BRCA1 c.2501G>A, p.(Gly834Glu)

Reasoning

BS1_Supporting is met under the ENIGMA BRCA1 v1.2 population thresholds. The gnomAD v4.1 group-maximum FAF is 5.137e-05, which is above 0.00002 and at or below 0.0001. The gnomAD v2.1 group-maximum FAF is below the supporting interval, but the VCEP rule permits application when the threshold is met in gnomAD v2.1 and/or gnomAD v3.1; the available gnomAD v4.1 value independently falls in the specified supporting range.

Rule

Filter allele frequency (FAF) is above 0.002% (FAF > 0.00002) and less than or equal to 0.01% (FAF <= 0.0001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s).

Evidence considered

• The ENIGMA BRCA1 and BRCA2 specification v1.2 defines BS1_Supporting as FAF >0.00002 and <=0.0001 in gnomAD v2.1 and/or gnomAD v3.1 non-cancer, non-founder populations.
• gnomAD v4.1 reports group-maximum FAF 5.137e-05 and highest observed South Asian AF 9.88121e-05, placing the group-maximum FAF within the ENIGMA BS1_Supporting interval.
• gnomAD v2.1 reports group-maximum FAF 1.082e-05, total AF 7.96724e-06, and zero homozygotes; this does not independently reach the BS1_Supporting lower threshold.

Still missing

• The case bundle provides gnomAD v4.1 rather than the VCEP-named gnomAD v3.1 dataset; confirm dataset-version equivalence if strict version matching is required.

Sources

Reasoning

The variant is a missense substitution at residue 834. The ENIGMA BRCA1 functional-domain list defines the clinically important domains as RING residues 2-101, coiled-coil residues 1391-1424, and BRCT residues 1650-1857; residue 834 lies outside all three. SpliceAI predicts no significant splice impact (maximum delta 0.001, ≤0.1). The BRCA1-specific BP1 rule therefore supports BP1_Strong.

Rule

Apply BP1_Strong for a missense or in-frame insertion, deletion, or delins variant outside a potentially clinically important functional domain and with no predicted splicing impact (SpliceAI ≤0.1). BRCA1 domains are RING aa 2-101, coiled-coil aa 1391-1424, and BRCT repeats aa 1650-1857.

Evidence considered

• ENIGMA BRCA1/2 specification v1.2 defines BP1_Strong for missense or in-frame variants outside a clinically important functional domain when no splicing is predicted (SpliceAI ≤0.1).
• The same specification defines BRCA1 clinically important functional domains as RING aa 2-101, coiled-coil aa 1391-1424, and BRCT repeats aa 1650-1857; p.Gly834 is outside every listed domain.
• The case variant is NP_009225.1:p.(Gly834Glu), and SpliceAI reports max_delta_score 0.001, satisfying the no-significant-splice-impact condition.

Sources

Reasoning

BRCA1 c.2501G>A (NM_007294.4) is a missense substitution, p.(Gly834Glu), not a nonsense, frameshift, initiation-codon, canonical splice-site, or exon-deletion variant. The ENIGMA PVS1 specification permits application to null variants, or to damaging mRNA-only functional evidence. SpliceAI shows no significant splice effect (maximum delta 0.001), and no variant-specific damaging mRNA assay is available in the evidence bundle. Therefore there is no predicted premature termination, NMD-triggering transcript, or RNA-based loss-of-function evidence to support PVS1.

Rule

ENIGMA BRCA1/BRCA2 v1.2 PVS1: null variant in a gene with loss of function as a known disease mechanism, weighted by the PVS1 flowchart; alternatively, well-established damaging mRNA-only functional evidence may support PVS1(RNA).

Evidence considered

• The ENIGMA BRCA1/BRCA2 v1.2 PVS1 rule applies to null variants (nonsense, frameshift, canonical donor/acceptor ±1/2, initiation codon, or exon deletion) with strength determined by its PVS1 flowchart; it also permits PVS1(RNA) only for well-established damaging mRNA-only functional evidence.
• The case annotation identifies NM_007294.4:c.2501G>A as NP_009225.1:p.(Gly834Glu), a single amino-acid substitution rather than a protein-truncating variant.
• SpliceAI maximum delta is 0.001 (DS_DG and DS_DL 0.001; all other delta scores 0), reported as no significant splice impact. No mRNA assay demonstrating a damaging transcript consequence is supplied.
• The exact-variant reports in PMID:26306726 and PMID:32438681 each identify c.2501G>A/p.G834E as a missense variant; neither supplies a variant-specific mRNA assay or a truncating consequence.

Sources

Reasoning

PM2 is not met because the variant is observed in the population databases used by the ENIGMA framework. It has 2 alleles in gnomAD v2.1 and 11 alleles in gnomAD v4.1, rather than being absent from controls. Both datasets report zero homozygotes; absence of homozygotes does not satisfy the VCEP PM2 absence requirement.

Rule

Absent from controls in an outbred population, from gnomAD v2.1 (non-cancer, exome only subset) and gnomAD v3.1 (non-cancer). Region around the variant must have an average read depth >=25.

Evidence considered

• The ENIGMA BRCA1 and BRCA2 specification v1.2 defines PM2_Supporting as absence from controls in an outbred population in gnomAD v2.1 non-cancer exome-only and gnomAD v3.1 non-cancer data, with average local read depth at least 25.
• gnomAD v2.1 reports 2/251028 alleles and zero homozygotes.
• gnomAD v4.1 reports 11/1613830 alleles and zero homozygotes. Because the allele is present, the absence-from-controls requirement for PM2 is not satisfied; the bundle does not provide a PM2-qualifying depth measure that could override this presence.

Sources

Reasoning

BRCA1 c.2501G>A encodes p.(Gly834Glu), a missense variant outside the ENIGMA-defined clinically important domains (RING aa 2-101, coiled-coil aa 1391-1424, BRCT aa 1650-1857). ENIGMA PP3 for protein impact is limited to missense/in-frame variants within those domains; its splice pathway requires SpliceAI >=0.2. SpliceAI maximum delta is 0.001, so the splice pathway is also not met.

Rule

ENIGMA BRCA1/2 VCEP v1.2 PP3: domain-limited missense/in-frame protein-impact path; splice path requires SpliceAI >=0.2.

Evidence considered

• ENIGMA BRCA1/2 VCEP v1.2 applies PP3 protein-prediction evidence only to missense/in-frame variants in BRCA1 RING aa 2-101, coiled-coil aa 1391-1424, or BRCT aa 1650-1857; p.Gly834Glu is outside these intervals.
• The ENIGMA PP3 splice pathway requires SpliceAI >=0.2. The case SpliceAI result has maximum delta 0.001 (DS_DG and DS_DL 0.001; DS_AG and DS_AL 0.0).

Sources

Reasoning

BA1 is not met. The ENIGMA BRCA1 v1.2 rule requires FAF >0.001 in gnomAD v2.1 and/or gnomAD v3.1 non-cancer, non-founder populations. The observed maximum group FAF in the available population data is 5.137e-05 in gnomAD v4.1, well below 0.001.

Rule

Filter allele frequency (FAF) is above 0.1% (FAF > 0.001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s).

Evidence considered

• The ENIGMA BRCA1 and BRCA2 specification v1.2 defines BA1 as FAF >0.001 in gnomAD v2.1 and/or gnomAD v3.1 non-cancer, non-founder populations.
• gnomAD v2.1 reports 2/251028 alleles, total AF 7.96724e-06, zero homozygotes, and group-maximum FAF 1.082e-05.
• gnomAD v4.1 reports 11/1613830 alleles, total AF 6.81608e-06, zero homozygotes, and group-maximum FAF 5.137e-05; the highest subpopulation AF is South Asian 9.88121e-05. These frequencies are below the BA1 threshold.

Sources

Reasoning

Although SpliceAI maximum delta is 0.001 (<=0.1), the ENIGMA BP4 missense pathway is limited to missense/in-frame variants inside a clinically important domain and requires both no predicted protein impact and no predicted splice impact. p.(Gly834Glu) is outside the RING, coiled-coil, and BRCT domains, so it does not meet the BP4 pathway. The benign low-prior pathway for this location is not BP4.

Rule

ENIGMA BRCA1/2 VCEP v1.2 BP4: missense/in-frame variants within a clinically important domain, with no predicted protein impact and SpliceAI <=0.1.

Evidence considered

• ENIGMA BRCA1/2 VCEP v1.2 restricts BP4 for missense/in-frame variants to clinically important functional domains and requires no predicted protein impact plus SpliceAI <=0.1.
• p.Gly834 is outside the VCEP-defined BRCA1 RING aa 2-101, coiled-coil aa 1391-1424, and BRCT aa 1650-1857 domains.
• The case SpliceAI maximum delta is 0.001, satisfying the no-predicted-splice-impact component but not overcoming the BP4 domain-scope requirement.

Sources

Reasoning

The variant is a missense substitution, p.Gly834Glu, and the ENIGMA BRCA1 specification permits PS1 when a previously classified pathogenic or likely pathogenic variant has the same protein change or qualifying matched splicing impact. The supplied evidence does not identify a pathogenic or likely pathogenic comparator with the same amino-acid change, and SpliceAI predicts no significant splice impact (maximum delta 0.001).

Rule

Apply PS1 for a predicted missense substitution when a previously classified pathogenic variant is considered to act via protein change and there is no confirmed or predicted effect on mRNA splicing (SpliceAI ≤0.1). Apply PS1_Moderate when the reference variant is previously classified likely pathogenic; qualifying splicing matches may be weighted by relative position and reference-variant confidence.

Evidence considered

• ENIGMA BRCA1/2 specification v1.2 defines PS1 for a missense substitution when a previously classified pathogenic variant acts through protein change, with no confirmed or predicted splice effect; PS1_Moderate is described for a previously classified likely pathogenic comparator.
• The case variant is NM_007294.4:c.2501G>A, NP_009225.1:p.(Gly834Glu), and SpliceAI reports a maximum delta score of 0.001, below the specification's 0.1 splice-impact threshold.
• No same-amino-acid pathogenic or likely pathogenic comparator is present in the supplied PM5 candidate collection or paper-extract evidence.

Still missing

• No qualifying previously classified pathogenic comparator with the same amino-acid change was identified in the supplied evidence bundle.
• No variant-specific evidence establishing a qualifying matched splicing impact was identified.

Sources

Reasoning

The applicable ENIGMA BRCA1/2 VCEP specification (version 1.2) requires well-established, biologically relevant functional studies with calibrated assay interpretation for PS3 and directs review to Specifications Table 9. No Table 9 entry for NM_007294.4:c.2501G>A (p.Gly834Glu/p.G834E) was identified in the case bundle, and the summarized ST4 functional dataset contains other variants but not this variant. The variant-specific publications located in the bundle report detection in clinical cohorts but provide no functional assay result. Therefore, damaging functional evidence is insufficient to assess PS3.

Rule

ENIGMA BRCA1/2 VCEP v1.2 PS3: well-established in vitro or in vivo functional studies supportive of a damaging effect; apply PS3 for assays measuring protein effect alone or mRNA and protein combined, using calibrated published assay recommendations in Specifications Table 9.

Evidence considered

• The ENIGMA BRCA1/2 specification v1.2 states that PS3 is supported by well-established in vitro or in vivo studies showing a damaging effect, with protein-only or combined mRNA/protein assays evaluated using calibrated published assays in Specifications Table 9.
• The case-bundle summary of ENIGMA Specifications Table 9 does not list c.2501G>A, and its summarized ST4 functional assay results list c.285T>G and c.5247A>C rather than the target variant.
• PMID:26306726 reports BRCA1 c.2501G>A/p.G834E as a variant identified by next-generation sequencing and confirmed by Sanger sequencing, but does not report a functional assay result for it.
• PMID:32438681 reports BRCA1 c.2501G>A/p.(Gly834Glu) once in a clinical cohort and assigns it Class 3 (uncertain significance), but reports no variant-specific functional assay, segregation result, or other functional measurement.

Still missing

• No variant-specific calibrated protein-function or combined mRNA/protein assay result was identified.
• No assay controls, replicate data, biological-relevance validation, or ENIGMA Table 9 PS3 strength assignment was identified for this variant.

Sources

Reasoning

The ENIGMA BRCA1/2 specification requires a case-control study with p<=0.05, OR>=4, and lower confidence interval excluding 2.0. Two exact-variant reports identify c.2501G>A/p.(Gly834Glu) once each in ovarian cancer and breast/ovarian cancer cohorts, but neither provides a matched control comparison or a qualifying case-control statistic.

Rule

ENIGMA BRCA1/2 v1.2 PS4: case-control study p<=0.05 and OR>=4 with lower confidence interval excluding 2.0; do not use proband counting.

Evidence considered

• ENIGMA specifies PS4 only for case-control evidence with p<=0.05, OR>=4, and lower confidence interval excluding 2.0; proband counting is not used.
• PMID:26306726 reports c.2501G>A/p.G834E in one of 212 ovarian cancer samples, without variant-specific control data or a case-control association statistic.
• PMID:32438681 reports c.2501G>A/p.(Gly834Glu) once in a retrospective cohort of 2351 breast and/or ovarian cancer patients, without variant-specific control data or a case-control association statistic.

Still missing

• Ethnicity- and country-matched case and control counts for c.2501G>A.
• A qualifying case-control OR, confidence interval, and p-value.

Sources

Reasoning

The applicable ENIGMA BRCA1/2 specification limits PM3 to a patient with a phenotype consistent with BRCA1/2-related Fanconi anemia and co-occurring pathogenic or likely pathogenic variants in the same gene, with strength assigned from the Table 6 point system. The available case bundle documents the target variant in cancer cohorts but provides no Fanconi anemia phenotype, affected-proband evidence meeting the specification, second BRCA1 pathogenic/likely pathogenic variant, or parental/offspring testing establishing trans or cis phase. PM3 therefore cannot be assigned from the available evidence.

Rule

ENIGMA BRCA1/2 v1.2 PM3: apply for a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene; assign strength using the Table 6 point system (PM3 Supporting = 1 point, PM3 = 2 points, PM3 Strong = at least 4 points).

Evidence considered

• ENIGMA BRCA1/2 v1.2 specifies PM3 for a patient with a phenotype consistent with BRCA1/2-related Fanconi anemia and co-occurring variants in the same gene; PM3 strength is based on the Table 6 point system.
• PMID:26306726 reports BRCA1 c.2501G>A (p.G834E) in one ovarian-cancer patient identified by NGS and confirmed by Sanger sequencing, but reports no co-occurring pathogenic BRCA1 variant or phase information.
• PMID:32438681 reports BRCA1 c.2501G>A (p.Gly834Glu) once in a breast/ovarian cancer cohort and provides no variant-specific segregation, co-occurrence, or phase result.
• The case bundle contains no documented Fanconi anemia phenotype or chromosome-breakage result for the case.
• The case bundle provides no parental or offspring testing establishing cis/trans phase with another BRCA1 variant.

Still missing

• Documented BRCA1/2-related Fanconi anemia phenotype, including chromosome-breakage testing and qualifying clinical features
• A co-occurring pathogenic or likely pathogenic BRCA1 variant classified under the ENIGMA specification
• Phasing information demonstrating the two variants are in trans

Sources

Reasoning

PP1 requires co-segregation with disease in multiple affected family members assessed by a quantitative co-segregation method. The case bundle provides no affected-relative genotypes, phenotype-linked pedigree, meioses, segregation likelihood ratio, or other quantitative co-segregation result.

Rule

ENIGMA BRCA1/2 v1.2: apply PP1 only for co-segregation in multiple affected family members using a quantitative co-segregation method; Supporting LR >=2.08:1, Moderate LR >=4.3:1, Strong LR >=18.7:1, Very Strong LR >=350:1.

Evidence considered

• ENIGMA v1.2 requires quantitative co-segregation analysis for PP1; its thresholds are Supporting LR >=2.08:1, Moderate LR >=4.3:1, Strong LR >=18.7:1, and Very Strong LR >=350:1.
• The consolidated case-evidence bundle contains no family segregation observations or quantitative co-segregation analysis for NM_007294.4:c.2501G>A (p.Gly834Glu).

Still missing

• Affected-relative genotypes and phenotypes are not reported.
• Number of informative meioses and family structure are not reported.
• No quantitative co-segregation likelihood ratio or Bayes score is available, so no PP1 strength can be assigned.

Sources

Reasoning

For BRCA1-related cancer predisposition, ENIGMA restricts PP4 to a combined, clinically calibrated multifactorial likelihood ratio. No combined LR is supplied for c.2501G>A. The two cohort observations cannot substitute for that combined LR.

Rule

ENIGMA BRCA1/2 v1.2 PP4: apply only a combined clinically calibrated multifactorial LR; Supporting >=2.08, Moderate >=4.3, Strong >=18.7, Very Strong >=350.

Evidence considered

• ENIGMA permits PP4 only for a combined multifactorial clinical LR; thresholds are Supporting >=2.08, Moderate >=4.3, Strong >=18.7, and Very Strong >=350.
• The VCEP clinical-history LR resource is an allowable component for PP4/BP5 but does not provide a reported c.2501G>A LR in the supplied evidence bundle.
• The exact-variant cohort reports in PMID:26306726 and PMID:32438681 provide isolated observations, not a combined multifactorial LR.

Still missing

• Variant-specific combined multifactorial clinical LR, with reviewable component data and weights.

Sources

Reasoning

BS2 cannot be assessed from the available bundle. The ENIGMA BRCA1 v1.2 rule applies BS2 in the absence of Fanconi Anemia features and requires aggregation of qualifying proband points; no case-level homozygous or compound-heterozygous observations, Fanconi Anemia phenotype assessments, or BS2 point assignments are provided.

Rule

Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See Specifications Table 8 for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points.

Evidence considered

• The ENIGMA BRCA1 and BRCA2 specification v1.2 defines BS2 as evidence applied in the absence of Fanconi Anemia features and assigns strength by the summed BS2 points: Supporting = 1 point, Moderate = 2 points, and Strong = at least 4 points.
• The population data show zero homozygotes in gnomAD v2.1 and gnomAD v4.1, but this alone does not provide the required qualifying proband observations or establish the ENIGMA BS2 point total.

Still missing

• Case-level evidence is needed for qualifying homozygous or compound-heterozygous observations and the associated absence of Fanconi Anemia features, including the ENIGMA BS2 point count.

Sources

Reasoning

The applicable ENIGMA BRCA1/2 VCEP specification (version 1.2) requires well-established, biologically relevant functional studies demonstrating no damaging effect, with calibrated assay interpretation in Specifications Table 9. No Table 9 entry for NM_007294.4:c.2501G>A (p.Gly834Glu/p.G834E) was identified in the case bundle, and the summarized ST4 functional dataset does not contain this variant. The variant-specific publications located report clinical detection only and provide no benign functional assay result. Therefore, benign functional evidence is insufficient to assess BS3.

Rule

ENIGMA BRCA1/2 VCEP v1.2 BS3: well-established in vitro or in vivo functional studies showing no damaging effect on protein function; apply BS3 for assays measuring protein effect alone or mRNA and protein combined, using calibrated published assay recommendations in Specifications Table 9.

Evidence considered

• The ENIGMA BRCA1/2 specification v1.2 states that BS3 requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function, with protein-only or combined mRNA/protein assays evaluated using calibrated published assays in Specifications Table 9.
• The case-bundle summary of ENIGMA Specifications Table 9 does not list c.2501G>A, and its summarized ST4 functional assay results list c.285T>G and c.5247A>C rather than the target variant.
• PMID:26306726 reports BRCA1 c.2501G>A/p.G834E as a clinically detected variant but provides no functional assay evidence supporting retained protein function.
• PMID:32438681 reports BRCA1 c.2501G>A/p.(Gly834Glu) once and classifies it as Class 3, but provides no variant-specific functional assay or benign functional result.

Still missing

• No variant-specific calibrated protein-function or combined mRNA/protein assay result demonstrating retained function was identified.
• No assay controls, replicate data, biological-relevance validation, or ENIGMA Table 9 BS3 strength assignment was identified for this variant.

Sources

Reasoning

BS4 requires lack of segregation in affected family members assessed by a quantitative co-segregation method. The case bundle provides no affected-relative genotypes, non-segregation observations, meioses, or segregation likelihood ratio; therefore BS4 cannot be assigned.

Rule

ENIGMA BRCA1/2 v1.2: apply BS4 only for lack of segregation in affected family members using a quantitative co-segregation method; Supporting LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1, Very Strong LR <=0.00285:1.

Evidence considered

• ENIGMA v1.2 requires quantitative co-segregation analysis for BS4; its thresholds are Supporting LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1, and Very Strong LR <=0.00285:1.
• The consolidated case-evidence bundle contains no family non-segregation observations or quantitative co-segregation analysis for NM_007294.4:c.2501G>A (p.Gly834Glu).

Still missing

• Affected-relative genotypes and phenotypes are not reported.
• No non-segregation events or informative meioses are reported.
• No quantitative co-segregation likelihood ratio or Bayes score is available, so no BS4 strength can be assigned.

Sources

Reasoning

For BRCA1-related cancer predisposition, ENIGMA restricts BP5 to a combined, clinically calibrated multifactorial LR against pathogenicity and specifically excludes co-observation in another breast/ovarian cancer risk gene as a BP5 basis. No qualifying combined LR or other eligible BP5 evidence is supplied.

Rule

ENIGMA BRCA1/2 v1.2 BP5: apply only a combined clinically calibrated multifactorial LR against pathogenicity; Supporting <=0.48, Moderate <=0.23, Strong <=0.05, Very Strong <=0.00285.

Evidence considered

• ENIGMA permits BP5 only for a combined multifactorial clinical LR against pathogenicity; thresholds are Supporting <=0.48, Moderate <=0.23, Strong <=0.05, and Very Strong <=0.00285.
• The supplied VCEP clinical-history LR resource is an allowable PP4/BP5 component, but the bundle supplies no c.2501G>A LR and no combined multifactorial LR.

Still missing

• Variant-specific combined multifactorial clinical LR <=0.48 with reviewable component data and weights.

Sources

Reasoning

The routine ENIGMA BP7 pathway is for silent variants meeting BP4 or suitably deep intronic variants meeting BP4 and therefore does not apply to this missense change. ENIGMA additionally permits BP7_Strong (RNA) for a missense variant outside a clinically important domain when a well-established mRNA assay shows no damaging transcript effect. p.(Gly834Glu) is outside those domains, but no variant-specific mRNA assay demonstrating an unaffected transcript profile was retrieved; SpliceAI alone cannot establish BP7_Strong (RNA).

Rule

ENIGMA BRCA1/2 VCEP v1.2 BP7 and BP7_Strong (RNA).

Evidence considered

• ENIGMA BRCA1/2 VCEP v1.2 applies routine BP7 to silent variants meeting BP4 or intronic variants at or beyond +7/-21 meeting BP4.
• The VCEP repurposes BP7_Strong (RNA) for intronic, silent, and missense/in-frame variants outside a clinically important functional domain only when well-established mRNA evidence shows no damaging transcript effect.
• No retrieved variant-specific mRNA assay result establishes an unaffected transcript profile for c.2501G>A; computational SpliceAI is not a substitute for this RNA-evidence pathway.

Still missing

• No well-established, variant-specific mRNA splicing assay showing an unaffected transcript profile for BRCA1 c.2501G>A was present in the case evidence bundle or the extracted full-text findings.

Sources