13-32319074-T-G

Position:

• chr13-32319074-T-G

Variant summary

Our verdict is Likely benign. Variant got -4 ACMG points: 0P and 4B. BP4_Strong

The NM_000059.4(BRCA2):​c.68-3T>G variant causes a splice region, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.0000199 in 1,611,944 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 2/3 splice prediction tools predict no significant impact on normal splicing. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: 𝑓 0.00012 (0 hom., cov: 33)
  • Exomes 𝑓: 0.0000096 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_region, intron
• Scores: Splicing: ADA: 0.8999
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications U:8 B:1
• Conservation: PhyloP100: -0.111

Genes affected

BRCA2 (HGNC:1101): (BRCA2 DNA repair associated) Inherited mutations in BRCA1 and this gene, BRCA2, confer increased lifetime risk of developing breast or ovarian cancer. Both BRCA1 and BRCA2 are involved in maintenance of genome stability, specifically the homologous recombination pathway for double-strand DNA repair. The largest exon in both genes is exon 11, which harbors the most important and frequent mutations in breast cancer patients. The BRCA2 gene was found on chromosome 13q12.3 in human. The BRCA2 protein contains several copies of a 70 aa motif called the BRC motif, and these motifs mediate binding to the RAD51 recombinase which functions in DNA repair. BRCA2 is considered a tumor suppressor gene, as tumors with BRCA2 mutations generally exhibit loss of heterozygosity (LOH) of the wild-type allele. [provided by RefSeq, May 2020]

BRCA2 (HGNC:):

ACMG classification

Verdict is Likely_benign. Variant got -4 ACMG points.

• BP4: Computational evidence support a benign effect (BayesDel_noAF=-0.77).

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.68-3T>G		splice_region_variant, intron_variant		ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.68-3T>G		splice_region_variant, intron_variant		5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.-302-3T>G		splice_region_variant, intron_variant		1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.68-3T>G		splice_region_variant, intron_variant		2		ENSP00000506251.1

Frequencies

GnomAD3 genomes AF: 0.000118 AC: 18 AN: 152188 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.00

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00118

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.00

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000644 AC: 16 AN: 248470 Hom.: 0 AF XY: 0.0000445 AC XY: 6 AN XY: 134754

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.000465

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000959 AC: 14 AN: 1459756 Hom.: 0 Cov.: 32 AF XY: 0.00000689 AC XY: 5 AN XY: 726106

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.000314

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome AF: 0.000118 AC: 18 AN: 152188 Hom.: 0 Cov.: 33 AF XY: 0.000229 AC XY: 17 AN XY: 74366

Gnomad4 AFR AF: 0.00

Gnomad4 AMR AF: 0.00118

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.00

Gnomad4 OTH AF: 0.00

Bravo AF: 0.0000793

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Uncertain:8 Benign:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Jun 09, 2023	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) -
Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Oct 17, 2022	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. -

Breast-ovarian cancer, familial, susceptibility to, 2 Uncertain:1 Benign:1

Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Sep 05, 2023	- -
Likely benign, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Sep 26, 2024	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]. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Nov 20, 2023

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. -

Malignant tumor of breast Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

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. -

Familial cancer of breast Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Baylor Genetics

Jan 26, 2024

- -

Hereditary cancer-predisposing syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Ambry Genetics

Jul 04, 2024

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. -

Hereditary breast ovarian cancer syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Jan 18, 2024

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). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.77
CADD	Benign	22
DANN	Benign	0.70

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Benign	0.90
dbscSNV1_RF	Benign	0.70
SpliceAI score (max)		0.77		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.77		Position offset: 1
DS_AL_spliceai		0.67		Position offset: 3

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs786201770; hg19: chr13-32893211;