ClinVar Genomic variation as it relates to human health

The BRCA2 c.8350C>T; p.Arg2784Trp variant (rs80359075), also known as 8578C>T, is reported in the literature in several individuals and families affected with breast, ovarian, or lung cancer (Brandao 2011, Donner 2018, Gomez Garcia 2009, Kraus 2017, van der Hout 2006). This variant is also reported in ClinVar (Variation ID: 38155), but is only observed on two alleles in the Genome Aggregation Database, indicating it is not a common polymorphism. The arginine at codon 2784 is highly conserved, and computational analyses predict that this variant is deleterious (REVEL: 0.738). Functional analyses of the variant protein show impaired homology-directed DNA break repair activity (Farrugia 2008, Guidugli 2013, Hart 2019, Mesman 2019). Based on available information, this variant is considered to be likely pathogenic. References: Brandao RD et al. Characterisation of unclassified variants in the BRCA1/2 genes with a putative effect on splicing. Breast Cancer Res Treat. 2011 Oct;129(3):971-82. PMID: 21638052. Donner I et al. Germline mutations in young non-smoking women with lung adenocarcinoma. Lung Cancer. 2018 Aug;122:76-82. PMID: 30032850. Farrugia DJ et al. Functional assays for classification of BRCA2 variants of uncertain significance. Cancer Res. 2008 May 1;68(9):3523-31. PMID: 18451181. Gomez Garcia EB et al. A method to assess the clinical significance of unclassified variants in the BRCA1 and BRCA2 genes based on cancer family history. Breast Cancer Res. 2009;11(1):R8. PMID: 19200354. Guidugli L et al. A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity. Cancer Res. 2013 Jan 1;73(1):265-75. PMID: 23108138. Hart SN et al. Comprehensive annotation of BRCA1 and BRCA2 missense variants by functionally validated sequence-based computational prediction models. Genet Med. 2019 Jan;21(1):71-80. PMID: 29884841. Kraus C et al. Gene panel sequencing in familial breast/ovarian cancer patients identifies multiple novel mutations also in genes others than BRCA1/2. Int J Cancer. 2017 Jan 1;140(1):95-102. PMID: 27616075. Mesman RLS et al. The functional impact of variants of uncertain significance in BRCA2. Genet Med. 2019 Feb;21(2):293-302. PMID: 29988080. van der Hout AH et al. A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting. Hum Mutat. 2006 Jul;27(7):654-66. PMID: 16683254.

This sequence change replaces arginine, which is basic and polar, with tryptophan, which is neutral and slightly polar, at codon 2784 of the BRCA2 protein (p.Arg2784Trp). This variant is present in population databases (rs80359075, gnomAD 0.006%). This variant has been observed in individuals affected with breast, ovarian, and lung cancer (PMID: 16683254, 27616075, 30032850, 34350294). This variant may be associated with reduced penetrance of hereditary breast and ovarian cancer syndrome based on statistical analysis (external communication). In addition, this variant has been observed in individuals with Fanconi anemia (PMID: 21520333, external communication). In at least one individual the data is consistent with the variant being in trans (on the opposite chromosome) from a pathogenic variant. ClinVar contains an entry for this variant (Variation ID: 38155). Advanced modeling performed at Invitae incorporating data from internal and/or published experimental studies (PMID: 33609447) indicates that this missense variant is expected to disrupt BRCA2 function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects BRCA2 function (PMID: 18451181, 23108138, 29884841, 29988080, 33293522). In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic.

The p.R2784W pathogenic mutation (also known as c.8350C>T), located in coding exon 18 of the BRCA2 gene, results from a C to T substitution at nucleotide position 8350. The arginine at codon 2784 is replaced by tryptophan, an amino acid with dissimilar properties. This alteration was identified in several breast and ovarian cancer cohorts and segregates with disease (van der Hout AH et al. Hum. Mutat. 2006 Jul;27:654-66; Gómez García EB et al. Breast Cancer Res. 2009 Feb;11:R8; Kraus C et al. Int. J. Cancer. 2017 Jan;140(1):95-102; Ambry internal data). This alteration was also observed in trans with a pathogenic BRCA2 alteration in a patient with Fanconi Anemia (personal communication). Several independent assays find this variant non-functional including multiple homology-directed DNA repair assays; a centriole amplification assay and a mouse embryonic stem cell complementation assay (Farrugia DJ et al. Cancer Res. 2008 May;68:3523-31; Guidugli L et al. Cancer Res. 2013 Jan;73:265-75; Guidugli L et al. Am. J. Hum. Genet. 2018 Jan; Mesman RLS et al. Genet. Med. 2018 Jul). Internal structural analysis indicates that this variant is likely to disrupt BRCA2 binding to DSS1, a protein that has been shown to stabilize BRCA2 and participate in its clinically relevant functions (Ambry internal data; Yang H et al. Science 2002 Sep;297:1837-48). This amino acid position is highly conserved in available vertebrate species. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. However, because this variant is identified in one or more patients with Fanconi Anemia it may be hypomorphic and thus, carriers of this variant and their families may present with reduced risks, and not with the typical clinical characteristics of a high-risk pathogenic BRCA2 alteration. As risk estimates are unknown at this time, clinical correlation is advised.

This missense variant replaces arginine with tryptophan at codon 2784 in the DNA binding domain of the BRCA2 protein. 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 shown the mutant protein to be non-functional in homology-directed DNA repair activity (PMID: 18451181, 29394989, 29988080, 33609447, 35736817) and unable to complement BRCA2-deficiency in mouse embryonic stem cells (PMID: 29988080). This variant has been observed in over ten individuals and families affected with breast and ovarian cancer (PMID: 18451181, 16683254, 19200354, 21638052, 27153395, 31409081, 31843900, 31742824) and in individuals affected with early-onset or metastatic prostate cancer with family history of prostate and breast cancer (Color internal data). In addition, this variant has been observed in compound heterozygous state with a pathogenic variant in the same gene in at least one individual affected with autosomal recessive Fanconi anemia, indicating that this variant contributes to disease (PMID: 21520333; ClinVar variation ID: SCV000186727.6, SCV000073500.12). This variant has been identified in 2/251430 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic.

Published functional studies demonstrate a damaging effect: displayed reduced homologous recombination repair activity in multiple homology-directed DNA break repair assays as well as poor survival in mouse embryonic stem cell assay (PMID: 18451181, 23108138, 29988080, 29884841, 33293522, 35736817); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis indicates that this missense variant does not alter protein structure/function; Also known as 8578C>T; This variant is associated with the following publications: (PMID: 21638052, 25447315, 36721989, 31060523, 34687993, 18451181, 19200354, 16683254, 19043619, 25782689, 27616075, 30032850, 10923033, 18951461, 29988080, 12228710, 16205630, 26145171, 22194698, 24323938, 31409081, 33293522, 29884841, 34350294, 32719484, 33609447, 35665744, 38623065, 36387127, Akolkar2022[preprint], 33471991, 35736817, 36099812, 31742824, 35762214, 35753512, 23108138, 29394989)

Data used in classification: This variant is predicted deleterious on AlignGVGD (class: C65), SIFT (Deleterious), Polyphen2 HumVar (probably damaging) and CADD (20.3) (PP3_sup). The variant is in the DNA-binding domain of BRCA2 (PM1_sup). In the BRCA2 Homology-Directed Repair Activity assay for the DNA Binding Domain (Guidugli et al Cancer Res 2013;73:265-275,Couch Lab), the variant has a probability of pathogenicity of 1.0 (PS3_strong). This variant is classified on ClinVar as likely pathogenic by accredited USA diagnostic laboratory GeneDx, 2017 (PP5_sup). Data not used in classification: The variant was observed in 1 independent UK family undergoing clinical diagnostic BRCA1/BRCA2 testing for HBOC according to diagnostic criteria, the denominator of which dataset of clinical testing was 16,600. Case control comparison against ethnically matched population data (1/16,600 in familial cases against 1/55,836 gnomAD NFE controls) 2-sided Fishers exact: pexact= 0.4. The frequency of this variant is 2/67,272 individuals (remainder of the gnomAD population).

The frequency of this variant in the general population, 0.000008 (2/251430 chromosomes, http://gnomad.broadinstitute.org), is consistent with pathogenicity. It has been reported in families with breast and/or ovarian cancer in the published literature (PMIDs: 27616075 (2016), 16683254 (2006), 21638052 (2011), 31060523 (2019), and 33471991 (2021) see also LOVD (https://databases.lovd.nl/shared/variants/BRCA2)), as well as in one individual affected with lung cancer (PMID: 30032850 (2018)). Multiple functional studies have reported that this variant impairs homology-directed DNA break repair activity and shows reduced cell survival compared to wild type, however it does not affect centrosome amplification (PMIDs: 18451181 (2008), 23108138 (2013), 29394989 (2018), 29988080 (2018), and 33293522 (2020)). 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 likely pathogenic.

Variant summary: BRCA2 c.8350C>T (p.Arg2784Trp) results in a non-conservative amino acid change located in the BRCA2, OB1 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 8e-06 in 251430 control chromosomes. c.8350C>T has been reported in the literature in individuals affected with Breast and/or Ovarian Cancer (e.g. van der Hout_2006, Farrugia_2008, Garcia_2009, Vail_2015, Kraus_2017, daCosta_2019, Machackova_2019, Shao_2020, Wu_2021), Fanconi anemia (e.g. Radulovic_2023), or other cancers (e.g. Donner_2018, Sirak_2021, Xia_2022). Several publications report experimental evidence evaluating an impact on protein function (e.g. Farrugia_2008, Guidugli_2013, Guidugli_2018, Mesman_2019, Richardson_2021, Hu_2022) including significantly impaired homology directed repair activity meaured by HDR assays. The following publications have been ascertained in the context of this evaluation (PMID: 30032850, 18451181, 19200354, 29394989, 23108138, 24323938, 29884841, 35736817, 19043619, 27616075, 25447315, 31409081, 29988080, 36721989, 33609447, 31742824, 34970085, 25782689, 34350294, 35762214, 31060523, 30447919, 16683254). 19 submitters have cited clinical-significance assessments for this variant to ClinVar after 2014, classifying the variant as pathogenic (n=3), likely pathogenic (n=13), or uncertain significance (n=3). Based on the evidence outlined above, the variant was classified as likely pathogenic.

Reduced ES cell viability and drug hypersensitivity (severe impact on BRCA2 function) PMID: 33293522

The BRCA2 c.8350C>T variant is predicted to result in the amino acid substitution p.Arg2784Trp. This variant has been reported in an individual with triple-negative breast cancer (Table S4, Kraus et al. 2017. PubMed ID: 27616075), multiple families with a history of hereditary breast and/or ovarian cancer (HBOC) (van der Hout et al. 2006. PubMed ID: 16683254; Gómez García et al. 2009. PubMed ID: 19200354; Table S3, Casadei et al. 2019. PubMed ID: 31843900; Table S2, Shao et al. 2020. PubMed ID: 31742824), and in a non-smoking woman with lung adenocarcinoma (Donner et al. 2018. PubMed ID: 30032850). It has also been observed in an ovarian tumor (Additional file 4, Da Costa et al. 2019. PubMed ID: 31060523). Experimental studies have demonstrated that this variant reduces homology-directed DNA repair (HRD) (Table 2, Guidugli et al. 2013. PubMed ID: 23108138; Farrugia et al. 2008. PubMed ID: 18451181; Guidugli et al. 2018. PubMed ID: 29394989; Mesman et al. 2019. PubMed ID: 29988080). Recent studies have interpreted this variant as pathogenic based on HRD assay results (Hart et al. 2019. PubMed ID: 29884841). This variant has been reported in the gnomAD public population database in 2 of ~251,000 alleles and is interpreted as likely pathogenic by the vast majority of clinical laboratories in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/38155/). In summary, this variant is interpreted as likely pathogenic.

This variant yielded normal BRCA2 splicing when tested on RNA from a patient’s lymphoblast cell line using cBROCA. cBROCA evaluates changes in splicing due to germline mutations from participants' RNA and does not evaluate changes that alter function at the protein level. This variant might have an effect at the protein level.