13-32346896-G-T

Position:

• chr13-32346896-G-T

Variant summary

Our verdict is Pathogenic. Variant got 13 ACMG points: 13P and 0B. PM2 PM5 PP3 PP5_Very_Strong

The NM_000059.4(BRCA2):​c.7007G>T​(p.Arg2336Leu) variant causes a missense, splice region change. The variant allele was found at a frequency of 0.000000689 in 1,450,868 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R2336H) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 6.9e-7 (0 hom.)
• Consequence: BRCA2 NM_000059.4 missense, splice_region
• Scores: Splicing: ADA: 0.9992
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:10
• Conservation: PhyloP100: 4.66

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 Pathogenic. Variant got 13 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr13-32346896-G-A is described in Lovd as [Likely_pathogenic].
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. Scorers claiming Uncertain: max_spliceai. No scorers claiming Benign.
• PP5: Variant 13-32346896-G-T is Pathogenic according to our data. Variant chr13-32346896-G-T is described in ClinVar as [Likely_pathogenic]. Clinvar id is 52242.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr13-32346896-G-T is described in Lovd as [Pathogenic]. Variant chr13-32346896-G-T is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.7007G>T	p.Arg2336Leu	missense_variant, splice_region_variant	13/27	ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.7007G>T	p.Arg2336Leu	missense_variant, splice_region_variant	13/27	5	NM_000059.4	ENSP00000369497	A2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.89e-7 AC: 1 AN: 1450868 Hom.: 0 Cov.: 29 AF XY: 0.00 AC XY: 0 AN XY: 721902

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: 0.0000192

Gnomad4 NFE exome AF: 0.00

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:10

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:4

Likely pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Oct 02, 2023	This missense variant replaces arginine with leucine at codon 2336 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). This variant also changes the last nucleotide of exon 13 of the BRCA2 gene and splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies for this variant, different variants affecting the same splice donor site (c.7007G>A, c.7007G>C) have been shown to produce aberrant RNA transcripts and are considered to be disease-causing (ClinVar variation ID: 38077, 52241). It suggests that the reference sequence at this splice site is important for normal RNA splicing. This variant has been reported in individuals with personal and/or family history of breast and/or ovarian cancer (PMID: 22970155, 27157322, 28993434, 30078507, 31742824, 31782247, 35886069, Color internal data) and non-small cell lung cancer (PMID: 31565484). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge	Oct 02, 2015	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Human Genome Sequencing Center Clinical Lab, Baylor College of Medicine	Oct 05, 2018	This c.7007G>T (p.Arg2336Leu) variant in exon 13 of the BRCA2 gene is the last base of exon 13 and is predicted to alter splicing resulting in a frame shift. This variant has been reported in patients with breast cancer (PMID: 22970155) and is not observed in general population databases. Other nucleotide base substitutions at this same site have also been observed to alter splicing and are considered pathogenic (PMID: 18489799, 9150172). Therefore, the c.7007G>T (p.Arg2336Leu) variant in the BRCA2 gene is classified as likely pathogenic. -
Pathogenic, no assertion criteria provided	clinical testing	Breast Cancer Information Core (BIC) (BRCA2)	Feb 20, 2013	- -

not provided Pathogenic:2

Likely pathogenic, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The BRCA2 p.Arg2336Leu variant was identified in 4 of 2798 proband chromosomes (frequency: 0.001) from individuals or families with breast or ovarian cancer (Kwong 2012, Kwong 2016). The variant was also identified in dbSNP (ID: rs28897743) as “With Pathogenic allele ", in ClinVar (classified as likely pathogenic by Invitae, Ambry Genetics, Color Genomics; as pathogenic by CIMBA, COGR, BIC), MutDB, LOVD 3.0 (1x as pathogenic), BIC Database (2x with clinical importance), and in ARUP Laboratories (as Definitely pathogenic). The variant was not identified in GeneInsight-COGR, Cosmic, UMD-LSDB, and Zhejiang University Database. The variant was not identified in the following control databases: the 1000 Genomes Project, the NHLBI GO Exome Sequencing Project, the Exome Aggregation Consortium (August 8th 2016), or the Genome Aggregation Database (Feb 27, 2017). In UMD, 2 alternate substitutions at this same nucleotide position (c.7007G>A, p.Arg2336His and c.7007G>C, p.Arg2336Pro) have been classified as causal, with evidence showing the G>A substitution causes a 70 bp deletion in exon 13 resulting in a frameshift that introduces a premature stop codon 30 codons into exon 14. Lymphocyte RT-PCR analysis showed the alternate variant c.7007G>A caused exon 13 skipping, in agreement with bionformatic studies which conclude a weakening and disruption of the splice site (Sanz 2010). A bioinformatic tool applying information theory to analyze splicing variants found the natural donor site was weakened for an alternate substitution variant (c.7007G>A), concordant with other splicing models/testing (Mucaki 2010). The p.Arg2336 residue is not conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. The p.Arg2336Leu variant occurs in the last three bases of the exon. This position has been shown to be part of the splicing consensus sequence and variants involving this position sometimes affect splicing. In addition in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as likely pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Apr 06, 2021	In the published literature, this variant has been reported in individuals/families affected with breast and/or ovarian cancer ((PMIDs: 22970155 (2012), 27157322 (2016), 28724667 (2017), 28993434 (2018), 30078507 (2018), 31782247 (2020)), as well as non-small cell lung cancer (PMID: 31565484 (2019)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded conflicting predictions that this variant is disease causing or benign. Additional analysis using software algorithms for the prediction of the effect of nucleotide changes on splicing yielded predictions that this variant may affect proper BRCA2 mRNA splicing. This variant has not been reported in large, multi-ethnic general populations. Two other pathogenic or likely pathogenic variants affect the same amino acid. Based on the available information, this variant is classified as likely pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 26, 2023	The c.7007G>T variant (also known as p.R2336L), located in coding exon 12 of the BRCA2 gene, results from a G to T substitution at nucleotide position 7007. The amino acid change results in arginine to leucine at codon 2336, an amino acid with dissimilar properties. However, this change occurs in the last base pair of coding exon 12, which makes it likely to have some effect on normal mRNA splicing. This alteration has been reported in individuals with breast and/or ovarian cancer, predominantly from Chinese cohorts (Kwong A et al. PLoS ONE. 2012;7(9):e43994; Wei H et al. Oncol Lett 2018 Jun;15(6):9420-9428; Li A et al. Gynecol Oncol . 2018 Oct;151(1):145-152; Bhaskaran SP et al. Int J Cancer. 2019 Aug;145(4):962-973; Cheng J et al. J Cell Mol Med. 2020 Jan;24(2):1676-1683; Gao X et al. Hum Mutat. 2020 Mar;41(3):696-708). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration may weaken the native splice donor site; however direct evidence is insufficient. Alterations at this same position and impacting the same donor site (c.7007G>C and c.7007G>A) have been reported to result in aberrant splicing (Houdayer C et al. Hum. Mutat. 2012 Aug; 33(8):1228-38; Ambry internal data), and have been described in a compound heterozygous or homozygous state in individuals diagnosed with Fanconi anemia (Meng L et al. JAMA Pediatr. 2017 12;171(12):e173438; Ghazwani Y et al. Cancer Genet 2016 Apr;209(4):171-6). Based on the majority of available evidence to date, this variant is likely to be pathogenic. However, because variants at this location have been identified in patients with Fanconi Anemia, this alteration may be hypomorphic, and thus, carriers of this variant and their families may present with reduced risks, and not with the typical clinical characteristics of a high-risk pathogenic BRCA2 alteration. As risk estimates are unknown at this time, clinical correlation is advised. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Aug 23, 2023	This missense variant replaces arginine with leucine at codon 2336 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). This variant also changes the last nucleotide of exon 13 of the BRCA2 gene and splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies for this variant, different variants affecting the same splice donor site (c.7007G>A, c.7007G>C) have been shown to produce aberrant RNA transcripts and are considered to be disease-causing (ClinVar variation ID: 38077, 52241). It suggests that the reference sequence at this splice site is important for normal RNA splicing. This variant has been reported in individuals with personal and/or family history of breast and/or ovarian cancer (PMID: 22970155, 27157322, 28993434, 30078507, 31742824, 31782247, 35886069, Color internal data) and non-small cell lung cancer (PMID: 31565484). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic. -

Hereditary breast ovarian cancer syndrome Pathogenic:2

Pathogenic, no assertion criteria provided	research	Research Molecular Genetics Laboratory, Women's College Hospital, University of Toronto	Jan 31, 2014	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Nov 18, 2023	This sequence change replaces arginine, which is basic and polar, with leucine, which is neutral and non-polar, at codon 2336 of the BRCA2 protein (p.Arg2336Leu). This variant also falls at the last nucleotide of exon 13, which is part of the consensus splice site for this exon. This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with breast and/or ovarian cancer (PMID: 22970155, 27157322, 29446198, 31742824, 31825140). ClinVar contains an entry for this variant (Variation ID: 52242). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be tolerated. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. This variant disrupts the c.7007 nucleotide in the BRCA2 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 9150172, 21719596, 25447315). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.13
BayesDel_addAF	Uncertain	0.098	D
BayesDel_noAF	Benign	-0.10
CADD	Pathogenic	32
DANN	Uncertain	0.99
Eigen	Benign	-0.11
Eigen_PC	Benign	0.032
FATHMM_MKL	Uncertain	0.86	D
M_CAP	Uncertain	0.14	D
MetaRNN	Benign	0.35	T;T
MetaSVM	Benign	-0.69	T
MutationTaster	Benign	1.0	D;D
PrimateAI	Benign	0.21	T
PROVEAN	Benign	-0.010	N;N
REVEL	Uncertain	0.40
Sift	Benign	0.092	T;T
Sift4G	Benign	0.10	T;T
Vest4		0.44
MutPred		0.46		Loss of catalytic residue at R2336 (P = 0.033);Loss of catalytic residue at R2336 (P = 0.033);
MVP		0.90
MPC		0.025
ClinPred		0.77	D
GERP RS		5.0
gMVP		0.26

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.90
SpliceAI score (max)		0.38		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.38		Position offset: 0

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

Publications

LitVar

Other links and lift over

dbSNP: rs28897743; hg19: chr13-32921033;