Submissions for variant NM_000059.4(BRCA2):c.68-3T>G

gnomAD frequency: 0.00013  dbSNP: rs786201770

Total submissions: 9

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
Ambry Genetics	RCV000164231	SCV000214852	uncertain significance	Hereditary cancer-predisposing syndrome	2024-07-04	criteria provided, single submitter	clinical testing	The c.68-3T>G intronic variant results from a T to G substitution 3 nucleotides upstream from coding exon 2 in the BRCA2 gene. This nucleotide position is poorly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and will result in the creation or strengthening of a novel splice acceptor site. RNA studies have demonstrated that this alteration results in multiple abnormal splicing events in the set of samples tested (Ambry internal data) including a splice variant which results in a frameshift variant as well as one that results in skipping of coding exon 2 (also known as exon 3 in the literature). The loss of coding exon 2 is strongly associated with hereditary breast and ovarian cancer phenotype based on multifactorial analysis (Caputo SM et al. Oncotarget, 2018 Apr;9:17334-17348). However, downstream functional studies showed that this alteration was able to rescue the growth defect in BRCA2-null mouse embryonic stem cells and that these surviving cells maintained partial activity in a homology directed DNA repair assay (personal communication). This alteration is also identified in patients who collectively have a phenotype that is not consistent with a high risk BRCA2 pathogenic variant (Nix P et al. Fam Cancer, 2021 Jan). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. It cannot yet be ruled out that this variant may be hypomorphic and present with reduced risks and/or biallelic phenotype.
Labcorp Genetics (formerly Invitae), Labcorp	RCV000547977	SCV000635535	uncertain significance	Hereditary breast ovarian cancer syndrome	2024-11-24	criteria provided, single submitter	clinical testing	This sequence change falls in intron 2 of the BRCA2 gene. It does not directly change the encoded amino acid sequence of the BRCA2 protein. It affects a nucleotide within the consensus splice site. This variant is present in population databases (rs786201770, gnomAD 0.05%). 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: 184893). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant is associated with inconclusive levels of altered splicing (PMID: 33469799, 35979650). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV004562347	SCV000918886	uncertain significance	not specified	2023-11-20	criteria provided, single submitter	clinical testing	Variant summary: BRCA2 c.68-3T>G alters a non-conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: two predict the variant abolishes a 3' acceptor site, and three predict the variant creates a 3' acceptor site two nucleotides upstream, potentially leading to a frameshift. Several studies report experimental evidence supporting these predictions, finding that the variant leads to aberrant splicing (e.g., Machackova_2019, Nix_2022, Thomassen_2022). The variant allele was found at a frequency of 6.4e-05 in 248470 control chromosomes (gnomAD). This frequency is not significantly higher than estimated for a pathogenic variant in BRCA2 causing Hereditary Breast And Ovarian Cancer Syndrome (6.4e-05 vs 0.00075), allowing no conclusion about variant significance. c.68-3T>G has been reported in the literature in individuals affected with Hereditary Breast And Ovarian Cancer Syndrome (e.g., Neveling_2017, Machackova_2019, Nix_2022). However, these reports do not provide unequivocal conclusions about association of the variant with Hereditary Breast And Ovarian Cancer Syndrome. At least one study reports experimental evidence demonstrating an impact on protein function, finding that the variant results in 50% viability in a mouse embryonic stem cell-based viability assay and also results in 90% HDR activity relative to wild type (e.g., Thomassen_2022). These results in combination with the experimental evidence demonstrating aberrant splicing do not allow convincing conclusions about variant significance. The following publications have been ascertained in the context of this evaluation (PMID: 31409081, 27974384, 33469799, 32641407). Three submitters have reported clinical-significance assessments for this variant to ClinVar after 2014. All submitters classified the variant as uncertain significance. Based on the evidence outlined above, the variant was classified as uncertain significance.
GeneDx	RCV001566570	SCV001790112	uncertain significance	not provided	2023-06-09	criteria provided, single submitter	clinical testing	Published functional studies demonstrate homology-directed repair activity comparable to wildtype (Thomassen et al., 2022); In silico analysis supports a deleterious effect on splicing; History-weighting algorithm of multiple carriers suggests this variant is not pathogenic (Nix et al., 2021); Also known as 296-3T>G; This variant is associated with the following publications: (PMID: 27974384, 27060066, 29707112, 35979650, 33469799)
Baylor Genetics	RCV003474853	SCV004210457	uncertain significance	Familial cancer of breast	2024-01-26	criteria provided, single submitter	clinical testing
Quest Diagnostics Nichols Institute San Juan Capistrano	RCV001566570	SCV004220523	uncertain significance	not provided	2022-10-17	criteria provided, single submitter	clinical testing	The frequency of this variant in the general population, 0.00047 (16/34390 chromosomes in Latino/Admixed American subpopulation, http://gnomad.broadinstitute.org), is higher than would generally be expected for pathogenic variants in this gene. To the best of our knowledge, the variant has not been reported in the published literature in individuals with BRCA2-associated cancers. Experimental studies have concluded that this variant results in aberrant splicing with partially retained viability and homology directed repair functions in mouse embryonic stem cells (PMID: 33469799 (2021) and 35979650 (2022)). Analysis of this variant using software algorithms for the prediction of the effect of nucleotide changes on BRCA2 mRNA splicing yielded inconclusive findings. In addition, some algorithms predict that this variant may result in the gain of a cryptic splice site . Based on the available information, we are unable to determine the clinical significance of this variant.
All of Us Research Program, National Institutes of Health	RCV003995328	SCV004828520	uncertain significance	Breast-ovarian cancer, familial, susceptibility to, 2	2023-09-05	criteria provided, single submitter	clinical testing
Myriad Genetics, Inc.	RCV003995328	SCV005404938	likely benign	Breast-ovarian cancer, familial, susceptibility to, 2	2024-09-26	criteria provided, single submitter	clinical testing	This variant is considered likely benign. This variant is intronic and is not expected to impact mRNA splicing. This variant is strongly associated with less severe personal and family histories of cancer, typical for individuals without pathogenic variants in this gene [PMID: 25085752].
Department of Pathology and Laboratory Medicine, Sinai Health System	RCV001355586	SCV001550512	uncertain significance	Malignant tumor of breast		no assertion criteria provided	clinical testing	The BRCA2 c.68-3T>G variant was identified in 1 of 304 proband chromosomes (frequency: 0.004) from individuals or families with breast cancer (Neveling 2017). The variant was identified in dbSNP (rs786201770) as “with uncertain significance allele” and ClinVar (classified as uncertain significance by Invitae, Ambry Genetics and Integrated Genetics). The variant was not identified in LOVD 3.0 and UMD-LSDB. The variant was identified in control databases in 16 of 248,470 chromosomes at a frequency of 0.00006 (Genome Aggregation Database Feb 27, 2017). The variant was observed in the Latino population in 16 of 34,390 chromosomes (freq: 0.0005); it was not observed in the African, Ashkenazi Jewish, East Asian, Finnish, European, Other or South Asian populations. The c.68-3T>G variant is located in the 3' splice region but does not affect the invariant -1 and -2 positions. However, positions -3 and -5 to -12 are part of the splicing consensus sequence and variants involving these positions sometimes affect splicing. In addition, 3 of 4 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a greater than 10% difference in splicing. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance.