13-32326502-C-T

Variant summary

Our verdict is Uncertain significance. Variant got 4 ACMG points: 4P and 0B. PM1 PM2

The NM_000059.4(BRCA2):c.520C>T(p.Arg174Cys) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00000344 in 1,453,724 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 13/21 in silico tools predict a benign outcome for this variant. Variant has been reported in ClinVar as Uncertain significance (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R174H) has been classified as Likely benign.

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 0.0000034 (0 hom.)
• Consequence: BRCA2 NM_000059.4 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:11
• Conservation: PhyloP100: 0.282

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 4 ACMG points.

• PM1: In a hotspot region, there are 3 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 4 benign, 16 uncertain in NM_000059.4
• PM2: Very rare variant in population databases, with high coverage

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA2	NM_000059.4	c.520C>T	p.Arg174Cys	missense_variant	7/27	ENST00000380152.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA2	ENST00000380152.8	c.520C>T	p.Arg174Cys	missense_variant	7/27	5	NM_000059.4	A2

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD3 exomes AF: 0.00000398 AC: 1 AN: 251366 Hom.: 0 AF XY: 0.00000736 AC XY: 1 AN XY: 135852

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: 0.00000880

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000344 AC: 5 AN: 1453724 Hom.: 0 Cov.: 30 AF XY: 0.00000415 AC XY: 3 AN XY: 723698

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: 0.00000453

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 33

Alfa AF: 0.0000233 Hom.: 0

Bravo AF: 0.00000756

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:11

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

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

Uncertain significance, no assertion criteria provided	clinical testing	Department of Medical and Surgical Sciences, University of Bologna	Sep 01, 2023	PM2(Supporting)+PP3(Supporting)+BP5(Supporting) according to ACMG/AMP classification guidelines specified for BRCA1 & BRCA2 (Classification Criteria V1.0.0 2023-09-08 - https://cspec.genome.network/cspec/ui/svi/affiliation/50087) (PMID: 38160042) -
Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Nov 20, 2023	This missense variant replaces arginine with cysteine at codon 174 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). A functional RNA study reported this variant causes partial exon 7 skipping in minigene splicing assay and confirmed in patient RNA (PMID: 21673748). This variant has been reported in an individual affected with early-onset breast cancer and strong family history of disease (PMID: 21673748) and also in two unaffected controls in a breast cancer case-control study (PMID: 30287823). This variant has been identified in 1/251366 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, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	May 01, 2012	- -

not provided Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Jul 27, 2021	Not observed at a significant frequency in large population cohorts (Lek 2016); In silico analysis supports that this missense variant does not alter protein structure/function; Observed in at least two individuals with early-onset familial breast cancer (Thery 2011); RNA and minigene assays demonstrate partial exon 7 skipping, producing both full-length and truncated mRNA transcripts in varying proportions (Gaildrat 2012, Di Giacomo 2013). Exon 7 skipping has been reported to create a frameshift ultimately causing a premature nonsense codon, resulting in a truncated protein product that appears to undergo nonsense-mediated mRNA decay (Pyne 2000); This variant is associated with the following publications: (PMID: 32072338, 32486089, 32170000, 32123317, 30287823, 26761715, 11185744, 23983145, 22962691, 21673748) -
Uncertain significance, criteria provided, single submitter	clinical testing	Institute for Clinical Genetics, University Hospital TU Dresden, University Hospital TU Dresden	Nov 03, 2021	- -

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Aug 04, 2022	The p.R174C variant (also known as c.520C>T), located in coding exon 6 of the BRCA2 gene, results from a C to T substitution at nucleotide position 520. The arginine at codon 174 is replaced by cysteine, an amino acid with highly dissimilar properties. Multiple splicing assays have confirmed that the BRCA2 c.520C>T induces incomplete skipping of coding exon 6-also called exon 7 in published literature (Ambry internal data; Di Giacomo D et al, Hum. Mutat. 2013 Nov; 34(11):1547-57; Gaildrat P, et al. J Med Genet. 2012;49:609-617; Théry JC, et al. Eur J Hum Genet. 2011;19:1052-1058). In addition to this exon skipping event being incomplete, the molecular and clinical impact of loss of coding exon 6 is unclear (Mesman RLS et al. Genet. Med., 2020 May). This amino acid position is well conserved in primates, however, lower organisms do not have a reference at this position on multiple sequence 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. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jul 06, 2023	This missense variant replaces arginine with cysteine at codon 174 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). A functional RNA study reported this variant causes partial exon 7 skipping in minigene splicing assay and confirmed in patient RNA (PMID: 21673748). This variant has been reported in an individual affected with early-onset breast cancer and strong family history of disease (PMID: 21673748) and also in two unaffected controls in a breast cancer case-control study (PMID: 30287823). This variant has been identified in 1/251366 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 breast ovarian cancer syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Invitae	Nov 03, 2023	This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 174 of the BRCA2 protein (p.Arg174Cys). This variant is present in population databases (rs41293469, gnomAD 0.0009%). This missense change has been observed in individual(s) with breast or ovarian cancer (PMID: 21673748, 31907386). ClinVar contains an entry for this variant (Variation ID: 51818). 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%. Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. 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. -
Uncertain significance, criteria provided, single submitter	clinical testing	Cancer Variant Interpretation Group UK, Institute of Cancer Research, London	Feb 18, 2020	Data included in classification: Case control comparison of UK matched against ethnically matched population data (3/25,773) in familial cases against 1/56,837 GNOMAD NFE controls pexact= 0.093 (PS4_mod). Absent from the remainder of the gnomAD population (0/68,846) (PM2_mod). Assays reported by Thery 2011, Gaildrat 2012, Di Giacomo 2013 (PMID: 21673748, PMID: 22962691, PMID: 2398314) show 60-70% exon 7 skipping likely due to disruption of ESE. ESE prediction tools predict enhancer site. (PS3_mod). Variant reported in the homozygous form (1) and heterozygous form (1) on the BRCA2 Fanconi database from the Japanese Biobank. Homozygote age 60 years without personal or family history of cancer but had other complex disease (not specified). (BS2_sup). Additional data (not included in classification): The variant has also been observed in at least 2 additional UK families, 5 families tested by Ambry and is reported an additional 4 times on ClinVar. Variable segregation in UK families. None strong. Other classifications: Ambry VUS 2018; Gene Dx VUS 2017; Color VUS 2017; Invitae VUS 2017. In silico predictions mixed. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Apr 20, 2018

Variant summary: BRCA2 c.520C>T (p.Arg174Cys) results in a non-conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a benign effect of the variant on protein function. Though 5/5 computational tools predict no significant impact on normal splicing, one in silico study predicted potential effects by using 3 ESR-dedicated approaches (Soukarieh 2016). In vitro and in vivo studies indicated that the variant of interest induces partial exon 7 skipping (Thery 2011, Gaildrat 2012, DiGiacomo 2013). Exon 7 skipping is predicted to result in a frameshift, resulting in a truncated protein (that is subject to NMD), however, the in vivo consequence of partial exon 7 skipping is not known. Additionally, when transcribed properly, this variant results in a full length transcript with a missense alteration that could compound the in vivo consequence of this variant. The variant allele was found at a frequency of 4.1e-06 in 246130 control chromosomes. c.520C>T has been reported in the literature in individuals affected with Hereditary Breast and Ovarian Cancer (Thery 2011, Gaildrat 2012). These reports do not provide unequivocal conclusions about association of the variant with Hereditary Breast and Ovarian Cancer. Four clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as uncertain significance. Based on the evidence outlined above, the variant was classified as uncertain significance. -

Familial cancer of breast Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Baylor Genetics

Oct 25, 2023

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.073
BayesDel_addAF	Benign	-0.070	T
BayesDel_noAF	Benign	-0.34
Cadd	Benign	14
Dann	Benign	0.77
Eigen	Benign	-0.51
Eigen_PC	Benign	-0.53
FATHMM_MKL	Uncertain	0.84	D
M_CAP	Benign	0.019	T
MetaRNN	Benign	0.16	T;T
MetaSVM	Benign	-0.86	T
MutationTaster	Benign	0.94	N;N
PrimateAI	Benign	0.42	T
PROVEAN	Benign	-0.42	N;N
REVEL	Benign	0.19
Sift	Benign	0.18	T;T
Sift4G	Benign	0.18	T;T
Vest4		0.36
MutPred		0.32		Gain of ubiquitination at K178 (P = 0.0582);Gain of ubiquitination at K178 (P = 0.0582);
MVP		0.81
MPC		0.13
ClinPred		0.30	T
GERP RS		0.93
gMVP		0.27

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.27		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.27		Position offset: -3

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

Publications

LitVar

Other links and lift over

dbSNP: rs41293469; hg19: chr13-32900639; COSMIC: COSV66463658;