identified in Cosmic or the MutDB database. The variant was identified in control databases in 6 of 277092 chromosomes at a frequency of 0.00002 (Genome Aggregation Database Feb 27, 2017). It was observed in the European population in 6 of 126600 chromosomes (freq: 0.00005), but not in the African, Other, Latino, Ashkenazi Jewish, East Asian, Finnish, or South Asian populations. Functional studies utilizing mRNA from lymphoblastoid cell lines shows this variant affects splicing, resulting in in-frame skipping of exon 5 (Golmard 2013, Davy 2017). The c.706-2A>G variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. In addition, 5 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic.

Curator: Arleen D. Auerbach. Submitter to LOVD: Johan den Dunnen.