ClinVar Genomic variation as it relates to human health

IARC class based on posterior probability from multifactorial likelihood analysis, thresholds for class as per Plon et al. 2008 (PMID: 18951446). Class 5 based on posterior probability = 1

This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in affected individuals with breast and/or ovarian cancer (PMIDs: 16489001 (2006), 20104584 (2010), 25452441 (2015), 33758026 (2022), and 34399810 (2021)). In a large scale breast cancer association study, the variant was observed in control individuals, as well as among individuals with breast cancer (PMID: 33471991 (2021), see also LOVD (http://databases.lovd.nl/shared/genes/BRCA2)). Functional studies demonstrated aberrant splicing, increased naturally occurring exon 18 skipping, reduced homologous repair activity, and failure to rescue mouse embryonic stem cells (PMIDs: 18451181 (2008), 18607349 (2008), 20215541 (2010), 20513136 (2010), 28339459 (2017), and 30736279 (2019)). This variant was also determined to have a high probability of being disease-causing by multifactorial likelihood analyses (PMIDs: 17924331 (2007), 18451181 (2008), 20513136 (2010), and 21990134 (2012)). 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. Additional analysis using software algorithms for the prediction of the effect of nucleotide changes on BRCA2 mRNA splicing yielded predictions that this variant may result in the gain of a cryptic splice site without affecting the natural splice sites . Based on the available information, this variant is classified as pathogenic.

This sequence change replaces glutamic acid, which is acidic and polar, with valine, which is neutral and non-polar, at codon 2663 of the BRCA2 protein (p.Glu2663Val). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with personal and/or family history of breast/ovarian cancer (PMID: 16489001, 20104584, 21965345, 25452441). It has also been observed to segregate with disease in related individuals. This variant is also known as 8216A>T. ClinVar contains an entry for this variant (Variation ID: 52462). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 17924331, 18451181, 21990134). Experimental studies have shown that this missense change affects BRCA2 function (PMID: 18607349, 20513136). For these reasons, this variant has been classified as Pathogenic.

The p.Glu2663Val variant in BRCA2 has been reported in at least 15 individuals with BRCA2-associated cancers (BIC database: https://research.nhgri.nih.gov/projects/bic/, Szabo 2000, Chevenix-Trench 2006, Borg 2010, Akbari 2011, Color pers. comm., Ambry pers. comm.) and segregated with breast and ovarian cancer in at least 1 affected family member (Color pers. comm.). It was absent from large population studies. In vitro functional studies suggest that this variant impacts protein function (Kuznetsov 2008, Farrugia 2008, Sanz 2010, Walker 2010, Whiley 2014, Fraile-Bethencourt 2017). Computational prediction tools and conservation analysis also support that the p.Glu2663Val variant impacts the protein. In addition, this variant was classified as pathogenic on August 10, 2015 by the ClinGen-approved ENIGMA expert panel (ClinVar SCV000244478.1). In summary, the p.Glu2663Val variant meets criteria to be classified as pathogenic for autosomal dominant hereditary breast and ovarian cancer. ACMG/AMP Criteria applied (Richards 2015): PS4, PS3, PM2_Supporting, PP3.

The p.E2663V pathogenic mutation (also known as c.7988A>T), located in coding exon 17 of the BRCA2 gene, results from an A to T substitution at nucleotide position 7988. The glutamic acid at codon 2663 is replaced by valine, an amino acid with dissimilar properties. This alteration was identified 18 families in a worldwide study of BRCA1/2 mutation positive families (Rebbeck TR et al. Hum. Mutat.. 2018 05;39:593-620). In one study, RT-PCR analysis demonstrated that this alteration leads to an out-of-frame deletion of the 355-bp exon 18, resulting in a premature stop codon within exon 19 (Farrugia DJ et al. Cancer Res. 2008 May; 68(9):3523-31). Functional studies from other research groups have confirmed these findings; however, in addition to the transcript that skips only exon 18, they found that this alteration also leads to the transcription of an isoform that skips both exon 17 and 18 as well as a full-length variant transcript (Walker LC et al. Hum. Mutat. 2010 Jun; 31(6):E1484-505; Fraile-Bethencourt E et al. PLoS Genet. 2017 Mar;13:e1006691). Further studies assessed the function of the variant protein and demonstrated abrogated function consistent with pathogenicity; homolgous repair activity was reduced significantly compared to wildtype BRCA2. This alteration has been classified as pathogenic (p>0.99) by multifactorial analysis, which integrates the following lines of evidence to produce a quantitative likelihood of pathogenicity: in silico prediction models, segregation with disease, tumor characteristics, mutation co-occurrence, and functional assay results (Easton D et al. Am J Hum Genet. 2007;81:873-883; Vallee M et al. Hum Mutat. 2012 Jan;33(1):22-8). It has also been classified as likely deleterious based on a protein likelihood ratio (Karchin R et al. Cancer Inform. 2008 Apr;6:203-16). Of note, this alteration is also referred to as 8216A>T in some published literature. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

PM2_Supporting; PP1, PP3; PS3; Expert panel

The BRCA2 c.7988A>T; p.Glu2663Val variant (rs80359031), also known as 8216A>T, is reported in the literature in multiple individuals affected with breast and ovarian cancer (Akbari 2011, Borg 2010, Chenevix-Trench 2006, Couch 2015). Functional analyses demonstrate aberrant splicing, reduced protein function, and a failure to rescue a mouse knockout cell line (Farrugia 2008, Fraile-Bethencourt 2017, Kuznetsov 2008, Sanz 2010, Walker 2010). This variant is reported as pathogenic by multiple laboratories in ClinVar (Variation ID: 52462), and is absent from general population databases (Exome Variant Server, Genome Aggregation Database), indicating it is not a common polymorphism. The glutamic acid at codon 2663 is highly conserved, and computational analyses (SIFT, PolyPhen-2) predict that this variant is deleterious. Based on available information, this variant is considered to be pathogenic. References: Akbari MR et al. Clinical impact of unclassified variants of the BRCA1 and BRCA2 genes. J Med Genet. 2011 Nov;48(11):783-6. Borg A et al. Characterization of BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance in unilateral and bilateral breast cancer: the WECARE study. Hum Mutat. 2010 Mar;31(3):E1200-40. Chenevix-Trench G et al. Genetic and histopathologic evaluation of BRCA1 and BRCA2 DNA sequence variants of unknown clinical significance. Cancer Res. 2006 Feb 15;66(4):2019-27. Couch FJ et al. Inherited mutations in 17 breast cancer susceptibility genes among a large triple-negative breast cancer cohort unselected for family history of breast cancer. J Clin Oncol. 2015 Feb 1;33(4):304-11. Farrugia DJ et al. Functional assays for classification of BRCA2 variants of uncertain significance. Cancer Res. 2008 May 1;68(9):3523-31. Fraile-Bethencourt E et al. Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genet. 2017 Mar 24;13(3):e1006691. Kuznetsov SG et al. Mouse embryonic stem cell-based functional assay to evaluate mutations in BRCA2. Nat Med. 2008 Aug;14(8):875-81. Sanz DJ et al. A high proportion of DNA variants of BRCA1 and BRCA2 is associated with aberrant splicing in breast/ovarian cancer patients. Clin Cancer Res. 2010 Mar 15;16(6):1957-67. Walker LC et al. Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity. Hum Mutat. 2010 Jun;31(6):E1484-505.

Variant summary: BRCA2 c.7988A>T (p.Glu2663Val) results in a non-conservative amino acid change located in the Breast cancer type 2 susceptibility protein, helical domain (IPR015252) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. Several computational tools predict a significant impact on normal splicing: Three predict the variant creates a 5' donor site. Multiple publications report evidence from quantitative isoform-specific RT-PCR, cDNA sequencing, and single-cell RNA in situ hybridisation experiments demonstrating that this variant affects mRNA splicing (Farrugia,_2008 Fraile-Bethencourt_2017, Lattimore_2019). However, these reports suggest that c.7988A>T only affects splicing in a proportion of mRNA transcripts and the wild-type transcript is still produced. Several studies have provided experimental evidence on the impact of the variant on protein function. The p.Glu2663Val amino acid change was shown to disrupt BRCA2 homology-directed repair activity, increase centrosome amplification, and had an inability to rescue the lethality of BRCA2-deficient ES-cells (Kuznetsov,_2008, Walker_2010). The variant was absent in 250238 control chromosomes. c.7988A>T has been reported in the literature in multiple individuals affected with Hereditary Breast And Ovarian Cancer Syndrome and has segregated with the disease in several families (example, Kuznetsov,_2008). These data indicate that the variant is very likely to be associated with disease. Eight clinical diagnostic laboratories and two expert panels have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

The BRCA2 c.7988A>T variant is classified as Pathogenic (PS3, PS4, PP3, PP5) The BRCA2 c.7988A>T variant is a single nucleotide change in the BRCA2 gene, which is predicted to change the amino acid glutamic acid at position 2663 in the protein to valine. The variant has been reported in probands with a clinical presentation of breast and/or ovarian cancer (PMID:29446198) (PS4). Well-established functional studies show a deleterious effect of this variant (PMID:20513136) (PS3). Computational predictions support a deleterious effect on the gene or gene product (PP3). Splicing predicitors predict abberrant splicing (introduction of donor site), resulting in exon skipping (exon19). The variant has been reported in dbSNP (rs80359031) and has been reported as Pathogenic by other diagnostic laboratories (ClinVar Variation ID: 52462).

Multifactorial studies suggest this variant is associated with breast and ovarian cancer (Lindor et al., 2012); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant does not alter protein structure/function; Also known as 8216A>T; This variant is associated with the following publications: (PMID: 19043619, 20104584, 21965345, 18451181, 21702907, 21520273, 20513136, 18607349, 16489001, 17924331, 28339459, 25452441, 20215541, 23893897, 23242139, 24212087, 21638052, 27060066, 29446198, 30638113, 31263054, 30736279, 31447099, 32392735, 32322110, 34399810, 30787465, 33758026, 33808557, 33087929, 33471991, 33978741, 34906479, 12228710, 21990134)

This missense variant replaces glutamic acid with valine at codon 2663 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 incapable of complementing BRCA2-deficiency in mouse embryonic stem cells (PMID: 18607349) and to exhibit reduced homologous DNA repair activity (PMID: 20513136). In addition, multiple RNA studies have shown that this variant affects RNA splicing and results in the production of transcripts lacking the entire or partial exon 18, or both exons 17 and 18, all predicted to cause premature protein truncation (PMID: 18451181, 20215541, 20513136). The amount of full length transcript produced from the mutant allele was greater than that of aberrant transcripts. However, the ratio of aberrant transcript to full length transcripts was higher in carrier individuals than in non-carrier individuals of this variant (PMID: 20513136). These functional study results indicate that this variant inactivates BRCA2 gene function, both by disrupting protein function and by dysregulating RNA splicing. This variant has been reported in over ten individuals affected with breast cancer, ovarian cancer or pancreatic cancer (PMID: 16489001, 17924331, 18451181, 18446624, 20104584, 20513136, 21965345, 23242139, 25452441, 33808557, 33439686; Color internal data). The variant has been shown to segregate with cancer in multiple families (ClinVar SCV000592166.2) and determined to have a high probability of being disease-causing by multifactorial likelihood analysis using computational prediction, functional study data and genetic data (PMID: 17924331, 18451181, 21990134). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic.

This missense variant replaces glutamic acid with valine at codon 2663 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 incapable of complementing BRCA2-deficiency in mouse embryonic stem cells (PMID: 18607349) and to exhibit reduced homologous DNA repair activity (PMID: 20513136). In addition, multiple RNA studies have shown that this variant affects RNA splicing and results in the production of transcripts lacking the entire or partial exon 18, or both exons 17 and 18, all predicted to cause premature protein truncation (PMID: 18451181, 20215541, 20513136). The amount of full length transcript produced from the mutant allele was greater than that of aberrant transcripts. However, the ratio of aberrant transcript to full length transcripts was higher in carrier individuals than in non-carrier individuals of this variant (PMID: 20513136). These functional study results indicate that this variant inactivates BRCA2 gene function, both by disrupting protein function and by dysregulating RNA splicing. This variant has been reported in over ten individuals affected with breast cancer, ovarian cancer or pancreatic cancer (PMID: 16489001, 17924331, 18451181, 18446624, 20104584, 20513136, 21965345, 23242139, 25452441, 33808557, 33439686; Color internal data). The variant has been shown to segregate with cancer in multiple families (ClinVar SCV000592166.2) and determined to have a high probability of being disease-causing by multifactorial likelihood analysis using computational prediction, functional study data and genetic data (PMID: 17924331, 18451181, 21990134). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic.

PP1, PP3, PP4_strong, PM2, PS3

The BRCA2 p.Glu2663Val variant has been identified in 4/5646 proband chromosomes (frequency 0.001) of individuals with breast and/or ovarian cancer, and was not identified in 360 control chromosomes, increasing the likelihood this variant may have clinical significance (Akbari_2011_21965345, Borg_2010_20104584, Chenevix-Trench_2006_16489001, Sanz_2010_20215541, Walker_2010_20513136). The variant is listed in the dbSNP database (ID: rs80359031), but no frequency information was provided, and so the prevalence of this variant in the general population is not known. It has been reported in the BIC (9x) database as a variant of unknown significance. The p.Glu2663 residue is conserved across mammals and computational analyses (PolyPhen, SIFT, AlignGVGD) suggest that the p.Glu2663Val variant may impact the protein. Additionally, computational prediction software (SpliceSiteFinder, MaxEntScan, NNSPLICE, HumanSpliceFinder) predicts this variant could potentially generate a cryptic splice site; however, these in silico tools are not predictive enough to assume pathogenicity. Functional studies evaluating splicing predict this variant to be deleterious (Capanu_2011_21520273, Farrugia_2008_18451181, Karachin_2008_19043619, Kuznetsov_2008_16489001, Lindor_2012_21990134); Farrugia et al 2008 demonstrated this variant results in skiipping of exon 18 and is predicted to cause a premature truncation of the BRCA2 protein. However, one study evaluating LOH in tumours identified this variant on the lost allele suggesting neutrality (Chenevix-Trench_2006_16489001). Cosegregation analysis based on data compiled by Myriad Genetics calculated this variant to have odds of 233:1 in favour of causality (Easton_2007_17924331). The variant has been shown to segregate with cancer in multiple families in Myriad's internal data (personal communication). In summary, based on the above information, this variant is classified as pathogenic.