rs61757642

Positions:

• chr13-32394936-A-T

Variant summary

Our verdict is Benign. Variant got -12 ACMG points: 0P and 12B. BP4_Strong BP6_Very_Strong

The NM_000059.4(BRCA2):​c.9501+3A>T variant causes a splice region, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.0000991 in 1,613,894 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 Benign (★★★).

• Frequency:
  • Genomes: 𝑓 0.000072 (0 hom., cov: 32)
  • Exomes 𝑓: 0.00010 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_region, intron
• Scores: Splicing: ADA: 0.9886
• Clinical Significance: Benign reviewed by expert panel U:11 B:22
• Conservation: PhyloP100: 2.04

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 Benign. Variant got -12 ACMG points.

• BP4: Computational evidence support a benign effect (BayesDel_noAF=-0.51).
• BP6: Variant 13-32394936-A-T is Benign according to our data. Variant chr13-32394936-A-T is described in ClinVar as [Benign]. Clinvar id is 38242.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr13-32394936-A-T is described in Lovd as [Pathogenic]. Variant chr13-32394936-A-T is described in Lovd as [Likely_benign]. Variant chr13-32394936-A-T is described in Lovd as [Benign].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.9501+3A>T		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.9501+3A>T		splice_region_variant, intron_variant		5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.9132+3A>T		splice_region_variant, intron_variant		1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.*1559+3A>T		splice_region_variant, intron_variant		2		ENSP00000506251.1

Frequencies

GnomAD3 genomes AF: 0.0000723 AC: 11 AN: 152228 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.0000482

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.0000655

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: 0.000118

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.000116 AC: 29 AN: 251052 Hom.: 0 AF XY: 0.000125 AC XY: 17 AN XY: 135712

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.000174

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: 0.000203

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.000102 AC: 149 AN: 1461666 Hom.: 0 Cov.: 31 AF XY: 0.000102 AC XY: 74 AN XY: 727120

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.000179

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.0000187

Gnomad4 NFE exome AF: 0.000122

Gnomad4 OTH exome AF: 0.0000662

GnomAD4 genome AF: 0.0000723 AC: 11 AN: 152228 Hom.: 0 Cov.: 32 AF XY: 0.0000269 AC XY: 2 AN XY: 74384

Gnomad4 AFR AF: 0.0000482

Gnomad4 AMR AF: 0.0000655

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.000118

Gnomad4 OTH AF: 0.00

Alfa AF: 0.000156 Hom.: 0

Bravo AF: 0.0000831

EpiCase AF: 0.000109

EpiControl AF: 0.00

ClinVar

Significance: Benign

Submissions summary: Uncertain:11 Benign:22

Revision: reviewed by expert panel

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Uncertain:4 Benign:7

Uncertain significance, no assertion criteria provided	clinical testing	Laboratoire de Biologie et Génétique du Cancer, Centre François Baclesse	-	- -
Likely benign, criteria provided, single submitter	clinical testing	Genome Diagnostics Laboratory, University Medical Center Utrecht	Oct 09, 2014	- -
Likely benign, criteria provided, single submitter	clinical testing	Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	May 31, 2017	- -
Benign, no assertion criteria provided	clinical testing	Department of Medical and Surgical Sciences, University of Bologna	Sep 01, 2023	BS1(Strong)+BP5(Very Strong) according to ACMG/AMP classification guidelines specified for BRCA1 & BRCA2 (Classification Criteria V1.0.0 2023-09-08 - https://cspec.genome.network/cspec/ui/svi/affiliation/50087) (PMID: 38160042) -
Benign, no assertion criteria provided	clinical testing	BRCAlab, Lund University	Mar 02, 2020	- -
Benign, reviewed by expert panel	curation	Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA)	Jun 18, 2019	IARC class based on posterior probability from multifactorial likelihood analysis, thresholds for class as per Plon et al. 2008 (PMID: 18951446). Class 1 based on posterior probability = 4.85E-07 -
Uncertain significance, criteria provided, single submitter	clinical testing	Illumina Laboratory Services, Illumina	Apr 27, 2017	This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). Publications were found based on this search. However, the evidence from the literature, in combination with allele frequency data from public databases where available, was not sufficient to rule this variant in or out of causing disease. Therefore, this variant is classified as a variant of unknown significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Michigan Medical Genetics Laboratories, University of Michigan	Nov 03, 2014	- -
Uncertain significance, no assertion criteria provided	clinical testing	Breast Cancer Information Core (BIC) (BRCA2)	May 29, 2002	- -
Benign, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	Sep 16, 2011	- -
Benign, criteria provided, single submitter	clinical testing	Victorian Clinical Genetics Services, Murdoch Childrens Research Institute	Jul 17, 2023	Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Benign. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with BRCA2-associated cancers, complementation group D1 fanconi anemia (MIM#605724) and Wilms tumor (MIM#194070). 0108 - This gene is associated with both recessive and dominant disease (OMIM). (I) 0209 - Splice site variant proven to affect splicing of the transcript with uncertain effect on protein sequence. RT-PCR of total RNA from lymphoblastoid cell lines demonstrated exon 25 skipping (PMID: 21394826). And while various laboratories also reported aberrant splicing with this variant (PMID: 24212087), a minigene assay showed that only 13% of product had exon 25 skipping and 87% had normal splicing (PMID: 25382762). (N) 0304 - Variant is present in gnomAD <0.01 (v2: 31 heterozygotes, 0 homozygotes). (SP) 0506 - Abnormal splicing is not predicted and nucleotide is highly conserved. (I) 0805 - This variant has strong previous evidence of being benign in unrelated individuals. It has been classifised as benign by expert panel ENIGMA (ClinVar). (SB) 1004 - This variant has moderate functional evidence supporting normal protein function. In vitro studies demonstrated normal protein expression and mutant protein retained 90% of normal protein function based homology damage repair assays (PMID: 32398771). (SB) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -

not provided Uncertain:4 Benign:4

Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Sep 05, 2017	This variant is denoted BRCA2 c.9501+3A>T or IVS25+3A>T and consists of an A>T nucleotide substitution at the +3 position of intron 25 of the BRCA2 gene. This variant, also known as 9729+3A>T using alternate nomenclature, has been observed in breast and/or ovarian cancer families (Capalbo 2006, Papi 2009, Borg 2010, Gabald? Barrios 2017). In vitro and in vivo RNA studies report that BRCA2 c.9501+3A>T results in skipping of exon 25 (Bonnet 2008, Papi 2009, Borg 2010, Whiley 2011). In addition, the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Splicing Working Group concluded that BRCA2 c.9501+3A>T produces unequivocal splicing aberrations (Whiley 2014). However, a minigene splicing assay quantified the aberrant splicing and found that this variant results in less than 15% aberrant transcript, meaning that the full length transcript is predominant (Acedo 2015). Additionally, the adenine (A) nucleotide that is altered is not conserved. BRCA2 c.9501+3A>T was observed at an allele frequency of 0.02% (16/66,618) in individuals of European (Non-Finnish) ancestry in large population cohorts (Lek 2016). Despite BRCA2 c.9501+3A>T being proven to result in skipping of exon 25, we are unable to predict the clinical impact of this variant given that the normal transcript appears to be more abundant. Thus, we consider it to be a variant of uncertain significance. -
Likely benign, criteria provided, single submitter	clinical testing	ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	Sep 09, 2021	- -
Likely benign, no assertion criteria provided	clinical testing	Laboratory of Diagnostic Genome Analysis, Leiden University Medical Center (LUMC)	-	- -
Uncertain significance, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The BRCA2 c.9501+3A>T variant was identified in 3 of 4414 proband chromosomes (frequency: 0.001) from individuals or families with breast/ovarian cancers in 2 studies (Borg 2010, Papi 2009). Giannini et al. (2006) also identified the variant in an Italian cohort of breast/ovarian cancer families, frequency unspecified. Multiple functional assays using minigene reporters and/or RNA analysis have shown that the variant causes incomplete skipping of exon 25, with wildtype transcript present and the splicing alteration affecting only a fraction of the transcripts from the variant allele (Bonnet 2008, Whiley 2011, Acedo 2015, Houdayer 2012). The variant was also identified in dbSNP (ID: rs61757642) “With likely pathogenic allele”, in the Exome Variant Server project in 3 of 8600 European American alleles (frequency: 0.0003), the Exome Aggregation Consortium (ExAC) database (released Jan 13, 2015) in 18 of 121142 chromosomes (frequency: 0.0001) (or 16 individuals from a population of European (Non-Finnish) individuals and 2 individuals from a population of Latinos). It was also identified by our laboratory in 7 individuals with breast and ovarian cancer co-occuring with BRCA2 c.9976A>T in at least 3 individuals. The variant was identified in the Clinvitae database (4x), ARUP Laboratories BRCA Mutations Database (classification pathogenic), the ClinVar database with conflicting interpretations (classified as a benign variant by the Sharing Clinical Reports Project, derived from Myriad reports; classified as likely pathogenic by GeneDX and CHEO, and uncertain significance by Ambry Genetics, BIC and Invitae), GeneInsight COGR database (2x, classified as “likely pathogenic” and unclassified), the BIC database (15x with unknown clinical importance and pending classification), UMD (classified as likely casual 15x, including 1x co-occuring with a pathogenic mutation c.5796_5797delTA (p.His1932GlnfsX12)). The c.9501+3A>T variant is located in the 5' splice region but does not affect the invariant +1 and +2 positions. However, positions +3 to +6 are part of the splicing consensus sequence and variants involving these positions sometimes affect splicing. In addition, 4 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) 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 unknown significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Mayo Clinic Laboratories, Mayo Clinic	Aug 17, 2019	- -
Uncertain significance, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Feb 01, 2021	- -
Benign, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jul 26, 2023	- -
Likely benign, no assertion criteria provided	clinical testing	Clinical Genetics Laboratory, Department of Pathology, Netherlands Cancer Institute	-	- -

Hereditary breast ovarian cancer syndrome Uncertain:2 Benign:3

Likely benign, criteria provided, single submitter	research	Genetics Program, Instituto Nacional de Cancer	Nov 01, 2021	- -
Uncertain significance, no assertion criteria provided	research	Hereditary Cancer Genetics group, Vall d'Hebron Institute of Oncology	Mar 01, 2019	- -
Uncertain significance, criteria provided, single submitter	clinical testing	National Health Laboratory Service, Universitas Academic Hospital and University of the Free State	Apr 19, 2022	- -
Benign, criteria provided, single submitter	clinical testing	Mendelics	Aug 22, 2023	- -
Benign, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Feb 01, 2024	- -

not specified Benign:4

Benign, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -
Benign, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Dec 12, 2022	Variant summary: BRCA2 c.9501+3A>T alters a 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: Three predict the variant weakens a canonical 5' donor site and one predicts the variant abolishes a canonical 5' splicing donor site. These predictions are supported by multiple functional studies that showed exon 25 was skipped (e.g. Bonnet_2008). However, several of these studies found that only a small fraction of transcript skipped exon 25, thereby calling into question the clinical relevance of the variant (e.g. Acedo_2014). In further contrast, a recently published study evaluating RNA genetic testing to re-classify splice variants has reported that this variant has no impact on splicing in accordance with the ACMG BS3 gudeline specification supporting its re-classification as likely benign (Karam_2019). The variant allele was found at a frequency of 0.00012 in 251052 control chromosomes, predominantly at a frequency of 0.0002 within the Non-Finnish European subpopulation in the gnomAD database. This frequency is not higher than expected for a pathogenic variant in BRCA2 causing Hereditary Breast And Ovarian Cancer Syndrome (0.00012 vs 0.00075), allowing no conclusion about variant significance. 6 heterozygous European American women over the age of 70 with no history of cancer (carrier frequency = 0.0008191) were found in a seperate dataset (FLOSSIES). c.9501+3A>T has been reported in the literature in individuals affected with breast cancer (e.g. Capalbo_2006, Whiley_2011, van der Hout_2006, Pajares_2018). In a recent report from Caputo_2021, the authors assigned an IARC classification of "1-benign" to the variant from their multifactorial likelihood analysis which incorporated co-segregation data from several unrelated French families. Multiple co-occurrences with other pathogenic variants have been reported in the UMD database and also observed in our laboratory and the literature (examples, UMD-BRCA2 c.5796_5797delTA, p.His1932GlnfsX12; BRCA2 c.2957dup, p.Asn986LysfsX2; BRCA1 c.1266T>G, p.Tyr422X; our laboratory-BRCA1 c.5153-2del; Karam_2019, an unspecified observation in trans with a pathogenic mutation in an individual without biallelic disease), providing supporting evidence for a benign role. Sixteen ClinVar submitters, including one expert panel (ENIGMA), have assessed the variant since 2014: nine classified the variant as uncertain significance, five as likely benign, and two as benign. Among these, the expert panel settled on a benign classification. Based on the evidence outlined above, the variant was classified as benign. -
Benign, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	Aug 15, 2023	- -
Benign, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -

Hereditary cancer-predisposing syndrome Benign:2

Likely benign, criteria provided, single submitter	clinical testing	Ambry Genetics	Mar 08, 2019	This alteration is classified as likely benign based on a combination of the following: seen in unaffected individuals, population frequency, intact protein function, lack of segregation with disease, co-occurrence, RNA analysis, in silico models, amino acid conservation, lack of disease association in case-control studies, and/or the mechanism of disease or impacted region is inconsistent with a known cause of pathogenicity. -
Likely benign, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 10, 2022	- -

Breast and/or ovarian cancer Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Foulkes Cancer Genetics LDI, Lady Davis Institute for Medical Research

Aug 23, 2013

- -

Medulloblastoma;C0346153:Familial cancer of breast;C0376358:Malignant tumor of prostate;C1838457:Fanconi anemia complementation group D1;C2675520:Breast-ovarian cancer, familial, susceptibility to, 2;C2751641:Glioma susceptibility 3;C3150546:Pancreatic cancer, susceptibility to, 2;CN033288:Wilms tumor 1 Benign:1

Benign, criteria provided, single submitter

clinical testing

Fulgent Genetics, Fulgent Genetics

Mar 30, 2022

- -

BRCA2-related disorder Benign:1

Likely benign, no assertion criteria provided

clinical testing

PreventionGenetics, part of Exact Sciences

Dec 13, 2022

This variant is classified as likely benign based on ACMG/AMP sequence variant interpretation guidelines (Richards et al. 2015 PMID: 25741868, with internal and published modifications). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.51
CADD	Benign	17
DANN	Benign	0.65

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Benign	0.70
SpliceAI score (max)		0.17		Details are displayed if max score is > 0.2

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

Publications

LitVar

Other links and lift over

dbSNP: rs61757642; hg19: chr13-32969073;