BRCA1 c.2597G>A, p.(Arg866His)

Reasoning

The variant is a missense substitution at BRCA1 residue 866. ENIGMA defines BP1_Strong for missense variants outside a clinically important functional domain when splicing is not predicted. Arg866 is outside the complete ENIGMA domain list of RING aa 2-101, coiled-coil aa 1391-1424, and BRCT repeats aa 1650-1857. SpliceAI max delta is 0.001, which is <=0.1, meeting the specification's no-predicted-splicing requirement. BP1_Strong is therefore met.

Rule

ENIGMA BRCA1/2 v1.2 BP1_Strong: missense or in-frame insertion, deletion, or delins variant outside a clinically important functional domain and no predicted splicing (SpliceAI <=0.1).

Evidence considered

• ENIGMA BP1_Strong applies to missense substitutions outside a clinically important functional domain when no splicing is predicted, defined by SpliceAI <=0.1.
• The ENIGMA complete BRCA1 clinically important domain list is RING aa 2-101, coiled-coil aa 1391-1424, and BRCT repeats aa 1650-1857; residue 866 is outside every listed domain.
• SpliceAI reports a maximum delta score of 0.001 for NM_007294.4:c.2597G>A, meeting the <=0.1 no-splicing threshold.

Sources

Reasoning

ENIGMA restricts BP5 to the combined clinical-data likelihood ratio against pathogenicity. The governing BP5 rules use the opposite direction from PP4: Supporting LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1, and Very Strong LR <=0.00285:1. The exact BRCA1 c.2597G>A clinical-history LR is 0.03014649625443013. For the BP5 Strong rule, the literal operator is <=: 0.03014649625443013 <= 0.05? Yes. The value does not meet the Very Strong threshold because 0.03014649625443013 <= 0.00285? No; therefore BP5 is met at Strong strength.

Rule

Use only combined likelihood ratio against pathogenicity from multifactorial clinical data; BP5 Strong requires LR <=0.05:1.

Evidence considered

• ENIGMA BP5 Strong rule literally states: "BP5_Strong - LR <=0.05:1"; BP5 Very Strong is LR <=0.00285:1.
• Li et al. clinical-history LR table exact match for BRCA1 c.2597G>A reports LOG(LR) = -3.501686573028564, N_Probands = 9, and LR = 0.03014649625443013.
• Explicit BP5 Strong comparison using its own operator: 0.03014649625443013 <= 0.05? Yes.
• Explicit BP5 Very Strong comparison: 0.03014649625443013 <= 0.00285? No; the strongest supported BP5 level is Strong.

Sources

Reasoning

Expert panel Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) classified as Benign.

Rule

Apply only when an exact-variant ClinVar expert-panel classification is Benign or Likely benign; Benign supports BP6 at Supporting strength.

Evidence considered

• ClinVar exact match NM_007294.4(BRCA1):c.2597G>A (p.Arg866His), variation 54613, reports expert-panel classification Benign by ENIGMA.
• The expert-panel classification is exact-variant evidence; laboratory submissions and aggregate ClinVar labels were excluded from this criterion.
• ClinVar expert panel classification

Sources

Reasoning

The variant is the missense substitution BRCA1 p.Arg866His. ENIGMA PS1 requires a previously classified pathogenic or likely pathogenic variant producing the same amino-acid change at the same codon, with no confirmed or predicted splicing effect. The available same-residue comparator p.Arg866Cys is assigned BS3 Strong/neutral in the ENIGMA functional tables, while p.Arg866Gln is reported as an unclassified variant rather than pathogenic or likely pathogenic; therefore no qualifying pathogenic same-codon comparator supports PS1. SpliceAI max delta is 0.001, satisfying the ENIGMA no-significant-splicing condition, but the required pathogenic comparator is absent.

Rule

ENIGMA BRCA1/2 v1.2 PS1: apply for predicted missense substitutions when a previously classified pathogenic variant is considered to act via protein change, with no confirmed or predicted effect on mRNA splicing (SpliceAI <=0.1); PS1 Moderate uses a previously classified likely pathogenic variant.

Evidence considered

• The ENIGMA BRCA1/2 specification applies PS1 to a missense variant when a previously classified pathogenic variant has the same protein change and is not acting through splicing; PS1 Moderate permits a previously classified likely pathogenic comparator.
• For the same residue, ENIGMA Table 9 assigns p.Arg866Cys (c.2596C>T) BS3 Strong and describes no functional impact, while the available clinical record for p.Arg866Gln (c.2716G>A) describes it as an unclassified variant rather than pathogenic or likely pathogenic.
• SpliceAI max delta is 0.001, below the ENIGMA no-significant-splicing cutoff of 0.1, so the missing pathogenic comparator—not splicing prediction—is decisive.

Sources

Reasoning

The ENIGMA BRCA1 v1.2 specification requires PS3 for calibrated functional studies showing a damaging protein effect, or for combined mRNA-and-protein assays; mRNA-only splicing evidence is directed to PVS1 (RNA), not PS3. For NM_007294.4:c.2597G>A (p.Arg866His), the ENIGMA supplementary functional/splicing tables record a minigene result of no aberration, with no calibrated damaging protein assay or Table 9 PS3 assignment identified. The relevant full-text paper extraction also did not establish a damaging result for this exact variant. Therefore, PS3 is not met.

Rule

ENIGMA BRCA1/2 v1.2 PS3: apply for well-established functional studies supporting a damaging effect measured via protein only or mRNA and protein combined; mRNA-only damaging evidence is applied as PVS1 (RNA).

Evidence considered

• ENIGMA BRCA1/2 specification v1.2 states that PS3 is used for well-established assays showing a damaging effect measured by protein alone or by mRNA and protein combined; damaging mRNA-only evidence is assigned through PVS1 (RNA), not PS3.
• The ENIGMA supplementary-table exact-match row for BRCA1 c.2597G>A (p.R866H) records a minigene assay with no aberration, assay category 'missense outside domain, minigene,' and PMID 18273839.
• ENIGMA Table 9 is the calibrated lookup for variant-specific PS3/BS3 assignments; its extracted exact-match result is for the nearby distinct c.2596C>T (p.Arg866Cys), not c.2597G>A.
• The extracted PMID:18273839 finding describes a BRCA1 exon 10-12 minigene splicing study and does not establish a damaging functional result for c.2597G>A/p.Arg866His.

Still missing

• No variant-specific calibrated protein-function assay or combined mRNA-and-protein assay supporting a damaging effect was identified.

Sources

Reasoning

The ENIGMA BRCA1/2 VCEP v1.2 permits PM2 Supporting only when the variant is absent from controls in an outbred population in gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer genomes, with regional average read depth at least 25. This variant is observed in both specified datasets—4/236516 alleles in gnomAD v2.1 non-cancer exomes and 2/147936 alleles in gnomAD v3.1 non-cancer genomes—so the required absence condition fails; read-depth review cannot change that conclusion.

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

• ENIGMA BRCA1/2 VCEP v1.2 specifies PM2 Supporting for absence from controls in gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer genomes, with average regional read depth >=25.
• The gnomAD v2.1 non-cancer exome record observes the variant at 4/236516 alleles (AF 1.6912175074836376e-05) with 0 homozygotes.
• The gnomAD v3.1 non-cancer genome record observes the variant at 2/147936 alleles (AF 1.3519359723123514e-05) with 0 homozygotes; therefore PM2's absence requirement is not satisfied.

Sources

Reasoning

The variant is missense (BRCA1 p.Arg866His), so PP3 is in scope. The governing ENIGMA BRCA1 v1.2 specification takes precedence over generic REVEL thresholds: its protein-impact PP3 rule requires a missense variant inside a potentially clinically important domain with BayesDel no-AF >=0.28, while its splice-impact path requires SpliceAI >=0.2. p.Arg866His is outside the specified RING, coiled-coil, and BRCT domains; BayesDel no-AF is 0.145553 and SpliceAI maximum delta is 0.001, so neither specified PP3 path is met. The ENIGMA supplementary splicing table records no aberration for this exact variant, consistent with the low SpliceAI score.

Rule

ENIGMA BRCA1 v1.2 PP3: missense protein-impact path requires an in-domain variant and BayesDel no-AF >=0.28; splice-impact path requires SpliceAI >=0.2.

Evidence considered

• The variant is NM_007294.4:c.2597G>A, BRCA1 p.(Arg866His), a missense substitution.
• ENIGMA BRCA1/2 specification v1.2 defines PP3 for missense protein impact inside a potentially clinically important domain with BayesDel no-AF >=0.28, or for predicted splicing with SpliceAI >=0.2.
• The specified BRCA1 clinically important domains are RING amino acids 2-101, coiled-coil amino acids 1391-1424, and BRCT repeats amino acids 1650-1857; Arg866 is outside these domains.
• The BayesDel no-AF score is 0.145553 and the SpliceAI maximum delta score is 0.001, both below the ENIGMA PP3 thresholds.
• ENIGMA Supplementary Table 2 records c.2597G>A/p.R866H as a missense variant with no aberration in the cited minigene splicing assessment, and Supplementary Table 3 links that result to PMID:18273839.

Sources

Reasoning

ENIGMA restricts PP4 to the combined clinical-data likelihood ratio. Its supporting threshold is LR >=2.08:1, moderate is LR >=4.3:1, strong is LR >=18.7:1, and very strong is LR >=350:1. For BRCA1 c.2597G>A, the Li et al. clinical-history table reports LR = 0.03014649625443013; the governing PP4 supporting comparison is 0.03014649625443013 >= 2.08? No, so PP4 is not met.

Rule

Use only combined likelihood ratio toward pathogenicity from multifactorial clinical data; PP4 Supporting requires LR >=2.08:1.

Evidence considered

• ENIGMA PP4 supporting rule literally states: "PP4 - LR >=2.08:1"; higher-strength thresholds are LR >=4.3:1, >=18.7:1, and >=350:1.
• Li et al. clinical-history LR table exact match for BRCA1 c.2597G>A reports LOG(LR) = -3.501686573028564, N_Probands = 9, and LR = 0.03014649625443013.
• Explicit comparison using the PP4-specific operator: 0.03014649625443013 >= 2.08? No.

Sources

Reasoning

The exact ClinVar variant record has an expert-panel classification of Benign by ENIGMA, not Pathogenic or Likely pathogenic. Under the requested exact-variant expert-panel rule, PP5 is therefore not met; ordinary laboratory submissions and aggregate labels were not used.

Rule

Apply only when an exact-variant ClinVar expert-panel classification is Pathogenic or Likely pathogenic; otherwise do not apply PP5.

Evidence considered

• ClinVar exact match NM_007294.4(BRCA1):c.2597G>A (p.Arg866His), variation 54613, reports expert-panel classification Benign by ENIGMA.
• The expert-panel classification is exact-variant evidence; laboratory submissions and aggregate ClinVar labels were excluded from this criterion.

Sources

Reasoning

The ENIGMA BRCA1/2 VCEP v1.2 requires BA1 when non-founder population filter allele frequency is above 0.001 in gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer genomes. The observed gnomAD v2.1 non-cancer exome group-max FAF is 3.24e-06, and the gnomAD v3.1 non-cancer genome group-max FAF is 0; both are below 0.001, so BA1 is not met.

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 BRCA1/2 VCEP v1.2 specifies BA1 for non-founder gnomAD v2.1 non-cancer exome and/or gnomAD v3.1 non-cancer genome FAF >0.001.
• The gnomAD v2.1 non-cancer exome record reports AF 1.6912175074836376e-05 (4/236516 alleles), with group-max FAF 3.24e-06 and zero homozygotes.
• The gnomAD v3.1 non-cancer genome record reports AF 1.3519359723123514e-05 (2/147936 alleles), with group-max FAF 0 and zero homozygotes.

Sources

Reasoning

The ENIGMA BRCA1/2 VCEP v1.2 specifies BS1 Strong for non-founder non-cancer FAF >0.0001 and BS1 Supporting for FAF >0.00002 through 0.0001 in gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer genomes. The highest applicable group-max FAF is 3.24e-06 in gnomAD v2.1 non-cancer exomes, while gnomAD v3.1 non-cancer genomes reports 0; neither reaches 0.00002, so BS1 is not met at any strength.

Rule

Filter allele frequency (FAF) is above 0.01% (FAF > 0.0001) for BS1 Strong, or above 0.002% and less than or equal to 0.01% (FAF > 0.00002 and FAF <= 0.0001) for BS1 Supporting, in gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer genomes, non-founder populations.

Evidence considered

• ENIGMA BRCA1/2 VCEP v1.2 specifies BS1 Strong for FAF >0.0001 and BS1 Supporting for FAF >0.00002 through 0.0001 using non-founder gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer genomes.
• The gnomAD v2.1 non-cancer exome record reports group-max FAF 3.24e-06, with AF 1.6912175074836376e-05 from 4 of 236516 alleles.
• The gnomAD v3.1 non-cancer genome record reports group-max FAF 0, with AF 1.3519359723123514e-05 from 2 of 147936 alleles.

Sources

Reasoning

The ENIGMA BRCA1/2 VCEP v1.2 applies BS2 in the absence of features of recessive disease, namely Fanconi Anemia, and assigns strength from counts of qualifying unaffected homozygotes. The available population records show zero homozygotes for c.2597G>A in gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer genomes, so no BS2 points are established and BS2 is not met.

Rule

Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype; BS2 Supporting = 1 point, BS2 Moderate = 2 points, and BS2 Strong = >=4 points.

Evidence considered

• ENIGMA BRCA1/2 VCEP v1.2 defines BS2 for absence of Fanconi Anemia features and requires qualifying unaffected homozygote points; BS2 Supporting requires 1 point, Moderate 2 points, and Strong at least 4 points.
• The gnomAD v2.1 non-cancer exome record reports 0 homozygotes for c.2597G>A.
• The gnomAD v3.1 non-cancer genome record reports 0 homozygotes for c.2597G>A.

Sources

Reasoning

The variant is missense, so BP4 is in scope. Under the governing ENIGMA BRCA1 v1.2 specification, the missense BP4 rule applies to variants inside a potentially clinically important domain and requires BayesDel no-AF <=0.15 together with SpliceAI <=0.1. Although the scores meet those numerical cutoffs (0.145553 and 0.001), p.Arg866His is outside the specified RING, coiled-coil, and BRCT domains; therefore the VCEP BP4 rule is not satisfied. The low SpliceAI score and the exact-variant ENIGMA no-aberration splicing record do not overcome the domain requirement.

Rule

ENIGMA BRCA1 v1.2 BP4: in-domain missense variant with BayesDel no-AF <=0.15 and SpliceAI <=0.1.

Evidence considered

• The variant is NM_007294.4:c.2597G>A, BRCA1 p.(Arg866His), a missense substitution.
• ENIGMA BRCA1/2 specification v1.2 defines BP4 for an in-domain missense variant with BayesDel no-AF <=0.15 and SpliceAI <=0.1.
• Arg866 lies outside the ENIGMA-defined BRCA1 clinically important domains: RING amino acids 2-101, coiled-coil amino acids 1391-1424, and BRCT repeats amino acids 1650-1857.
• The BayesDel no-AF score is 0.145553 and the SpliceAI maximum delta score is 0.001; the numerical cutoffs are met, but the required domain condition is not.
• ENIGMA Supplementary Table 2 records no aberration for the exact c.2597G>A/p.R866H minigene assessment, with Supplementary Table 3 citing PMID:18273839.

Sources

Reasoning

The ENIGMA BRCA1 specification requires a case-control study with p-value <=0.05, OR >=4, and a lower confidence interval excluding 2.0. The available evidence reports the variant in multiple affected individuals but provides no case-control OR, confidence interval, or p-value, and the specification states that proband counting should not be used; therefore PS4 cannot be assessed from the available evidence.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Case-control studies; p-value <=0.05 and OR >=4 (lower confidence interval excludes 2.0).

Evidence considered

• ENIGMA BRCA1/2 specification v1.2: "Case-control studies; p-value <=0.05 and OR >=4 (lower confidence interval excludes 2.0)."
• The case evidence describes multiple affected individuals but does not provide the required case-control statistics; ENIGMA specifically says not to use proband counting as originally described.

Still missing

• A suitable ethnicity- and country-matched case-control dataset with variant counts, OR, confidence interval, and p-value is required.

Sources

Reasoning

The governing ENIGMA BRCA1/2 VCEP specification restricts PM3 to a sufficiently rare variant co-occurring with a pathogenic or likely pathogenic BRCA1 variant in a patient with a phenotype consistent with BRCA1-related Fanconi anemia. The specification assigns 2 points when the variants are shown or reliably inferred to be in trans (or the variant is homozygous), and 1 point when phase is unknown; final strength is Supporting at 1 point, Moderate at 2 points, and Strong at 4 or more points. The available case evidence provides no affected-proband observation with a second pathogenic or likely pathogenic BRCA1 variant, no phase information, and no Fanconi-anemia phenotype or chromosome-breakage result, so a PM3 point value and strength cannot be assigned.

Rule

ENIGMA BRCA1/2 VCEP v1.2 PM3: apply only for a sufficiently rare variant co-occurring 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 points by trans/homozygous versus unknown phase and sum across unrelated probands.

Evidence considered

• ENIGMA BRCA1/2 VCEP v1.2 states that PM3 is for a rare variant co-occurring with a pathogenic or likely pathogenic variant in the same gene in a patient with a phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia.
• ENIGMA Table 6 assigns 2 points for a pathogenic or likely pathogenic variant in trans or a homozygote and 1 point when phase is unknown; final PM3 strength is Supporting at 1 point, Moderate at 2 points, and Strong at 4 or more points.
• The case evidence contains no documented affected-proband co-occurrence with a second pathogenic or likely pathogenic BRCA1 variant, phase result, or Fanconi-anemia phenotype/chromosome-breakage result for NM_007294.4:c.2597G>A.

Still missing

• A documented affected proband with a second pathogenic or likely pathogenic BRCA1 variant is needed.
• Phase information establishing trans status, or a valid VCEP-supported inference of trans status, is needed.
• Fanconi-anemia phenotype evidence, including chromosome-breakage testing and qualifying clinical features, is needed.

Sources

Reasoning

The ENIGMA specification permits PP1 only for co-segregation with disease in multiple affected family members measured by a quantitative co-segregation method, with Supporting evidence requiring LR >=2.08:1, Moderate LR >=4.3:1, Strong LR >=18.7:1, and Very Strong LR >=350:1. The available case evidence provides no family members, genotype-phenotype segregation observations, meioses, or quantitative co-segregation likelihood ratio for this variant, so PP1 cannot be assessed.

Rule

Apply PP1 by quantitative co-segregation analysis in multiple affected family members: Supporting LR >=2.08:1; Moderate LR >=4.3:1; Strong LR >=18.7:1; Very Strong LR >=350:1.

Evidence considered

• The ENIGMA specification requires co-segregation in multiple affected family members assessed quantitatively for PP1.
• The ENIGMA PP1 thresholds are Supporting LR >=2.08:1, Moderate LR >=4.3:1, Strong LR >=18.7:1, and Very Strong LR >=350:1.
• No proband-relative genotype, phenotype, meiosis, or quantitative segregation result is available in the reviewed case evidence.

Still missing

• Affected and unaffected relatives with verified genotypes and phenotypes are not provided.
• No informative meioses or quantitative co-segregation likelihood ratio is provided.

Sources

Reasoning

The ENIGMA specification permits BS4 only for lack of segregation in affected family members measured by a quantitative co-segregation method, with Supporting evidence requiring LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1, and Very Strong LR <=0.00285:1. The available case evidence provides no affected relatives who lack the variant, non-segregation observations, informative meioses, or quantitative likelihood ratio, so BS4 cannot be assessed.

Rule

Apply BS4 by quantitative lack of segregation in affected family members: Supporting LR <=0.48:1; Moderate LR <=0.23:1; Strong LR <=0.05:1; Very Strong LR <=0.00285:1.

Evidence considered

• The ENIGMA specification requires quantitative lack of segregation in affected family members for BS4.
• The ENIGMA BS4 thresholds are Supporting LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1, and Very Strong LR <=0.00285:1.
• No affected-relative genotype, phenotype, non-segregation observation, meiosis, or quantitative segregation result is available in the reviewed case evidence.

Still missing

• Affected relatives without the variant and their validated phenotypes are not provided.
• No informative non-segregating meioses or quantitative co-segregation likelihood ratio is provided.

Sources