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This variantScenario
BenignPathogenic
Likely benign.A likely benign classification under the recorded framework.
In plain language
Gene-level background from the recorded interpretation.
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About BRCA2
BRCA2 encodes a DNA repair protein that maintains genome stability by repairing double-strand breaks through homologous recombination and by protecting DNA replication forks. It acts as a tumor suppressor, and inherited loss-of-function changes cause hereditary breast and ovarian cancer syndrome, with elevated lifetime risks of breast, ovarian, prostate, and pancreatic cancers; biallelic changes cause Fanconi anemia complementation group D1. Reduced or altered BRCA2 activity is implicated in multiple tumor types, and PARP inhibitors are an approved treatment for BRCA2-associated ovarian and breast cancers.
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01The reasoning
How it was classified
The recorded framework is ClinGen BRCA2 VCEP v1.2. The criteria and strengths shown below come from the saved assessment; this page does not recalculate the classification.
Recorded applied criteria
The applied criteria and their strengths are shown as assessed under the stated framework.
OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. BRCA2, a tumor suppressor involved in the DNA damage response, is mutated in various cancer types.
ACMG/AMP criteria under ClinGen BRCA2 VCEP v1.2, with the data and sources behind each one.
28 criteria with recorded statuses: 2 met · 9 not met · 6 not assessed · 11 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
PVS1: not_applicable. c.4183G>T is a missense substitution (p.Ala1395Ser) in exon 11 of the canonical BRCA2 transcript (NM_000059.4); it is not a null variant (no nonsense, frameshift, canonical +/-1,2 splice-site, initiation codon, or single/multi-exon deletion) and SpliceAI predicts no splicing impact (max delta 0.00). No mRNA assay evidence exists for this variant, so neither PVS1 nor PVS1(RNA) applies.
PM4: not_applicable. Declared Not Applicable by ENIGMA BRCA1/BRCA2 Specification v1.2; additionally, a single-nucleotide missense substitution does not cause protein length change.
BP3: not_applicable. Declared Not Applicable by ENIGMA BRCA1/BRCA2 Specification v1.2; additionally, the variant is not an in-frame indel in a repetitive region.
Governing framework: ClinGen ENIGMA BRCA1 and BRCA2 Expert Panel Specifications v1.2 (CSPEC doc 1535438831) - applicable VCEP found, so generic ACMG/AMP is not used.
PM1 and PP2 are declared Not Applicable by ENIGMA v1.2 and are recorded as not_applicable.
PM5 in ENIGMA v1.2 is PTC-specific (PM5_PTC) and does not apply to this missense variant; the classic same-residue missense PM5 is not used by this VCEP and no same-residue comparator was found (0 candidates).
PS1 is not met because no previously classified pathogenic or likely pathogenic variant producing the same amino acid change (p.Ala1395Ser) exists in ClinVar variation 96805 submissions, the pm5_candidates collection, or the retrieved literature; SpliceAI gate (0.00) is satisfied but the P/LP comparator is absent.
BP1 is met at Strong (BP1_Strong): p.Ala1395 is outside the BRCA2 clinically important functional domains (PALB2 binding aa 10-40; DNA binding aa 2481-3186) and SpliceAI predicts no splicing impact (max delta 0.00 <= 0.1). This is the only codon/residue/domain-group criterion met.
No criterion in this group was inferred from another source's final classification; ClinVar classifications were used only to establish the absence of same-residue P/LP comparators.
Governing framework: ClinGen ENIGMA BRCA1/2 Expert Panel v1.2 (CSPEC present, version 1.2). PS2/PM6 are not defined in ENIGMA v1.2, so generic ACMG/AMP (PMID:25741868) applies for those; PP1/BS4 follow ENIGMA v1.2 quantitative co-segregation LR rules (Appendix I).
BRCA2 c.4183G>T (p.Ala1395Ser) is a ClinVar VUS (13 germline submissions; one 2023 'likely benign' submission). No criterion in this group was inferred from any of those classifications.
PS2/PM6: the case bundle contains no parental testing results, no de novo occurrence documentation, and no proband phenotype or pedigree data - both criteria not_assessed (insufficient evidence).
PP1/BS4: no segregation or non-segregation data and no co-segregation LR for this variant. BRCA2 c.4183G>T is absent from ENIGMA Parsons 2019 SuppT1 (a BRCA1 c.4183G>T row exists for the same HGVS protein, legacy 4411G>T) and from the ST7 reference set; SuppT2 shows zero co-segregation families - both criteria not_assessed (insufficient evidence).
All four criteria remain not_assessed pending collection of parental/de novo testing data and family segregation data.
PS3: not_met. No well-established functional study supportive of a damaging effect exists for BRCA2 c.4183G>T (p.Ala1395Ser): ENIGMA Specification Table 9 (the calibrated PS3/BS3 variant list, 4,734 rows) has no entry; ENIGMA Supplementary Tables ST4/ST9/ST6 have no entry; the ENIGMA multifactorial resource (PMID:31131967) lists the variant with clinical LR data but empty functional-assay fields; OncoKB reports no variant-specific functional evidence; no downloaded publication mentions the variant. Absence of evidence is definitive, so PS3 is not met rather than not assessed.
BS3: not_met. No well-established functional study showing no damaging effect exists for this variant, for the same reasons as PS3. Notably, PMID:31911673 (coldspots), cited by the single ClinVar 'Likely benign' submitter, does not mention c.4183G>T / p.Ala1395Ser in its full text and therefore cannot support BS3; non-functional evidence (gnomAD rarity, REVEL/BayesDel scores) belongs to other criteria (BS1/BA1/BP4) and does not satisfy BS3.
Governing framework: ClinGen ENIGMA BRCA1 and BRCA2 Expert Panel Specifications v1.2 (CSPEC doc 1535438831); generic ACMG/AMP was not needed.
Variant: BRCA2 NM_000059.4:c.4183G>T (p.Ala1395Ser), missense, exon 11. PP4/BP5 and PS4 were adjudicated exclusively with ENIGMA quantitative multifactorial/case-control rules.
PP4 Supporting is met: ENIGMA combined multifactorial likelihood LR = 2.225 (Parsons 2019, PMID:31131967 SuppT1) >= 2.08 supporting threshold; the Li 2020 clinical-history LR (0.793, 1 proband) is in the neutral zone and was not double-counted.
BP5 not met: both the combined LR (2.225) and clinical-history LR (0.793) exceed the BP5 supporting threshold of <=0.48.
PS4 not assessed: no case-control study (p<=0.05, OR>=4, lower CI >2) exists for this variant in the evidence bundle; ENIGMA disallows proband counting; ClinVar-cited Brazilian cohort papers could not be verified to report this variant.
PP5 and BP6 not applicable: ENIGMA v1.2 removes both criteria, and no ClinVar expert-panel (3-star) classification exists for this variant (0 expert panel submissions); the aggregate 1-star ClinVar label and the single 'Likely benign' laboratory curation cannot trigger either criterion.
The governing framework is the ClinGen ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (CSPEC, precedence rank 1), so its allelic-criterion definitions control; generic ACMG/AMP is not substituted where the specification rules.
ENIGMA v1.2 defines PM3 only for patients with a BRCA1-/BRCA2-related Fanconi Anemia phenotype who carry co-occurrent variants in the same gene. This case is a dominant HBOC/BRCA2-cancer-predisposition context (all 13 ClinVar submissions list cancer/hereditary-cancer or not-provided conditions; no FA condition anywhere), and no second BRCA2 variant is documented. PM3 is therefore not applicable, and no points can be assigned.
ENIGMA v1.2 explicitly marks BP2 as Not Applicable for BRCA1/BRCA2. Additionally, no phase or co-occurrence data exists in the case bundle to which a generic BP2 could attach. BP2 is therefore not applicable.
Neither criterion contributes pathogenic or benign weight to the final classification. Any future re-evaluation of PM3 would require documented FA phenotype features (chromosome breakage and/or FA clinical features per ENIGMA Table 6) plus identification of a co-occurring second BRCA2 variant with phase information.
No full-text publication in the case packet mentions c.4183G>T / p.Ala1395Ser (targeted grep across all downloaded full texts), so no publication-level allelic evidence (trans/cis observations, FA probands, compound heterozygotes) could be cited.
Governing framework: ClinGen ENIGMA BRCA1 and BRCA2 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BRCA2 Version 1.2 (CSPEC doc 1535438831, registry key 'cspec'). An applicable gene-specific VCEP specification exists, so generic ACMG/AMP was not used.
Variant: NM_000059.4:c.4183G>T, p.(Ala1395Ser) - missense substitution in BRCA2 exon 11 (codon 1395), located outside the (potentially) clinically important functional domains defined by ENIGMA v1.2 (PALB2-binding aa 10-40; DNA-binding aa 2481-3186).
PP3 not met: SpliceAI max delta = 0.00 < 0.2 (splice path, applicable to missense irrespective of location), and the BayesDel no-AF protein-change path (>= 0.30) both fails the domain-location gate (variant outside domains) and the score threshold (-0.321456).
BP4 not met: SpliceAI (0.00 <= 0.1) and BayesDel no-AF (-0.321456 <= 0.18) satisfy the no-predicted-impact components, but the ENIGMA v1.2 BP4 missense rule additionally requires location inside a clinically important functional domain; p.Ala1395Ser is outside the defined domains. ENIGMA Figure 1A routes the benign in-silico signal to BP1 (outside-domain, no splice impact) rather than BP4.
BP7 not applicable: ENIGMA v1.2 BP7 supporting is restricted to silent/intronic variants; this is a missense. BP7_Strong (RNA) would require mRNA-only functional assay evidence, which is absent for this variant.
This group contributes no met criteria: PP3 not_met, BP4 not_met, BP7 not_applicable.
VCEP spreadsheets (Specifications Table 9, Supplementary Table ST4, HUMU-40-1557-s001/Parsons 2019) contain no pre-assigned PP3/BP4/BP7 code for c.4183G>T and no variant-level splice or functional assay data.
BA1 not met: gnomAD v2.1 absent; gnomAD v4.1 joint grpmax FAF 2.8e-07 (0.000028%) and total AF 1.24747e-06 are far below the ENIGMA v1.2 BA1 threshold of FAF > 0.1%.
BS1 not met: observed FAF (grpmax 2.8e-07) is below both the BS1 Strong threshold (FAF > 0.01%) and the BS1 Supporting threshold (FAF > 0.002%).
BS2 not assessed: ENIGMA v1.2 BS2 is a Fanconi Anemia phenotype points criterion (Specifications Table 8), not a frequency criterion; no proband phenotype data exist in the case bundle (human review required).
PM2 not met: variant is present in gnomAD v4.1 in the non-founder European (non-Finnish) population (2 alleles, AF 1.70e-06), so strict absence from outbred controls - required for ENIGMA PM2_Supporting - is not demonstrated; v3.1 not separately queried and read depth >= 25 not verified (human review flagged).
No population-frequency or BS2-relevant threshold is met; the population group contributes no benign stand-alone/strong/supporting evidence for this variant.
No criteria match.
Met · 2
PP4SupportingMet (Supporting): ENIGMA combined multifactorial likelihood LR 2.225 for this exact variant meets the >=2.08 threshold.
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Reasoning
Under ENIGMA v1.2, PP4 is used ONLY to capture combined LR towards pathogenicity based on multifactorial likelihood clinical data, with PP4 Supporting at LR >=2.08 (neutral zone >0.48 and <2.08). The ENIGMA multifactorial likelihood resource, Parsons et al. 2019 (PMID:31131967) Supplementary Table 1, reports for the exact variant BRCA2 c.4183G>T (p.Ala1395Ser): pathology LR 1.07, co-occurrence LR 1.0246, family-history LR 2.0295, case-control LR (not available), Combined LR (Odds for Causality) = 2.225, prior probability of pathogenicity 0.02, posterior probability 4.34%, IARC class 'Uncertain'. Combined LR 2.225 >= 2.08, so PP4 is met at Supporting strength per the VCEP threshold. The clinical-history-only LR from Li et al. 2020 (PMID:31853058), 0.793 (1 proband), lies in the ENIGMA neutral zone and was not applied; it is a family/personal-history-type component already subsumed in the combined multifactorial evidence direction and was therefore not double-counted. Determination is borderline (combined LR only ~7% above the supporting threshold) and rests on a single ENIGMA quantitative source, hence medium confidence.
Rule
ENIGMA BRCA1/BRCA2 v1.2 PP4: combined LR towards pathogenicity from multifactorial likelihood clinical data; Supporting LR >=2.08, Moderate >=4.3, Strong >=18.7, Very Strong >=350; combined LR >0.48 and <2.08 is neutral and PP4 does not apply.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (CSPEC 1535438831) PP4 rule: 'Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data. PP4 - LR >=2.08' (Supporting); neutral zone 'Combined LR >0.48 and <2.08 doesn't provide supporting evidence in either direction (PP4 and BP5 not applicable)'.
Parsons et al. 2019 (PMID:31131967) Supplementary Table 1 (HUMU-40-1557-s001.xlsx, sheet 1): BRCA2 c.4183G>T, p.Ala1395Ser - pathology LR 1.07, co-occurrence LR 1.0246153856, family history LR 2.0294847967, Combined LR (Odds for Causality) 2.2250022419453646, prior 0.02, posterior 4.3435864217950808E-2 (IARC 'Uncertain'). Combined LR >=2.08 -> PP4 Supporting.
Li et al. 2020 (PMID:31853058) clinical-history LR table (vcep_pmid_31853058_brca2_clinical_history_lr): c.4183G>T LR = 0.7929228517802025 (N=1 proband) - neutral zone, component not applied to avoid double counting family/personal history already integrated in the combined LR.
Still missing
Combined LR 2.225 exceeds the PP4 supporting threshold (2.08) by only ~7%; a single ENIGMA multifactorial source underlies this value.
The clinical-history component (Li 2020, PMID:31853058) is based on 1 proband and is in the neutral zone (LR 0.793); additional probands or updated ENIGMA multifactorial data would increase confidence.
No segregation LR component was available for this variant in the Parsons table (cell blank).
BP1StrongMet (Strong): missense p.Ala1395Ser lies outside BRCA2's functional domains with SpliceAI max delta 0.00.
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Reasoning
ENIGMA BRCA1/BRCA2 VCEP v1.2 BP1_Strong applies to 'silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI <= 0.1)'. Clinically important functional domains for BRCA2 are defined as the PALB2 binding domain aa 10-40 and the DNA binding domain aa 2481-3186. p.Ala1395 (residue 1395) lies outside both domains, and SpliceAI reports a max delta score of 0.00 (<= 0.1), i.e., no predicted splicing impact. Both conditions of the BP1_Strong rule are satisfied for this missense variant. Supporting context (not required for the rule): Dines et al. 2020 (PMID:31911673) define BRCA2 exons 10-11 as a missense 'coldspot' with zero pathogenic/likely pathogenic missense variants (odds <0.01, 95% CI 0.0-0.01); c.4183G>T is located in BRCA2 exon 11, though the paper does not mention this exact variant. BP1 is therefore met at Strong strength under the governing VCEP.
Rule
ENIGMA BRCA1/BRCA2 VCEP v1.2 BP1_Strong: 'Apply BP1_Strong for silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI <= 0.1).' Clinically important functional domains: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186 (Specifications Figure 1A, Appendix J).
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (cspec, doc 1535438831): BP1_Strong rule - missense variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI <= 0.1); domains defined as BRCA2 PALB2 binding domain aa 10-40 and BRCA2 DNA binding domain aa 2481-3186.
SpliceAI lookup (spliceai): max delta score = 0.00 <= 0.1 - no predicted splicing impact.
Variant normalization (prefetch.json / case_summary.json): p.Ala1395Ser; residue 1395 is not within aa 10-40 or aa 2481-3186.
PMID:31911673 (Dines et al. 2020, Genetics in Medicine): BRCA2 exons 10 and 11 constitute a coldspot with no pathogenic/likely pathogenic missense variants (0/34; odds <0.01, 95% CI 0.0-0.01) - region-level context only; the exact variant is not mentioned in this paper.
Still missing
The BP1_Strong rule is fully satisfied with the available data (domain location + SpliceAI <= 0.1); no additional gap for the rule itself. The coldspot paper (PMID:31911673) provides region-level (exon 11) support but does not mention the exact variant, so it is cited only as context, not as variant-level evidence.
PS1Not met: no pathogenic or likely pathogenic variant producing the same p.Ala1395Ser change exists (0 comparator candidates).
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Reasoning
ENIGMA BRCA1/BRCA2 VCEP v1.2 PS1 requires a previously classified pathogenic (PS1 Strong) or likely pathogenic (PS1_Moderate) variant producing the same amino acid change (or the same predicted splicing event) as the variant under assessment, with no confirmed or predicted splicing effect (SpliceAI <= 0.1). For NM_000059.4:c.4183G>T (p.Ala1395Ser), the SpliceAI gate is satisfied (max delta = 0.00), but no pathogenic or likely pathogenic comparator at residue p.Ala1395 was identified in any available source: ClinVar variation 96805 has 13 submissions all VUS except one 'Likely benign' curation (SCV003848748, UW), zero expert-panel (ENIGMA) submissions, and the pm5_candidates.json same-residue candidate collection found 0 candidates (total_candidates_seen = 0). No full-text paper in the case bundle mentions c.4183G>T or p.Ala1395Ser, so no literature comparator exists. Without a previously classified P/LP variant yielding the identical amino acid change, PS1 cannot be applied.
Rule
ENIGMA BRCA1/BRCA2 VCEP v1.2 PS1: 'Apply PS1, for predicted missense substitutions, where a previously classified pathogenic variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI <= 0.1))'; PS1_Moderate for a previously classified likely pathogenic variant. See Specifications Table 5 and Appendices E, J, K.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (cspec, doc 1535438831): PS1 rule - Strong for same amino acid change as a previously classified pathogenic variant acting via protein change with SpliceAI <= 0.1; Moderate if the reference variant is only likely pathogenic.
SpliceAI lookup (spliceai): max delta score = 0.00, satisfying the PS1 no-splicing gate.
ClinVar variation 96805 (clinvar): 13 submissions, 12 Uncertain significance and 1 Likely benign (SCV003848748); no pathogenic/likely pathogenic submission at this residue; 0 expert panel submissions.
pm5_candidates.json (pm5_candidates): total_candidates_seen = 0, same_residue_candidates = 0; no comparator variant at residue 1395 collected.
Still missing
No previously classified pathogenic or likely pathogenic variant at p.Ala1395 (any nucleotide change) was found in ClinVar variation 96805 submissions, the pm5_candidates artifact, or the retrieved literature. A curated ENIGMA VCEP database query (e.g., ENIGMA LOVD) for same-residue P/LP comparators would be the definitive check; if one exists, PS1 (Strong/Moderate) would need re-evaluation.
PS3Not met: no well-established functional studies supporting a damaging effect exist for this variant.
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Reasoning
PS3 (ENIGMA BRCA1/BRCA2 VCEP Specification v1.2) requires well-established in vitro or in vivo functional studies supportive of a damaging effect, applied for assays measuring effect via protein only or mRNA+protein combined, with per-variant code recommendations in ENIGMA Specification Table 9 (calibrated assays; Figure 1C / Appendix E). No such evidence exists for NM_000059.4:c.4183G>T (p.Ala1395Ser). (1) ENIGMA Specification Table 9 (the authoritative curated PS3/BS3 variant list, 4,734 rows, incl. 531 BRCA2 rows) contains no entry for c.4183G>T / p.Ala1395Ser / legacy 4411G>T, so no pre-assigned PS3 code or supporting calibrated-study PMID exists; the only codon-1395 row is a different variant (BRCA1 c.4184A>G p.Gln1395Arg) whose row itself records 'PS3 and BS3 not met'. (2) ENIGMA Supplementary Tables v1.2 ST4 (functional assay results, 4,304 rows), ST9 (BRCA2 functional assays) and ST6 (yeast functional studies) do not include the variant. (3) In the ENIGMA multifactorial resource (Parsons et al. 2019, PMID:31131967, SuppT1) the variant is present with clinical likelihood-ratio data (Combined LR 2.225, prior 0.02, posterior 0.043, IARC 'Uncertain') but its 'Functional Paper/s' and 'Functional Category Assigned' fields are empty, and SuppT4 (functional assay data) does not list the variant — i.e., the ENIGMA resource itself assigns no functional assay category. (4) OncoKB reports no variant-specific functional evidence ('Unknown Oncogenic Effect', not reviewed). (5) None of the downloaded full-text publications (PMID:31131967, 31911673, 31853058, 26467025, 25741868, 17508274, 19305347) mentions c.4183G>T / p.Ala1395Ser. (6) Two ClinVar submitters' assertions state 'to our knowledge, functional studies have not been reported for this variant' (no submitter classification was used). PS3 is therefore not met; there is no functional evidence, and consequently no conflicting functional results requiring the Figure 1C conflict-resolution clause.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 PS3: well-established in vitro or in vivo functional studies supportive of a damaging effect (protein-only or mRNA+protein assays), per-variant code recommendations from calibrated published assays in Specifications Table 9; Figure 1C and Appendix E. Not met when no calibrated functional assay data exist for the variant.
Evidence considered
ENIGMA BRCA1/BRCA2 Specification v1.2 PS3 rule (Table 1): 'Well-established in vitro or in vivo functional studies supportive of a damaging effect. Apply PS3 for assays measuring effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.'
ENIGMA Specification Table 9 (calibrated functional assay PS3/BS3 variant list, 4,734 rows incl. 531 BRCA2 rows): no row for BRCA2 c.4183G>T / p.Ala1395Ser / legacy 4411G>T; the sole codon-1395 row is BRCA1 c.4184A>G p.(Gln1395Arg), which records 'PS3 and BS3 not met'.
ENIGMA Supplementary Tables v1.2: ST4 'Fxn assay results' (4,304 rows) has no entry for this variant; ST9 'BRCA2 FA' (59 rows) and ST6 'Yeast Fxn Studies' have no entry.
Parsons et al. 2019 (PMID:31131967) SuppT1 row for BRCA2 c.4183G>T p.(Ala1395Ser): Combined LR 2.225, prior 0.02, posterior 0.0434, IARC class 'Uncertain'; 'Functional Paper/s' and 'Functional Category Assigned' columns empty; variant absent from SuppT4 functional assay table.
OncoKB (BRCA2 A1395S): no variant-specific functional evidence, 'Unknown Oncogenic Effect', not reviewed.
ClinVar variation 96805 submission audit: no ENIGMA expert-panel submission; All of Us (SCV005424424) and Color (SCV001736201) assertions state 'to our knowledge, functional studies have not been reported for this variant'; no submitter final classification used.
SpliceAI: max delta score 0.00 (no predicted splicing impact), so mRNA-only assay codes (PVS1(RNA)/BP7(RNA)) are not in play; PS3 would require a protein-function assay, none available.
Still missing
No calibrated functional assay data for this variant: ENIGMA Specification Table 9 has no entry and the ENIGMA multifactorial resource assigns no functional category, so a well-established protein-function assay (e.g., Mesman/Bouwman/Fernandes/Petitalot-style, or the forthcoming full BRCA2 saturation genome editing dataset) would be required before PS3 could be applied.
PM2Not met: present in gnomAD v4.1 (AF 1.7e-06), so the strict absence requirement of PM2 is not demonstrated.
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Reasoning
ENIGMA BRCA1/BRCA2 VCEP v1.2 PM2_Supporting requires the variant to be absent from controls in an outbred population, from gnomAD v2.1 (non-cancer, exome only subset) and gnomAD v3.1 (non-cancer), with average read depth >= 25 in the surrounding region (Appendix G). The variant is absent from gnomAD v2.1, but it IS observed in gnomAD v4.1: 2 alleles in the European (non-Finnish) non-founder population (AF 1.70022e-06; joint grpmax FAF 2.8e-07; 0 homozygotes; both alleles are genome-only, genome AC 2/152,112). Because the variant is present in an outbred, non-founder population in a current gnomAD release, the strict absence condition of PM2_Supporting is not demonstrated, and PM2 is not met. Human review is flagged because the ENIGMA-specified dataset (gnomAD v3.1) was not separately queried - the pipeline used v4.1, whose genome cohort overlaps v3.1 - and the read-depth >= 25 requirement could not be verified from the available data.
Rule
PM2 (Supporting): 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. ENIGMA BRCA1/BRCA2 VCEP v1.2.
Evidence considered
cspec: ENIGMA BRCA1/BRCA2 VCEP Specifications v1.2 (ClinGen CSPEC doc 1535438831), PM2 rule - absent from controls in an outbred population, from gnomAD v2.1 (non-cancer, exome only subset) and gnomAD v3.1 (non-cancer); region average read depth >= 25; Appendix G
gnomad_v2: absent from gnomAD v2.1 (non-cancer, exome subset)
gnomad_v4: present - total AF 1.24747e-06 (2/1,603,248 alleles), European (non-Finnish) AF 1.70022e-06 (2/1,176,318 alleles), 0 homozygotes, joint grpmax FAF 2.8e-07; observed alleles are genome-only (exome AC 0, genome AC 2/152,112)
gnomad_canada: absent from gnomAD-Canada v1.0 (HostSeq genomes)
Still missing
gnomAD v3.1 (non-cancer) absence was not directly verified; gnomAD v4.1 shows the variant present (2 alleles, NFE), which is evidence against the required strict absence.
Average read depth >= 25 across the region around the variant (ENIGMA PM2_Supporting requirement) could not be verified from the case bundle data.
PP3Not met: BayesDel no-AF -0.32 falls below the 0.30 threshold and SpliceAI max delta 0.00 below 0.2.
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Reasoning
Variant NM_000059.4:c.4183G>T (p.Ala1395Ser) is a missense substitution in BRCA2 exon 11 at codon 1395. Under the governing ClinGen ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, PP3 has two paths. (1) Protein-change path: requires the missense to lie inside a (potentially) clinically important functional domain (BRCA2 PALB2-binding domain aa 10-40; BRCA2 DNA-binding domain aa 2481-3186) AND BayesDel no-AF score >=0.30; p.Ala1395Ser is outside both defined domains, and BayesDel no-AF = -0.321456 is far below 0.30, so this path fails. (2) Splice path: applies to missense/in-frame variants irrespective of location when SpliceAI >=0.2; SpliceAI max delta score = 0.00 (DS_AG/DS_AL/DS_DG/DS_DL all 0.0) is below 0.2, so this path also fails. No other computational evidence is determinative: REVEL 0.099 is recorded but the ENIGMA v1.2 framework defines no REVEL threshold (generic SVI REVEL calibration is not applied where an applicable VCEP specification exists), and no VCEP lookup spreadsheet pre-assigns PP3 for this variant. PP3 is not met.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, PP3 (Pathogenic Supporting): 'Apply PP3 for missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel no-AF score ≥0.30)... Apply PP3 for predicted splicing (SpliceAI ≥0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of donor and acceptor 1,2 sites.' (potentially) clinically important functional domains: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure 1A and Appendix J.
Evidence considered
SpliceAI max delta score = 0.00 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.0, DS_DL 0.0), SpliceAI Lookup (source_registry key 'spliceai'). ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (ClinGen CSPEC doc 1535438831, source_registry key 'cspec') PP3 splice path requires SpliceAI >= 0.2 for missense variants irrespective of domain location; 0.00 < 0.2, so the splice path is not met.
BayesDel no-AF score = -0.321456, Local BayesDel predictor lookup (source_registry key 'bayesdel'). ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (source_registry key 'cspec') PP3 protein-change path requires BayesDel no-AF >= 0.30 AND location inside a (potentially) clinically important functional domain (BRCA2 PALB2-binding aa 10-40; BRCA2 DNA-binding aa 2481-3186). p.Ala1395Ser is outside both domains and the score is far below 0.30; the protein-change path is not met.
REVEL score = 0.099, Local REVEL predictor lookup (source_registry key 'revel'). Recorded for completeness only; not used to call PP3 because the governing ENIGMA v1.2 specification (source_registry key 'cspec') calibrates missense prediction on BayesDel no-AF, not REVEL.
BA1Not met: gnomAD FAF 2.8e-07 is far below the >0.1% BA1 threshold.
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Reasoning
ENIGMA BRCA1/BRCA2 VCEP v1.2 BA1 (Stand Alone) requires filter allele frequency (FAF) > 0.1% (FAF > 0.001) in gnomAD v2.1 (non-cancer, exome only) and/or gnomAD v3.1 (non-cancer), non-founder population(s). The variant is absent from gnomAD v2.1. In gnomAD v4.1 (queried in place of v3.1) the joint grpmax FAF is 2.8e-07 (0.000028%) and the total AF is 1.24747e-06 (2/1,603,248 alleles; 0 homozygotes), with the highest population AF in European (non-Finnish) at 1.70022e-06. These values are orders of magnitude below the 0.1% BA1 threshold, so BA1 is not met. The grpmax FAF is the appropriate gnomAD filter-allele-frequency metric per the ENIGMA Appendix G approach.
Rule
BA1 (Stand Alone): Filter allele frequency (FAF) > 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). ENIGMA BRCA1/BRCA2 VCEP v1.2.
gnomad_v2: absent from gnomAD v2.1 (non-cancer, exome subset)
gnomad_v4: present in gnomAD v4.1, total AF 1.24747e-06 (2/1,603,248 alleles, 0 homozygotes), joint grpmax FAF 2.8e-07, highest population AF European (non-Finnish) 1.70022e-06
gnomad_canada: absent from gnomAD-Canada v1.0 (HostSeq genomes)
Still missing
gnomAD v3.1 (non-cancer) was not directly queried; gnomAD v4.1 was used instead. The v4.1 grpmax FAF (2.8e-07) is so far below the 0.1% threshold that this version difference cannot change the conclusion.
BS1Not met: gnomAD FAF 2.8e-07 is below even the 0.002% BS1-Supporting threshold.
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Reasoning
ENIGMA BRCA1/BRCA2 VCEP v1.2 BS1 Strong requires FAF > 0.01% (FAF > 0.0001) and BS1 Supporting requires 0.002% < FAF <= 0.01% (FAF > 0.00002 and <= 0.0001) in gnomAD v2.1 (non-cancer, exome only) and/or gnomAD v3.1 (non-cancer), non-founder population(s). The variant is absent from gnomAD v2.1, and the gnomAD v4.1 joint grpmax FAF is 2.8e-07 (0.000028%) - below even the BS1 Supporting threshold of 0.002% (2e-05). The highest observed population frequency (European non-Finnish AF 1.70022e-06) is likewise below the Supporting threshold. BS1 is therefore not met at any strength.
gnomad_v2: absent from gnomAD v2.1 (non-cancer, exome subset)
gnomad_v4: joint grpmax FAF 2.8e-07; total AF 1.24747e-06 (2/1,603,248 alleles, 0 homozygotes); highest population AF European (non-Finnish) 1.70022e-06
gnomad_canada: absent from gnomAD-Canada v1.0 (HostSeq genomes)
Still missing
gnomAD v3.1 (non-cancer) was not directly queried; gnomAD v4.1 was used instead. The v4.1 grpmax FAF (2.8e-07) is below the BS1 Supporting threshold by two orders of magnitude, so this version difference cannot change the conclusion.
BS3Not met: no well-established functional assay demonstrates no damaging effect on protein function.
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Reasoning
BS3 (ENIGMA BRCA1/BRCA2 VCEP Specification v1.2) requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function (protein-only or mRNA+protein assays), with per-variant code recommendations in ENIGMA Specification Table 9 (Figure 1C / Appendix E). No such evidence exists for NM_000059.4:c.4183G>T (p.Ala1395Ser): (1) ENIGMA Specification Table 9 (4,734 rows incl. 531 BRCA2 rows) has no entry for this variant, so no pre-assigned BS3 code or supporting calibrated-study PMID exists; the only codon-1395 row is BRCA1 c.4184A>G p.Gln1395Arg, itself annotated 'PS3 and BS3 not met'. (2) ENIGMA Supplementary Tables v1.2 ST4/ST9/ST6 contain no entry for the variant. (3) The ENIGMA multifactorial resource (PMID:31131967) lists the variant in SuppT1 with clinical LR data but empty functional-assay fields and no SuppT4 entry, i.e., no functional category. (4) OncoKB has no variant-specific functional evidence. (5) No downloaded publication mentions the variant; in particular PMID:31911673 (Dines et al. 'coldspots'), cited by the single ClinVar 'Likely benign' submitter, does not name c.4183G>T / p.Ala1395Ser anywhere in its full text (0 occurrences), so it provides no variant-level coldspot/functional support for BS3. (6) Population frequency (gnomAD v4.1 AF 1.25e-06, FAF 2.8e-07) and in-silico scores (REVEL 0.099, BayesDel no-AF -0.32) are non-functional evidence that other criteria (BS1/BA1/BP4) address, not BS3. BS3 is therefore not met; absence of any assay means no well-established study demonstrates no damaging effect.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 BS3: well-established in vitro or in vivo functional studies showing no damaging effect on protein function (protein-only or mRNA+protein assays), per-variant code recommendations from calibrated published assays in Specifications Table 9; Figure 1C and Appendix E. Not met when no calibrated functional assay data exist for the variant.
Evidence considered
ENIGMA BRCA1/BRCA2 Specification v1.2 BS3 rule (Table 1): 'Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. Assay measures effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.'
ENIGMA Specification Table 9: no row for BRCA2 c.4183G>T / p.Ala1395Ser / legacy 4411G>T among 4,734 rows (531 BRCA2 rows); nearest codon-1395 row (BRCA1 c.4184A>G p.Gln1395Arg) is annotated 'PS3 and BS3 not met'.
ENIGMA Supplementary Tables v1.2 ST4/ST9/ST6: variant absent from all functional assay result tables.
Parsons et al. 2019 (PMID:31131967) SuppT1: variant present with multifactorial clinical LR data (Combined LR 2.225, posterior 0.043, IARC 'Uncertain') but no functional assay category; SuppT4 functional assay table does not include the variant.
PMID:31911673 (Dines et al. 2020 coldspots): full text screened; 0 occurrences of c.4183G>T / p.Ala1395Ser / A1395S / '1395'; provides no variant-level BS3 (coldspot/functional) evidence.
OncoKB (BRCA2 A1395S): no variant-specific functional evidence, 'Unknown Oncogenic Effect', not reviewed.
ClinVar variation 96805: 13 submissions, no ENIGMA expert-panel submission; two submitters state 'to our knowledge, functional studies have not been reported for this variant'; one 'Likely benign' submission (SCV003848748, UW, curation method, weak trust tier) cites PMID:31911673, which does not mention this variant; no submitter classification used as evidence.
SpliceAI: max delta score 0.00 (no splicing impact); BS3 via protein-function assay cannot be met without assay data.
Still missing
No calibrated functional assay demonstrating no damaging effect exists for this variant (ENIGMA Specification Table 9 no entry; ENIGMA multifactorial resource functional fields empty; no functional paper mentions the variant). A well-established neutral functional assay result (protein-only or mRNA+protein) would be required to revisit BS3.
A future complete BRCA2 saturation genome editing release (Dace & Findlay interim sheet in Table 9 currently covers only early-exon positions, not c.4183) could provide this variant's function score; re-review BS3 if it becomes available.
Under the governing ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, the BP4 (Supporting) rule for missense variants requires the variant to be INSIDE a (potentially) clinically important functional domain AND to show no predicted impact via protein change or splicing (BayesDel no-AF <= 0.18 AND SpliceAI <= 0.1). c.4183G>T (p.Ala1395Ser) is a missense at codon 1395, outside the defined BRCA2 clinically important domains (PALB2-binding aa 10-40; DNA-binding aa 2481-3186), so the domain-location requirement fails and BP4 is not met. The no-impact components do hold (SpliceAI max delta = 0.00 <= 0.1; BayesDel no-AF = -0.321456 <= 0.18), but per ENIGMA Figure 1A the framework captures this benign in-silico signal through BP1 (BP1_Strong: 'silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI <=0.1)'), which is a criterion outside this group's ownership. VCEP lookup spreadsheets (Specifications Table 9, Supplementary Table ST4, HUMU-40-1557-s001/Parsons 2019) contain no pre-assigned BP4 code and no variant-level splice or functional assay row for c.4183G>T. REVEL 0.099 is not determinative because ENIGMA v1.2 defines no REVEL threshold. BP4 is not met.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, BP4 (Benign Supporting): 'Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1). Silent variant inside a (potentially) clinically important functional domain, if no predicted impact via splicing (SpliceAI ≤0.1). Intronic variants outside of the native donor and acceptor splice sites (i.e. not +/- 1,2 positions) AND no predicted impact via splicing (SpliceAI ≤0.1).' (potentially) clinically important functional domains: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure 1A and Appendix J.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (source_registry key 'cspec') BP4 (Supporting) missense rule: 'Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1)'. p.Ala1395Ser (codon 1395) is outside the defined BRCA2 clinically important domains (PALB2-binding aa 10-40; DNA-binding aa 2481-3186); the location gate fails, so BP4 is not met.
SpliceAI max delta = 0.00 (<= 0.1) and BayesDel no-AF = -0.321456 (<= 0.18) (source_registry keys 'spliceai', 'bayesdel'): the no-predicted-impact components of the ENIGMA v1.2 BP4 rule are satisfied, but ENIGMA Figure 1A routes outside-domain missense variants with no splice impact to BP1 (BP1_Strong rule, source_registry key 'cspec') rather than BP4.
VCEP lookup spreadsheets checked for a pre-assigned BP4 code for c.4183G>T: Specifications Table 9 (source_registry key 'vcep_specifications_table9_v1_2_2024_11_18'), Supplementary Tables ST4 (source_registry key 'vcep_supplementarytables_v1_2_2024_11_18'), and HUMU-40-1557-s001 SuppT1 (Parsons et al. 2019, source_registry key 'vcep_humu_40_1557_s001'): no BP4/PP3/BP7 pre-assignment and no splice or functional assay row for this variant.
BP5Not met: combined LR 2.225 is in the pathogenic direction, above the <=0.48 BP5 threshold.
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Reasoning
Under ENIGMA v1.2, BP5 is used ONLY to capture combined LR against pathogenicity from multifactorial likelihood clinical data, with BP5 Supporting at LR <=0.48 (not applicable for co-observation). Both available quantitative measures for BRCA2 c.4183G>T fall above the BP5 threshold: the ENIGMA combined multifactorial LR (Parsons et al. 2019, PMID:31131967, SuppT1) is 2.225 (pathogenic direction), and the clinical-history-only LR (Li et al. 2020, PMID:31853058 companion table) is 0.793, which lies in the ENIGMA neutral zone (>0.48 and <2.08) where 'PP4 and BP5 not applicable'. No benign-direction clinical data (co-occurrence/segregation LR <=0.48) are available for this variant. BP5 is therefore not met.
Rule
ENIGMA BRCA1/BRCA2 v1.2 BP5: combined LR against pathogenicity from multifactorial likelihood clinical data; Supporting LR <=0.48, Moderate <=0.23, Strong <=0.05, Very Strong <=0.00285; not applicable for co-observation; combined LR >0.48 and <2.08 is neutral and BP5 does not apply.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (CSPEC 1535438831) BP5 rule: 'Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data. BP5 - LR <=0.48' (Supporting); 'Not applicable for co-observation'; neutral zone >0.48 and <2.08.
Parsons et al. 2019 (PMID:31131967) Supplementary Table 1: BRCA2 c.4183G>T Combined LR (Odds for Causality) = 2.2250022419453646, which is >0.48 (BP5 not met).
Li et al. 2020 (PMID:31853058) clinical-history LR table: c.4183G>T LR = 0.7929228517802025 (N=1 proband) - in neutral zone >0.48 and <2.08, BP5 not applicable from this component.
Still missing
No quantitative evidence with combined LR <=0.48 for this variant; benign-direction clinical data (e.g., co-occurrence with pathogenic variant, segregation against causality) would be required to consider BP5.
Clinical-history LR (Li 2020) of 0.793 is based on a single proband and remains in the neutral zone.
PS2Not assessed: no parental testing or documentation of de novo occurrence was available.
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Reasoning
No de novo evidence of any kind exists for BRCA2 c.4183G>T (p.Ala1395Ser). ENIGMA BRCA1/2 v1.2 (governing CSPEC) does not define PS2, so generic ACMG/AMP PS2 applies (Richards et al. 2015, PMID:25741868): a variant shown to have arisen de novo with both parental samples confirmed by identity testing to be the biological parents, phenotype consistent with the disease, and no family history. The case bundle contains no parental testing results, no documentation of de novo occurrence, and no proband phenotype or family-history data; all 13 ClinVar submissions are germline VUS records with no de novo flag. Evidence insufficient - criterion not assessed.
Rule
Generic ACMG/AMP PS2 (PMID:25741868): de novo (paternity and maternity confirmed) in a patient with the disease and no family history. No ENIGMA BRCA1/2 v1.2 specification for PS2.
Evidence considered
Richards et al. 2015 (ACMG/AMP standards), PMID 25741868: PS2 requires a de novo occurrence with both parental samples shown by identity testing to be the biological parents; no such data exists in this case bundle
Still missing
No parental testing results (maternity/paternity identity confirmation) for the proband
No documentation that the variant arose de novo (no negative parental testing observed or reported)
No proband phenotype/family-history data in the case bundle to satisfy the PS2 phenotype-consistency and no-family-history requirements
PS4Not assessed: no case-control or prevalence data for this exact variant was available.
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Reasoning
Governing framework is the ClinGen ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (CSPEC doc 1535438831), whose PS4 rule requires case-control evidence with p-value <=0.05 AND OR >=4 (lower confidence interval excludes 2.0), and explicitly states 'Do not use Proband Counting as originally described'. No case-control dataset for BRCA2 c.4183G>T (p.Ala1395Ser) is available in the evidence bundle: the Parsons et al. 2019 (PMID:31131967) Supplementary Table 1 Case-Control LR cell for this variant is blank; the variant is absent from the ENIGMA ST7 reference set (all 16 sheets of SupplementaryTables_V1.2_2024-11-18.xlsx); and none of the locally available full texts mention the variant. The ClinVar-cited Brazilian cohort papers (PMID:28651617, PMID:35264596, PMID:36329109) have no local full text and their PubMed abstracts do not mention c.4183G>T / Ala1395Ser, so prevalence/case-control enrichment cannot be verified from them. Multiple non-expert ClinVar laboratory observations (13 submissions, all single-submitter/curation) cannot substitute for case-control data under ENIGMA PS4. Evidence insufficient to assess PS4; human follow-up recommended to locate any case-control or prevalence study reporting this exact variant.
Rule
ENIGMA BRCA1/BRCA2 v1.2 PS4: prevalence of the variant in affected individuals significantly increased vs controls in case-control studies; p-value <=0.05 and OR >=4 (lower confidence interval excludes 2.0); proband counting not used (Appendix F).
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (CSPEC 1535438831) PS4 rule: 'Case-control studies; p-value <=0.05 and OR >=4 (lower confidence interval excludes 2.0)' and instruction 'Do not use Proband Counting as originally described'.
Parsons et al. 2019 (PMID:31131967) Supplementary Table 1: BRCA2 c.4183G>T Case-Control LR cell is blank (no case-control LR available).
ENIGMA SupplementaryTables_V1.2_2024-11-18.xlsx (ST7 reference set, 16 sheets): variant c.4183G>T / A1395S absent - no ENIGMA reference-set posterior probability evidence for PS4.
ClinVar variation 96805: 13 submissions, 0 expert panel submissions, 1-star review status; no case-control assertion among submissions.
Still missing
No case-control study reporting p-value and OR with confidence interval for the exact variant BRCA2 c.4183G>T (p.Ala1395Ser) was identified; ENIGMA v1.2 PS4 requires p<=0.05 and OR>=4 with lower CI excluding 2.0.
ClinVar-cited cohort/prevalence papers PMID:28651617, PMID:35264596, PMID:36329109 are not available as local full text and their abstracts do not mention this variant - variant-level prevalence in affected Brazilian HBOC cohorts could not be verified.
Proband counting is explicitly disallowed by ENIGMA v1.2 PS4, so the 13 ClinVar laboratory observations cannot be used as PS4 evidence.
PM6Not assessed: no evidence that the variant arose de novo.
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Reasoning
No evidence that the variant arose de novo even without parentage confirmation. ENIGMA v1.2 does not define PM6; generic ACMG/AMP PM6 applies (PMID:25741868) when a de novo origin is assumed but parental identity is not confirmed. The case bundle contains no observation or report of de novo occurrence (no parental samples tested or reported, no indication the variant is absent in parents), so not even an 'assumed de novo' basis exists. All ClinVar submissions report germline origin without any de novo status. Evidence insufficient - criterion not assessed.
Rule
Generic ACMG/AMP PM6 (PMID:25741868): assumed de novo, but without confirmation of paternity and maternity. No ENIGMA BRCA1/2 v1.2 specification for PM6.
Evidence considered
Richards et al. 2015 (ACMG/AMP standards), PMID 25741868: PM6 applies when a de novo origin is assumed but parental identity is not confirmed; application requires at least an observed/assumed de novo occurrence, which is absent here
Still missing
No parental genotype data (whether parents were tested is not reported)
No observation or report of de novo occurrence of the variant
No family history information to support or exclude a de novo inheritance pattern
PP1Not assessed: no family segregation data or quantitative co-segregation likelihood ratio was available.
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Reasoning
ENIGMA BRCA1/2 v1.2 (governing CSPEC) requires a quantitative co-segregation likelihood ratio (LR) from a co-segregation analysis method (COOL/CAL-Leiden, Appendix I) to apply PP1: LR >=2.08 supporting, >=4.3 moderate, >=18.7 strong, >=350 very strong; LR 0.48-2.08 is neutral. No family segregation data exists in the case bundle: no pedigrees, no testing of affected family members, and no co-segregation LR for BRCA2 c.4183G>T. The ENIGMA multifactorial dataset (Parsons et al. 2019, PMID:31131967) has no BRCA2 row for this variant in SuppT1 (the only c.4183G>T/p.Ala1395Ser row is BRCA1, legacy 4411G>T), and SuppT2 records empty co-segregation family counts for BRCA2 c.4183G>T. Easton et al. 2007 (PMID:17924331) was reviewed as methodology context only, per VCEP index guidance that it must not be used alone for PP1. Evidence insufficient - criterion not assessed.
Rule
ENIGMA BRCA1/2 v1.2 PP1: co-segregation with disease in multiple affected family members measured by quantitative co-segregation analysis; LR >=2.08 supporting, >=4.3 moderate, >=18.7 strong, >=350 very strong (Appendix I).
Parsons et al. 2019, PMID 31131967, Supplementary Table 1 (HUMU-40-1557-s001.xlsx): no BRCA2 c.4183G>T row with a combined/segregation LR; Supplementary Table 2 lists the variant with empty Co-Segregation Family Count (Easton 2007 dataset; Edinir Palmero sources) - no LR computable
Still missing
No segregation analysis of affected family members (no meioses/pedigree data)
No quantitative co-segregation LR (COOL/CAL-Leiden) for this variant
No variant-level LR in ENIGMA multifactorial tables (Parsons 2019 SuppT1) for BRCA2 c.4183G>T
BS2Not assessed: no proband phenotype data to score the ENIGMA BS2 points table.
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Reasoning
Under ENIGMA BRCA1/BRCA2 VCEP v1.2, BS2 is NOT a population-frequency criterion. It is applied in the absence of features of recessive disease (Fanconi Anemia phenotype) and is scored per proband using Specifications Table 8: BS2_Supporting = 1 point, BS2_Moderate = 2 points, BS2_Strong = >= 4 points (Appendix H). The case bundle contains no proband-level phenotype data, no Fanconi Anemia assessment, and no extract of Specifications Table 8 scoring, so the point total cannot be computed. gnomAD homozygote count is 0 for this variant, but homozygous observations in gnomAD are not the ENIGMA BS2 metric. BS2 is therefore not assessed and requires human input of the Table 8 phenotype points.
Rule
BS2: Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype; points per proband per Specifications Table 8 (BS2_Supporting = 1 point, BS2_Moderate = 2 points, BS2_Strong = >= 4 points). ENIGMA BRCA1/BRCA2 VCEP v1.2.
Evidence considered
cspec: ENIGMA BRCA1/BRCA2 VCEP Specifications v1.2 (ClinGen CSPEC doc 1535438831), BS2 rule - applied in absence of features of recessive disease (Fanconi Anemia phenotype); points per proband per Specifications Table 8 (BS2_Supporting = 1 point, BS2_Moderate = 2 points, BS2_Strong = >= 4 points); Appendix H
gnomad_v4: total homozygotes = 0 (2/1,603,248 alleles) - noted for context only, not an ENIGMA BS2 metric
Still missing
ENIGMA v1.2 BS2 requires Specifications Table 8 points per proband based on absence of Fanconi Anemia features; no proband phenotype data and no Table 8 extract are present in the case bundle.
No biallelic (homozygous or compound heterozygous) Fanconi Anemia observations are available for this variant.
BS4Not assessed: no co-segregation or non-segregation likelihood ratio data was available.
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Reasoning
ENIGMA BRCA1/2 v1.2 BS4 requires a quantitative co-segregation LR demonstrating lack of segregation: LR <=0.48 supporting, <=0.23 moderate, <=0.05 strong, <=0.00285 very strong (LR 0.48-2.08 neutral). No non-segregation evidence exists in the case bundle: no testing of affected family members, no segregation analysis, and no LR for BRCA2 c.4183G>T. The ENIGMA multifactorial dataset (Parsons 2019, PMID:31131967) contains no BRCA2 SuppT1 row for this variant and records zero co-segregation families in SuppT2. Per instructions, BS4 was not inferred from other sources' final classifications (e.g., one ClinVar 'likely benign' submission or gnomAD population presence belong to other criteria groups). Evidence insufficient - criterion not assessed.
Rule
ENIGMA BRCA1/2 v1.2 BS4: lack of segregation in affected members of a family measured by quantitative co-segregation analysis; LR <=0.48 supporting, <=0.23 moderate, <=0.05 strong, <=0.00285 very strong (Appendix I).
Parsons et al. 2019, PMID 31131967, Supplementary Tables 1-2 (HUMU-40-1557-s001.xlsx): no BRCA2 c.4183G>T LR row; co-segregation family count empty - no lack-of-segregation evidence retrievable
Still missing
No segregation analysis showing lack of segregation among affected family members
No quantitative co-segregation LR <=0.48 for this variant
No pedigree data for affected relatives (phenocopy assessment impossible)
Not applicable · 11· recorded as not applicableShow reasons ▾Hide reasons ▴PVS1N/ANot applicable: missense substitution, not a loss-of-function variant class, with no predicted splicing impact (SpliceAI max delta 0.00).
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Reasoning
NM_000059.4:c.4183G>T is a single-nucleotide substitution predicted to cause a missense change, NP_000050.3:p.(Ala1395Ser), in exon 11 of the MANE Select BRCA2 transcript (NM_000059.4; ClinVar variation 96805 uses the same canonical nomenclature). Under the governing ENIGMA BRCA1/BRCA2 Specification v1.2 (ClinGen CSPEC doc 1535438831), PVS1 applies to null variants (nonsense, frameshift, canonical splice-site donor/acceptor +/-1,2, initiation codon, single or multi-exon deletion) or, as PVS1(RNA), to variants with a well-established mRNA assay demonstrating a damaging transcript effect. This variant is none of these: it is not a nonsense/frameshift/initiation-codon variant, it is exonic and not at a canonical +/-1,2 splice position, and SpliceAI predicts no splicing impact (max delta score = 0.00). No variant-level mRNA assay data was identified in any reviewed source (no downloaded publication mentions c.4183G>T/p.Ala1395Ser; OncoKB reports no variant-specific functional evidence), so PVS1(RNA) cannot be applied either. PVS1 is therefore not applicable to this variant. BRCA2 LOF is an established disease mechanism (ENIGMA applies PVS1 for the gene), but the variant class excludes PVS1 here.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 PVS1: null variant (nonsense, frameshift, splice site donor/acceptor +/-1,2, initiation codon, single or multi-exon deletion) in a gene where LOF is a known mechanism of disease, applied at appropriate strength per the PVS1 flowchart; alternatively PVS1(RNA) for a well-established mRNA-assay-proven damaging transcript effect. Not applicable to missense substitutions with no predicted or demonstrated splicing effect.
Evidence considered
ENIGMA BRCA1/BRCA2 Specification v1.2 PVS1 rule: 'Null variant (nonsense, frameshift, splice site (donor/acceptor +/-1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease', or well-established mRNA assay supportive of a damaging transcript effect applied as PVS1 (RNA). The assessed variant is a missense substitution and meets neither condition.
Variant consequence from canonical normalization: NM_000059.4:c.4183G>T -> NP_000050.3:p.(Ala1395Ser), exonic (exon 11), single-nucleotide substitution; not a null-variant class.
SpliceAI lookup for NM_000059.4:c.4183G>T: max delta score = 0.00 (no predicted impact on splicing).
ClinVar variation 96805 lists this variant as NM_000059.4(BRCA2):c.4183G>T (p.Ala1395Ser), confirming the missense consequence on the canonical transcript.
Still missing
No variant-level mRNA splicing assay (e.g., RNA-seq or minigene) data is available; if such data became available and demonstrated a damaging transcript profile, PVS1(RNA) would need re-evaluation.
PM1VCEPNot applicable: the ENIGMA VCEP does not use PM1 for BRCA2, capturing domain analysis through PP3/BP4 instead.
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Reasoning
Under the governing ENIGMA BRCA1/BRCA2 VCEP specification v1.2, PM1 is declared Not Applicable for BRCA2 ('Considered as component of bioinformatic analysis (PP3/BP4)'). The VCEP defines the (potentially) clinically important functional domains used in its framework (BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding domain aa 2481-3186) and applies them through PP3/BP4/BP1 rather than through a PM1 hotspot criterion. Because an applicable VCEP specification exists, generic ACMG/AMP PM1 (mutational hotspot / critical functional domain) is not used. Accordingly PM1 is not assessed as met or not_met; it is not applicable in this framework.
VCEP rule
ENIGMA BRCA1/BRCA2 VCEP v1.2 PM1: Not applicable (component of bioinformatic analysis PP3/BP4). Clinically important functional domains for BRCA2 defined as PALB2 binding domain aa 10-40 and DNA binding domain aa 2481-3186 (Specifications Figure 1A, Appendix J).
None for this criterion: the VCEP explicitly marks PM1 not applicable; any domain-based evidence is captured under PP3/BP4/BP1, which are adjudicated by the bioinformatic/consequence groups.
PM3N/ANot applicable: no Fanconi anemia phenotype or second co-occurring BRCA2 variant is documented.
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Reasoning
Governing framework is the ClinGen ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (cspec), which defines PM3 exclusively for patients with a BRCA1-/BRCA2-related Fanconi Anemia (FA) phenotype who carry co-occurrent variants in the same gene (points-based: PM3_Supporting = 1 point, PM3_Moderate = 2 points, PM3_Strong = >=4 points, per Specifications Table 6 / Appendix H). Neither precondition is documented for this case: (1) the variant was reported in ClinVar (variation 96805) under breast/ovarian cancer and hereditary cancer-predisposition conditions with autosomal-dominant BRCA2-related cancer predisposition context — no Fanconi Anemia phenotype, no chromosome breakage data, and no FA clinical features are present anywhere in the case bundle; (2) no second (co-occurring) BRCA2 variant is reported in any submission, so there is no biallelic/compound-heterozygote observation to weigh. Because the ENIGMA specification restricts PM3 to the FA context and that context is absent, the criterion is not applicable here; generic ACMG PM3 (variant in trans with a pathogenic variant in a recessive disorder) also does not apply because this is a dominantly inherited HBOC/BRCA2-cancer-predisposition setting, not a recessive FA setting. No variant-level allelic evidence was found in any full-text paper in the case packet (targeted search for c.4183G>T / p.Ala1395Ser across all downloaded full texts returned no hits).
Rule
Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene. PM3_Supporting = 1 point, PM3_Moderate = 2 points, PM3_Strong = >=4 points (ENIGMA BRCA1/BRCA2 Specification v1.2, Specifications Table 6, Appendix H).
Evidence considered
ENIGMA BRCA1/BRCA2 v1.2 specification (cspec, CSPEC ruleset): PM3 rule text restricts application to FA-consistent phenotype with co-occurrent variants in the same gene; PM3 point thresholds (1/2/>=4) per Specifications Table 6 and Appendix H
ClinVar variation 96805 (clinvar): 13 submissions, all under BRCA2-related cancer predisposition / HBOC / breast cancer / not-provided conditions; no Fanconi Anemia condition, no expert panel submission, no co-occurring variant or phase data reported
Still missing
No proband-level phenotype documentation (e.g., chromosome breakage studies DEB/MMC/spontaneous, FA physical features) exists in the case bundle; FA status is inferred as absent only from ClinVar conditions — if FA were suspected, PM3 would need re-evaluation under ENIGMA Table 6/Appendix H
No co-occurring second BRCA2 variant identified in any ClinVar submission or literature; biallelic/compound-heterozygote status of the proband is unknown
No phase (in trans / in cis) information is available for this variant relative to any other variant
PM4VCEPNot applicable: single-nucleotide missense with no protein length change, and ENIGMA excludes PM4 for BRCA1/BRCA2.
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Reasoning
The governing ENIGMA BRCA1/BRCA2 Specification v1.2 declares PM4 'Not applicable' for BRCA1/BRCA2 classification (instructions to use: 'Considered as component of bioinformatic analysis (PP3/BP4)'; all strength rules are marked Not applicable). Independently of the VCEP declaration, PM4 addresses protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants; c.4183G>T is a single-nucleotide missense substitution (p.Ala1395Ser) that does not alter protein length, so PM4 would not be met under generic ACMG/AMP either. PM4 is therefore not applicable in this case.
VCEP rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2: PM4 declared Not Applicable for this VCEP (captured within bioinformatic analysis PP3/BP4).
Evidence considered
ENIGMA BRCA1/BRCA2 Specification v1.2 PM4 entry: applicability 'Not applicable'; original ACMG summary 'Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants'.
Variant consequence: single-nucleotide substitution with no protein length change (no in-frame insertion/deletion, no stop-loss).
PM5N/ANot applicable: ENIGMA applies PM5 only to protein-termination-codon variants, not missense changes.
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Reasoning
In ENIGMA BRCA1/BRCA2 VCEP v1.2, PM5 is repurposed exclusively for protein termination codon (PTC) variants: 'Only applied to genomic PTC changes (not splicing)', with exon-specific PM5_PTC weights from Specifications Table 4. The variant under assessment is a missense substitution (p.Ala1395Ser), not a PTC, so PM5_PTC is not applicable. The classic ACMG PM5 (novel missense at a residue where a different pathogenic missense has been seen) is explicitly not used by this VCEP (pm5_candidates.json: pm5_mode = 'ptc_gene_specific', eligible_for_classic_pm5_search = false). Independently, the pm5_candidates artifact collected 0 same-residue comparator variants, so even under a classic reading no comparator exists. PM5 is therefore not applicable to this variant.
Rule
ENIGMA BRCA1/BRCA2 VCEP v1.2 PM5: 'Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before' - applied only to PTC changes, per Specifications Table 4 / Appendix D.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (cspec, doc 1535438831): PM5 instructionsToUse = 'Only applied to genomic PTC changes (not splicing). Weight determined by exon where the termination codon occurs... See Specifications Table 4 for PM5_PTC codes.'
Specifications Table 4 v1.2 (vcep_specifications_table4_v1_2_2024_11_18): PM5_PTC applicable exons for BRCA2 listed per exon; PM5_N/A exons are E12, E27, E6 (exon 11 not excluded). Not applied here because the variant is missense, not a PTC.
Still missing
None for this variant type: PM5 in ENIGMA v1.2 applies only to PTC variants, and c.4183G>T is a missense substitution. Should a future assertion change the consequence to a termination-codon event, exon-specific PM5_PTC weight (Table 4) would need to be checked against exon 11.
PP2VCEPNot applicable: the ENIGMA VCEP does not use PP2 for BRCA2.
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Reasoning
Under the governing ENIGMA BRCA1/BRCA2 VCEP specification v1.2, PP2 is declared Not Applicable for BRCA2. The VCEP notes the high frequency of benign missense variation in BRCA1/BRCA2 and does not use the generic ACMG PP2 ('missense variant in a gene with a low rate of benign missense variation and where missense is a common mechanism of disease'). Because an applicable VCEP specification exists, generic ACMG/AMP PP2 is not used, and PP2 is not assessed as met or not_met.
VCEP rule
ENIGMA BRCA1/BRCA2 VCEP v1.2 PP2: Not applicable (high frequency of benign missense variation in BRCA2).
PP5VCEPNot applicable: ENIGMA VCEP does not use PP5, and no expert-panel pathogenic classification exists.
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Reasoning
PP5 is formally 'Not Applicable for this VCEP' in the governing ENIGMA BRCA1/BRCA2 v1.2 specification ('This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee'). Independently of the VCEP removal, PP5 may only be triggered by an exact-variant ClinVar expert-panel classification of Pathogenic/Likely Pathogenic; none exists for this variant: ClinVar variation 96805 has 0 expert panel submissions, review stars = 1, and an aggregate classification of Uncertain significance (11 laboratory submissions) with a conflicting single 'Likely benign' laboratory curation (SCV003848748). Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger PP5, so no PP5 evidence exists.
VCEP rule
PP5 applies only for an exact-variant ClinVar expert-panel (3-star) classification of Pathogenic/Likely Pathogenic; ENIGMA BRCA1/BRCA2 v1.2 declares PP5 not applicable for this VCEP.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (CSPEC 1535438831) PP5 applicability: 'Not Applicable for this VCEP' - 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee'.
ClinVar variation 96805 submission audit: expert_panel_submissions = 0, review_stars = 1 (criteria provided, conflicting classifications), aggregate Uncertain significance; no expert-panel Pathogenic/Likely Pathogenic classification for this exact variant.
Still missing
No ClinVar expert-panel (3-star) classification exists for BRCA2 c.4183G>T (p.Ala1395Ser) - 0 expert panel submissions; re-evaluate if ENIGMA or another ClinGen expert panel publishes a formal classification.
Screened guideline papers tagged as PP5 candidates (PMID:17508274, PMID:19305347, PMID:23788249) are generic practice guidelines with no variant-level assertion and cannot support PP5.
BP2VCEPNot applicable: the ENIGMA VCEP does not apply BP2 to BRCA2.
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Reasoning
Under the governing ClinGen ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (cspec), BP2 is explicitly marked 'Not Applicable' in the CSPEC ruleset, i.e., the VCEP does not apply BP2 (variant observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or in cis with a pathogenic variant in any inheritance pattern) to BRCA1/BRCA2 classification. The specification exists and governs, so generic ACMG BP2 is not substituted. Independently, even if generic BP2 were considered, it could not be applied: no phase information, no co-occurrence observation, and no documentation of the variant in trans with a pathogenic BRCA2 variant (or in cis) in an unaffected individual exist anywhere in the case bundle (ClinVar submissions, evidence files, or full texts — none of the downloaded papers mention c.4183G>T / p.Ala1395Ser).
VCEP rule
BP2: Not Applicable (ENIGMA BRCA1/BRCA2 Specification v1.2, CSPEC ruleset).
ClinVar variation 96805 (clinvar): submissions contain no co-occurring variant or phase information that would support generic BP2
Still missing
No phase (in trans / in cis) data documented for this variant relative to any pathogenic BRCA2 variant
No co-occurrence observation (variant present alongside a second BRCA2 variant in an individual) reported in any ClinVar submission or literature source in the case packet
BP3VCEPNot applicable: the ENIGMA VCEP does not apply BP3; this is a missense, not an in-frame indel.
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Reasoning
The governing ENIGMA BRCA1/BRCA2 Specification v1.2 declares BP3 'Not applicable' for BRCA1/BRCA2 classification (instructions to use: 'Captured by bioinformatic tool prediction, and domain analysis. See Appendix J'; all strength rules are marked Not applicable). Independently of the VCEP declaration, generic ACMG BP3 concerns in-frame deletions/insertions in a repetitive region without a known function; c.4183G>T is a single-nucleotide missense substitution (p.Ala1395Ser) with no repeat-region or in-frame indel context, so BP3 would not be met under generic ACMG/AMP either. BP3 is therefore not applicable in this case.
VCEP rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2: BP3 declared Not Applicable for this VCEP (captured by bioinformatic tool prediction and domain analysis).
Evidence considered
ENIGMA BRCA1/BRCA2 Specification v1.2 BP3 entry: applicability 'Not applicable'; original ACMG summary 'In frame-deletions/insertions in a repetitive region without a known function'.
Variant consequence: single-nucleotide substitution, not an in-frame insertion/deletion in a repetitive region.
BP6VCEPNot applicable: ENIGMA VCEP does not use BP6, and no expert-panel benign classification exists.
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Reasoning
BP6 is formally 'Not Applicable for this VCEP' in the governing ENIGMA BRCA1/BRCA2 v1.2 specification ('This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee'). Independently of the VCEP removal, BP6 may only be triggered by an exact-variant ClinVar expert-panel classification of Benign/Likely Benign; none exists for this variant (0 expert panel submissions, 1-star review status). The single 'Likely benign' submission (SCV003848748, University of Washington, method 'curation', criteria provided - single submitter) is an ordinary laboratory/curation assertion, not an expert-panel classification, and must never trigger BP6.
VCEP rule
BP6 applies only for an exact-variant ClinVar expert-panel (3-star) classification of Benign/Likely Benign; ENIGMA BRCA1/BRCA2 v1.2 declares BP6 not applicable for this VCEP.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP specification v1.2 (CSPEC 1535438831) BP6 applicability: 'Not Applicable for this VCEP' - 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee'.
ClinVar variation 96805 submission audit: expert_panel_submissions = 0, review_stars = 1; SCV003848748 'Likely benign' is a single-submitter laboratory curation (non-expert), not an expert-panel classification.
Still missing
No ClinVar expert-panel (3-star) Benign/Likely Benign classification exists for BRCA2 c.4183G>T (p.Ala1395Ser); re-evaluate if an expert panel publishes a formal classification.
The only benign-direction assertion (SCV003848748 'Likely benign', laboratory curation) is non-expert and explicitly excluded from BP6.
BP7N/ANot applicable: BP7 applies to silent or intronic variants, not this missense substitution.
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Reasoning
Under the governing ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, BP7 (Supporting) applies only to (a) silent variants inside a (potentially) clinically important functional domain IF BP4 is met, and (b) intronic variants located outside conserved donor/acceptor motif positions (at or beyond positions +7/-21) IF BP4 is met. c.4183G>T is a missense substitution (p.Ala1395Ser), so neither supporting condition applies. BP7_Strong (RNA) - well-established in vitro/in vivo mRNA-only assay showing no damaging effect - is in principle available for missense/in-frame variants outside a clinically important domain, but no mRNA-based functional assay data exist for this variant: ClinVar submissions state functional studies have not been reported, ENIGMA Specification Table 9 and Supplementary Table ST4 contain no row for c.4183G>T, and the Parsons 2019 (HUMU-40-1557-s001) splicing-result columns are empty. BP7 is therefore not applicable with the available evidence.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, BP7 (Benign Supporting): 'Silent variant inside a (potentially) clinically important functional domain, IF BP4 met. Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.' BP7_Strong (RNA): 'Well-established in vitro or in vivo functional studies shows no damaging effect on protein function as measured by effect on mRNA transcript profile - mRNA assay only. Apply as BP7_Strong (RNA) for intronic and silent variants, as well as missense/in-frame variants located outside a (potentially) clinically important functional domain.' See Specifications Figure 1B and Appendix E.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (source_registry key 'cspec') BP7 (Supporting) rule: 'Silent variant inside a (potentially) clinically important functional domain, IF BP4 met. Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.' c.4183G>T is missense, so BP7 supporting is not applicable.
ENIGMA v1.2 BP7_Strong (RNA) requires well-established mRNA-only functional assay evidence of no damaging effect for missense/in-frame variants outside a clinically important domain (Specifications Figure 1B / Appendix E, source_registry key 'cspec'). No such data exist for c.4183G>T: ClinVar submissions state 'to our knowledge, functional studies have not been reported for this variant' (source_registry key 'clinvar'; SCV005424424 All of Us, SCV001736201 Color); no row in Specifications Table 9 (source_registry key 'vcep_specifications_table9_v1_2_2024_11_18') or Supplementary Table ST4 (source_registry key 'vcep_supplementarytables_v1_2_2024_11_18'); splicing-result and functional columns empty in HUMU-40-1557-s001 SuppT1 (source_registry key 'vcep_humu_40_1557_s001').
No pre-assigned BP7 code for c.4183G>T found in any VCEP lookup spreadsheet (Table 9, ST4, HUMU-40-1557-s001).
Still missing
No mRNA-based functional assay data available for c.4183G>T; a BP7_Strong (RNA) code would require well-established mRNA-only assay evidence of no damaging effect (ENIGMA v1.2, Specifications Figure 1B / Appendix E). Absent from ClinVar submissions, ENIGMA Table 9, Supplementary Table ST4, and Parsons 2019 SuppT1 splicing-result columns.
AF 1.2e-06 · 2 / 1,603,248 alleles · 0 hom · FAF 2.8e-07
European (non-Finnish)2 / 1,176,318
1.7e-06
0 alternate alleles recorded in 9 other groups: Remaining individuals, Admixed American, European (Finnish), Amish, East Asian, Middle Eastern, South Asian, Ashkenazi Jewish, African/African American.
Statements
gnomAD v4.1
This variant is present in gnomAD v4.1 (AF= 1.24747e-06; MAF= 0.00012%, 2/1603248 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.70022e-06; MAF= 0.00017%, 2/1176318 alleles, homozygotes = 0); grpmax FAF= 2.8e-07.
gnomAD v2.1
Absent from gnomAD v2.1.
gnomAD-Canada
Absent from gnomAD-Canada v1.0.
SpliceAI and Pangolin
SpliceAI acceptor lossat -69 bp
0.000
SpliceAI acceptor gainat -128 bp
0.000
SpliceAI donor lossat +471 bp
0.000
SpliceAI donor gainat +155 bp
0.000
Pangolin splice lossmagnitude shown
-0.000
Pangolin splice gain
0.000
Other predictors
REVEL
0.099
BayesDel
-0.321
How the predictions were used
PP3Not met: BayesDel no-AF -0.32 falls below the 0.30 threshold and SpliceAI max delta 0.00 below 0.2.
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Reasoning
Variant NM_000059.4:c.4183G>T (p.Ala1395Ser) is a missense substitution in BRCA2 exon 11 at codon 1395. Under the governing ClinGen ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, PP3 has two paths. (1) Protein-change path: requires the missense to lie inside a (potentially) clinically important functional domain (BRCA2 PALB2-binding domain aa 10-40; BRCA2 DNA-binding domain aa 2481-3186) AND BayesDel no-AF score >=0.30; p.Ala1395Ser is outside both defined domains, and BayesDel no-AF = -0.321456 is far below 0.30, so this path fails. (2) Splice path: applies to missense/in-frame variants irrespective of location when SpliceAI >=0.2; SpliceAI max delta score = 0.00 (DS_AG/DS_AL/DS_DG/DS_DL all 0.0) is below 0.2, so this path also fails. No other computational evidence is determinative: REVEL 0.099 is recorded but the ENIGMA v1.2 framework defines no REVEL threshold (generic SVI REVEL calibration is not applied where an applicable VCEP specification exists), and no VCEP lookup spreadsheet pre-assigns PP3 for this variant. PP3 is not met.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, PP3 (Pathogenic Supporting): 'Apply PP3 for missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel no-AF score ≥0.30)... Apply PP3 for predicted splicing (SpliceAI ≥0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of donor and acceptor 1,2 sites.' (potentially) clinically important functional domains: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure 1A and Appendix J.
Evidence considered
SpliceAI max delta score = 0.00 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.0, DS_DL 0.0), SpliceAI Lookup (source_registry key 'spliceai'). ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (ClinGen CSPEC doc 1535438831, source_registry key 'cspec') PP3 splice path requires SpliceAI >= 0.2 for missense variants irrespective of domain location; 0.00 < 0.2, so the splice path is not met.
BayesDel no-AF score = -0.321456, Local BayesDel predictor lookup (source_registry key 'bayesdel'). ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (source_registry key 'cspec') PP3 protein-change path requires BayesDel no-AF >= 0.30 AND location inside a (potentially) clinically important functional domain (BRCA2 PALB2-binding aa 10-40; BRCA2 DNA-binding aa 2481-3186). p.Ala1395Ser is outside both domains and the score is far below 0.30; the protein-change path is not met.
REVEL score = 0.099, Local REVEL predictor lookup (source_registry key 'revel'). Recorded for completeness only; not used to call PP3 because the governing ENIGMA v1.2 specification (source_registry key 'cspec') calibrates missense prediction on BayesDel no-AF, not REVEL.
Under the governing ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, the BP4 (Supporting) rule for missense variants requires the variant to be INSIDE a (potentially) clinically important functional domain AND to show no predicted impact via protein change or splicing (BayesDel no-AF <= 0.18 AND SpliceAI <= 0.1). c.4183G>T (p.Ala1395Ser) is a missense at codon 1395, outside the defined BRCA2 clinically important domains (PALB2-binding aa 10-40; DNA-binding aa 2481-3186), so the domain-location requirement fails and BP4 is not met. The no-impact components do hold (SpliceAI max delta = 0.00 <= 0.1; BayesDel no-AF = -0.321456 <= 0.18), but per ENIGMA Figure 1A the framework captures this benign in-silico signal through BP1 (BP1_Strong: 'silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI <=0.1)'), which is a criterion outside this group's ownership. VCEP lookup spreadsheets (Specifications Table 9, Supplementary Table ST4, HUMU-40-1557-s001/Parsons 2019) contain no pre-assigned BP4 code and no variant-level splice or functional assay row for c.4183G>T. REVEL 0.099 is not determinative because ENIGMA v1.2 defines no REVEL threshold. BP4 is not met.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, BP4 (Benign Supporting): 'Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1). Silent variant inside a (potentially) clinically important functional domain, if no predicted impact via splicing (SpliceAI ≤0.1). Intronic variants outside of the native donor and acceptor splice sites (i.e. not +/- 1,2 positions) AND no predicted impact via splicing (SpliceAI ≤0.1).' (potentially) clinically important functional domains: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure 1A and Appendix J.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (source_registry key 'cspec') BP4 (Supporting) missense rule: 'Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1)'. p.Ala1395Ser (codon 1395) is outside the defined BRCA2 clinically important domains (PALB2-binding aa 10-40; DNA-binding aa 2481-3186); the location gate fails, so BP4 is not met.
SpliceAI max delta = 0.00 (<= 0.1) and BayesDel no-AF = -0.321456 (<= 0.18) (source_registry keys 'spliceai', 'bayesdel'): the no-predicted-impact components of the ENIGMA v1.2 BP4 rule are satisfied, but ENIGMA Figure 1A routes outside-domain missense variants with no splice impact to BP1 (BP1_Strong rule, source_registry key 'cspec') rather than BP4.
VCEP lookup spreadsheets checked for a pre-assigned BP4 code for c.4183G>T: Specifications Table 9 (source_registry key 'vcep_specifications_table9_v1_2_2024_11_18'), Supplementary Tables ST4 (source_registry key 'vcep_supplementarytables_v1_2_2024_11_18'), and HUMU-40-1557-s001 SuppT1 (Parsons et al. 2019, source_registry key 'vcep_humu_40_1557_s001'): no BP4/PP3/BP7 pre-assignment and no splice or functional assay row for this variant.
BP7N/ANot applicable: BP7 applies to silent or intronic variants, not this missense substitution.
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Reasoning
Under the governing ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, BP7 (Supporting) applies only to (a) silent variants inside a (potentially) clinically important functional domain IF BP4 is met, and (b) intronic variants located outside conserved donor/acceptor motif positions (at or beyond positions +7/-21) IF BP4 is met. c.4183G>T is a missense substitution (p.Ala1395Ser), so neither supporting condition applies. BP7_Strong (RNA) - well-established in vitro/in vivo mRNA-only assay showing no damaging effect - is in principle available for missense/in-frame variants outside a clinically important domain, but no mRNA-based functional assay data exist for this variant: ClinVar submissions state functional studies have not been reported, ENIGMA Specification Table 9 and Supplementary Table ST4 contain no row for c.4183G>T, and the Parsons 2019 (HUMU-40-1557-s001) splicing-result columns are empty. BP7 is therefore not applicable with the available evidence.
Rule
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, BP7 (Benign Supporting): 'Silent variant inside a (potentially) clinically important functional domain, IF BP4 met. Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.' BP7_Strong (RNA): 'Well-established in vitro or in vivo functional studies shows no damaging effect on protein function as measured by effect on mRNA transcript profile - mRNA assay only. Apply as BP7_Strong (RNA) for intronic and silent variants, as well as missense/in-frame variants located outside a (potentially) clinically important functional domain.' See Specifications Figure 1B and Appendix E.
Evidence considered
ENIGMA BRCA1/BRCA2 VCEP Specification v1.2 (source_registry key 'cspec') BP7 (Supporting) rule: 'Silent variant inside a (potentially) clinically important functional domain, IF BP4 met. Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.' c.4183G>T is missense, so BP7 supporting is not applicable.
ENIGMA v1.2 BP7_Strong (RNA) requires well-established mRNA-only functional assay evidence of no damaging effect for missense/in-frame variants outside a clinically important domain (Specifications Figure 1B / Appendix E, source_registry key 'cspec'). No such data exist for c.4183G>T: ClinVar submissions state 'to our knowledge, functional studies have not been reported for this variant' (source_registry key 'clinvar'; SCV005424424 All of Us, SCV001736201 Color); no row in Specifications Table 9 (source_registry key 'vcep_specifications_table9_v1_2_2024_11_18') or Supplementary Table ST4 (source_registry key 'vcep_supplementarytables_v1_2_2024_11_18'); splicing-result and functional columns empty in HUMU-40-1557-s001 SuppT1 (source_registry key 'vcep_humu_40_1557_s001').
No pre-assigned BP7 code for c.4183G>T found in any VCEP lookup spreadsheet (Table 9, ST4, HUMU-40-1557-s001).
Still missing
No mRNA-based functional assay data available for c.4183G>T; a BP7_Strong (RNA) code would require well-established mRNA-only assay evidence of no damaging effect (ENIGMA v1.2, Specifications Figure 1B / Appendix E). Absent from ClinVar submissions, ENIGMA Table 9, Supplementary Table ST4, and Parsons 2019 SuppT1 splicing-result columns.
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). REVEL score = 0.099. BayesDel score = -0.321456.
8 paper records listed. 3 cited in the classification.
Cited in the classification
Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.
Parsons MT et al. · 2019 Sep
ENIGMA large-scale multifactorial likelihood analysis (Parsons et al. 2019). Direct lookup of BRCA2 c.4183G>T (p.Ala1395Ser) in the accompanying supplementary workbook (HUMU-40-1557-s001.xlsx): no BRCA2 row exists in Supplementary Table 1 'All Data' - the only c.4183G>T / p.(Ala1395Ser) row is BRCA1 (legacy description 4411G>T, combined LR 2.225, prior 0.02, posterior 0.043, IARC Uncertain). Supplementary Table 2 'Data Sources' lists BRCA2 c.4183G>T twice (source 'Easton et al 2007 dataset' and source 'Edinir Palmero') with EMPTY co-segregation family counts (one family-history-score count and one breast-tumour count respectively). No combined LR or co-segregation LR is therefore available for BRCA2 c.4183G>T, so PP1/BS4 cannot be quantified from this resource.
Source: 'Easton et al 2007 dataset', Gene: 'BRCA2', Variant: 'c.4183G>T', Co-Segregation Family Count: (empty), Breast Tumour Count: (empty), Family History Score Family Count: '1' | Source: 'Edinir Palmero', Gene: 'BRCA2', Variant: 'c.4183G>T', Co-Segregation Family Count: (empty), Breast Tumour Count: '1', Family History Score Family Count: (empty)
Location HUMU-40-1557-s001.xlsx, Supplementary Table 2 'Data Sources' rows 708 and 1263 (columns: Source, Gene, Variant, Co-Segregation Family Count, Breast Tumour Count, Family History Score Family Count); absence of a BRCA2 row verified across Supplementary Table 1 'All Data'
Context ENIGMA multifactorial likelihood model for BRCA1/BRCA2 missense and splice variants combining segregation, pathology, co-occurrence, family history and case-control components with prior probability of pathogenicity and IARC 5-class output
Classification of variants of uncertain significance in BRCA1 and BRCA2 using personal and family history of cancer from individuals in a large hereditary cancer multigene panel testing cohort.
Li H et al. · 2020 Apr
Li et al. 2020 derives variant-level clinical-history likelihood ratios for BRCA1/BRCA2 VUS using logistic regression on personal and family cancer history in ~138,000 multigene panel-tested individuals. The main article text does not mention BRCA2 c.4183G>T (0 hits for 4183/Ala1395/A1395); the variant-level LR for this variant is provided in the companion MOESM3-derived table (vcep_pmid_31853058_brca2_clinical_history_lr), reporting LR 0.793 from 1 proband, which lies in the ENIGMA neutral zone. The paper provides methodology/context for using clinical-history LRs as a PP4/BP5 component.
Likelihood ratios for individual variants were derived using a logistic regression equation based on the characteristics of the personal and family histories of the individuals with known pathogenic variants (largely loss of function [LOF]) compared with individuals who did not have BRCA1 or BRCA2 pathogenic variants or VUS.
Context Clinical-history (personal and family cancer history) logistic regression model in 138,342 eligible multigene panel-tested individuals; per-variant log-LR and LR.
Systematic misclassification of missense variants in BRCA1 and BRCA2 "coldspots".
Dines JN et al. · 2020 May
Dines et al. (2020) evaluated 5,720 BRCA1/BRCA2 missense variants reported in ClinVar and used a Bayesian approach to identify 'coldspots' - regions without essential functions that tolerate variation. BRCA1 exon 11 and BRCA2 exons 10 and 11 (~65% of the BRCA2 coding sequence) were identified as coldspots: of 34 pathogenic/likely pathogenic missense variants in BRCA2, none fall in exons 10-11 (odds <0.01, 95% CI 0.0-0.01). The authors propose that location within a coldspot is strong evidence that a BRCA1/BRCA2 missense variant is benign. The paper does not mention c.4183G>T or p.Ala1395Ser specifically; it is relevant to this group only as region-level context: the variant under assessment lies in BRCA2 exon 11, which the paper defines as a coldspot.
Of 34 P or LP missense variants in BRCA2, none are in exons 10-11 (odds <0.01, 95% CI 0.0-0.01).
Location Abstract, Results section
Context Retrospective analysis of 5,720 BRCA1/BRCA2 missense variants reported in ClinVar (accessed December 2018); Bayesian modeling of variant classification to define coldspot regions.
Other recorded publications
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
ACMG/AMP 2015 joint consensus standards (Richards et al.) defining the de novo criteria PS2 and PM6. Consulted because ENIGMA BRCA1/2 v1.2 does not specify PS2/PM6, making the generic rules governing. Establishes that PS2 requires a documented de novo occurrence with both parental samples confirmed via identity testing and phenotype consistent with the disease, and that PM6 covers an assumed de novo occurrence without parentage confirmation. No variant-specific content; used solely as the framework rule source to conclude neither criterion is supportable with current case data.
A variant observed to have arisen de novo (parental samples test negative), is considered strong support for pathogenicity if the following conditions are met: Both parental samples were shown through identity testing to be the biological parents of the patient. Note: PM6 applies if identity is assumed but not confirmed.
Available full-text extraction, section 'PS2 PM6 De novo variants'
A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders.
The evidence statements recorded by the interpretation pipeline. They have not been reinterpreted by the page.
gnomAD v4.1
This variant is present in gnomAD v4.1 (AF= 1.24747e-06; MAF= 0.00012%, 2/1603248 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.70022e-06; MAF= 0.00017%, 2/1176318 alleles, homozygotes = 0); grpmax FAF= 2.8e-07.
gnomAD v2.1
Absent from gnomAD v2.1.
gnomAD-Canada
Absent from gnomAD-Canada v1.0.
ClinVar
This variant has been reported in ClinVar as Uncertain significance (10 clinical laboratories) and as Uncertain Significance (1 clinical laboratory). (ClinVarID = 96805)
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). REVEL score = 0.099. BayesDel score = -0.321456.
Functional
OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. BRCA2, a tumor suppressor involved in the DNA damage response, is mutated in various cancer types.
Somatic
This variant does not lie in a statistically significant hotspot. This variant has not previously been reported in somatic cancers (COSMIC).
Cancer Hotspots
This variant does not lie in a statistically significant hotspot.
This variant was interpreted by pipeline 7.1.0.
The current version is 11.0.07.
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