13-32325186-T-C

Variant names:

• chr13-32325186-T-C
• rs876661045
• NM_000059.4:c.425+2T>C

Variant summary

Our verdict is Pathogenic. Variant got 11 ACMG points: 11P and 0B. PVS1 PM2 PP5

The NM_000059.4(BRCA2):​c.425+2T>C variant causes a splice donor, intron change. The variant allele was found at a frequency of 0.000000725 in 1,379,436 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 Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 7.2e-7 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_donor, intron
• Scores: Splicing: ADA: 0.9934
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:5 U:2
• Conservation: PhyloP100: 3.92

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 11 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, LoF is a know mechanism of disease, No cryptic splice site detected. Exon removal results in frameshift change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 13-32325186-T-C is Pathogenic according to our data. Variant chr13-32325186-T-C is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 234487.We mark this variant Likely_pathogenic, oryginal submissions are: {Likely_pathogenic=4, Uncertain_significance=2, Pathogenic=1}. Variant chr13-32325186-T-C is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.425+2T>C		splice_donor_variant, intron_variant	Intron 4 of 26	ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.425+2T>C		splice_donor_variant, intron_variant	Intron 4 of 26	5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.56+2T>C		splice_donor_variant, intron_variant	Intron 4 of 26	1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.425+2T>C		splice_donor_variant, intron_variant	Intron 3 of 25	2		ENSP00000506251.1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 7.25e-7 AC: 1 AN: 1379436 Hom.: 0 Cov.: 26 AF XY: 0.00000145 AC XY: 1 AN XY: 690408

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: 9.63e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:5 Uncertain:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Hereditary breast ovarian cancer syndrome Pathogenic:2

Feb 23, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change affects a donor splice site in intron 4 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 breast and/or ovarian cancer (PMID: 32438681, 33287145). ClinVar contains an entry for this variant (Variation ID: 234487). Studies have shown that disruption of this splice site results in skipping of exon 4 and introduces a premature termination codon (Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic. -

Mar 20, 2024

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: BRCA2 c.425+2T>C is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes the canonical 5' splicing donor site. One predict the variant weakens the canonical 5' splicing donor site. However, to our knowledge, these predictions have yet to be confirmed by published peer-reviewed functional studies. However, at-least two well recognized peers within the data sharing community have reported a splicing impact. The variant was absent in 247740 control chromosomes. To our knowledge, this variant has not been reported as a single germline variant in isolation in an individual affected with Breast and/or Ovarian Cancer. c.425+2T>C has been reported in the literature as a germine variant that was transmitted in double heterozygosity with a different BRCA1 variant, c.3756_3759delGTCT (p.Ser1253Argfs*10) in a 36 year old female with Ovarian Cancer (Vietri_2020, overlaping citations in Santonocito_2020, Franzese_2020, De Falco_2020) (Vietri_2020). The proband's mother who also harbored the double heterozygous genotype was unaffected at age 70 and her maternal male cousin also harboring the same double heterozygous genotype was affected with colorectal cancer at age 37. Double heterozygosity in BRCA1 and BRCA2 genes, although extremely rare, have been reported (cited in Vietri_2020). At-least one additional co-occurrence with another pathogenic BRCA1 variant(s) has been observed at our laboratory (BRCA1 c.5266dup, p.Gln1756fs). To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publications have been ascertained in the context of this evaluation (PMID: 33057101, 32234730, 31850198, 32438681, 33287145). ClinVar contains an entry for this variant (Variation ID: 234487). Based on the evidence outlined above, the variant was classified as likely pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:1 Uncertain:1

Sep 26, 2024

Ambry Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.425+2T>C intronic variant results from a T to C substitution two nucleotides after coding exon 3 in the BRCA2 gene. This alteration has been identified in an individual diagnosed with breast and/or ovarian cancer (Santonocito C et al. Cancers (Basel), 2020 May;12:). 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 in the set of samples tested (Ambry internal data). RNA studies of a close-match alteration, BRCA2 c.425G>T, have demonstrated that it results in complete abnormal splicing in the set of samples tested (Ambry internal data; Brandão R et al. Breast Cancer Res. Treat. 2011 Oct;129(3):971-82; Nix P et al. Fam Cancer, 2021 Jan). BRCA2 c.425G>T, which has the same splicing profile as this alteration (Ambry internal data), was able to rescue the growth defect in BRCA2-null mouse embryonic stem cells and these surviving cells maintained partial activity in a homology directed DNA repair functional assay (Mesman R et al. Genet Med 2020 Aug;22(8):1355-1365). BRCA2 c.425G>T was also identified in patients who collectively have a phenotype that is not consistent with a high risk BRCA2 pathogenic variant (Nix P et al. Fam Cancer, 2021 Jan). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. -

Feb 06, 2020

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant causes a T>C nucleotide substitution at the +2 position of intron 4 of the BRCA2 gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies, this variant is expected to result in an absent or disrupted protein product. This variant has not been reported in individuals affected with hereditary cancer in the literature. 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 Likely Pathogenic. -

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:1

Feb 20, 2018

Counsyl

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Familial cancer of breast Pathogenic:1

May 26, 2022

Baylor Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

not provided Uncertain:1

Jul 21, 2021

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Canonical splice site variant with an unclear effect on protein function, as an exonic splice variant at this splice junction, c.425G>T, results in the production of multiple transcripts, including potentially functional in-frame transcripts (Brandao 2011, Mesman 2020, Nix 2021); Observed in a woman with ovarian cancer who also harbored a BRCA1 pathogenic variant (Vietri 2020); Not observed at significant frequency in large population cohorts (Lek 2016); Also known as 653+2T>C; This variant is associated with the following publications: (PMID: 33287145) -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.37	D
BayesDel_noAF	Pathogenic	0.29
CADD	Pathogenic	26
DANN	Uncertain	0.99
Eigen	Pathogenic	1.0
Eigen_PC	Pathogenic	0.90
FATHMM_MKL	Uncertain	0.88	D
GERP RS		5.8

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Benign	0.68
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.99		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: rs876661045; hg19: chr13-32899323;