rs81002893

Positions:

• chr13-32376795-A-G
• chr13-32376795-A-T

Variant summary

Our verdict is Pathogenic. Variant got 11 ACMG points: 11P and 0B. PM2 PP3 PP5_Very_Strong

The NM_000059.4(BRCA2):​c.8754+4A>G variant causes a splice donor region, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.000000684 in 1,461,666 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. Variant has been reported in ClinVar as Pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
• Consequence: BRCA2 NM_000059.4 splice_donor_region, intron
• Scores: Splicing: ADA: 0.9969
• Clinical Significance: Pathogenic reviewed by expert panel P:12 U:1
• Conservation: PhyloP100: 3.47

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 11 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 13-32376795-A-G is Pathogenic according to our data. Variant chr13-32376795-A-G is described in ClinVar as [Pathogenic]. Clinvar id is 52669.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr13-32376795-A-G is described in Lovd as [Pathogenic]. Variant chr13-32376795-A-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.8754+4A>G		splice_donor_region_variant, intron_variant		ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.8754+4A>G		splice_donor_region_variant, intron_variant		5	NM_000059.4	ENSP00000369497	A2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.84e-7 AC: 1 AN: 1461666 Hom.: 0 Cov.: 32 AF XY: 0.00 AC XY: 0 AN XY: 727118

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: 8.99e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:12 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:4 Uncertain:1

Pathogenic, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	May 01, 2012	- -
Pathogenic, reviewed by expert panel	curation	Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA)	Jun 18, 2019	Allele-specific assay on patient-derived mRNA demonstrated that the variant allele produces only predicted non-functional transcripts. Variant allele produces r.8754_8755ins8754+1_8754+46 transcript (PMID: 22505045). A mRNA splicing mini-gene assay has concordant results (PMID: 25382762). -
Pathogenic, criteria provided, single submitter	clinical testing	Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge	Oct 02, 2015	- -
Uncertain significance, no assertion criteria provided	clinical testing	Breast Cancer Information Core (BIC) (BRCA2)	Feb 20, 2004	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Aug 19, 2016	- -

Hereditary breast ovarian cancer syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Dec 08, 2023	This sequence change falls in intron 21 of the BRCA2 gene. It does not directly change the encoded amino acid sequence of the BRCA2 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individual(s) with ovarian, prostate and breast cancer (PMID: 17011978, 20927582, 27433846, 28199346). This variant is also known as IVS21+4A>G. ClinVar contains an entry for this variant (Variation ID: 52669). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 17924331, 21990134, 31131967). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant alters mRNA splicing and is expected to lead to the loss of protein expression (PMID: 17011978, 22505045, 25382762). For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Apr 07, 2016	Variant Summary: The variant of interest is located at a conserved intronic position, in which 5/5 splicing prediction programs via Alamut predict a loss (or weakening) effect on a canonical RNA splicing donor site. The variant of interest was not observed in controls (ExAC, 1000 Gs, ESP), but has been reported in multiple affected individuals via publications. Multiple independent functional studies show that this variant can cause a loss of intron 21 donor site, which leads to consequent activation of a cryptic splice site at position IVS21+46. The resulting transcript retained a 46-bp fragment of intron 21 and generates a stop codon at position c.IVS21+9, resulting in a truncated protein and consequently, suggesting a deleterious effect of this variant (Bontti_2006, Acedo_2014, and Houdayer_2012). In addition, multiple clinical laboratories/databases cite the variant with a classification of "Pathogenic/Causal." Therefore, taking all available lines of evidence into consideration, the variant of interest is classified as Pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	May 15, 2023	The c.8754+4A>G variant in BRCA2 has been reported in over 9 individuals with BRCA2-associated cancers, including at least 1 male with breast cancer, and segregated with disease in at least 4 affected relatives from one family (Rashid 2019 PMID: 31528241, Vietri 2012 PMID: 23096105, Pritchard 2016 PMID: 27433846, Houdayer 2012 PMID: 22505045, Ding 2011 PMID: 20927582, Lee 2008 PMID: 18284688, Bonatti 2006 PMID: 17011978). It was absent from large population studies. This variant is located in the 5' splice region. Computational tools predict a splicing impact. This is corroborated by multiple studies show that this variant results in the loss of the original donor site and the creation of a cryptic splice site that interferes with normal splicing, resulting in an aberrant transcript that is subjected to nonsense-mediated decay (Acedo 2015 PMID: 25382762, Houdayer 2012 PMID: 22505045, Bonatti 2006 PMID: 17011978). Additionally, in vitro cell viability assay studies show that this variant displayed full complementation to its wildtype counterpart, but a homology directed repair (HDR) assay shows that its HDR capacity is severely impaired (Mesman 2020 PMID: 32398771). Additionally, this variant was classified as pathogenic on June 18, 2019 by the ClinGen-approved ENIGMA expert panel (Variation ID 52669). In summary, this variant meets criteria to be classified as likely pathogenic for autosomal dominant hereditary breast and ovarian cancer. ACMG/AMP Criteria applied: PS4, PM2_Supporting, PS3_Moderate, PP1, PP3. -

not provided Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Jan 10, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Oct 09, 2020	This variant has been reported in individuals with breast, ovarian, male breast and prostate cancers in the published literature (PMID: 31528241 (2019), 27433846 (2016), 23096105 (2012), 18284688 (2008), 18779604 (2008)). Functional data indicated that this variant creates a cryptic splice site and interferes with normal BRCA2 mRNA splicing (PMID: 25382762 (2015), 22505045 (2012), 17011978 (2006)). This variant has not been reported in large, multi-ethnic general populations. Variant is predicted to negatively affect a known splice site Therefore, the variant is classified as pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Oct 27, 2023	This variant causes an A to G nucleotide substitution at the +4 position of intron 21 of the BRCA2 gene. RNA studies have shown that this variant causes use of a cryptic splice site, resulting in premature truncation (PMID: 17011978, 22505045, 25382762). In a functional study in Brca2-deficient mouse embryonic stem cells, this variant displayed full complementation, but impaired homology-directed DNA repair (PMID: 32398771). This variant has been reported in at least 4 unrelated individuals affected with breast or ovarian cancer (PMID: 20927582, 23096105, 31528241, 34072659), and in 1 individual affected with prostate cancer (PMID: 27433846). This variant has been reported in a multifactorial analysis with segregation, tumor pathology, co-occurrence and family history likelihood ratios for pathogenicity of 4.868, 3.133, 1.339, and 2.222, respectively (PMID: 31131967). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of BRCA2 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Sep 05, 2023	The c.8754+4A>G intronic pathogenic mutation results from an A to G substitution 4 nucleotides after coding exon 20 in the BRCA2 gene. Functional splicing assays have shown this alteration to result in loss of the native donor site and the subsequent activation of a cryptic splice site at the c.8754+46 position, resulting in a transcript subject to nonsense-mediated decay (Bonatti F et al. Cancer Genet. Cytogenet. 2006 Oct; 170(2):93-101; Acedo A et al. Hum. Mutat. 2015 Feb; 36(2):210-21; Ambry internal data). This alteration has also been classified as likely pathogenic by multifactorial analyses, which integrate multiple lines of evidence to produce a quantitative likelihood of pathogenicity (Easton DF et al. Am. J. Hum. Genet. 2007 Nov;81:873-83; Lindor NM et al. Hum. Mutat. 2012 Jan;33:8-21; Vallee MP et al. Hum. Mutat. 2012 Jan;33:22-8; Parsons MT et al. Hum. Mutat., 2019 09;40:1557-1578). Protein functional data has shown that this variant results in significantly impaired homology-directed repair activity (Mesman RLS et al. Genet Med 2020 Aug;22(8):1355-1365). Of note, this alteration is also designated as IVS21+4A>G in published literature. This variant was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). In silico splice site analysis predicts that this alteration will weaken the native splice donor site and will result in the creation or strengthening of a novel splice donor site. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Malignant tumor of breast Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The BRCA2 c.8754+4A>G variant was identified by Houdayer (2012) in an individual referred for BRCA1/2 analysis based on personal or family history. The variant was also identified in dbSNP (ID: rs81002893) “With other allele”, HGMD as “disease causing mutation”, LOVD, COSMIC, ClinVar database, the BIC database (7X with unknown clinical importance), and UMD (2X as a causal variant). The variant was classified as a pathogenic variant by the Sharing Clinical Reports Project (SCRP) (submitted within the ClinVar database and derived from Myriad reports). The c.8754+4A>G variant is located in the 5' splice region but does not affect the invariant +1 and +2 positions. However, positions +3 to +6 are part of the splicing consensus sequence and variants involving these positions sometimes affect splicing. Five in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing, and two studies using mRNA analysis demonstrated abnormal splicing with a loss of the intron 21 donor site and consequent activation of a cryptic splice site downstream (Bonatti 2006, Houdayer 2012). In addition, two multifactorial probability-based models classified the variant as likely causal or pathogenic (Easton 2007, Lindor 2012). In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.33
CADD	Benign	19
DANN	Benign	0.93

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.76
SpliceAI score (max)		0.74		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.74		Position offset: 42
DS_DL_spliceai		0.55		Position offset: -4

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

Publications

LitVar

Other links and lift over

dbSNP: rs81002893; hg19: chr13-32950932;