ClinVar Genomic variation as it relates to human health

ACMG classification criteria: PS3 supporting, PS4 strong, PP1 strong

PS3, PM2, PP3

Variant summary: RAD51D c.620C>T (p.Ser207Leu) results in a non-conservative amino acid change located in the DNA recombination and repair protein Rad51-like, C-terminal and AAA+ ATPase domain of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 2.8e-05 in 253526 control chromosomes (gnomAD and publication). c.620C>T has been reported in the literature in multiple individuals affected with in multiple individuals affected with Breast, Endometrial, Ovarian cancer and Urothelial carcinoma (example, Loveday_2011, Wickramanayake_2012, Golmard_2013, Rivera_2017, Tung_2016, Tung_2015, Shirts_2016, Nassar_2020). In a conservative assessment of the transmission of this variant as ascertained from the literature, we captured at-least 12 transmissions of the variant allele and 2 transmissions of the reference allele to affected individuals. This variant was reported as being highly prevalent in French Canadians and as being associated with a high risk for ovarian high-grade serous carcinoma (HGSC) (3.8% cases vs. 0.2% controls), but was not associated with risk for breast, endometrial, pancreas or colorectal cancers (Rivera_2017). The authors conclude this variant as a bona fide pathogenic RAD51D missense cancer susceptibility allele and is one of the most frequently observed alleles conferring high risk of ovarian high-grade serous carcinoma (HGSC) in the French Canadian population of Quebec. These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function (Rivera_2017). The most pronounced variant effect results in a disruption the RAD51D-XRCC2 interaction, impaired homologous recombination and sensitivity to PARP-inhibitor therapies. Nine clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Multiple laboratories reported the variant with conflicting assessments (pathogenic, n=1; likely pathogenic, n=5; VUS, n=3). Some submitters cite overlapping evidence utilized in the context of this evaluation and have re-classified this variant to the pathogenic spectrum since its previous evaluation. Based on the evidence outlined above, the variant was classified as pathogenic.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with susceptibility to familial breast-ovarian cancer 4 (MIM#614291). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0112 - The condition associated with this gene has incomplete penetrance. This gene is described as having moderate penetrance (PMID: 26057125). (I) 0200 - Variant is predicted to result in a missense amino acid change from serine to leucine. (I) 0251 - This variant is heterozygous. (I) 0302 - Variant is present in gnomAD (v2) <0.001 for a dominant condition (8 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2) (3 heterozygotes, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0600 - Variant is located in the annotated Rad51 domain (PDB). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported multiple times as a VUS, likely pathogenic and pathogenic, where recent classifications are consistently in support of pathogenicity. This variant results in an increased risk of cancer development, and is enriched in patient cohorts with ovarian, endometrial, breast and urothelial cancers (ClinVar, PMID: 28646019). (SP) 1002 - This variant has moderate functional evidence supporting abnormal protein function. HEK293 cells transfected with this variant have demonstrated impaired homologous repair and interrupted RAD51D-XRCC2 interaction (PMID: 28646019). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

This variant is considered likely pathogenic. This variant has been reported in multiple individuals with clinical features of gene-specific disease [PMID: 28646019]. Functional studies indicate this variant impacts protein function [PMID: 28646019].

This sequence change replaces serine, which is neutral and polar, with leucine, which is neutral and non-polar, at codon 95 of the RAD51D protein (p.Ser95Leu). This variant is present in population databases (rs370228071, gnomAD 0.005%). This missense change has been observed in individual(s) with ovarian, endometrial cancer, peritoneal carcinoma, and breast cancer (PMID: 21822267, 22986143, 25186627, 26845104, 26976419, 28646019). It has also been observed to segregate with disease in related individuals. ClinVar lists this variant (Interpretation: Conflicting interpretations of pathogenicity; Pathogenic (4)|Likely pathogenic(10)|Uncertain significance(3); Variation ID: 142102).. A Published functional studies suggest a damaging effect: impaired homologous recombination activity (Rivera et al., 2017); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Experimental studies have shown that this missense change affects RAD51D function (PMID: 16717288, 28646019). For these reasons, this variant has been classified as Likely Pathogenic. Heterozygous germline variant in the RAD51D gene associated with susceptibility to familial breast-ovarian cancer type 4 . Loveday et al., 2011 investigated the role of RAD51D in cancer susceptibility and identified the risk of ovarian cancer for RAD51D variant carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 x 10(-6)). By contrast, the authors estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). They also demonstrated that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D variant carriers. According to the NCCN Guidelines V.3.2023. the absolute risk for breast cancer in individuals with pathogenic RAD51D variant is 20-40%, for epithelial ovarian cancer 10-20%, and uknown risk for other cancer types. Mode of Inheritance: Autosomal dominant (OMIM®: 614291).

Criteria applied: PS3,PS4_mod,PM5,PM2_SUP

The RAD51D c.620C>T (p.Ser207Leu) variant has been reported in the published literature in individuals with breast cancer (PMID: 35710434 (2022), 34606182 (2021), 33471991 (2021), 26976419 (2016), 25186627 (2015), 24139550 (2013)), ovarian cancer (PMID: 35565380 (2022), 31922703 (2019), 26845104 (2016)), and bladder cancer (PMID: 31844177 (2020)). It is also reported to be a prevalent variant in French Canadian women with breast and ovarian cancer (PMID: 28646019 (2017), 34923718 (2022)). Experimental studies indicate that the variant is deleterious to RAD51D protein function (PMID: 28646019 (2017)). The frequency of this variant in the general population, 0.000046 (6/129132 chromosomes (Genome Aggregation Database, http://gnomad.broadinstitute.org)), is uninformative in the assessment of its pathogenicity. Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, this variant is classified as pathogenic.

This missense variant replaces serine with leucine at codon 207 in the highly conserved Walker B motif of the RAD51D protein that is required for ATPase function and XRCC2 binding. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have reported that this variant protein disrupts normal XRCC2 binding and RAD51 foci formation and results in impaired homology-mediated DNA repair in cell-based assays (PMID: 28646019). This variant has been observed in multiple individuals affected with breast cancer, ovarian cancer, endometrial cancer and urothelial carcinoma (PMID: 21822267, 22986143, 24139550, 25186627, 28646019, 31844177, 34433815, 34606182, 35565380, 35710434) and has been reported to segregate with diseases in at least two families (PMID: 28646019). This variant also has been reported in a breast cancer case-control meta-analysis in 3/60466 cases and 1/53461 unaffected individuals (PMID: 33471991; Leiden Open Variation Database DB-ID RAD51D_000118). This variant has been identified in 8/282800 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic.

This sequence change replaces serine, which is neutral and polar, with leucine, which is neutral and non-polar, at codon 207 of the RAD51D protein (p.Ser207Leu). This variant is present in population databases (rs370228071, gnomAD 0.005%). This missense change has been observed in individual(s) with ovarian, endometrial cancer, peritoneal carcinoma, and breast cancer (PMID: 21822267, 22986143, 25186627, 26845104, 26976419, 28646019). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 142102). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt RAD51D protein function with a positive predictive value of 80%. Experimental studies have shown that this missense change affects RAD51D function (PMID: 16717288, 28646019). For these reasons, this variant has been classified as Pathogenic.

The RAD51D c.620C>T; p.Ser207Leu variant (rs370228071) is reported in the literature in several individuals and families affected with breast and/or ovarian cancer (Akbar 2022, Fu 2021, Golmard 2013, Loveday 2011, Rivera 2017, Wickramanayake 2012) and is a founder variant in the French-Canadian population (Boni 2022). This variant is reported in ClinVar (Variation ID: 142102), and is found in the general population with an overall allele frequency of 0.0028% (8/282,800 alleles) in the Genome Aggregation Database. Functional analyses of the variant protein show impaired RAD51D-XRCC2 binding and defects in DNA repair (Rivera 2017). Computational analyses predict that this variant is deleterious (REVEL: 0.819). Based on available information, this variant is considered to be likely pathogenic. References: Akbar F et al. Spectrum of germline pathogenic variants using a targeted next generation sequencing panel and genotype-phenotype correlations in patients with suspected hereditary breast cancer at an academic medical centre in Pakistan. Hered Cancer Clin Pract. 2022 Jun 16;20(1):24. PMID: 35710434. Boni J et al. A decade of RAD51C and RAD51D germline variants in cancer. Hum Mutat. 2022 Mar;43(3):285-298. PMID: 34923718. Fu F et al. Association between 15 known or potential breast cancer susceptibility genes and breast cancer risks in Chinese women. Cancer Biol Med. 2021 Oct 5;19(2):253–62. PMID: 34606182. Golmard L et al. Germline mutation in the RAD51B gene confers predisposition to breast cancer. BMC Cancer. 2013 Oct 19;13:484. PMID: 24139550. Loveday C et al. Germline mutations in RAD51D confer susceptibility to ovarian cancer. Nat Genet. 2011 Aug 7;43(9):879-882. PMID: 21822267. Rivera B et al. Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma. Cancer Res. 2017 Aug 15;77(16):4517-4529. PMID: 28646019. Wickramanayake A et al. Loss of function germline mutations in RAD51D in women with ovarian carcinoma. Gynecol Oncol. 2012 Dec;127(3):552-5. PMID: 22986143.

The p.S207L variant (also known as c.620C>T), located in coding exon 7 of the RAD51D gene, results from a C to T substitution at nucleotide position 620. The serine at codon 207 is replaced by leucine, an amino acid with dissimilar properties. This alteration has previously been reported in individuals with cancers including serous peritoneal, breast, ovarian, and urothelial carcinoma (Loveday C et al. Nat Genet. 2011 Aug 7;43(9):879-882; Wickramanayake A et al. Gynecol. Oncol. 2012 Dec;127:552-5; Tung N et al. J. Clin. Oncol. 2016 May;34:1460-8; Golmard L et al. BMC Cancer. 2013 Oct;13:484; Shirt BH et al. Genet Med. 2016 Oct;18(10):974-81; Nassar AH et al. Genet Med, 2020 Apr;22:709-718; Dorling et al. N Engl J Med. 2021 02;384:428-439; Fu F et al. Cancer Biol Med, 2021 Oct; Alenezi WM et al. Cancers (Basel), 2022 Apr;14; Akbar F et al. Hered Cancer Clin Pract, 2022 Jun;20:24). In a study by Rivera et al., this alteration showed segregation with disease in two families with ovarian high grade serous carcinoma (HGSC) and in two other families with breast cancer. A case control analysis found strong association with ovarian HGSC (P=2x10-6), but no significant association with breast cancer. This same group conducted numerous functional studies and found impaired homologous recombination, XRCC2 binding, and RAD51 foci formation (Rivera B et al. Cancer Res. 2017 Aug;77:4517-4529). This alteration is located in the highly conserved Walker B motif of the RAD51D protein that is required for ATPase function and XRCC2 binding (Rivera B et al. Cancer Res. 2017 Aug;77:4517-4529; Wiese C et al. Nucleic Acids Res. 2006 May;34:2833-43). Structural analysis predicts that this alteration would disrupt important hydrogen bonding and introduce steric clashes that would interfere with protein function (Rivera B et al. Cancer Res. 2017 Aug;77:4517-4529). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the majority of available evidence to date, this variant is likely to be pathogenic.

Observed in individuals with primary peritoneal, breast, and/or ovarian cancer and co-segregated with ovarian cancer in at least two families (PMID: 21822267, 22986143, 24139550, 25186627, 26845104, 28646019); Published functional studies suggest a damaging effect: impaired homologous recombination activity (PMID: 28646019); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Not observed at significant frequency in large population cohorts (gnomAD); Also known as c.680C>T; p.(S227L); This variant is associated with the following publications: (PMID: 16717288, 26845104, 22986143, 21822267, 28646019, 24139550, 26976419, 25186627, 31844177, 31922703, 32322110, 34606182, 34433815, 34298626, 35565380, 34923718, 26057125, 33471991, 34326862, 35710434, 36969410, 14704354, 21111057, 38003901, KwongA2024[article], 38661557, 36969007)

RAD51D: PS4, PS3:Moderate, PP3

The missense variant NM_002878.4(RAD51D):c.620C>T (p.Ser207Leu) causes the same amino acid change as a previously established pathogenic variant. The p.Ser207Leu variant is observed in 1/18,394 (0.0054%) alleles from individuals of gnomAD East Asian background in gnomAD. The p.Ser207Leu variant is novel (not in any individuals) in 1kG. There is a large physicochemical difference between serine and leucine, which is likely to impact secondary protein structure as these residues differ in polarity, charge, size and/or other properties. The p.Ser207Leu missense variant is predicted to be damaging by both SIFT and PolyPhen2. The serine residue at codon 207 of RAD51D is conserved in all mammalian species. The nucleotide c.620 in RAD51D is predicted conserved by GERP++ and PhyloP across 100 vertebrates. For these reasons, this variant has been classified as Likely Pathogenic.

__The RAD51D p.Ser207Leu variant was identified in 13 of 682 proband chromosomes (frequency: 0.0190615835777126) from individuals or families with __and was present in 2 of 1860 control chromosomes (frequency: 0.0010752688172043) from healthy individuals (Rivera_2017_PMID: 28646019). The variant was identified in dbSNP (ID: rs370228071) as conflicting interpretations of pathogenicity and likely pathogenic, ClinVar (Conflicting interpretations of pathogenicity. Likely pathogenic by: Counsyl in 2018, Ambry in 2018, Integrated Genetics in 2018, GeneDx in 2018, Quest Diagnostics in 2019, Color in 2018, Invitae in 2019. VUS by: Institute for biomarker research in 2017, University of Washington in 2015, Fulgent in 2017), and LOVD 3.0 (one entry, not classified, effect unknown) databases. The variant was not identified in the Cosmic database. The variant was identified in control databases in 8 of 282800 chromosomes at a frequency of 0.00002829 (Genome Aggregation Database March 6, 2019, v2.1.1). The variant was observed in the following populations: Other in 1 of 7224 chromosomes (freq: 0.000138), East Asian in 1 of 19954 chromosomes (freq: 0.00005), European (non-Finnish) in 6 of 129132 chromosomes (freq: 0.000046), but was not observed in the African, Latino, Ashkenazi Jewish, European (Finnish), or South Asian populations. The p.Ser207 residue is conserved in mammals but not in more distantly related organisms, and four out of five computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. This variant is located in the RAD51C binding domain and the Walker B motif, which is important in homologous repair (Wiese_2006_16717288). The c.620C>T variant was shown to disrupt the interaction between RAD51D and XRCC2, which cause impaired homologous recombination (Rivera_2017_PMID: 28646019). __Rivera et al. (2017) identified this variant in 3.81% of high grade serous ovarian cancer cases and 0.2% of unaffected controls in the French Canadian population (Rivera_2017_PMID: 28646019). __The variant segregated with ovarian high-grade serous carcinoma in 3 families (Rivera_2017_PMID: 28646019). In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as likely pathogenic.

The RAD51D c.620C>T variant is predicted to result in the amino acid substitution p.Ser207Leu. This variant has been reported in multiple individuals with breast and/or ovarian cancer (Loveday et al. 2011. PubMed ID: 21822267; Tung et al. 2016. PubMed ID: 26976419; Rivera et al. 2017. PubMed ID: 28646019; Alenezi et al. 2022. PubMed ID: 35565380; Akbar et al. 2022. PubMed ID: 35710434), peritoneal carcinoma (Wickramanyake et al. 2012. PubMed ID: 22986143), and urothelial carcinoma (Nassar et al. 2020. PubMed ID: 31844177). It has also been observed in multiple individuals with a personal or family history of breast/ovarian cancer tested at PreventionGenetics, although some of these individuals were also found to have an additional pathogenic variant in the RECQL gene (internal data). This variant has been shown to co-segregate with ovarian cancer in at least two families and was significantly associated with increased ovarian cancer risk compared to controls; however, this and other studies have not found a significant association between this variant and an increased risk of breast cancer (Rivera et al. 2017. PubMed ID: 28646019; Breast Cancer Association Consortium et al. 2021. PubMed ID: 33471991). In vitro experiments found this variant disrupted the accumulation of RAD proteins at the site of double strand breaks and prevented protein-protein interactions between RAD51D and XRCC2, thereby abolishing RAD-mediated DNA repair activity by homologous recombination (Rivera et al. 2017. PubMed ID: 28646019). An additional experiment also found this variant may increase sensitivity to PARP inhibitors (Rivera et al. 2017. PubMed ID: 28646019). This variant is reported in 0.0050% of alleles in individuals of East Asian descent in gnomAD and may be a founder variant in French Canadian populations (Rivera et al. 2017. PubMed ID: 28646019; Alenezi et al. 2022. PubMed ID: 35565380). This variant has varying interpretations in ClinVar; several institutions have classified it as a variant of uncertain significance, although the vast majority interpret it as either likely pathogenic or pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/142102/). Taken together, this variant is interpreted as pathogenic, especially in the context of an increased risk of ovarian cancer.

Variant interpreted as Likely pathogenic and reported on 02-12-2021 by Lab Ambry Genetics. GenomeConnect-Invitae Patient Insights Network assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. Registry team members make no attempt to reinterpret the clinical significance of the variant. Phenotypic details are available under supporting information.