13-32332449-G-C

Variant summary

Our verdict is Likely benign. Variant got -3 ACMG points: 0P and 3B. BP4_Moderate BP6

The NM_000059.4(BRCA2):c.971G>C(p.Arg324Thr) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.0000402 in 1,591,946 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 13/19 in silico tools predict a benign outcome for this variant. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R324K) has been classified as Likely benign.

• Frequency:
  • Genomes: 𝑓 0.0000066 (0 hom., cov: 32)
  • Exomes 𝑓: 0.000044 (0 hom.)
• Consequence: BRCA2 NM_000059.4 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications U:10 B:2
• Conservation: PhyloP100: 1.58

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 Likely_benign. Variant got -3 ACMG points.

• BP4: Computational evidence support a benign effect (MetaRNN=0.21475512).
• BP6: Variant 13-32332449-G-C is Benign according to our data. Variant chr13-32332449-G-C is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 38261.We mark this variant Likely_benign, oryginal submissions are: {Likely_benign=2, Uncertain_significance=9}.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA2	NM_000059.4	c.971G>C	p.Arg324Thr	missense_variant	10/27	ENST00000380152.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA2	ENST00000380152.8	c.971G>C	p.Arg324Thr	missense_variant	10/27	5	NM_000059.4	A2

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152092 Hom.: 0 Cov.: 32

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

GnomAD3 exomes AF: 0.0000215 AC: 5 AN: 232690 Hom.: 0 AF XY: 0.0000239 AC XY: 3 AN XY: 125782

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: 0.0000461

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.0000438 AC: 63 AN: 1439854 Hom.: 0 Cov.: 31 AF XY: 0.0000447 AC XY: 32 AN XY: 715590

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: 0.0000552

Gnomad4 OTH exome AF: 0.0000337

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152092 Hom.: 0 Cov.: 32 AF XY: 0.0000135 AC XY: 1 AN XY: 74294

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

Alfa AF: 0.0000283 Hom.: 0

Bravo AF: 0.0000151

ExAC AF: 0.0000165 AC: 2

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Uncertain:10 Benign:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Counsyl	Jun 14, 2017	- -
Uncertain significance, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	Nov 02, 2011	- -
Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Dec 13, 2023	This missense variant replaces arginine with threonine at codon 324 of the BRCA2 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). Experimental functional studies have reported conflicting results for this variant. In one study, the variant was shown to result in increased radial chromosome formation, percentage of cells in G2/M, a partial rescue of cell viability following DNA damage treatment (PMID: 31721781). However, another study reported no sensitivity to PARP inhibitors or carboplatin in cell viability assays (PMID: 32444794). This variant has been reported in individuals affected with breast cancer, ovarian cancer, and T-ALL (PMID: 11802209, 23269703, 31721781), the individual affected with ovarian cancer was also found to carry a pathogenic BRCA1 variant, which could explain the observed phenotype (PMID: 23269703). In a large breast cancer case-control meta analysis conducted by the BRIDGES consortium, this variant was reported in 2/60466 cases and 3/53461 unaffected controls (PMID: 33471991; Leiden Open Variation Database DB-ID BRCA2_002337). This variant has been identified in 5/232690 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. -

Hereditary cancer-predisposing syndrome Uncertain:2 Benign:1

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Jun 15, 2023	The p.R324T variant (also known as c.971G>C), located in coding exon 9 of the BRCA2 gene, results from a G to C substitution at nucleotide position 971. The arginine at codon 324 is replaced by threonine, an amino acid with similar properties. This variant was reported in a large hereditary breast and ovarian cancer family in which the proband was compound heterozygous for biallelic pathogenic mutations in BRCA1 (Domchek SM et al. Cancer Discov 2013; 3:399-405). This alteration was also detected in 1/989 unrelated individuals from a cohort of German breast/ovarian cancer families (Meindl A et al. Int. J. Cancer, 2002 Feb;97:472-80). This alteration was also identified in an individual with a family history of pancreatic cancer (Murali K et al. Hered Cancer Clin Pract, 2021 Aug;19:33). This amino acid position is poorly conserved on species alignment. In addition, this alteration is predicted to be tolerated by in silico analysis. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear. -
Likely benign, criteria provided, single submitter	curation	University of Washington Department of Laboratory Medicine, University of Washington	Mar 23, 2023	Missense variant in a coldspot region where missense variants are very unlikely to be pathogenic (PMID:31911673). -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jun 07, 2023	This missense variant replaces arginine with threonine at codon 324 of the BRCA2 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). Experimental functional studies have reported conflicting results for this variant. In one study, the variant was shown to result in increased radial chromosome formation, percentage of cells in G2/M, a partial rescue of cell viability following DNA damage treatment (PMID: 31721781). However, another study reported no sensitivity to PARP inhibitors or carboplatin in cell viability assays (PMID: 32444794). This variant has been reported in individuals affected with breast cancer, ovarian cancer, and T-ALL (PMID: 11802209, 23269703, 31721781), the individual affected with ovarian cancer was also found to carry a pathogenic BRCA1 variant, which could explain the observed phenotype (PMID: 23269703). In a large breast cancer case-control meta analysis conducted by the BRIDGES consortium, this variant was reported in 2/60466 cases and 3/53461 unaffected controls (PMID: 33471991; Leiden Open Variation Database DB-ID BRCA2_002337). This variant has been identified in 5/232690 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. -

not provided Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Oct 15, 2018	- -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Sep 30, 2022	Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis, which includes protein predictors and evolutionary conservation, supports that this variant does not alter protein structure/function; Observed in individuals with personal and/or family history of breast, ovarian, and/or pancreatic cancer (Meindl et al., 2002; Domchek et al., 2013; Murali et al., 2021); This variant is associated with the following publications: (PMID: 25348012, 11802209, 29435075, 23269703, 34399810) -

not specified Uncertain:1 Benign:1

Uncertain significance, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Feb 07, 2022	Variant summary: BRCA2 c.971G>C (p.Arg324Thr) results in a non-conservative amino acid change in the encoded protein sequence. Three of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 2.1e-05 in 232690 control chromosomes (gnomAD). The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.971G>C has been reported in the literature in individuals affected with Breast, Ovarian, and Pancreatic cancer (example: Domcheck_2013, Meindl_2002, Dorling_2021, Murali_2021). However, in one of the reported families the variant was found to co-occur with two separate pathogenic/likely pathogenic BRCA1 variants, c.2457delC and c.5207T>C. In addition, the variant seemed to not segregate with the disease in this family (Domchek_2013). At least two publications report experimental evidence evaluating an impact on protein function: One Fanconi-deficient rescue assay demonstrated that the variant causes partial loss of function (Pouliot_2019), and one BRCA2 null cell survival and proliferation assay following PARP inhibition demonstrated that the variant had normal function (Ikegami_2020). Six ClinVar submitters have assessed the variant since 2014: all submitters classified the variant as of uncertain significance. Based on the evidence outlined above, the variant was classified as uncertain significance. -
Likely benign, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	Aug 15, 2023	- -

BRCA2-related disorder Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

PreventionGenetics, part of Exact Sciences

Jul 27, 2023

The BRCA2 c.971G>C variant is predicted to result in the amino acid substitution p.Arg324Thr. This variant was reported in an individual with family history of breast cancer (Meindl et al. 2002. PubMed ID: 11802209) and in an individual with family history of pancreatic cancer who had also variant of uncertain significance in BRCA1, p. Ala5Val (Murali et al. 2021. PubMed ID: 34399810). Functional studies show this variant has no effect on protein function (Ikegami et al. 2020. PubMed ID: 32444794) and intermediate effect on protein function (Pouliot et al. 2019. PubMed ID: 31721781). This variant is reported in 0.0046% of alleles in individuals of European (Non-Finnish) descent in gnomAD (http://gnomad.broadinstitute.org/variant/13-32906586-G-C). At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence. -

Hereditary breast ovarian cancer syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Jan 16, 2024

This sequence change replaces arginine, which is basic and polar, with threonine, which is neutral and polar, at codon 324 of the BRCA2 protein (p.Arg324Thr). This variant is present in population databases (rs397507435, gnomAD 0.005%). This missense change has been observed in individual(s) with breast cancer and/or family history of pancreatic cancer (PMID: 11802209, 34399810). ClinVar contains an entry for this variant (Variation ID: 38261). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is not expected to disrupt BRCA2 protein function with a negative predictive value of 95%. Experimental studies have shown that this missense change does not substantially affect BRCA2 function (PMID: 31721781, 32444794). 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.12
BayesDel_addAF	Benign	-0.19	T
BayesDel_noAF	Benign	-0.37
Cadd	Benign	14
Dann	Benign	0.88
Eigen	Benign	-0.51
Eigen_PC	Benign	-0.51
FATHMM_MKL	Benign	0.43	N
M_CAP	Uncertain	0.19	D
MetaRNN	Benign	0.21	T;T
MetaSVM	Benign	-0.88	T
MutationTaster	Benign	1.0	N;N
PrimateAI	Benign	0.32	T
PROVEAN	Benign	-0.84	N;N
REVEL	Benign	0.14
Sift	Uncertain	0.0040	D;D
Sift4G	Uncertain	0.030	D;D
Vest4		0.23
MutPred		0.24		Loss of catalytic residue at R324 (P = 0.0379);Loss of catalytic residue at R324 (P = 0.0379);
MVP		0.85
MPC		0.032
ClinPred		0.091	T
GERP RS		2.9
gMVP		0.23

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

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

Publications

LitVar

Other links and lift over

dbSNP: rs397507435; hg19: chr13-32906586; COSMIC: COSV99061653;