chr13-32326283-G-T

Position:

• chr13-32326283-G-T

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.516+1G>T variant causes a splice donor, intron change. The variant allele was found at a frequency of 0.00000137 in 1,459,762 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 Likely pathogenic (★★).

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_donor, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:8
• Conservation: PhyloP100: 6.49

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 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 0.0038997757 fraction of the gene. Cryptic splice site detected, with MaxEntScore 7.6, offset of -39, new splice context is: gtgGTatgt. 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 13-32326283-G-T is Pathogenic according to our data. Variant chr13-32326283-G-T is described in ClinVar as [Likely_pathogenic]. Clinvar id is 267649.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr13-32326283-G-T is described in Lovd as [Pathogenic]. Variant chr13-32326283-G-T is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.516+1G>T		splice_donor_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.516+1G>T		splice_donor_variant, intron_variant		5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.147+1G>T		splice_donor_variant, intron_variant		1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.516+1G>T		splice_donor_variant, intron_variant		2		ENSP00000506251.1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome AF: 0.00000137 AC: 2 AN: 1459762 Hom.: 0 Cov.: 31 AF XY: 0.00000275 AC XY: 2 AN XY: 726320

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: 0.00

GnomAD4 genome Cov.: 33

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:8

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

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

Pathogenic, criteria provided, single submitter	clinical testing	Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	Sep 21, 2015	- -
Pathogenic, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Genome Diagnostics Laboratory, University Medical Center Utrecht	Jul 28, 2017	- -
Pathogenic, criteria provided, single submitter	clinical testing	Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge	Oct 02, 2015	- -

Hereditary breast ovarian cancer syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Apr 06, 2023	For these reasons, this variant has been classified as Pathogenic. Studies have shown that disruption of this splice site alters mRNA splicing and is expected to lead to the loss of protein expression (PMID: 30883759). 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: 21990134). ClinVar contains an entry for this variant (Variation ID: 267649). Disruption of this splice site has been observed in individual(s) with breast and/or ovarian cancer (PMID: 27767231, 29446198). This variant is not present in population databases (gnomAD no frequency). This sequence change affects a donor splice site in intron 6 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Oct 14, 2019	The c.516+1G>T variant in BRCA2 has been reported in the literature in at least 1 individual with ovarian cancer (Weren 2017), and has also been reported in individuals with hereditary breast and ovarian cancer (HBOC) in ClinVar (Variation ID: 267649). It was absent from large population studies. This variant occurs within the canonical splice site (+/- 1,2) and is predicted to cause altered splicing leading to an abnormal or absent protein. Furthermore, in vitro functional studies support that this variant leads to abnormal splicing (Whiley 2011). In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant HBOC. ACMG/AMP Criteria applied: PVS1, PM2, PS3_Supporting. -

not provided Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

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

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Hereditary cancer-predisposing syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

May 21, 2024

The c.516+1G>T intronic variant results from a G to T substitution one nucleotide after coding exon 5 of the BRCA2 gene. 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. This alteration was identified in a large, worldwide study of BRCA1/2 mutation positive families (Rebbeck TR et al. Hum Mutat, 2018 05;39:593-620). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. RNA studies have demonstrated that this alteration results in abnormal splicing (Ambry internal data; Fraile-Bethencourt E et al. J Pathol, 2019 08;248:409-420; Mesman RLS et al. Genet Med, 2020 08;22:1355-1365). In addition, this alteration was unable to complement cell growth in a BRCA2-null mouse embryonic stem cell assay (Mesman RLS et al. Genet Med, 2020 08;22:1355-1365). 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.37	D
BayesDel_noAF	Pathogenic	0.29
CADD	Pathogenic	33
DANN	Uncertain	1.0
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.91
FATHMM_MKL	Pathogenic	0.99	D
GERP RS		5.2

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.98		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.98		Position offset: -1

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

Publications

LitVar

Other links and lift over

dbSNP: rs397507762; hg19: chr13-32900420;