BRCA2 c.516+1G>A, p.Splice_Site
NM_000059.4:c.516+1G>A
Pathogenic
This BRCA2 canonical splice site variant c.516+1G>A is classified as Pathogenic based on PVS1 (Very Strong) supported by absence from controls (PM2 Supporting), strong in silico splice predictions (PP3 Supporting), and reputable pathogenic assertions (PP5 Supporting).
ACMG/AMP Criteria Applied
PVS1
PM2
PP3
PP5
Genetic Information
Gene & Transcript Details
Gene
BRCA2
Transcript
NM_000059.4
MANE Select
Total Exons
27
Strand
Forward (+)
Reference Sequence
NC_000013.10
Alternative Transcripts
| ID | Status | Details |
|---|---|---|
| NM_000059.2 | Alternative | 27 exons | Forward |
| NM_000059.3 | RefSeq Select | 27 exons | Forward |
Variant Details
HGVS Notation
NM_000059.4:c.516+1G>A
Protein Change
Splice
Location
Exon 6
(Exon 6 of 27)
5'Exon Structure (27 total)3'
Functional Consequence
Loss of Function
Related Variants
Variant interpretation based on transcript NM_000059.4
Genome Browser
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HGVS InputNM_000059:c.516+1G>A
Active Tracks
ConservationRefSeqClinVargnomAD
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Clinical Data
Population Frequency
Global Frequency
0.0 in 100,000
Extremely Rare
Global: 0.0%
0%
0.05%
0.1%
1%
5%
10%+
ACMG Criteria Applied
PM2
This variant is not present in gnomAD (PM2 criteria applies).
Classification
3 publications
Pathogenic
Based on 5 submitter reviews in ClinVar
Submitter Breakdown
5 Path
Pathogenic
Likely Path.
VUS
Likely Benign
Benign
Publications (3)
The c.516+1G>A intronic pathogenic mutation results from a G to A substitution one nucleotide after coding exon 5 of the BRCA2 gene. This alteration has been previously detected in multiple individuals in six unrelated Belgian families with breast cancer; RNA studies showed that this alteration led to skipping of multiple exons (Claes K et al. Genes Chromosomes Cancer. 2003 Jul;37:314-20; Claes K et al. Br. J. Cancer. 2004 Mar;90(6):1244-51; Machackova E et al. BMC Cancer. 2008 May;8:140). This alteration has also been detected in 1/313 unselected Chinese breast cancer patients (Li G. et al J. Cancer Res. Clin. Oncol. 2017 Oct;143(10):2011-2024). Of note, this alteration is also designated as IVS6+1G>A in published literature. 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 will weaken the native splice donor site. In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as a disease-causing mutation.
This variant is not present in population databases (ExAC no frequency). This variant has been reported in the literature in individuals affected with breast cancer (PMID: 12759930, 28664449). ClinVar contains an entry for this variant (Variation ID: 51786). Experimental studies using patient lymphoblastoid cells have shown that this intronic change leads to multiple alternative transcripts, skipping exons 5, 6, and 5 and 6, resulting in truncated protein products (PMID: 12759930, 18489799). Donor and acceptor splice site variants typically lead to a loss of protein function (PMID: 16199547), and loss-of-function variants in BRCA2 are known to be pathogenic (PMID: 20104584). For these reasons, this variant has been classified as Pathogenic. This sequence change affects a donor splice site in intron 6 of the BRCA2 gene. It is expected to disrupt RNA splicing and likely results in an absent or disrupted protein product.
Clinical Statement
This variant has been reported in ClinVar as Pathogenic (5 clinical laboratories).
Functional Impact
Functional Domain
Hotspot Status
Not a hotspot
Domain Summary
This variant is not located in a mutational hotspot or critical domain (0 mutations).
Related Variants in This Domain
Computational Analysis
Pathogenicity Predictions
Predictor Consensus
Mixed/VUS
PP3 Applied
No
Additional Predictors
Benign:
CADD: 5.97
VCEP Guidelines
Applied ACMG/AMP Criteria (VCEP Specific) VCEP Guidelines
PVS1
PVS1 (Very Strong)
According to VCEP guidelines, the rule for PVS1 is: 'Very Strong Null variant (splice site (donor/acceptor +/−1,2)...) in a gene where LOF is a known mechanism of disease.' The evidence for this variant shows: NM_000059.4:c.516+1G>A alters the canonical +1 splice donor site in BRCA2. Therefore, this criterion is applied at Very Strong strength because loss of function is the established mechanism and this is a canonical splice site variant.
PS1
PS1 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PS1 is: 'Strong: for predicted missense substitutions where a previously classified pathogenic variant has the same amino acid change.' The evidence for this variant shows: NM_000059.4:c.516+1G>A is a splice site variant, not a missense change. Therefore, PS1 is not applied.
PS2
PS2 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PS2 is: 'Strong: de novo (both maternity and paternity confirmed).' No de novo data are provided for this variant. Therefore, PS2 is not applied.
PS3
PS3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PS3 is: 'Strong: well-established in vitro or in vivo functional studies supportive of a damaging effect.' The evidence for this variant shows: no functional studies have been reported. Therefore, PS3 is not applied.
PS4
PS4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PS4 is: 'Strong: prevalence in affected individuals significantly increased compared to controls (case-control OR ≥4, p≤0.05).' No case-control data are provided. Therefore, PS4 is not applied.
PM1
PM1 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM1 is: 'Moderate: located in a mutational hot spot or critical functional domain without benign variation.' The evidence for this variant shows: it is a splice site change, not within a defined missense hotspot or domain. Therefore, PM1 is not applied.
PM2
PM2 (Supporting) Strength Modified
According to VCEP guidelines, the rule for PM2 is: 'Supporting: Absent from controls in an outbred population, from gnomAD v2.1 (non-cancer) and gnomAD v3.1.' The evidence for this variant shows: MAF = 0% and it is not present in gnomAD. Therefore, PM2 is applied at Supporting strength because it is absent from population controls.
PM3
PM3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PM3 is: 'Supportive to Strong: observed in trans with a pathogenic variant in patients with Fanconi anemia.' No Fanconi anemia phenotype or phase data are provided. Therefore, PM3 is not applied.
PM4
PM4 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM4 is: 'Moderate: protein length changes due to in-frame indels or stop-loss.' The evidence for this variant shows: it is a splice site change, not an in-frame indel. Therefore, PM4 is not applied.
PM5
PM5 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM5 is: 'Moderate: different pathogenic missense change at same residue or PTC in same exon.' The evidence for this variant shows: no data on other PTCs in the same exon. Therefore, PM5 is not applied.
PM6
PM6 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM6 is: 'Moderate: assumed de novo without confirmation of paternity/maternity.' No de novo data are provided. Therefore, PM6 is not applied.
PP1
PP1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PP1 is: 'Supporting to Strong: co-segregation in multiple affected family members.' No segregation data are provided. Therefore, PP1 is not applied.
PP2
PP2 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PP2 is: 'Supporting: missense variant in a gene with low rate of benign missense variation.' The evidence for this variant shows: it is a splice site change, not a missense. Therefore, PP2 is not applied.
PP3
PP3 (Supporting)
According to VCEP guidelines, the rule for PP3 is: 'Supporting: Apply PP3 for predicted splicing (SpliceAI ≥0.2).' The evidence for this variant shows: SpliceAI donor loss 0.98 and acceptor loss 0.95, predicting significant splice disruption. Therefore, PP3 is applied at Supporting strength because computational tools strongly predict a deleterious splicing effect.
PP4
PP4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PP4 is: 'Supporting to Strong: phenotype specific to the gene based on multifactorial likelihood.' No phenotype data are provided. Therefore, PP4 is not applied.
PP5
PP5 (Supporting)
According to standard ACMG guidelines, the rule for PP5 is: 'Supporting: reputable source reports variant as pathogenic without available evidence.' The evidence for this variant shows: ClinVar lists it as Pathogenic by five clinical labs. Therefore, PP5 is applied at Supporting strength because a reputable source reports pathogenicity without detailed evidence.
BA1
BA1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BA1 is: 'Stand Alone: allele frequency >0.1% in gnomAD.' The evidence for this variant shows: MAF = 0%, below threshold. Therefore, BA1 is not applied.
BS1
BS1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS1 is: 'Strong: allele frequency >0.01% in gnomAD.' The evidence for this variant shows: MAF = 0%, below threshold. Therefore, BS1 is not applied.
BS2
BS2 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS2 is: 'Supporting to Strong: observed in healthy individuals without Fanconi anemia.' No data on healthy individuals are provided. Therefore, BS2 is not applied.
BS3
BS3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS3 is: 'Strong: well-established functional studies show no damaging effect.' No such studies exist for this variant. Therefore, BS3 is not applied.
BS4
BS4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS4 is: 'Strong: lack of segregation in affected family members.' No segregation data are provided. Therefore, BS4 is not applied.
BP1
BP1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BP1 is: 'Supporting: silent or missense outside functional domain with no splicing impact.' The evidence for this variant shows: it is a canonical splice site change. Therefore, BP1 is not applied.
BP2
BP2 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP2 is: 'Supporting: observed in trans with a pathogenic variant in a dominant disease without recessive phenotype.' No such data are provided. Therefore, BP2 is not applied.
BP3
BP3 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP3 is: 'Supporting: in-frame indel in repetitive region without functional impact.' The evidence for this variant shows: it is a splice site change. Therefore, BP3 is not applied.
BP4
BP4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BP4 is: 'Supporting: no predicted impact via protein or splicing (BayesDel ≤0.18 and SpliceAI ≤0.1).' The evidence for this variant shows: SpliceAI >0.9, predicting disruption. Therefore, BP4 is not applied.
BP5
BP5 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP5 is: 'Supporting: variant found in a case with an alternate molecular basis for disease.' No such data are provided. Therefore, BP5 is not applied.
BP6
BP6 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP6 is: 'Supporting: reputable source reports variant as benign without evidence.' No benign assertions exist. Therefore, BP6 is not applied.
BP7
BP7 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BP7 is: 'Supporting: silent or intronic outside consensus splice sites with no impact.' The evidence for this variant shows: it affects the canonical +1 splice site. Therefore, BP7 is not applied.