ClinVar Genomic variation as it relates to human health

The BRCA2 c.9004G>A; p.Glu3002Lys variant (rs80359152) has been reported in several individuals and families with breast and/or ovarian cancer (Belanger 2015, Cavallone 2010, Cote 2012, Palmero 2016, Salazar 2006). Functional studies show that this variant is unable to rescue BRCA2 null embryonic stem cells, has reduced homology-directed repair activity, and affects cytokinesis (Biswas 2012, Guidugli 2013, Mondal 2012). This variant is listed in ClinVar (Variation ID: 38201), and is absent from the general population databases (1000 Genomes Project, Exome Variant Server, Genome Aggregation Database). The glutamate at codon 3002 is a highly conserved residue in the DNA-binding domain, and computational algorithms (SIFT, PolyPhen2, MutationTaster, Align GVGD, Prior Probabilities) predict this variant to be damaging to the protein. Taken together, this variant is considered pathogenic. REFERENCES Link to ClinVar database for p.Glu3002Lys: https://www.ncbi.nlm.nih.gov/clinvar/variation/38201/ Belanger MH et al. A targeted analysis identifies a high frequency of BRCA1 and BRCA2 mutation carriers in women with ovarian cancer from a founder population. J Ovarian Res. 2015 Mar 27;8:1. Biswas K et al. Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet. 2012 Sep 15;21(18):3993-4006. Cavallone L et al. Comprehensive BRCA1 and BRCA2 mutation analyses and review of French Canadian families with at least three cases of breast cancer. Fam Cancer. 2010 Dec;9(4):507-17. Cote S et al. The BRCA2 c.9004G>A (E2002K) [corrected] variant is likely pathogenic and recurs in breast and/or ovarian cancer families of French Canadian descent. Breast Cancer Res Treat. 2012 Jan;131(1):333-40. 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. Mondal G et al. BRCA2 localization to the midbody by filamin A regulates cep55 signaling and completion of cytokinesis. Dev Cell. 2012 Jul 17;23(1):137-52. Palmero EI et al. Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil. Genet Mol Biol. 2016 May 24;39(2):210-22. Salazar R et al. BRCA1-2 mutations in breast cancer: identification of nine new variants of BRCA1-2 genes in a population from central Western Spain. Cancer Lett. 2006 Feb 20;233(1):172-7.

PM2, PS3, PS4_moderate

This sequence change replaces glutamic acid, which is acidic and polar, with lysine, which is basic and polar, at codon 3002 of the BRCA2 protein (p.Glu3002Lys). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with breast and/or ovarian cancer (PMID: 20694749, 21947752, 22678057, 25884701, 27223485). It has also been observed to segregate with disease in related individuals. This variant is also known as c.9232G>A. ClinVar contains an entry for this variant (Variation ID: 38201). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on BRCA2 protein function. Experimental studies have shown that this missense change affects BRCA2 function (PMID: 22678057, 22771033, 23108138). RNA analysis performed to evaluate the impact of this missense change on mRNA splicing indicates it does not significantly alter splicing (Invitae). For these reasons, this variant has been classified as Pathogenic.

The p.E3002K variant (also known as c.9004G>A), located in coding exon 22 of the BRCA2 gene, results from a G to A substitution at nucleotide position 9004. The glutamic acid at codon 3002 is replaced by lysine, an amino acid with similar properties. This alteration has been identified in several unrelated families affected with breast, ovarian and/or pancreatic cancer, and has been reported to segregate with disease in at least two unrelated families (Salazar R et al. Cancer Lett. 2006 Feb;233(1):172-7; Cavallone L et al. Fam. Cancer 2010 Dec;9(4):507-17; Biswas K et al. Hum. Mol. Genet. 2012 Sep;21(18):3993-4006; Cote S et al. Breast Cancer Res. Treat. 2012 Jan;131(1):333-40; Belanger MH et al. J. Ovarian Res. 2015 Mar;8:1; Gostimir M et al. BMC Cancer 2016 10;16(1):786; Pinto P et al. Breast Cancer Res. Treat. 2016 Sep;159:245-56; Hu C et al. JAMA, 2018 Jun;319:2401-2409; Palmero EI et al. Sci Rep. 2018 Jun 15;8(1):9188; Parsons MT et al. Hum Mutat, 2019 Sep;40:1557-1578). This alteration has been predicted to be deleterious by multiple functional analyses (Biswas K et al. Hum. Mol. Genet. 2012 Sep;21(18):3993-4006; Mondal G et al. Dev. Cell 2012 Jul;23(1):137-52; Guidugli L et al. Cancer Res. 2013 Jan;73(1):265-75; Guidugli L et al. Am. J. Hum. Genet. 2018 Jan; Mesman RLS et al. Genet. Med. 2019 02;21:293-302). Of note, this alteration is also designated as 9232G>A in published literature. 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 supporting evidence, this alteration is interpreted as a disease-causing mutation.

The p.Glu3002Lys variant in BRCA2 has been reported in >15 individuals with brea st or ovarian cancer and segregated with disease in 2 affected relatives from 1 family, including 1 obligate carrier (Salazar 2006, Cavallon 2010, Cote 2010, Be langer 2015, BIC database). It was also absent from large population studies. In vitro functional studies provide some evidence that the p.Glu3002Lys may impact protein function (Biswas 2012, Guidugli 2013). Computational prediction tools a nd conservation analysis suggest that the p.Glu3002Lys variant may impact the pr otein, though this information is not predictive enough to determine pathogenici ty. In summary, this variant meets criteria to be classified as pathogenic for h ereditary breast and ovarian cancer in an autosomal dominant manner based upon p roband count, segregation studies, absence from controls, functional evidence. A CMG/AMP Criteria applied: PS4, PM2, PS3_Moderate, PP1, PP3.

In the published literature, this variant has been reported in individuals and families with breast and/or ovarian cancer (PMID: 19043619 (2008), 20694749 (2010), 22678057 (2012), 21947752 (2012), 25884701 (2015), 29161300 (2017), 29907814 (2018), and 30322717 (2018)). Also, experimental studies indicate that this variant has a deleterious effect on BRCA2 protein function (PMID: 22678057 (2012), 22771033 (2012), 29394989 (2018), and 29988080 (2018)). Based on the available information, this variant is classified as pathogenic.

Data used in classification: The frequency of this variant is 0/138,632 individuals (gnomAD) (PM2_mod). This variant is predicted deleterious on AlignGVGD (class: C55), SIFT (Deleterious), Polyphen2 HumVar (probably damaging) and CADD (36) (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 as pathogenic by 8 submitters on ClinVar, including by accredited USA diagnostic laboratories GeneDx (2018) and Color (2016) (PP5_sup). Data not used in classification: There are additional reports of this variant in ClinVar (5), UMD (3), BIC (9), and BRCA2 LOVD (2).

Variant summary: BRCA2 c.9004G>A (p.Glu3002Lys) results in a conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 250784 control chromosomes. c.9004G>A has been reported in the literature in multiple affected individuals and has been shown to co-segregate with disease in multiple independent families (Biswas_2012, Cote_2012, Cavallone_2010, etc.). These data indicate that the variant is very likely to be associated with disease. An extensive array of functional studies have shown E3002K to significantly interfere with homology-directed DNA repair (HDR) activity (Guidugli_2012, Mondal_2012) and ability to rescue BRCA2 -/- cells (Biswas_2012); additionally, E3002K disrupts interaction with Filamin A, localization of BRCA2 to the central spindle/midbody, and the integrity of cytokinesis (Mondal_2012). 12 clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

Not observed in large population cohorts (Lek 2016); In silico analysis supports that this missense variant does not alter protein structure/function; Also known as 9232G>A; This variant is associated with the following publications: (PMID: 27724927, 24123850, 22632462, 25628955, 27553368, 22771033, 29387975, 25348012, 23704879, 12228710, 25884701, 21947752, 15876480, 26137147, 27223485, 19043619, 23108138, 20694749, 22678057, 25085752, 29108258, 28651617, 29161300, 29394989, 29988080, 18284688, 29907814, 30415210, 31131967, 29884841, 31447099)

ACMG classification criteria: PS3, PS4, PM2, PP1

This missense variant replaces glutamic acid with lysine at codon 3002 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 reported that this variant impacts BRCA2 function in homology-mediated repair assays and the rescue of Brca2-deficient mouse embryonic stem cells (PMID: 22678057, 22771033, 23108138, 29394989, 29884841, 29988080, 35736817). This variant has been detected in over 20 individuals and families affected with breast, ovarian and pancreatic cancer (PMID: 15876480, 18284688, 20694749, 22678057, 25628955, 25884701, 27223485, 27724927, 30322717, 30415210, 33471991; Leiden Open Variation Database DB-ID BRCA2_000392, 35411189, 36119527). This variant also has been reported in five families with a combined segregation likelihood ratio for pathogenicity of 30.2646 (PMID: 31131967) and is a recurrent mutation in French-Canadian hereditary breast and ovarian cancer families (PMID: 32300229). 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 lysine at codon 3002 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 reported that this variant impacts BRCA1 function in homology-mediated repair assays and the rescue of Brca2-deficient mouse embryonic stem cells (PMID: 22678057, 22771033, 23108138, 29394989, 29884841, 29988080, 35736817). This variant has been detected in over 20 individuals and families affected with breast, ovarian and pancreatic cancer (PMID: 15876480, 18284688, 20694749, 22678057, 25628955, 25884701, 27223485, 27724927, 30322717, 30415210, 33471991; Leiden Open Variation Database DB-ID BRCA2_000392, 35411189, 36119527). This variant also has been reported in five families with a combined segregation likelihood ratio for pathogenicity of 30.2646 (PMID: 31131967) and is a recurrent mutation in French-Canadian hereditary breast and ovarian cancer families (PMID: 32300229). 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.

The c.9004G>A (p.Glu3002Lys) variant in the BRCA2 gene is located on the exon 23 and is predicted to replace glutamic acid with lysine at codon 3002 (p.Glu3002Lys). The variant has been reported in more than 15 unrelated individuals with breast, ovarian, or prostate cancer (PMID: 21947752, 25884701, 36119527, 20694749, 15876480, 29982661). This variant segregates with disease in multiple families (PMID: 34597585, 21947752). The negative functional impact of the variant has been confirmed by the HDR assay (PMID: 23108138) and mouse ES-cell based assay (PMID: 22678057). This variant is absent in the general population database (gnomAD). Therefore, the c.9004G>A (p.Glu3002Lys) variant of BRCA2 has been classified as pathogenic.

The p.Glu3002Lys variant has been reported in the literature in 10/2398 proband chromosomes of individuals with hereditary breast cancer. It was not, however, detected in any of the 200 control chromosomes tested (Biswas_2012, Cavallone_2010, Cote_2012, Mondal_2012, Salazar_2006). It has also been reported in the UMD (x2), LOVD, BIC (x9) and BOCs databases. It is listed in the dbSNP database (ID#:rs80359152) but no frequency information was provided, and so the prevalence of this variant in the general population is not known. The p.Glu3002 residue is conserved across mammals and computational analyses (PolyPhen, SIFT, AlignGVGD) suggest that the p.Glu3002Lys variant may impact the protein. However, this information is not predictive enough to assume pathogenicity. Yet, functional studies have shown that compared to the wild-type protein, the variant affects the integrity of cytokinesis during the cell cycle, and that the DNA repair capacity of the mutant allele is compromised (Biswas_2012, Mondal_2012). 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 predicted pathogenic.