NM_000059.4:c.8332-2A>G

Variant names:

• chr13-32370400-A-G
• rs587782774
• NM_000059.4:c.8332-2A>G

Variant summary

Our verdict is Pathogenic. The variant received 12 ACMG points: 12P and 0B. PVS1_Moderate PM2 PP5_Very_Strong

The NM_000059.4(BRCA2):​c.8332-2A>G variant causes a splice acceptor, intron change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★).

• Frequency: Genomes: not found (cov: 32)
• Consequence: BRCA2 NM_000059.4 splice_acceptor, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:13
• Conservation: PhyloP100: 9.08
• 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: Pathogenic. The variant received 12 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.015209125 fraction of the gene. Cryptic splice site detected, with MaxEntScore 4.9, offset of 14, new splice context is: gtccggatttctgctaacAGtac. Cryptic site results in frameshift change. If cryptic site found is not functional and variant results in exon loss, it results in inframe change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 13-32370400-A-G is Pathogenic according to our data. Variant chr13-32370400-A-G is described in ClinVar as Pathogenic/Likely_pathogenic. ClinVar VariationId is 142861.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000059.4. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	BRCA2	NM_000059.4	MANE Select	c.8332-2A>G		splice_acceptor intron	N/A	NP_000050.3
	BRCA2	NM_001432077.1		c.8332-2A>G		splice_acceptor intron	N/A	NP_001419006.1
	BRCA2	NM_001406720.1		c.8332-2A>G		splice_acceptor intron	N/A	NP_001393649.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	BRCA2	ENST00000380152.8	TSL:5 MANE Select	c.8332-2A>G		splice_acceptor intron	N/A	ENSP00000369497.3
	BRCA2	ENST00000544455.6	TSL:1	c.8332-2A>G		splice_acceptor intron	N/A	ENSP00000439902.1
	BRCA2	ENST00000530893.7	TSL:1	c.7963-2A>G		splice_acceptor intron	N/A	ENSP00000499438.2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 32

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:13

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:3

Feb 14, 2017

Counsyl

Significance: Likely pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

This submission and the accompanying classification are no longer maintained by the submitter. For more information on current observations and classification, please contact variantquestions@myriad.com.

Jun 06, 2023

KCCC/NGS Laboratory, Kuwait Cancer Control Center

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.8332-2A>G intronic variant results from an A to G substitution two nucleotides upstream from coding exon 18 in the BRCA2 gene. This nucleotide position is highly conserved on sequence alignment. Using the BDGP and ESEfinder splice site prediction tools, this alteration is predicted to abolish the native splice acceptor site. Alterations that disrupt the canonical splice acceptor site are typically deleterious in nature (ACMG Recommendations for Standards for Interpretation and Reporting of Sequence Variations. Revision 2007. Genet Med. 2008; 10:294). In-silico prediction show pathogenic computational verdict based on 6 pathogenic predictions from BayesDel_addAF, DANN, EIGEN, FATHMM-MKL, MutationTaster and scSNV-Splicing vs no benign predictions. ClinVar classifies this variant as Pathogenic, rated 2 stars, with 5 submissions and no conflicts. Therefore, the c.8332-2A>G variant is classified as pathogenic.

Oct 02, 2015

Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

not provided Pathogenic:3

Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

May 08, 2024

GeneDx

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Canonical splice site variant demonstrated to result in a null allele in a gene for which loss of function is a known mechanism of disease (PMID: 31343793); Published functional study demonstrates inability to rescue cell viability in a complementation assay (PMID: 32398771); Not observed at significant frequency in large population cohorts (gnomAD); Also known as 8560-2A>G; This variant is associated with the following publications: (PMID: 28152038, 29446198, 31343793, 12228710, 32398771)

Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

Hereditary cancer-predisposing syndrome Pathogenic:3

Mar 25, 2025

Ambry Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.8332-2A>G intronic variant results from an A to G substitution two nucleotides upstream from coding exon 18 in the BRCA2 gene. This nucleotide position is highly 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 abnormal splicing (Ambry internal data; Mesman RLS et al. Genet Med, 2020 Aug;22:1355-1365; Montalban G et al. Hum Mutat, 2019 Dec;40:2296-2317). The results from two saturation genome editing-based studies, including a haploid cell-survival assay and a humanized mouse embryonic stem cell line assay of drug response and survival, are discordant for this nucleotide substitution (Huang H et al. Nature. 2025 Feb;638(8050):528-537; Sahu S et al. Nature. 2025 Feb;638(8050):538-545). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the majority of available evidence to date, this variant is likely to be pathogenic.

Feb 25, 2025

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant causes an A to G nucleotide substitution at the -2 position of intron 18 of the BRCA2 gene. RNA studies have shown that this variant and a similar variant disrupting the intron 18 splice acceptor site, resulted in the out-of-frame deletion of 14 bases in exon 19 (c.8332_8345del) and the in-frame deletion of exon 19 (c.8332_8487del) impacting the functionally important DNA binding domain of the BRCA2 protein (PMID: 21735045, 31343793). A functional study has reported that this variant impacted BRCA2 function in a haploid cell proliferation assay (PMID: 39779857). This variant has been reported in at least seven suspected hereditary breast and ovarian cancer families (PMID: 29446198, 31343793, 32614418). A multifactorial analysis has reported a likelihood ratio for pathogenicity based on personal and family history of 0.847 from log(LR)=-0.072239541 in two carriers (PMID: 31853058). 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.

Nov 26, 2024

Molecular Diagnostics Laboratory, Catalan Institute of Oncology

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

PVS1 (RNA), PM2_Supporting c.8332-2A>G, located in a canonic splicing site of the BRCA2 gene, is predicted to alter splicing. Studies have shown that disruption of this splice site results in partial deletion of exon 19 and skipping of exon 19 and introduces a premature termination codon (r.[8332_8345del, r.8332_8487del]; p.[Ile2778Tyrfs*15, p.Ile2778_Gln2829del]) (PMID: 31343793) (PVS1(RNA). It is not present in the population database gnomAD v2.1.1, non cancer dataset (PM2_supporting). To our knowledge, functional studies have not been reported for this variant. In addition, the variant has been identified in the ClinVar database (6x pathogenic, 4x likely pathogenic), in the LOVD database (8x pathogenic) and BRCA Exchange database, (‘not yet reviewed’).Based on currently available information, the variant c.8332-2A>G is classified as a likely pathogenic variant according to ClinGen- BRCA1 and BRCA2 Guidelines version 1.0.0.

Hereditary breast ovarian cancer syndrome Pathogenic:2

Mar 01, 2019

Hereditary Cancer Genetics group, Vall d'Hebron Institute of Oncology

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: research

Apr 26, 2023

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

ClinVar contains an entry for this variant (Variation ID: 142861). Disruption of this splice site has been observed in individual(s) with breast and/or ovarian cancer (PMID: 29446198, 31343793). This variant is not present in population databases (gnomAD no frequency). This sequence change affects an acceptor splice site in intron 18 of the BRCA2 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and may result in an absent or disrupted protein product. Studies have shown that disruption of this splice site results in partial deletion of exon 19 and skipping of exon 19 and introduces a premature termination codon (PMID: 21735045, 31343793). 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.

Malignant tumor of breast Pathogenic:1

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

The c.8332-2A>G variant has not been previously identified in the literature nor by our laboratory and is of the type which is expected to cause the disorder. The c.8332-2A>G variant is located in the 3' splice region, and is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant -1 and -2 positions of the splice consensus sequence. In summary, based on the information above this variant is Pathogenic.

Familial cancer of breast Pathogenic:1

Sep 07, 2022

Baylor Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.41	D
BayesDel_noAF	Pathogenic	0.35
CADD	Pathogenic	34
DANN	Uncertain	1.0
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	1.0	D
PhyloP100		9.1
GERP RS		5.3
Mutation Taster		=0/100	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
Splicevardb		3.0
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.55		Position offset: 16
DS_AL_spliceai		0.99		Position offset: 2

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

Other links and lift over

dbSNP: rs587782774; hg19: chr13-32944537;