BRCA2 c.3941A>G, p.(Lys1314Arg)

Reasoning

The variant is a missense substitution at Lys1314. Under ENIGMA BRCA2 v1.2, BP1_Strong applies to missense variants outside a potentially clinically important functional domain when splicing is not predicted. Lys1314 is outside the specified PALB2-binding domain (amino acids 10-40) and DNA-binding domain (amino acids 2481-3186), and SpliceAI predicts no significant splice impact (maximum delta score 0.001, below the 0.1 threshold).

Rule

Apply BP1_Strong for a missense variant outside a potentially clinically important BRCA2 functional domain when SpliceAI is <=0.1.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BP1_Strong for missense variants outside the clinically important domains when SpliceAI is at or below 0.1.
• The variant changes BRCA2 p.Lys1314Arg; residue 1314 is outside the framework-defined domains at amino acids 10-40 and 2481-3186.
• SpliceAI reports DS_AG 0.0, DS_AL 0.0, DS_DG 0.001, DS_DL 0.0, with maximum delta score 0.001.

Sources

Reasoning

NM_000059.4:c.3941A>G is a missense substitution, NP_000050.3:p.(Lys1314Arg), in BRCA2 exon 11, not a nonsense, frameshift, initiation-codon, canonical splice-site, or exon-deletion variant. ENIGMA PVS1 applies to null variants or qualifying mRNA-assay evidence; neither is present. SpliceAI has a maximum delta score of 0.001, providing no predicted splice consequence. As no premature termination or aberrant transcript is established, NMD and exon-level truncation considerations are not engaged.

Rule

ENIGMA BRCA2 v1.2 PVS1: apply at an appropriate PVS1 strength for a null variant in BRCA2, with strength guided by the PVS1 flowchart and clinically important functional domains; qualifying mRNA-only functional evidence may also be evaluated as PVS1 (RNA).

Evidence considered

• The ENIGMA BRCA2 v1.2 PVS1 specification limits this code to null variants (including nonsense, frameshift, canonical donor/acceptor ±1,2, initiation-codon, and exon-deletion variants) or qualifying mRNA-assay evidence.
• The normalized consequence is NP_000050.3:p.(Lys1314Arg), a single amino-acid substitution in exon 11; it does not create a protein termination codon or alter protein length.
• SpliceAI reports max delta 0.001 (DS_DG 0.001; all other delta scores 0), with no significant predicted splice impact.
• PMID:16683254 reports the exact variant as c.3941A>G (p.Lys1314Arg) and designates its table effect as unknown; it does not report a truncating or RNA-splicing consequence.

Sources

Reasoning

PM2 is not met under the ENIGMA BRCA2 v1.2 rule because the variant is observed in controls rather than absent from controls. It is present in gnomAD v2.1 (1 allele; AF 4.51076e-06) and gnomAD v4.1 (4 alleles; AF 2.53330e-06), with zero homozygotes in both datasets. The observed rarity does not satisfy this VCEP's absence requirement; no read-depth failure was reported, but read depth cannot convert a present allele into PM2 evidence.

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 average read depth >=25.

Evidence considered

• ENIGMA BRCA2 Specification v1.2 defines PM2 Supporting as absence from controls in gnomAD v2.1 non-cancer exome-only and gnomAD v3.1 non-cancer datasets, with average read depth >=25 around the variant.
• gnomAD v2.1 shows the variant in 1/221,692 alleles (AF 4.51076e-06) with zero homozygotes.
• gnomAD v4.1 shows the variant in 4/1,578,966 alleles (AF 2.53330e-06) with zero homozygotes; therefore the variant is not absent from population controls.

Sources

Reasoning

Not met under the governing ClinGen ENIGMA BRCA2 v1.2 specification. This is a missense variant, p.(Lys1314Arg), so it was assessed only on the VCEP protein-impact path. PP3 requires a missense/in-frame variant within the BRCA2 PALB2-binding domain (aa 10-40) or DNA-binding domain (aa 2481-3186) and BayesDel no-AF >=0.30. Residue 1314 is outside both domains and the reported BayesDel no-AF score is -0.521046.

Rule

ClinGen ENIGMA BRCA2 v1.2 PP3: missense/in-frame variant within aa 10-40 or aa 2481-3186 AND BayesDel no-AF >=0.30.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 PP3 rule requires both location in the PALB2-binding domain (aa 10-40) or DNA-binding domain (aa 2481-3186) and BayesDel no-AF >=0.30 for a missense/in-frame variant.
• The normalized consequence is NP_000050.3:p.(Lys1314Arg); residue 1314 is outside both VCEP-defined domains. The reported BayesDel no-AF score is -0.521046.

Sources

Reasoning

PP5 is not met because ClinVar has no exact-variant expert-panel assertion. The exact matched ClinVar record has zero expert-panel submissions and only non-expert laboratory/curation submissions (predominantly uncertain significance, with one likely benign assertion); none can trigger PP5.

Rule

Apply PP5 only for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic.

Evidence considered

• The exact ClinVar record for NM_000059.4:c.3941A>G (p.Lys1314Arg) reports zero expert-panel submissions. Ordinary laboratory and non-expert curation assertions were not used for PP5.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion would be required for PP5.

Sources

Reasoning

BA1 is not met. The ENIGMA BRCA2 v1.2 rule requires FAF >0.001 (0.1%) in an applicable non-cancer, non-founder gnomAD population. The variant is far below this threshold: gnomAD v2.1 AF 4.51076e-06, highest reported subpopulation AF 9.73539e-06, and gnomAD v4.1 AF 2.53330e-06 with grpmax FAF 8.0e-07.

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

• ENIGMA BRCA2 Specification v1.2 defines BA1 as FAF >0.001 (0.1%) in gnomAD v2.1 non-cancer exome and/or gnomAD v3.1 non-cancer non-founder populations.
• gnomAD v2.1 reports 1/221,692 alleles, AF 4.51076e-06, with highest observed European non-Finnish AF 9.73539e-06 and zero homozygotes.
• gnomAD v4.1 reports 4/1,578,966 alleles, AF 2.53330e-06, European non-Finnish AF 3.43350e-06, grpmax FAF 8.0e-07, and zero homozygotes.

Sources

Reasoning

BS1 is not met. The ENIGMA BRCA2 v1.2 thresholds are FAF >0.0001 for BS1 Strong or >0.00002 and <=0.0001 for BS1 Supporting. Observed population frequencies are below the supporting threshold in both available gnomAD datasets, including the highest reported subpopulation AFs (v2.1 9.73539e-06; v4.1 3.43350e-06).

Rule

Filter allele frequency (FAF) is above 0.01% (FAF > 0.0001) for BS1 Strong, or above 0.002% (FAF > 0.00002) and less than or equal to 0.01% (FAF <= 0.0001) for BS1 Supporting, in specified non-cancer, non-founder gnomAD populations.

Evidence considered

• ENIGMA BRCA2 Specification v1.2 defines BS1 Strong as FAF >0.0001 and BS1 Supporting as FAF >0.00002 and <=0.0001 in specified non-cancer, non-founder gnomAD populations.
• gnomAD v2.1 reports AF 4.51076e-06 overall and 9.73539e-06 in the highest observed subpopulation, both below 0.00002.
• gnomAD v4.1 reports AF 2.53330e-06 overall, 3.43350e-06 in the highest observed subpopulation, and grpmax FAF 8.0e-07, all below 0.00002.

Sources

Reasoning

Not met under the governing ClinGen ENIGMA BRCA2 v1.2 specification. For missense/in-frame variants, BP4 is restricted to the VCEP-defined functional domains. p.(Lys1314Arg) is outside the PALB2-binding domain (aa 10-40) and DNA-binding domain (aa 2481-3186), so BP4 cannot be applied on the missense protein-impact path.

Rule

ClinGen ENIGMA BRCA2 v1.2 BP4: missense/in-frame variant within aa 10-40 or aa 2481-3186 with the VCEP-specified benign protein-impact conditions.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 BP4 rule restricts missense/in-frame BP4 to variants inside the PALB2-binding domain (aa 10-40) or DNA-binding domain (aa 2481-3186); its protein-prediction component is BayesDel no-AF <=0.18.
• The normalized consequence is NP_000050.3:p.(Lys1314Arg); residue 1314 is outside both VCEP-defined domains. The reported BayesDel no-AF score is -0.521046, but the required domain condition is not satisfied.

Sources

Reasoning

BP6 is not met because ClinVar has no exact-variant expert-panel assertion. The exact matched ClinVar record has zero expert-panel submissions; the non-expert likely benign assertion cannot trigger BP6.

Rule

Apply BP6 only for an exact-variant ClinVar expert-panel classification of Benign or Likely benign.

Evidence considered

• The exact ClinVar record for NM_000059.4:c.3941A>G (p.Lys1314Arg) reports zero expert-panel submissions. The reported non-expert likely benign submission was not used for BP6.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign assertion would be required for BP6.

Sources

Reasoning

Not met. The supporting BP7 rules are limited to silent variants in a VCEP-defined functional domain or qualifying intronic variants when BP4 is met, neither of which describes this missense variant. BP7_Strong (RNA) would require a well-established mRNA-only assay showing no damaging transcript effect; no such variant-specific assay is provided.

Rule

ClinGen ENIGMA BRCA2 v1.2 BP7: supporting only for specified silent/intronic contexts with BP4; strong RNA evidence requires a well-established mRNA-only assay showing no damaging effect.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 BP7 supporting rules apply to silent or qualifying intronic variants in conjunction with BP4, not to this missense substitution.
• Under the same specification, BP7_Strong (RNA) requires a well-established in vitro or in vivo mRNA-only assay showing no damaging transcript effect; the case bundle provides no such result for p.(Lys1314Arg).

Still missing

• No variant-specific well-established mRNA-only assay result is available to assess BP7_Strong (RNA).

Sources

Reasoning

The variant is a missense substitution, p.Lys1314Arg, and SpliceAI predicts no meaningful splice impact (maximum delta score 0.001). However, the reviewed exact-variant report describes c.3941A>G/p.Lys1314Arg as an unclassified variant observed in one family, not as a previously classified pathogenic or likely pathogenic same-amino-acid substitution. No qualifying pathogenic or likely pathogenic same-amino-acid comparator was identified, so PS1 cannot be applied.

Rule

For predicted missense substitutions, apply PS1 when a previously classified pathogenic or likely pathogenic variant has the same protein change, acts through protein change, and has no confirmed or predicted splice effect; strength depends on the comparator classification.

Evidence considered

• ENIGMA BRCA2 v1.2 applies PS1 to a predicted missense substitution only when a previously classified pathogenic variant has the same protein change and no confirmed or predicted splice effect; the likely pathogenic comparator is eligible for PS1 at Moderate strength.
• The case variant is NM_000059.4:c.3941A>G, NP_000050.3:p.(Lys1314Arg), and SpliceAI reports a maximum delta score of 0.001.
• PMID:16683254 reports c.3941A>G/p.Lys1314Arg in one family as an unclassified variant with an unknown effect category, not as a pathogenic or likely pathogenic comparator.

Still missing

• A previously classified pathogenic or likely pathogenic BRCA2 variant producing the same amino-acid substitution, with evidence that its effect is through the protein rather than splicing, is not available.

Sources

Reasoning

The ENIGMA BRCA2 VCEP framework applies PS3 to well-established functional studies measuring protein function, or mRNA and protein together, and directs reviewers to calibrated assay recommendations in Table 9. The curated Table 9 material supplied for this case does not contain NM_000059.4:c.3941A>G (p.Lys1314Arg). The only variant-specific publication identified reports detection of the variant in one family as an unclassified variant; it does not provide a validated protein-function assay or a damaging functional result. Therefore PS3 cannot be applied.

Rule

ENIGMA BRCA2 v1.2 PS3: apply for well-established in vitro or in vivo functional studies supporting a damaging effect; use protein-only or combined mRNA/protein assays and calibrated published assay recommendations in Table 9.

Evidence considered

• The ENIGMA BRCA2 specification states that PS3 requires well-established in vitro or in vivo studies supporting a damaging effect and, for protein-only or combined mRNA/protein assays, recommends calibrated published assays in Specification Table 9.
• The supplied ENIGMA Table 9 extraction lists calibrated assay rows but has no row for c.3941A>G or p.Lys1314Arg.
• van der Hout et al. reports c.3941A>G (also shown as c.4169A>G) p.Lys1314Arg in one family as an unclassified variant with effect category B, defined in the table as unknown; this is not a damaging functional assay result.

Still missing

• No calibrated, variant-specific protein-function or combined mRNA/protein assay result was identified.
• No assay control data or validated functional-effect calibration for p.Lys1314Arg was identified.

Sources

Reasoning

PS4 cannot be assigned. The applicable ENIGMA BRCA2 v1.2 specification requires a case-control comparison with p<=0.05, OR>=4, and lower confidence interval excluding 2.0. No such exact-variant analysis is supplied. PMID:16683254 documents p.Lys1314Arg in one family, which is not a case-control enrichment result.

Rule

ENIGMA BRCA2 v1.2 PS4: case-control evidence requires p<=0.05 and OR>=4 with the lower confidence interval excluding 2.0.

Evidence considered

• ENIGMA BRCA2 v1.2 applies PS4 only when prevalence in affected individuals is significantly increased over controls: p<=0.05, OR>=4, and lower confidence interval excludes 2.0.
• The supplied ENIGMA supplementary-table material was checked for the exact variant/protein change and provides no exact-variant posterior-probability or case-control result for c.3941A>G / p.Lys1314Arg.
• PMID:16683254 reports c.3941A>G / p.Lys1314Arg in one family, without affected-versus-control counts, an odds ratio, confidence interval, or p value.

Still missing

• An exact-variant case-control study reporting affected and control counts, OR, 95% confidence interval, and p value is required.

Sources

Reasoning

The ENIGMA BRCA2 VCEP specification makes PM3 applicable only when the variant under assessment is sufficiently rare and is co-occurrent with a pathogenic or likely pathogenic BRCA2 variant in a patient with a phenotype consistent with BRCA1/2-related Fanconi anemia, with strength assigned from Table 6 points. The available case evidence provides no documented second pathogenic/likely pathogenic BRCA2 variant, phase (trans/cis), affected-proband observations meeting the Fanconi anemia phenotype requirements, chromosome-breakage result, or PM3 point total. The variant was reported in one family as an unclassified variant, but that observation does not establish PM3.

Rule

ENIGMA BRCA2 VCEP v1.2 PM3: apply for a sufficiently rare variant co-occurrent with a pathogenic or likely pathogenic variant in the same gene in a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia; assign PM3 strength by the Table 6 point total (Supporting = 1 point, Moderate = 2 points, Strong = at least 4 points).

Evidence considered

• The ENIGMA BRCA2 VCEP specification states that PM3 requires a sufficiently rare variant co-occurrent with a pathogenic or likely pathogenic variant in the same gene, and requires a patient phenotype consistent with BRCA1/2-related Fanconi anemia; strength is assigned from Specifications Table 6 points.
• The case contains no documented pathogenic/likely pathogenic BRCA2 co-variant, phase information, chromosome-breakage result, qualifying Fanconi anemia clinical features, or PM3 point calculation.
• PMID:16683254 reports c.3941A>G (also listed as c.4169A>G) causing p.Lys1314Arg in one family and labels it unclassified/unknown; it does not report a pathogenic BRCA2 allele in trans or the Fanconi anemia phenotype required by the VCEP rule.

Still missing

• Identify a co-occurrent pathogenic or likely pathogenic BRCA2 variant in the same proband.
• Establish phase, specifically that the variants are in trans; parental or offspring testing is needed.
• Document the affected proband's Fanconi anemia phenotype, including chromosome-breakage testing and qualifying clinical features.
• Apply ENIGMA BRCA2 Specifications Table 6 to calculate PM3 points and assign strength.

Sources

Reasoning

PP1 is applicable under the ENIGMA BRCA1/2 VCEP specification, but the bundle does not provide a quantitative cosegregation likelihood ratio, affected-relative genotypes, phenotype details, number of informative meioses, or a documented segregation analysis. PMID:16683254 reports the variant in one family, but the available extraction gives no affected-relative testing or segregation result; therefore PP1 cannot be assigned at Supporting, Moderate, or Strong strength.

Rule

Apply PP1 only from quantitative cosegregation with disease: Supporting LR >=2.08:1, Moderate LR >=4.3:1, Strong LR >=18.7:1; LR >0.48 and <2.08 is not supporting evidence in either direction.

Evidence considered

• ENIGMA BRCA1 and BRCA2 Specification v1.2 requires quantitative cosegregation analysis for PP1 and assigns Supporting at LR >=2.08:1, Moderate at LR >=4.3:1, and Strong at LR >=18.7:1; LR >0.48 and <2.08 provides no supporting evidence in either direction.
• The extracted report for PMID:16683254 lists NM_000059.4:c.3941A>G (also reported as c.4169A>G) / p.Lys1314Arg once and labels the effect B, defined as unknown; it does not document segregation of the variant with disease in affected relatives.
• The report context is BRCA2 mutation screening by DGGE with sequence analysis of aberrant fragments; the variant was reported in one family, without a usable family-level segregation LR in the supplied evidence.

Still missing

• Quantitative cosegregation LR or equivalent CAL-Leiden/COOL analysis is missing.
• The number and disease status of informative relatives and meioses are missing.
• Genotypes and phenotype consistency for affected and unaffected relatives are missing.
• Phase, testing methodology, and any correction for phenocopies or alternative pathogenic variants are missing.

Sources

Reasoning

PP4 cannot be assigned. Under ENIGMA BRCA2 v1.2, breast cancer/family history is genetically heterogeneous and PP4 may only capture a combined multifactorial clinical-data LR toward pathogenicity. The exact-variant BRCA2 clinical-history LR table has no c.3941A>G row, and no alternative combined clinical LR is supplied.

Rule

ENIGMA BRCA2 v1.2 PP4: use only a combined multifactorial clinical-data LR toward pathogenicity; Supporting LR>=2.08, Moderate LR>=4.3, Strong LR>=18.7, Very Strong LR>=350.

Evidence considered

• ENIGMA BRCA2 v1.2 permits PP4 only for a combined LR from multifactorial clinical data because breast cancer is common and genetically heterogeneous; the supporting, moderate, strong, and very-strong thresholds are LR>=2.08, >=4.3, >=18.7, and >=350, respectively.
• The supplied Li et al. BRCA2 clinical-history LR spreadsheet was queried for exact Gene=BRCA2 and HGVS_Nucleotide=c.3941A>G; no matching row was present, so it provides no variant-specific clinical-history LR.

Still missing

• A reviewed combined multifactorial clinical LR for c.3941A>G is required: >=2.08 for Supporting, >=4.3 for Moderate, >=18.7 for Strong, or >=350 for Very Strong.

Sources

Reasoning

BS2 cannot be assessed from the case bundle. ENIGMA BRCA2 v1.2 applies BS2 using the absence of Fanconi anemia features and assigns strength from a Table 8 point total, but the bundle provides no eligible proband-level phenotype, chromosome-breakage, or clinical-feature data for this assessment.

Rule

Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype; assign strength using ENIGMA Specifications Table 8 and Appendix H point rules.

Evidence considered

• ENIGMA BRCA2 Specification v1.2 states that BS2 is applied in the absence of features of recessive disease, namely Fanconi anemia phenotype, with strength based on the Table 8 point total: Supporting 1 point, Moderate 2 points, and Strong >=4 points.
• The case evidence bundle contains no proband-level Fanconi anemia phenotype or chromosome-breakage information suitable for assigning BS2 points.

Still missing

• Eligible proband-level data needed for ENIGMA Table 8 BS2 point assignment, including Fanconi anemia phenotype assessment and relevant clinical or chromosome-breakage findings.

Sources

Reasoning

The ENIGMA BRCA2 VCEP framework applies BS3 to well-established functional studies showing no damaging effect on protein function, with calibrated assay recommendations in Table 9. The curated Table 9 material supplied for this case does not contain NM_000059.4:c.3941A>G (p.Lys1314Arg). The variant-specific publication reports the variant as unclassified and gives no benign functional assay result, assay controls, or validated calibration. The available evidence is therefore insufficient to apply BS3.

Rule

ENIGMA BRCA2 v1.2 BS3: apply for well-established in vitro or in vivo functional studies showing no damaging effect on protein function; use calibrated published assay recommendations in Table 9.

Evidence considered

• The ENIGMA BRCA2 specification states that BS3 requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function and directs reviewers to calibrated published assays in Specification Table 9.
• The supplied ENIGMA Table 9 extraction lists calibrated assay rows but has no row for c.3941A>G or p.Lys1314Arg.
• van der Hout et al. reports c.3941A>G (also shown as c.4169A>G) p.Lys1314Arg in one family as an unclassified variant with effect category B, defined in the table as unknown; this is not evidence of preserved protein function.

Still missing

• No calibrated, variant-specific assay demonstrating preserved protein function was identified.
• No validated benign assay controls or quantitative functional calibration for p.Lys1314Arg was identified.

Sources

Reasoning

BS4 is applicable under the ENIGMA BRCA1/2 VCEP specification, but the case bundle contains no quantitative cosegregation LR or documented non-segregation in affected family members. The report of the variant in one family does not establish lack of segregation. Therefore BS4 cannot be assigned at Supporting, Moderate, or Strong strength.

Rule

Apply BS4 only from quantitative lack of segregation in affected family members: Supporting LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1; LR >0.48 and <2.08 is not supporting evidence in either direction.

Evidence considered

• ENIGMA BRCA1 and BRCA2 Specification v1.2 requires quantitative cosegregation analysis for BS4 and assigns Supporting at LR <=0.48:1, Moderate at LR <=0.23:1, and Strong at LR <=0.05:1; LR >0.48 and <2.08 provides no supporting evidence in either direction.
• The extracted report for PMID:16683254 lists the variant in one family but provides no affected-relative genotype data or evidence that the variant fails to segregate with disease; its table label B is defined as unknown.
• Because no non-segregating affected individuals or quantitative benign-direction LR are documented, the available family observation cannot support BS4.

Still missing

• Quantitative cosegregation LR or equivalent CAL-Leiden/COOL analysis is missing.
• Affected relatives who lack the variant, their phenotype status, and the number of informative meioses are not reported.
• Potential phenocopies, alternative pathogenic variants, and testing quality/coverage are not reported.

Sources

Reasoning

BP5 cannot be assigned. Under ENIGMA BRCA2 v1.2, BP5 may only capture a combined multifactorial clinical-data LR against pathogenicity and is not applicable to co-observation with a pathogenic variant in another breast-ovarian cancer risk gene. The exact-variant BRCA2 clinical-history LR table has no c.3941A>G row, and no combined benign-direction LR is supplied.

Rule

ENIGMA BRCA2 v1.2 BP5: use only a combined multifactorial clinical-data LR against pathogenicity; Supporting LR<=0.48, Moderate LR<=0.23, Strong LR<=0.05, Very Strong LR<=0.00285. Do not apply for co-observation in another breast-ovarian cancer risk gene.

Evidence considered

• ENIGMA BRCA2 v1.2 permits BP5 only for a combined multifactorial clinical-data LR against pathogenicity; the supporting, moderate, strong, and very-strong thresholds are LR<=0.48, <=0.23, <=0.05, and <=0.00285, respectively.
• The supplied Li et al. BRCA2 clinical-history LR spreadsheet was queried for exact Gene=BRCA2 and HGVS_Nucleotide=c.3941A>G; no matching row was present, so it provides no variant-specific clinical-history LR.

Still missing

• A reviewed combined multifactorial clinical LR for c.3941A>G is required: <=0.48 for Supporting, <=0.23 for Moderate, <=0.05 for Strong, or <=0.00285 for Very Strong.

Sources