NM_000059.4:c.9648+1G>C

Variant names:

• chr13-32397045-G-C
• rs730881573
• NM_000059.4:c.9648+1G>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>C variant causes a splice donor, intron change. The variant allele was found at a frequency of 0.00000657 in 152,138 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: 𝑓 0.0000066 (0 hom., cov: 33)
• Consequence: BRCA2 NM_000059.4 splice_donor, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:6 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 0.014331676 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-C is Pathogenic according to our data. Variant chr13-32397045-G-C is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 182265.We mark this variant Likely_pathogenic, oryginal submissions are: {Likely_pathogenic=2, Uncertain_significance=1, Pathogenic=4}.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.9648+1G>C		splice_donor_variant, intron_variant	Intron 26 of 26	ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.9648+1G>C		splice_donor_variant, intron_variant	Intron 26 of 26	5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.9279+1G>C		splice_donor_variant, intron_variant	Intron 26 of 26	1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.*1706+1G>C		splice_donor_variant, intron_variant	Intron 25 of 25	2		ENSP00000506251.1

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152138 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.00

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.0000147

Gnomad OTH AF: 0.00

GnomAD4 exome Cov.: 31

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152138 Hom.: 0 Cov.: 33 AF XY: 0.00 AC XY: 0 AN XY: 74310

Gnomad4 AFR AF: 0.00

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.0000147

Gnomad4 OTH AF: 0.00

Bravo AF: 0.00000378

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:6 Uncertain:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Pathogenic:2

Sep 28, 2022

GeneDx

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Canonical splice site variant predicted to result in an in-frame deletion of a critical region, which is part of the DNA binding domain (Yang et al., 2002); Not observed at a significant frequency in large population cohorts (gnomAD); Observed in an individual referred for clinical testing for inherited cancer (Susswein et al., 2016); Also known as 9876+1G>C; This variant is associated with the following publications: (PMID: 12228710, 26681312) -

Apr 26, 2019

Revvity Omics, Revvity

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Hereditary breast ovarian cancer syndrome Pathogenic:2

Jan 28, 2025

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.9648+1G>C is located in the last 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 pathogenic variants have been observed in the last exon in our laboratory. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing and loss of BRCA2 function. Several computational tools predict a significant impact on normal splicing: Four predict the variant abolishes a 5' splicing donor site. However, these predictions have yet to be confirmed by functional studies. The variant was absent in 251328 control chromosomes. To our knowledge, no occurrence of c.9648+1G>C in individuals affected with Hereditary Breast And Ovarian Cancer Syndrome and no experimental evidence demonstrating its impact on protein function have been reported. ClinVar contains an entry for this variant (Variation ID: 182265). Based on the evidence outlined above, the variant was classified as likely pathogenic. -

Aug 12, 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 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). This variant has not been reported in the literature in individuals affected with BRCA2-related conditions. ClinVar contains an entry for this variant (Variation ID: 182265). 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: 32398771). This variant disrupts a region of the BRCA2 protein in which other variant(s) (p.Tyr3308*) have been determined to be pathogenic (PMID: 18593900, 18607349). This suggests that this is a clinically significant region of the protein, and that variants that disrupt it are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. -

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

Aug 25, 2015

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Familial cancer of breast Pathogenic:1

Feb 28, 2023

Baylor Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Hereditary cancer-predisposing syndrome Uncertain:1

Feb 03, 2025

Ambry Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.9648+1G>C intronic variant results from a G to C substitution one nucleotide after coding exon 25 of the BRCA2 gene. Alterations that disrupt the canonical splice site are expected to result in aberrant splicing. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. The resulting transcript is predicted to be in-frame and is not expected to trigger nonsense-mediated mRNAdecay. 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 (Ambry internal data). A close match alteration (c.9648+1G>A) has been shown to result in the same splicing profile and has also been confirmed in trans with a pathogenic finding in this same gene in two brothers with no reported features of Fanconi anemia (Ambry internal data; Gay-Bellile M et al. Clin Genet, 2020 Apr;97:668-669). Based on the available evidence, the clinical significance of this variant remains unclear. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.36	D
BayesDel_noAF	Pathogenic	0.28
CADD	Pathogenic	31
DANN	Uncertain	0.99
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.94
FATHMM_MKL	Pathogenic	0.99	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;