13-32376796-G-C

Position:

• chr13-32376796-G-C

Variant summary

Our verdict is Uncertain significance. Variant got 3 ACMG points: 3P and 0B. PM2 PP3

The NM_000059.4(BRCA2):​c.8754+5G>C variant causes a splice donor 5th base, intron change involving the alteration of a non-conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a benign 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)
• Consequence: BRCA2 NM_000059.4 splice_donor_5th_base, intron
• Scores: Splicing: ADA: 0.9951
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:2 U:1
• Conservation: PhyloP100: 2.49

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]

ACMG classification

Verdict is Uncertain_significance. Variant got 3 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, max_spliceai. No scorers claiming Uncertain. Scorers claiming Benign: dbscSNV1_RF.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA2	NM_000059.4	c.8754+5G>C		splice_donor_5th_base_variant, intron_variant		ENST00000380152.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA2	ENST00000380152.8	c.8754+5G>C		splice_donor_5th_base_variant, intron_variant		5	NM_000059.4	A2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 32

GnomAD4 genome Cov.: 32

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:2 Uncertain:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Aug 11, 2023

The c.8754+5G>C intronic variant results from a G to C substitution 5 nucleotides after coding exon 20 in the BRCA2 gene. RNA studies have shown that this alteration results in the retention of 46 nucleotides at the end of coding exon 20 and results in a premature termination codon (Ambry internal data; Wangensteen T et al. Hered Cancer Clin Pract, 2019 May;17:14). Another alteration at the same position, c.8754+5G>A, has been shown to have a similar splicing impact and functional studies show that mouse embryonic stem cells containing this variant are unable to perform complementation (Hendriks G et al. Hum Mutat, 2014 Nov;35:1382-91; Vreeswijk MP et al. Hum Mutat, 2009 Jan;30:107-14). This nucleotide position is highly conserved in available vertebrate species. This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). In silico splice site analysis predicts that this alteration will weaken the native splice donor site and will result in the creation or strengthening of a novel splice donor site. Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Hereditary breast ovarian cancer syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Oct 03, 2023

This sequence change falls in intron 21 of the BRCA2 gene. It does not directly change the encoded amino acid sequence of the BRCA2 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individual(s) with BRCA2-related conditions (PMID: 31143303). ClinVar contains an entry for this variant (Variation ID: 940222). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in activation of a cryptic splice site and introduces a premature termination codon (Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. This variant disrupts the c.8754+5 nucleotide in the BRCA2 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 18693280, 25146914). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. -

not provided Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Quest Diagnostics Nichols Institute San Juan Capistrano

Sep 16, 2022

It has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported to result in aberrant splicing and retention of 46 base pairs if the 5’ end of intron 21 (PMID: 31143303 (2019)). Further studies are required to determine the clinical significance of this finding. Analysis of this variant using software algorithms for the prediction of the effect of nucleotide changes on splicing yielded predictions that this variant may affect proper BRCA2 mRNA splicing . Based on the available information, we are unable to determine the clinical significance of this variant. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.30
CADD	Benign	21
DANN	Benign	0.96

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Benign	0.65
SpliceAI score (max)		0.94		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.76		Position offset: 41
DS_DL_spliceai		0.94		Position offset: -5

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

Publications

LitVar

Other links and lift over

dbSNP: rs81002813; hg19: chr13-32950933;