ClinVar Genomic variation as it relates to human health

The c.517G>C variant (also known as p.G173R) is located in coding exon 6 of the BRCA2 gene. This variant results from a G to C substitution at nucleotide position 517. The glycine at codon 173 is replaced by arginine, an amino acid with dissimilar properties. This change occurs in the first base pair of coding exon 6. This nucleotide position is highly conserved in available vertebrate species. This variant has been detected in breast cancer cohorts (Tung N et al. Cancer, 2015 Jan;121:25-33; Dorling et al. N Engl J Med. 2021 02;384:428-439). In silico splice site analysis predicts that this alteration may weaken the native splice acceptor site. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data; Baert A et al. Hum. Mutat. 2017 Dec). Another nucleotide substitution at the same position (BRCA2 c.517G>T) was also shown to have a similar severe splice defect in multiple minigene assays (Gaildrat P et al. J. Med. Genet. 2012 Oct;49:609-17; Fraile-Bethencourt E et al. J Pathol, 2019 08;248:409-420). Yet another close-match alteration at this same acceptor site, BRCA2 c.517-2A>G, has been identified in a compound heterozygous and homozygous state in patients with Fanconi Anemia (Leach M et al. Practical Flow Cytometry in Hematology: 100 worked examples. 321-324; 2015; Muramatsu H et al. Genet. Med., 2017 07;19:796-802). BRCA2 c.517-2A>G also demonstrates an intermediate effect in a mouse embryonic stem cell survival and subsequent homology-directed DNA repair assays. Clinical and functional data collectively support BRCA2 c.517-2A>G as a likely hypomorphic variant (Mesman RLS et al. Genet Med, 2020 08;22:1355-1365). BRCA2 c.517-2A>G has a similar splice defect as this variant (Ambry internal data; Houdayer C et al. Hum Mut. 2012; 33:1228–38; Fraile-Bethencourt E et al. J. Pathol. 2019 08;248(4):409-420). Based on the majority of available evidence to date, this variant is likely to be pathogenic. However, because a variant with a similar splicing profile as this variant is functionally hypomorphic and has been identified in multiple patients with Fanconi Anemia it may be similarly 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.

DNA sequence analysis of the BRCA2 gene demonstrated a sequence change, c.517G>C, in exon 7 that results in an amino acid change, p.Gly173Arg. In vitro mRNA analysis showed that the c.517G>C variant induces aberrant splicing (skipping of exon 7) due to loss of the natural splice site leading to a frameshift, r.517_631del; p.(Gly173Serfs*19) (PMID: 29280214). This change is predicted to result in degradation of the transcript. This sequence change has been previously described in patients with breast cancer in two studies but no additional information was provided (PMIDs: 29446198, 21702907). This sequence change has not been described in the population databases (ExAC and gnomAD). The p.Gly173Arg change affects a poorly conserved amino acid residue located in a domain of the BRCA2 protein that is known to be functional. In-silico pathogenicity prediction tools (SIFT, PolyPhen2, Align GVGD, REVEL, in silico splice prediction tools) provide contradictory results for the p.Gly173Arg substitution. A different variant affecting the same nucleotide, c.517G>T(p.Gly173Cys), has been reported to lead to abnormal splicing of the BRCA2 gene by functional splicing minigene assay and was predicted to result in a frame shift (p.Gly173Serfs*19) (PMID: 22962691). These collective evidences indicate that this sequence change is likely pathogenic.

It has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in a triple-negative breast tumor tissue specimen (PMID: 24963051 (2014)). In a large scale breast cancer association study, this variant was observed in a breast cancer case and not in unaffected study control individuals (PMID: 33471991 (2021), ). In addition, this variant has been observed in individuals with breast cancer (Quest internal data). Experimental studies report this variant causes aberrant splicing resulting in protein truncation based on cDNA analysis of carrier lymphocytes (PMID: 29280214 (2018)), and impaired DNA repair via a functional ex vivo assay (PMID: 24963051 (2014)). Based on the available information, this variant is classified as likely pathogenic.

This variant changes the first nucleotide in exon 7 of the BRCA2 gene and is predicted to impact RNA splicing. This variant is also known as p.Gly173Arg based on predicted change at the protein level. An RNA study using peripheral blood cells from a carrier has shown that this variant results in out-of-frame skipping of exon 7, causing a frameshift and premature translation stop signal (PMID: 29280214). This variant has been reported in an individual affected with breast cancer (Leiden Open Variation Database DB-ID BRCA2_001018; PMID: 33471991). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of BRCA2 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Likely Pathogenic.

Exonic splice variant demonstrated to result in abnormal splicing, leading to out-of-frame skipping of exon 7 (Baert et al., 2017); Observed in an individual with breast cancer (Dorling et al., 2021); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis is inconclusive as to whether the variant alters gene splicing. In the absence of RNA/functional studies, the actual effect of this sequence change is unknown.; In silico analysis, which includes protein predictors and evolutionary conservation, supports that this variant does not alter protein structure/function; Also known as 745G>C; This variant is associated with the following publications: (PMID: 24963051, 22962691, 21702907, 29446198, 28726806, 30139880, 30883759, 33471991, 29280214)

PS4_MOD, PM2_SUP, PVS1_STR

This sequence change replaces glycine, which is neutral and non-polar, with arginine, which is basic and polar, at codon 173 of the BRCA2 protein (p.Gly173Arg). RNA analysis indicates that this missense change induces altered splicing and may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). This variant has not been reported in the literature in individuals affected with BRCA2-related conditions. ClinVar contains an entry for this variant (Variation ID: 182177). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Studies have shown that this missense change results in skipping of exon 7 and introduces a premature termination codon (PMID: 29280214; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. 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.Gly173Arg variant was not identified in the literature however it is listed in dbSNP (rs397507768) as pathogenic/uncertain significance, but no frequency information was provided, thus the prevalence of this variant in the general population could not be determined. This variant was also identified by ClinVar (4 submissions, Pathogenic 1x by CIMBA, and Uncertain significance 3x, by GeneDx, Genetics Diagnostic, Ambry Genetics), Clinvitae (3x as Uncertain significance, 2 x Pathogenic) and GeneInsight - COGR database (as Unknown significance, by CHEO). This variant was not identified in any of the following databases: UMD, LOVD, BIC, COSMIC, the 1000 Genomes Project, the NHLBI GO Exome Sequencing Project and The Exome Aggregation Consortium database (August 8, 2016). The p.Gly173 residue is conserved in mammals but it is not conserved across other organisms. Computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. The c.517G>C variant occurs in the first base of the exon. This position has been shown to be part of the splicing consensus sequence and variants involving this position sometimes affect splicing. 2 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing, this is not very predictive of pathogenicity. Although the p.Gly173Arg variant was not identified in the literature, a different amino acid change at this position, c.517G>T, p.Gly173Cys, has been shown to alter the natural splice site (Gaildrat 2014). The study performed splicing minigene assays and analyses of patient RNA showed that the c.517G>T variant induced total exon skipping (RNA r.517_631del), a defect that results in a frame shift (p.Gly173SerfsX19). 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.