13-32319074-T-G

Variant names:

• chr13-32319074-T-G
• rs786201770
• NM_000059.4:c.68-3T>G

Variant summary

Our verdict is Likely benign. The variant received -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
• Publications: 3 publications found

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 Gene-Disease associations (from GenCC):

• breast-ovarian cancer, familial, susceptibility to, 2

  Inheritance: AD Classification: DEFINITIVE, STRONG Submitted by: Ambry Genetics, Genomics England PanelApp, Labcorp Genetics (formerly Invitae), ClinGen
• Fanconi anemia complementation group D1

  Inheritance: AR Classification: DEFINITIVE, STRONG Submitted by: Labcorp Genetics (formerly Invitae), Ambry Genetics, ClinGen, G2P
• pancreatic cancer, susceptibility to, 2

  Inheritance: AD Classification: STRONG Submitted by: Genomics England PanelApp
• sarcoma

  Inheritance: AD Classification: MODERATE Submitted by: Genomics England PanelApp
• hereditary breast ovarian cancer syndrome

  Inheritance: AD Classification: SUPPORTIVE Submitted by: Orphanet
• Fanconi anemia

  Inheritance: AR Classification: SUPPORTIVE Submitted by: Orphanet
• medulloblastoma

  Inheritance: AD Classification: LIMITED Submitted by: Ambry Genetics

ACMG classification

Our verdict: Likely_benign. The variant received -4 ACMG points.

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

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.68-3T>G		splice_region_variant, intron_variant	Intron 2 of 26	ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.68-3T>G		splice_region_variant, intron_variant	Intron 2 of 26	5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.-302-3T>G		splice_region_variant, intron_variant	Intron 2 of 26	1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.68-3T>G		splice_region_variant, intron_variant	Intron 1 of 25	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

GnomAD2 exomes AF: 0.0000644 AC: 16 AN: 248470 AF XY: 0.0000445

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 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

African (AFR) AF: 0.00 AC: 0 AN: 33362

American (AMR) AF: 0.000314 AC: 14 AN: 44544

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 26110

East Asian (EAS) AF: 0.00 AC: 0 AN: 39672

South Asian (SAS) AF: 0.00 AC: 0 AN: 85678

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53258

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5680

European-Non Finnish (NFE) AF: 0.00 AC: 0 AN: 1111126

Other (OTH) AF: 0.00 AC: 0 AN: 60326

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

African (AFR) AF: 0.00 AC: 0 AN: 41454

American (AMR) AF: 0.00118 AC: 18 AN: 15272

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 3470

East Asian (EAS) AF: 0.00 AC: 0 AN: 5206

South Asian (SAS) AF: 0.00 AC: 0 AN: 4828

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 10612

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 316

European-Non Finnish (NFE) AF: 0.00 AC: 0 AN: 68028

Other (OTH) AF: 0.00 AC: 0 AN: 2090

Alfa AF: 0.00 Hom.: 0

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

Oct 17, 2022

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: 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.

Jun 09, 2023

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: 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)

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

Sep 26, 2024

Myriad Genetics, Inc.

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: 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].

Sep 05, 2023

All of Us Research Program, National Institutes of Health

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

not specified Uncertain:1

Nov 20, 2023

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

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: 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.

Malignant tumor of breast Uncertain:1

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: 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.

Familial cancer of breast Uncertain:1

Jan 26, 2024

Baylor Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Hereditary cancer-predisposing syndrome Uncertain:1

Aug 01, 2025

Ambry Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: 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). Based on data from gnomAD, the frequency for this variant is above the maximum credible frequency for a disease-causing variant in this gene based on internally established thresholds (Karczewski et al. Nature. 2020 May;581(7809):434-443; Whiffin et al. Genet Med. 2017 10;19:1151-1158). 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

Nov 24, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: 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.

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.77
CADD	Benign	22
DANN	Benign	0.70
PhyloP100		-0.11

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

Other links and lift over

dbSNP: rs786201770; hg19: chr13-32893211;