13-32329440-C-G

Variant summary

Our verdict is Pathogenic. Variant got 12 ACMG points: 12P and 0B. PM2 PP3_Moderate PP5_Very_Strong

The NM_000059.4(BRCA2):c.632-3C>G variant causes a splice region, splice polypyrimidine tract, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00000251 in 1,590,500 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign 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: 𝑓 0.000013 (0 hom., cov: 32)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_region, splice_polypyrimidine_tract, intron
• Scores: Splicing: ADA: 0.9999
• Clinical Significance: Likely pathogenic reviewed by expert panel P:8 U:2
• Conservation: PhyloP100: -0.0370

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]

ACMG classification

Verdict is Pathogenic. Variant got 12 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 13-32329440-C-G is Pathogenic according to our data. Variant chr13-32329440-C-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 219896.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr13-32329440-C-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA2	NM_000059.4	c.632-3C>G		splice_region_variant, splice_polypyrimidine_tract_variant, intron_variant		ENST00000380152.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA2	ENST00000380152.8	c.632-3C>G		splice_region_variant, splice_polypyrimidine_tract_variant, intron_variant		5	NM_000059.4	A2

Frequencies

GnomAD3 genomes AF: 0.0000131 AC: 2 AN: 152154 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.0000482

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

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: 0.00000411 AC: 1 AN: 243142 Hom.: 0 AF XY: 0.00 AC XY: 0 AN XY: 131370

Gnomad AFR exome AF: 0.0000668

Gnomad AMR exome AF: 0.00

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: 0.00000139 AC: 2 AN: 1438228 Hom.: 0 Cov.: 27 AF XY: 0.00 AC XY: 0 AN XY: 716226

Gnomad4 AFR exome AF: 0.0000607

Gnomad4 AMR exome AF: 0.00

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: 0.0000131 AC: 2 AN: 152272 Hom.: 0 Cov.: 32 AF XY: 0.00 AC XY: 0 AN XY: 74450

Gnomad4 AFR AF: 0.0000481

Gnomad4 AMR AF: 0.00

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: 0.00000378

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:8 Uncertain:2

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2 Uncertain:1

Likely pathogenic, criteria provided, single submitter	curation	Sema4, Sema4	Sep 11, 2021	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 19, 2020	This variant causes a C to G nucleotide substitution at the -3 position of intron 7 of the BRCA2 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. Functional RNA studies have shown that this variant causes the use of an alternative splice acceptor site 2-nucleotide upstream of the reference acceptor site, creating premature translation stop signal in the RNA transcripts (PMID: 22505045, 30883759). The aberrant transcripts are expected to result in an absent or non-functional protein product. This variant has been reported in individuals affected with breast and/or ovarian cancer (PMID: 22505045, Color internal data). In addition, a multifactorial likelihood model using health history and tumor pathology data has suggested this variant have a high probability of being pathogenic (PMID: 31131967). This variant has been identified in 1/243142 chromosomes 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. -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Nov 09, 2023	The c.632-3C>G intronic variant results from a C to G substitution 3 nucleotides upstream from coding exon 7 in the BRCA2 gene. This nucleotide position is well 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. This alteration results in use of this novel cryptic acceptor site two nucleotides upstream of the native site resulting in a frameshifting transcript (Ambry internal data; Houdayer C et al. Hum. Mutat. 2012 Aug;33(8):1228-38; Fraile-Bethencourt E et al. J. Pathol., 2019 08;248:409-420). In addition, this alteration has been shown to express an in-frame transcript, known as 6q39_8 in the literature (Ambry internal data). This transcript has been shown to be able to perform homology directed repair in protein functional studies at a near wild-type level (Mesman, RLS et al. Genet Med 2020 08;22(8):1355-1365). This alteration was identified as homozygous in a fetus with features consistent with Fanconi anemia (Malric, A et al. Pediatr Blood Cancer 2015 Mar;62(3):463-70). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. -

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

Likely pathogenic, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	Dec 06, 2011	- -
Likely pathogenic, reviewed by expert panel	curation	ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel, ClinGen	Apr 23, 2024	The c.632-3C>G variant is an intronic variant occurring in intron 7 of the BRCA2 gene. This variant is present in gnomAD v2.1 (exomes only, non-cancer subset) or gnomAD v3.1 (non-cancer subset) but is below the ENIGMA BRCA1/2 VCEP threshold >0.00002 for BS1_Supporting (PM2_Supporting, BS1, and BA1 are not met). This variant is reported to result in aberrant mRNA splicing. RT-PCR and Mini-gene assays demonstrated that the variant impacts splicing by creating a new acceptor splice site, resulting in retention of 2bp from intron 7, which leads to a frameshift and generates a premature stop codon (PMIDs: 30883759, 32123317, 22505045). Reduced expression of aberrant transcript reported by one assay suggesting potential incomplete expression of the aberrant transcript (Ambry internal contributor). Appropriate code strength determined by comparison of results to PVS1 decision tree PVS1 (RNA) was downgraded to PVS1_Moderate (RNA). This variant has been detected in 2 siblings with phenotype consistent with BRCA2-Fanconi Anemia (FA). At least two clinical features of FA (physical features, pathology findings and cancer diagnosis <=5yr) and confirmed chromosome breakage, are seen in these individuals. Both siblings were homozygous for the variant. Total points equated to 2 (PM3 met; PMID: 25381700). Multifactorial likelihood ratio analysis using clinically calibrated data produced a combined LR for this variant of 12.477 (based on Pathology LR=1.07; Co-occurrence LR=1.0498; Family History LR=11.107), within the thresholds for Moderate evidence towards pathogenicity (LR >4.3 & <=18.7) (PP4_Moderate met; PMID: 31131967, 31853058). In summary, this variant meets the criteria to be classified as a Likely pathogenic variant for BRCA2-related cancer predisposition based on the ACMG/AMP criteria applied as specified by the ENIGMA BRCA1/2 VCEP (PVS1_Moderate (RNA), PM3, PP4_Moderate). -

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Jul 01, 2022	BRCA2: PVS1, PM2, PS3:Supporting -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jul 28, 2023	Non-canonical splice site variant demonstrated to result in loss-of-function (Houdayer et al., 2012; Fraile-Bethencourt et al., 2019); Multifactorial likelihood analysis suggests this variant is pathogenic (Parsons et al., 2019); Not observed at significant frequency in large population cohorts (gnomAD); Also known as 860-3C>G; This variant is associated with the following publications: (PMID: 26913838, 22505045, 29750258, 25416802, 32123317, 33466630, 30883759, 33646313, 25381700, 31131967) -

Familial cancer of breast Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Oct 10, 2022

- -

Hereditary breast ovarian cancer syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jan 26, 2024

This sequence change falls in intron 7 of the BRCA2 gene. It does not directly change the encoded amino acid sequence of the BRCA2 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or disrupted protein product. This variant is present in population databases (rs568027879, gnomAD 0.007%). This variant has been observed in individual(s) with a personal and/or family history of breast and/or ovarian cancer (PMID: 22505045, 33466630, 33646313). ClinVar contains an entry for this variant (Variation ID: 219896). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 31131967). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in introduction of a cryptic splice acceptor site and introduces a premature termination codon (PMID: 22505045, 30883759, 32123317; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Quest Diagnostics Nichols Institute San Juan Capistrano

Feb 28, 2017

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.46
Cadd	Uncertain	23
Dann	Benign	0.63

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.97
SpliceAI score (max)		0.95		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.95		Position offset: 1
DS_AL_spliceai		0.77		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: rs568027879; hg19: chr13-32903577;