rs730881573

Positions:

• chr13-32397045-G-A
• chr13-32397045-G-C

Variant summary

Our verdict is Uncertain significance. Variant got 5 ACMG points: 5P and 0B. PVS1_Moderate PM2 PP5

The NM_000059.4(BRCA2):​c.9648+1G>A variant causes a splice donor change. The variant allele was found at a frequency of 0.000000684 in 1,461,148 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: 6.8e-7 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_donor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:3 U:1
• Conservation: PhyloP100: 5.58

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 5 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.0142341815 fraction of the gene. Cryptic splice site detected, with MaxEntScore 3.9, offset of -20, new splice context is: ctgGTacag. Cryptic site results in frameshift 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
• PP5: Variant 13-32397045-G-A is Pathogenic according to our data. Variant chr13-32397045-G-A is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 487418.We mark this variant Likely_pathogenic, oryginal submissions are: {Likely_pathogenic=2, Uncertain_significance=1, Pathogenic=1}.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.9648+1G>A		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.9648+1G>A		splice_donor_variant		5	NM_000059.4	ENSP00000369497	A2

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome AF: 6.84e-7 AC: 1 AN: 1461148 Hom.: 0 Cov.: 31 AF XY: 0.00000138 AC XY: 1 AN XY: 726952

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: 9.00e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 33

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:3 Uncertain:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

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

Likely pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Jan 16, 2017	- -
Pathogenic, criteria provided, single submitter	clinical testing	Hereditary Cancer Unit, Hospital Universitario 12 de Octubre	Dec 12, 2022	This variant is absent in the populations databases and has already been reported in scientific literature in a woman from a cohort of 7051 Japanese breast cancer patients (Yukihide Momozawa, 2018). Additionally, functional studies performed in our laboratory conclude that c.9648+1G>A variant leads to the skipping of exon 26 of BRCA2 protein. A mouse embryonic stem cell (mESC)-based assay revealed that BRCA2 c.9648+1G>A causes exon 26 skipping (human BRCA2-containing BAC), and that in-frame exon 26 skipping does not complement removal of the conditional mBrca2 allele, supporting loss-of-function (Mesman RLS, 2020). This change is classified as pathogenic (Class 5) following the qualitative ACMG criteria since (i) it is absent in several population databases (PM2_Supporting) (ii) it is a GT-AG variant causing an in-frame alteration targeting a region critical to protein function (PVS1_O_Strong) and (iii) a well-stablished functional assay supports damage on gene product (PS3). Additionally, this change is classified as likely pathogenic (Class 4) following the quantitative ACMG point system in which PVS1_strong, PS3 and PM2_Supporting add up to 9 points (likely pathogenic: 6-9 point range). In summary, this variant meets our criteria to be classified as likely pathogenic. -

Hereditary breast ovarian cancer syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Nov 08, 2023

This sequence change affects a donor splice site in intron 26 of the BRCA2 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in a shortened 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 colorectal cancer (PMID: 31875949). ClinVar contains an entry for this variant (Variation ID: 487418). Studies have shown that disruption of this splice site alters BRCA2 gene expression (PMID: 32398771). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that disruption of this splice site results in skipping of exon 26, but is expected to preserve the integrity of the reading-frame (PMID: 31875949, 32398771). In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. -

Hereditary cancer-predisposing syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Ambry Genetics

Mar 01, 2023

The c.9648+1G>A intronic variant results from a G to A substitution one nucleotide after coding exon 25 of the BRCA2 gene. Alterations that disrupt the canonical splice site are expected to cause aberrant splicing. 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 a transcript predicted to lead to a protein with an in-frame deletion of 49 amino acids (Gay-Bellile M et al. Clin Genet, 2020 Apr;97:668-669; Mesman RLS et al. Genet Med, 2020 Aug;22:1355-1365; Ambry internal data), however, the exact functional impact of the deleted amino acids is unknown at this time (Ambry internal data). This variant has been detected in conjunction with a pathogenic finding in this same gene and confirmed in trans in two brothers with no reported features of Fanconi anemia (Gay-Bellile M et al. Clin Genet, 2020 Apr;97:668-669), suggesting that this allele may retain some normal BRCA2 activity. However, another study reported this alteration as non-functional, as it did not complement conditional loss of Brca2 activity in a cell viability assay performed in murine embryonic stem cells (mESCs) (Mesman RLS et al. Genet Med, 2020 Aug;22:1355-1365). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.41	D
BayesDel_noAF	Pathogenic	0.34
CADD	Pathogenic	30
DANN	Uncertain	0.99
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.94
FATHMM_MKL	Pathogenic	0.99	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)		1.0		Details are displayed if max score is > 0.2
DS_DL_spliceai		1.0		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: rs730881573; hg19: chr13-32971182; COSMIC: COSV104700968; COSMIC: COSV104700968;