13-32363535-T-C

Position:

• chr13-32363535-T-C

Variant summary

Our verdict is Pathogenic. Variant got 12 ACMG points: 12P and 0B. PVS1_Moderate PM2 PP5_Very_Strong

The NM_000059.4(BRCA2):​c.8331+2T>C variant causes a splice donor change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000206 in 1,457,672 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000021 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_donor
• Scores: Splicing: ADA: 0.9921
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:17
• Conservation: PhyloP100: 7.67

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.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.034513015 fraction of the gene. Cryptic splice site detected, with MaxEntScore 3.2, offset of 21, new splice context is: ttgGTaaaa. Cryptic site results in inframe change. If cryptic site found is not functional and variant results in exon loss, it results in frameshift change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 13-32363535-T-C is Pathogenic according to our data. Variant chr13-32363535-T-C is described in ClinVar as [Likely_pathogenic]. Clinvar id is 267692.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr13-32363535-T-C is described in Lovd as [Pathogenic]. Variant chr13-32363535-T-C is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.8331+2T>C		splice_donor_variant		ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.8331+2T>C		splice_donor_variant		5	NM_000059.4	ENSP00000369497	A2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 0.00000206 AC: 3 AN: 1457672 Hom.: 0 Cov.: 30 AF XY: 0.00000138 AC XY: 1 AN XY: 725464

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: 0.00000180

Gnomad4 OTH exome AF: 0.0000166

GnomAD4 genome Cov.: 32

Bravo AF: 0.00000756

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:17

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

not provided Pathogenic:5

Likely pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Dec 24, 2020	Canonical splice site variant expected to result in aberrant splicing, however published functional and RNA studies are conflicting showing both aberrant splicing resulting in skipping of exon 18 and normal splicing, as well as both aberrant and wildtype transcript expression (Fraile-Bethencourt 2017, Gelli 2019, Wangensteen 2019, Wai 2020); Not observed in large population cohorts (Lek 2016); Also known as 8559+2T>G; Observed in individuals with a personal and/or family history of breast and/or ovarian cancer referred for genetic testing at GeneDx and in published literature (Cunningham 2014, Tung 2015, Kwong 2018, Fanale 2020, Rumford 2020); This variant is associated with the following publications: (PMID: 29487695, 32123317, 25186627, 28339459, 30832263, 31143303, 24504028, 29339979, 29446198, 30702160, 31131967, 32854451, 32098980, 31209999, 32380732) -
Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Jan 10, 2023	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	Aug 15, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jan 20, 2023	This variant disrupts a canonical splice-donor site and interferes with normal BRCA2 mRNA splicing. This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in an individual with ovarian cancer (PMID: 24504028 (2014)), and in individuals with breast cancer (PMID: 25186627 (2015), 29487695 (2018), 30130155 (2018), 30702160 (2019), 32854451 (2020), 35464868 (2022)). In large breast cancer association studies, this variant was found in individuals affected with breast cancer as well as unaffected individuals (PMID: 33471991 (2021), https://databases.lovd.nl/shared/variants/BRCA2). Functional splicing assays demonstrate this this variant causes aberrant splicing and skipping of exon 18 and/or partial exon 17 and exon 18, creating a premature stop codon (PMID: 28339459 (2017), 30832263 (2019), 31143303 (2019), 33469799 (2021)). However, production of the normal transcript was also demonstrated (PMID: 30832263 (2019), 32123317 (2020), 33469799 (2021)). Based on the available information, this variant is classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Clinical Genetics Laboratory, Skane University Hospital Lund	May 08, 2023	- -

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:4

Pathogenic, criteria provided, single submitter	clinical testing	Department of Medical Genetics, Oslo University Hospital	Nov 12, 2015	- -
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, no assertion criteria provided	clinical testing	BRCAlab, Lund University	Aug 26, 2022	- -
Pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Oct 05, 2023	This variant disrupts a canonical splice site and is predicted to result in abnormal splicing. This prediction has been confirmed by functional studies (PMID: 28339459, 31143303). Aberrant splicing and/or loss of function is an established mechanism of disease. This variant has been reported in multiple individuals with hereditary breast and ovarian cancer syndrome (PMID: 24504028, 29339979, 24728189, 29487695). This variant is absent from large population databases, including the Genome Aggregation Database (http://gnomad.broadinstitute.org/). -

Hereditary breast ovarian cancer syndrome Pathogenic:4

Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Jan 16, 2024	This sequence change affects a donor splice site in intron 18 of the BRCA2 gene. It is expected to disrupt RNA splicing. Variants that disrupt the donor or acceptor splice site typically lead to a loss of protein function (PMID: 16199547), and loss-of-function variants in BRCA2 are known to be pathogenic (PMID: 20104584). This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individual(s) with clinical features of hereditary breast and ovarian cancer syndrome (PMID: 24504028, 25186627, 29339979, 29446198, 29487695). ClinVar contains an entry for this variant (Variation ID: 267692). Studies have shown that disruption of this splice site alters mRNA splicing and is expected to lead to the loss of protein expression (PMID: 20104584). For these reasons, this variant has been classified as Pathogenic. -
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	National Health Laboratory Service, Universitas Academic Hospital and University of the Free State	Nov 16, 2021	- -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Oct 14, 2019	The c.8331+2T>C variant in BRCA2 has been reported in at least 2 probands with BRCA2-related cancer (Cunningham 2014, Tung 2015). It was absent from large population studies, but has been reported in ClinVar (Variation ID: 267692). This variant occurs within the canonical splice site (+/- 1,2) and is predicted to cause altered splicing leading to an abnormal or absent protein. In vitro functional studies confirm that this variant leads to abnormal splicing (Fraile-Bethencourt 2017). In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant hereditary breast and ovarian cancer (HBOC). ACMG/AMP Criteria applied: PVS1, PM2, PS4_Supporting. -

Hereditary cancer-predisposing syndrome Pathogenic:3

Likely pathogenic, criteria provided, single submitter	curation	Sema4, Sema4	Apr 05, 2021	- -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Dec 05, 2023	This variant causes a T>C nucleotide substitution at the +2 position of intron 18 splice donor site of the BRCA2 gene. RNA studies have shown that this variant results in the production of several aberrant transcripts that lack (i) exon 18, (ii) exon 17 and 18, or (iii) partial exon 17 and exon 18 (PMID: 28339459, 30832263, 33469799). These transcripts are not expected to produce a functional protein, however, it was noted that the slicing defect may be incomplete (PMID: 30832263, 33469799; ClinVar SCV001178672.2). This variant has been reported in over ten individuals affected with breast and/or ovarian cancer (PMID: 18779604, 24728189, 25186627, 28339459, 30832263, 31131967, 33469799, 35464868; communication with an external laboratory). 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. Incomplete splicing defect attributed to this variant suggests that this variant may be associated with reduced penetrance. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Mar 14, 2024	The c.8331+2T>C intronic variant results from a T to C substitution two nucleotides after coding exon 17 in the BRCA2 gene. This alteration has been reported in multiple hereditary breast and/or ovarian cohorts (Cunningham JM et al. Sci Rep, 2014 Feb;4:4026; Song H et al. Hum. Mol. Genet. 2014 Sep;23(17):4703-9; Kwong A et al. Oncotarget 2018 Jan;9(8):7832-7843; Heramb C et al. Hered Cancer Clin Pract 2018 Jan;16:3). This nucleotide position is highly conserved in available vertebrate species. Several variants at this splice donor site demonstrated substantial but incomplete abnormal splicing in multiple different RNA analyses (Ambry internal data; Fraile-Bethencourt E et al. PLoS Genet, 2017 Mar;13:e1006691; Gelli E et al. Cancers (Basel), 2019 Mar;11; Wangensteen T et al. Hered Cancer Clin Pract, 2019 May;17:14; Nix P et al. Fam Cancer, 2022 Jan;21:7-19). However, at least one recent RNA study has reported no aberrant splicing in association with this variant (Wai HA. Genet Med. 2020 Jun;22(6):1005-1014). In addition, this alteration was observed in individuals who collectively do not present with a clinical history seen in typical high-risk hereditary breast and ovarian cancer (HBOC) variant carriers (Nix P et al. Fam Cancer, 2022 Jan;21:7-19). However, these data cannot rule out the possibility of a hypomorphic variant with atypical risks. Based on the majority of available evidence to date, this variant is likely to be pathogenic. However, 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. -

Familial cancer of breast Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Apr 12, 2021

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Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.38	D
BayesDel_noAF	Pathogenic	0.31
CADD	Pathogenic	29
DANN	Uncertain	1.0
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.97
FATHMM_MKL	Pathogenic	1.0	D
MutationTaster	Benign	1.0	D;D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Benign	0.66
SpliceAI score (max)		0.93		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.93		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: rs398122602; hg19: chr13-32937672;