rs769152395

chr13-32319075-A-Gchr13-32319075-A-T

Variant summary

Our verdict is Likely pathogenic. Variant got 9 ACMG points: 9P and 0B. PVS1_Moderate PM2 PP3_Strong PP5

The NM_000059.4(BRCA2):c.68-2A>G variant causes a splice acceptor change. The variant allele was found at a frequency of 0.00000137 in 1,458,660 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 Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_acceptor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:2 U:4
• Conservation: PhyloP100: 6.59

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 Likely_pathogenic. Variant got 9 ACMG points.

• PVS1: Splicing variant, NOT destroyed by nmd, known LOF gene, truncates exone, which is 0.02417861 fraction of the gene. Cryptic splice site detected, with MaxEntScore 7.8, offset of 6, new splice context is: ttttttttaaatggatttAGgac. Cryptic site results in inframe change. If cryptic site found is not functional and variant results in exon loss, it results in inframe change.
• PM2: Very rare variant in population databases, with high coverage
• 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 13-32319075-A-G is Pathogenic according to our data. Variant chr13-32319075-A-G is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 187538.We mark this variant Likely_pathogenic, oryginal submissions are: {Uncertain_significance=4, Likely_pathogenic=2}.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA2	NM_000059.4	c.68-2A>G		splice_acceptor_variant		ENST00000380152.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA2	ENST00000380152.8	c.68-2A>G		splice_acceptor_variant		5	NM_000059.4	A2

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD3 exomes AF: 0.00000404 AC: 1 AN: 247642 Hom.: 0 AF XY: 0.00000744 AC XY: 1 AN XY: 134330

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00000889

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000137 AC: 2 AN: 1458660 Hom.: 0 Cov.: 32 AF XY: 0.00000138 AC XY: 1 AN XY: 725568

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

ExAC AF: 0.00000824 AC: 1

EpiCase AF: 0.00

EpiControl AF: 0.0000593

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:2 Uncertain:4

Revision: criteria provided, conflicting classifications

Submissions by phenotype

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

Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	May 04, 2023	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Mendelics	May 28, 2019	- -

Hereditary breast ovarian cancer syndrome Pathogenic:1 Uncertain:1

Uncertain significance, criteria provided, single submitter	clinical testing	Invitae	Jan 24, 2023	Disruption of this splice site has been observed in individual(s) undergoing genetic testing. An analysis comparing the clinical and personal history of variant carriers with that of carriers of known benign or pathogenic variants showed inconclusive results for this variant (PMID: 33469799). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. Studies have shown that disruption of this splice site results in the activation of a cryptic splice site in exon 3 (PMID: 33469799; Invitae). ClinVar contains an entry for this variant (Variation ID: 187538). This variant is present in population databases (rs769152395, gnomAD 0.0009%). This sequence change affects an acceptor splice site in intron 2 of the BRCA2 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in the loss of 2 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Aug 12, 2019	Variant summary: BRCA2 c.68-2A>G 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: 4/5 tools predict that the variant abolishes a 3' acceptor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 4e-06 in 247642 control chromosomes (gnomAD). To our knowledge, c.68-2A>G has not been reported in the literature in individuals affected with Hereditary Breast and Ovarian Cancer and no experimental evidence demonstrating an impact on protein function has been reported. Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar (after 2014). All laboratories classified the variant as likely pathogenic. Based on the evidence outlined above, the variant was classified as likely pathogenic. -

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Sep 02, 2022	This variant causes an A to G nucleotide substitution at the -2 position of intron 2 of the BRCA2 gene. Splice site prediction tools indicate that this variant may have a significant impact on RNA splicing. RNA studies have confirmed that the variant disrupts splicing of the full-length transcript, however, the predominant product is a 6-basepair deletion in exon 3, resulting in the in-frame deletion of two amino acids p.Asp23_Leu24del, and an increase in the skipping of exon 3 (PMID: 33469799, 35979650; ClinVar accession ID: SCV000218118.6, SCV000831230.5). Functional studies have reported that this variant does not impact BRCA2 function in the complementation of Brca2-deficient mouse embryonic stem cells and in a homology-directed repair assay (PMID: 35979650). A carrier health history analysis for this variant compared against known benign and pathogenic variant carriers has reported that the pathogenicity of this variant was inconclusive but trending towards benign (PMID: 33469799). While a multifactorial analysis has reported likelihood ratios for pathogenicity based on tumor pathology, segregation and family history of 0.57, 0.28 and 0.79, respectively (PMID: 35979650). This variant has been identified in 1/247642 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Oct 25, 2021	The c.68-2A>G intronic variant results from an A to G substitution two nucleotides before coding exon 2 of the BRCA2 gene. This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and will result in the creation or strengthening of a novel splice acceptor site. RNA studies have demonstrated that this alteration results in substantial expression of multiple abnormal transcripts including a splice variant which is predicted to result in an in-frame loss of two amino acids as well as one that results in skipping of coding exon 2 (also known as exon 3 in the literature); (Ambry internal data; Nix P et al. Fam Cancer, 2021 Jan; personal communication). The loss of coding exon 2 of is strongly associated with hereditary breast and ovarian cancer phenotype based on multifactorial analysis (Caputo SM et al. Oncotarget, 2018 Apr;9:17334-17348); however the functional and clinical impact of the small in-frame loss is unknown. Downstream functional studies showed that this alteration was able to rescue the growth defect in BRCA2-null mouse embryonic stem cells and that these surviving cells maintained partial activity in a homology directed DNA repair assay (personal communication). This alteration is 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. It cannot yet be ruled out that this variant may be hypomorphic and present with reduced risks and/or biallelic phenotype. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.25	D
BayesDel_noAF	Uncertain	0.13
Cadd	Pathogenic	35
Dann	Uncertain	0.99
Eigen	Pathogenic	1.0
Eigen_PC	Pathogenic	0.89
FATHMM_MKL	Pathogenic	0.98	D
MutationTaster	Benign	1.0	D;D
GERP RS		5.7

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.97		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.95		Position offset: 8
DS_AL_spliceai		0.97		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: rs769152395; hg19: chr13-32893212;