13-32326615-T-G

Variant summary

Our verdict is Pathogenic. Variant got 20 ACMG points: 20P and 0B. PVS1 PP3_Strong PP5_Very_Strong

The NM_000059.4(BRCA2):c.631+2T>G variant causes a splice donor change. The variant allele was found at a frequency of 0.00000509 in 1,572,838 control chromosomes in the GnomAD database, with no homozygous occurrence. 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 Pathogenic (★★★).

• Frequency:
  • Genomes: 𝑓 0.0000066 (0 hom., cov: 33)
  • Exomes 𝑓: 0.0000049 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_donor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:22
• Conservation: PhyloP100: 5.38

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 20 ACMG points.

• PVS1: Splicing variant, LoF is a know mechanism of disease, No cryptic splice site detected. Exon removal results in frameshift change.
• 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 13-32326615-T-G is Pathogenic according to our data. Variant chr13-32326615-T-G is described in ClinVar as [Pathogenic]. Clinvar id is 9349.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr13-32326615-T-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA2	NM_000059.4	c.631+2T>G		splice_donor_variant		ENST00000380152.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA2	ENST00000380152.8	c.631+2T>G		splice_donor_variant		5	NM_000059.4	A2

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152218 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.00

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: 0.0000147

Gnomad OTH AF: 0.00

GnomAD4 exome AF: 0.00000493 AC: 7 AN: 1420620 Hom.: 0 Cov.: 29 AF XY: 0.00000564 AC XY: 4 AN XY: 708736

Gnomad4 AFR exome AF: 0.00

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: 0.00000651

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152218 Hom.: 0 Cov.: 33 AF XY: 0.00 AC XY: 0 AN XY: 74366

Gnomad4 AFR AF: 0.00

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: 0.0000147

Gnomad4 OTH AF: 0.00

Alfa AF: 0.0000588 Hom.: 0

TwinsUK AF: 0.000270 AC: 1

ALSPAC AF: 0.00 AC: 0

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:22

Revision: reviewed by expert panel

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:7

Pathogenic, 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 5 based on posterior probability = 0.998311 -
Pathogenic, criteria provided, single submitter	clinical testing	Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge	Oct 02, 2015	- -
Pathogenic, criteria provided, single submitter	research	HudsonAlpha Institute for Biotechnology, HudsonAlpha Institute for Biotechnology	Jan 22, 2019	- -
Pathogenic, criteria provided, single submitter	clinical testing	Michigan Medical Genetics Laboratories, University of Michigan	Apr 21, 2016	- -
Pathogenic, no assertion criteria provided	clinical testing	Breast Cancer Information Core (BIC) (BRCA2)	May 29, 2002	- -
Pathogenic, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	Jan 08, 2013	- -
Pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Jun 14, 2016	- -

not provided Pathogenic:4

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Mar 22, 2023	Observed in the heterozygous state in individuals with a personal or family history of breast, ovarian, or other cancers (Evans et al., 2003; Wong-Brown et al., 2015; Ellingson et al., 2015; Maxwell et al., 2017); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports a deleterious effect on splicing; Also known as 859+2T>G; This variant is associated with the following publications: (PMID: 20455026, 26659639, 15070707, 25525159, 21548014, 25682074, 16115142, 26834852, 26296701, 24259538, 26920070, 25085752, 28831036, 29753700, 11185744, 16825431, 15645491, 30787465, 21719596, 32398771, 20927582, 33087929, 32719484, 12960223, 31131967) -
Pathogenic, criteria provided, single submitter	clinical testing	ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	May 06, 2021	The BRCA2 c.631+2T>G variant (rs81002899), also known as IVS7+2T>G, is reported in the literature in multiple individuals with a personal or family history of breast and/or ovarian cancer (Pyne 2000, Wagner 2004). In addition, this variant has been described in the homozygous state or in trans to another pathogenic BRCA2 variant in individuals affected with Fanconi anemia (Meyer 2005, Wagner 2004). The c.631+2T>G variant is absent from general population databases (Exome Variant Server, Genome Aggregation Database), indicating it is not a common polymorphism. This variant abolishes the canonical splice donor site of intron 7, which is likely to disrupt gene function. Indeed, functional assays suggest this variant causes skipping of exon 7, resulting in mRNA degradation and a lack of detectable protein (Pyne 2000, Meyer 2005). Based on available information, this variant is considered to be pathogenic. References: Meyer S et al. A cross-linker-sensitive myeloid leukemia cell line from a 2-year-old boy with severe Fanconi anemia and biallelic FANCD1/BRCA2 mutations. Genes Chromosomes Cancer. 2005 Apr;42(4):404-15. Pyne MT et al. The BRCA2 genetic variant IVS7 + 2T-->G is a mutation. J Hum Genet. 2000;45(6):351-7. Wagner JE et al. Germline mutations in BRCA2: shared genetic susceptibility to breast cancer, early onset leukemia, and Fanconi anemia. Blood. 2004 Apr 15;103(8):3226-9. -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jun 20, 2023	The BRCA2 c.631+2T>G variant is located in a canonical splice-donor site and interferes with normal BRCA2 mRNA splicing. This variant has been reported in the published literature in individuals with breast and/or ovarian cancer (PMIDs: 33471991 (2021) see also LOVD (https://databases.lovd.nl/shared/variants/BRCA2), 31090900 (2019), 29625052 (2018), 26689913 (2015), 25682074 (2015), 26296701 (2015), 11185744 (2000)). Additionally, the variant is reported to be damaging to BRCA2 function by causing the premature truncation of exon 7 (PMIDs: PMIDs: 31131967 (2019), 21719596 (2011), 11185744 (2000)). This variant has not been reported in large, multi-ethnic general populations (Genome Aggregation Database, http://gnomad.broadinstitute.org). Based on the available information, this variant is classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Jun 05, 2019	- -

Hereditary breast ovarian cancer syndrome Pathogenic:4

Pathogenic, no assertion criteria provided	research	Research Molecular Genetics Laboratory, Women's College Hospital, University of Toronto	Jan 31, 2014	- -
Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Jan 31, 2024	This sequence change affects a donor splice site in intron 7 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. This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individual(s) with BRCA2-related conditions (PMID: 11185744, 15070707, 15645491, 16825431, 21719596, 25682074, 26296701). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. This variant is also known as IVS7+2T>G. ClinVar contains an entry for this variant (Variation ID: 9349). Studies have shown that disruption of this splice site results in skipping of exon 7 and introduces a premature termination codon (PMID: 11185744, 21719596; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Jul 05, 2016	Variant summary: The BRCA2 c.631+2T>G variant involves the alteration of a conserved intronic nucleotide located at the invariable splice acceptor site in intron 7 of BRCA2. One in silico tool predicts a damaging outcome for this variant along with 5/5 splice site tools predicting the variant to result in the elimination of the splice donor site in intron 7. These predictions were confirmed by Pyne_J Hum Genet_2000 demonstrating that an RNA splicing product that deletes exon 7 was produced by the chromosome that carries the variant of interest. The deletion of exon 7 from the RNA alters the open reading frame by removing residues 249287 and incorporating 18 abnormal amino acids before terminating with an opal stop codon. This variant is absent in 121164 control chromosomes while it was reported in several HBOC spectrum patients. Furthermore, multiple clinical diagnostic laboratories and reputable databases classified this variant as Pathogenic. Taken together, this variant is classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Feb 20, 2018	The c.631+2T>G variant in BRCA2 has been reported in >15 individuals with BRCA2- associated cancers (Pyne 2000, Wong-Brown 2015, Breast Cancer Information Core ( BIC) database). It was absent from large population studies. The c.631+2T>G vari ant occurs in the invariant region (+/- 1,2) of the splice consensus sequence an d has been shown to cause altered splicing leading to an absent protein (Pyne 20 00, Meyer 2005, Biswas 2011). Heterozygous loss of function of the BRCA2 gene is an established disease mechanism in hereditary breast and ovarian cancer (HBOC) . In summary, this variant meets criteria to be classified as pathogenic for her editary breast and ovarian cancer syndrome in an autosomal dominant manner. ACMG /AMP criteria applied: PS4, PM2, PVS1. -

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 23, 2023	This variant causes a T to G nucleotide substitution at the +2 position of intron 7 of the BRCA2 gene. An RNA study has detected the out-of-frame skipping of exon 7 in carrier RNA (PMID: 11185744). To our knowledge, functional studies have not been reported for this variant. This variant has been detected in at least ten individuals affected with breast and ovarian cancer (PMID: 11185744, 19620486, 20927582, 25682074, 28831036, 33471991; Leiden Open Variation Database DB-ID BRCA2_000039) and is reported to have segregation likelihood ratio for pathogenicity of 22.5147 (PMID: 31131967). This variant also has been reported in four homozygous and heterozygous carriers from three unrelated families who were affected with the recessive disease, Fanconi anemia (PMID: 15070707, 15645491, 21548014). 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 Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	May 23, 2022	The c.631+2T>G intronic pathogenic mutation results from a T to G substitution two nucleotides after coding exon 6 in the BRCA2 gene. This mutation has been reported in multiple families with hereditary breast and ovarian cancer (HBOC) syndrome (Pyne MT et al. J. Hum. Genet. 2000;45:351-7; Wong-Brown MW et al. Breast Cancer Res. Treat. 2015 Feb;150:71-80). It has also been reported in the homozygous state and in the compound heterozygous state with other deleterious BRCA2 mutations in individuals who were either clinically diagnosed with or had symptoms of Fanconi anemia (Wagner JE et al. Blood. 2004 Apr;103:3226-9; Alter, BP et al. J. Med. Genet. 2007 Jan;44(1):1-9; Myers, K et al. Pediatr Blood Cancer 2012 Mar;58(3):462-5). This alteration results in substantial aberrant splicing in the set of samples tested (Ambry internal data). However, this alteration was able to fully complement the growth defect in Brca2-null mouse embryonic stem cells and surviving cells harboring this alteration retained substantial amounts of homology-directed DNA repair function (Mesman RLS et al. Genet Med, 2020 08;22:1355-1365). In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as a disease-causing mutation. However, because this variant is identified in one or more patients with Fanconi Anemia it may be hypomorphic and thus, carriers of this variant and their families may present with reduced risks, and not with the typical clinical characteristics of a high-risk pathogenic BRCA2 alteration. As risk estimates are unknown at this time, clinical correlation is advised. -

Breast and/or ovarian cancer Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

CHEO Genetics Diagnostic Laboratory, Children's Hospital of Eastern Ontario

Sep 19, 2017

- -

Fanconi anemia Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Feb 20, 2018

The c.631+2T>G variant in BRCA2 has been identified in the homozygous or compoun d heterozygous state in 5 individuals with Fanconi anemia (Myer 2005, Myers 2012 , Wagner 2004), and segregated with disease in 2 individuals from 2 families. It was absent from large population studies. The c.631+2T>G variant occurs in the invariant region (+/- 1,2) of the splice consensus sequence and has been shown t o cause altered splicing leading to an absent protein (Pyne 2000, Meyer 2005, Bi swas 2011). In summary, this variant meets criteria to be classified as pathogen ic for Fanconi anemia in an autosomal recessive manner. ACMG/AMP criteria applie d: PVS1, PM3_VeryStrong, PS4_Moderate, PM2, PP1. -

Fanconi anemia complementation group D1 Pathogenic:1

Pathogenic, no assertion criteria provided

literature only

OMIM

Jan 01, 2007

- -

Malignant tumor of breast Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The c.631+2T>G variant was identified in the literature associated with Fanconi Anaemia in a compound heterozygous state and with breast cancer in a heterozygous state (Pyne 2000, Biswas 2011). The variant was identified in dbSNP (ID: rs81002899) with “Pathogenic/untested allele,” and was not found in NHLBI Exome Sequencing Project (Exome Variant Server) or Exome Aggregation Consortium (ExAC) databases. The variant was also identified in the ClinVar database 6X and classified as a pathogenic variant by the Sharing Clinical Reports Project, derived from Myriad reports, Invitae, Ambry Genetics, GeneDx, BIC and OMIM. The c.631+2T>G variant was identified in the BIC database 18X with clinical importance and in ARUP Laboratories 1X classified as “definitely pathogenic”, The Fanconi Anaemia (FA) LOVD database identified the variant 3X classified as “predicted deleterious”. The c.631+2T>G variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. 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. Biochemical and genetic analysis demonstrates an RNA splicing product that deletes exon 7 was produced by the chromosome that carries BRCA2 c.631+2T>G. The deletion of exon 7 from the RNA alters the open reading frame by removing residues 249–287 and incorporating 18 abnormal amino acids before stop codon terminating, this is strong evidence that this variant is deleterious (Pyne 2000). In addition studies of functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell–based assay, the c.631+2T>G allele produces an alternatively spliced transcript lacking exons 4-7, encoding an in-frame BRCA2 protein with an internal deletion of 105 amino acids (BRCA2∆105). Evaluation of this transcript in normal and leukemia cells suggests that BRCA2∆105 is proficient in homologous recombination-mediated DNA repair as measured by different functional assays. The authors concluded that the BRCA2∆105 transcript may lead to a milder FA phenotype, however further evidence would be required to confirm this finding (Biswas 2011). In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic. -

Familial cancer of breast Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Dec 09, 2022

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.28	D
BayesDel_noAF	Pathogenic	0.16
Cadd	Pathogenic	30
Dann	Uncertain	0.98
Eigen	Pathogenic	0.94
Eigen_PC	Pathogenic	0.78
FATHMM_MKL	Pathogenic	0.99	D
MutationTaster	Benign	1.0	A;A
GERP RS		4.5

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.82		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.82		Position offset: -2

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

Publications

LitVar

Other links and lift over

dbSNP: rs81002899; hg19: chr13-32900752;