BAP1 c.126T>C, p.(Pro42=)

Reasoning

PM2 (supporting pathogenic) is met when the variant is absent from population databases or present at an extremely low frequency; this pipeline's non-VCEP gnomAD operating threshold is PM2 met when AF < 0.1%. The variant is absent from gnomAD v2.1 and present in gnomAD v4.1 at total AF 3.10757e-06 (0.00031%; 5/1,608,972 alleles) with grpmax FAF 4.43e-06 and zero homozygotes; even the highest subpopulation AF (African/African American, 2.66987e-05 = 0.00267%) is more than 30-fold below the 0.1% threshold. The frequency determination is unambiguous, so PM2 is met at supporting strength. Confidence is moderate rather than high because the variant is technically present (5 alleles) rather than literally absent in gnomAD v4.1, and because PM2's evidentiary interpretation for a synonymous variant in a dominant-disorder context carries some judgment; the criterion-level frequency determination itself is solid.

Rule

generic_acmg_2015_population (PMID 25741868): PM2 met when absent or at extremely low frequency in population databases; pipeline non-VCEP gnomAD operating threshold PM2 < 0.1%

Evidence considered

• gnomAD v4.1 (gnomad_v4): total AF 3.10757e-06 (0.00031%; 5/1,608,972 alleles), exome AF 2.74589e-06, genome AF 6.56806e-06, grpmax FAF 4.43e-06, 0 homozygotes; highest subpopulation AF 2.66987e-05 (AFR, 0.00267%)
• gnomAD v2.1 (gnomad_v2): variant absent
• PM2 per ACMG/AMP 2015 (PMID 25741868): absent in controls or at extremely low frequency; pipeline non-VCEP operating threshold PM2 < 0.1% — all observed frequencies are >30-fold below the threshold

Still missing

• gnomAD-Canada v1.0 (HostSeq genomes) query failed with HTTP 500, so no independent Canadian population data was available; an additional large population source (e.g., TOPMed) would further firm the ultra-rare frequency determination, though the conclusion is unchanged

Sources

Reasoning

PP3 (multiple lines of computational evidence support a deleterious effect on the gene or gene product) is not met. The splice-impact path was evaluated with SpliceAI: max delta score = 0.01 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.0), far below the recommended clinical threshold of delta >= 0.2 (Jaganathan et al., Nature Genetics 2019, PMID 30661751), so no splice-altering potential is predicted. The missense in-silico path is not evaluable: the variant is synonymous (NP_004647.1:p.(Pro42=)), so missense predictors do not apply to it, and REVEL returned no score for this variant (prefetch found=false). BayesDel also returned no score and no verified published threshold is available to this pipeline, so it is treated as insufficiently calibrated and cannot support PP3. No computational predictor provides any evidence of a deleterious effect; the single available prediction (SpliceAI) actually argues against splice impact. No BAP1 VCEP/CSPEC exists (generic ACMG/AMP 2015, PMID 25741868, applies).

Rule

Generic ACMG/AMP 2015 (PMID 25741868) PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product. Not met - no predictor supports pathogenicity.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'): max delta score 0.01 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.0) - below the recommended 0.2 delta threshold for suspected splice impact (Jaganathan et al., Nature Genetics 2019, PMID 30661751); no PP3 support
• REVEL: score not found for this variant (prefetch.json revel.found=false) - no REVEL-based PP3 evidence available
• BayesDel: score not found (prefetch.json bayesdel.found=false) and no verified published threshold is available to this pipeline - treated as insufficiently calibrated, not usable for PP3
• Generic ACMG/AMP 2015 rules (source_registry key 'generic_acmg_combination_rules', source_pmid 25741868): PP3 requires computational evidence of a deleterious effect; no such evidence exists for this variant

Still missing

• REVEL score not found for c.126T>C in the local REVEL v1.3 data; a missense-path PP3/BP4 evaluation is impossible for this synonymous variant regardless
• BayesDel score not found and, per pipeline rule, no verified published threshold is available - BayesDel treated as insufficiently calibrated for PP3/BP4
• No BAP1-specific VCEP/CSPEC computational-evidence calibration (e.g., gene-specific PP3/BP4 lookup) exists; generic ACMG/AMP 2015 thresholds used

Sources

Reasoning

PP5 may be applied at supporting strength only when an exact-variant ClinVar expert-panel classification is Pathogenic/Likely pathogenic. The ClinVar record VCV000515124 contains zero expert-panel submissions (expert_panel_submissions = 0; review status 'criteria provided, single submitter' / 'criteria provided, multiple submitters, no conflicts', 1-2 stars) and the aggregate classification is Benign/Likely benign from four ordinary clinical-testing laboratories (GeneDx, Ambry, Myriad, Labcorp). No expert-panel Pathogenic/Likely pathogenic classification exists for this exact variant, so PP5 is not met. The aggregate ClinVar label and individual laboratory assertions are explicitly excluded from triggering PP5.

Rule

PP5 per phenotype/prevalence group rule: supporting (PP5) only for an exact-variant ClinVar expert-panel classification of Pathogenic/Likely pathogenic; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions never trigger PP5. Framework: generic ACMG/AMP 2015 (PMID 25741868).

Evidence considered

• ClinVar VCV000515124 aggregate classification is 'Benign/Likely benign' (3 Likely benign + 1 Benign clinical laboratory submissions), review status 'criteria provided' (1-2 stars), 0 expert panel submissions.
• Submission audit (SCV000727119 GeneDx, SCV001170879 Ambry, SCV005406131 Myriad, SCV005692622 Labcorp): all are ordinary clinical testing submissions; none is an expert panel classification.
• Per the governing rule for this group, ordinary laboratory submissions and aggregate ClinVar labels must never trigger PP5.

Still missing

• No ClinVar expert-panel (3-star) Pathogenic/Likely pathogenic classification exists for NM_004656.4:c.126T>C; only single-submitter clinical laboratory assertions are available.

Sources

Reasoning

BA1 (stand-alone benign) requires an allele frequency too high to be consistent with any disease-causing allele: AF > 5% per ACMG/AMP 2015 (PMID 25741868), and AF > 1% per this pipeline's non-VCEP gnomAD operating threshold. The highest observed frequency of NM_004656.4:c.126T>C is in gnomAD v4.1 African/African American individuals (AF = 2.66987e-05; 0.00267%; 2/74,910 alleles); the total AF is 3.10757e-06 (0.00031%; 5/1,608,972 alleles) with grpmax FAF 4.43e-06, and the variant is absent from gnomAD v2.1. The maximum subpopulation AF is ~375-fold below the 1% operating threshold and ~1,800-fold below the ACMG 2015 5% threshold, so BA1 is clearly not met.

Rule

generic_acmg_2015_population (PMID 25741868): BA1 requires AF > 0.05; pipeline non-VCEP gnomAD operating threshold BA1 > 1%

Evidence considered

• gnomAD v4.1 (gnomad_v4): total AF 3.10757e-06 (5/1,608,972 alleles; exome 4/1,456,720; genome 1/152,252); highest subpopulation AF 2.66987e-05 (African/African American, 2/74,910); grpmax FAF 4.43e-06; 0 homozygotes
• gnomAD v2.1 (gnomad_v2): variant absent
• BA1 threshold AF > 5% per ACMG/AMP 2015 (PMID 25741868); pipeline non-VCEP operating threshold BA1 > 1% — neither approached by any observed population frequency

Sources

Reasoning

BS1 (supporting benign) requires an allele frequency greater than expected for the disorder. BAP1 tumor predisposition syndrome (BAP1-TPDS) is a rare, autosomal dominant, high-penetrance cancer predisposition syndrome, so disease-causing alleles are expected at frequencies far below 0.1% in the general population. The observed maximum subpopulation AF is 2.66987e-05 (0.00267%, African/African American) and the total AF is 3.10757e-06 (0.00031%), which is roughly two orders of magnitude below any reasonable 'greater than expected' threshold, including this pipeline's non-VCEP operating threshold of BS1 > 0.3%. BS1 is not met.

Rule

generic_acmg_2015_population (PMID 25741868): BS1 requires AF greater than expected for the disorder; pipeline non-VCEP gnomAD operating threshold BS1 > 0.3%

Evidence considered

• gnomAD v4.1 (gnomad_v4): maximum subpopulation AF 2.66987e-05 (AFR, 0.00267%) and total AF 3.10757e-06 (0.00031%) — far below any 'greater than expected for disorder' frequency for a rare autosomal dominant cancer syndrome
• gnomAD v2.1 (gnomad_v2): variant absent, inconsistent with a BS1-level population frequency
• BS1 per ACMG/AMP 2015 (PMID 25741868): AF greater than expected for the disorder; pipeline non-VCEP operating threshold BS1 > 0.3% — not approached

Sources

Reasoning

BS2 (strong benign) requires observation of the variant in healthy individuals in a pattern inconsistent with disease expression: homozygosity for a recessive disorder, or presence in healthy adults for a dominant disorder with complete (or near-complete) penetrance. gnomAD v4.1 reports zero homozygotes across all populations (0/1,608,972 alleles), the total AF is only 3.10757e-06, and no healthy-adult cohort enrichment or homozygote observation exists for this variant. Moreover, gnomAD is not a phenotypically screened healthy-adult cohort, so it cannot by itself support BS2. BS2 is not met.

Rule

generic_acmg_2015_population (PMID 25741868): BS2 requires observation in healthy adults (homozygotes for recessive, or healthy heterozygotes for dominant) inconsistent with disease

Evidence considered

• gnomAD v4.1 (gnomad_v4): 0 homozygotes in every population (total 5/1,608,972 alleles); no BS2-supportive observation of the variant in healthy homozygotes or at a frequency inconsistent with disease
• BS2 per ACMG/AMP 2015 (PMID 25741868): requires observation in healthy adults inconsistent with disease penetrance — no such observation available

Sources

Reasoning

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) BP2 requires the variant to have been observed in trans with a pathogenic variant for a fully dominant disease/gene, or observed in cis with a pathogenic variant in any inheritance pattern. All retrieved sources were examined for such allelic/phase observations: (1) ClinVar variation 515124 has 4 submissions (GeneDx SCV000727119 Likely benign, Ambry SCV001170879 Likely benign, Myriad SCV005406131 Benign, Labcorp SCV005692622 Likely benign) - none contains a phase/segregation annotation or reports the variant in trans or cis with a pathogenic variant; (2) the three retrieved publications were checked - PMID:25394175 (full text searched: no mention of c.126T>C, no allelic data), PMID:27748099 (title-level metadata only; no variant-specific content), PMID:28492532 (Sherloc methodology paper; no case-level variant data). No source reports the required trans/cis observation, so BP2 is not met. Per instruction, the aggregate ClinVar classifications (3x Likely benign, 1x Benign) were not used to infer this or any criterion.

Rule

ACMG/AMP 2015 (Richards et al., PMID 25741868): BP2 - 'Observed in trans with a pathogenic variant for a fully dominant disease/gene; or observed in cis with a pathogenic variant in any inheritance pattern' (supporting, benign).

Evidence considered

• generic_acmg_combination_rules (Richards et al. 2015, PMID 25741868): BP2 requires observation in trans with a pathogenic variant (fully dominant disease/gene) or in cis with a pathogenic variant
• clinvar (variation 515124, VCV000515124): 4 submissions reviewed; no phase/segregation annotations, no report of trans/cis arrangement with a pathogenic variant
• PMID:25394175 (full text, publications/25394175.txt): referral-indication guideline; searched for 'c.126T>C', 'c.126', 'p.Pro42=', 'P42' - no matches, no allelic data
• PMID:27748099 (GeneReviews BAP1-TPDS chapter): only title-level metadata available in this case; no variant-specific or phase content retrievable
• PMID:28492532 (Sherloc): abstract-level methodology paper; no mention of c.126T>C, no case-level allelic observations

Still missing

• No phase/segregation data available: no source reports the variant observed in trans with a pathogenic variant (dominant context) or in cis with a pathogenic variant; no family segregation studies were retrieved for this case.

Sources

Reasoning

BP4 (multiple lines of computational evidence suggest no impact on gene or gene product) is not met because only a single line of computational evidence is available. SpliceAI predicts no splice impact (max delta score = 0.01, all four delta scores <= 0.01; below the recommended 0.2 threshold, Jaganathan et al. 2019, PMID 30661751), which is concordant with a benign impact, but generic ACMG/AMP 2015 BP4 explicitly requires MULTIPLE lines of computational evidence. REVEL returned no score for this variant and BayesDel is both unavailable and insufficiently calibrated in this pipeline, so no second concordant predictor could be obtained. The single SpliceAI prediction was not double-counted: it is noted here as the one available line (insufficient for BP4) and is the same prediction that would be consumed by BP7, so BP4 is not awarded from it. ClinVar laboratory classifications (3x Likely benign, 1x Benign) were not used as BP4 evidence per the rule against inferring a criterion from another source's final classification.

Rule

Generic ACMG/AMP 2015 (PMID 25741868) BP4: Multiple lines of computational evidence suggest no impact on gene or gene product. Not met - only a single computational line (SpliceAI) available.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'): max delta score 0.01 (all delta scores <= 0.01), below the recommended 0.2 delta threshold (Jaganathan et al., Nature Genetics 2019, PMID 30661751) - single concordant line, insufficient alone for BP4's multiple-lines requirement
• REVEL: score not found for this variant (prefetch.json revel.found=false) - could not provide a second concordant line
• BayesDel: score not found (prefetch.json bayesdel.found=false); no verified published threshold available to this pipeline - cannot provide a second concordant line
• Generic ACMG/AMP 2015 rules (source_registry key 'generic_acmg_combination_rules', source_pmid 25741868): BP4 requires multiple lines of computational evidence suggesting no impact; only one line (SpliceAI) exists here, so BP4 is not met

Still missing

• Only one in-silico line (SpliceAI) available; BP4 requires multiple lines of computational evidence - REVEL score not found and BayesDel unavailable/uncalibrated, so no second concordant predictor exists
• No BAP1 VCEP/CSPEC computational-evidence specification (e.g., gene-specific PP3/BP4 lookup spreadsheet) was available to pre-assign BP4
• A second validated splice/missense predictor result for c.126T>C would be needed to meet BP4 under generic ACMG/AMP 2015

Sources

Reasoning

BP6 may be applied at supporting strength only when an exact-variant ClinVar expert-panel classification is Benign/Likely benign. The ClinVar record VCV000515124 has zero expert-panel submissions (expert_panel_submissions = 0; review status 1-2 stars); all four submissions (GeneDx Likely benign, Ambry Likely benign, Myriad Benign, Labcorp Likely benign) are ordinary single-submitter clinical testing assertions. Although the aggregate label is Benign/Likely benign, per the governing rule for this group the aggregate ClinVar label and ordinary laboratory assertions must never trigger BP6. Therefore BP6 is not met.

Rule

BP6 per phenotype/prevalence group rule: supporting (BP6) only for an exact-variant ClinVar expert-panel classification of Benign/Likely benign; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions never trigger BP6. Framework: generic ACMG/AMP 2015 (PMID 25741868).

Evidence considered

• ClinVar VCV000515124: 4 clinical laboratory submissions (3 Likely benign, 1 Benign), review status 'criteria provided, single submitter' / 'criteria provided, multiple submitters, no conflicts' (1-2 stars), 0 expert panel submissions.
• Submission audit confirms is_expert_panel = false for all submissions (SCV000727119, SCV001170879, SCV005406131, SCV005692622).
• Per the governing rule for this group, ordinary laboratory submissions and aggregate ClinVar labels must never trigger BP6.

Still missing

• No ClinVar expert-panel (3-star) Benign/Likely benign classification exists for NM_004656.4:c.126T>C; the available Benign/Likely benign labels come exclusively from ordinary clinical laboratories and cannot be used for BP6.

Sources

Reasoning

No proband-level de novo evidence exists for BAP1 c.126T>C (p.Pro42=) in any source in this case. ClinVar VCV000515124 (4 single-submitter clinical laboratory submissions: 3 Likely benign, 1 Benign; no expert panel) contains no PS2 signal and no submission reports a de novo occurrence or parental testing. None of the available publications (PMID:25394175, PMID:27748099, PMID:28492532) mention the variant, and the only full-text paper (PMID:25394175) contains no de novo/segregation content (searched: c.126T>C, p.Pro42=, de novo, parental, paternity, maternity, segregation - zero matches). Generic ACMG/AMP 2015 PS2 (de novo, maternity and paternity confirmed, in a patient with the disease and no family history) cannot be applied because no trio/parental genotype data for this variant is present in the evidence bundle.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) PS2: de novo (paternity and maternity confirmed) in a patient with the disease and no family history. Applied because no BAP1 CSPEC/VCEP specification exists (framework_mode: generic_acmg).

Evidence considered

• ClinVar VCV000515124: 4 submissions (GeneDx LB, Ambry LB, Myriad B, Labcorp LB); criteria signals present only for PP3 in one submission (GeneDx SCV000727119); no de novo or parental-testing evidence reported in any submission.
• PMID:25394175 (full text, 117 KB): reviewed in full; no mention of c.126T>C or p.Pro42= and no de novo/segregation/parental-testing content; practice guideline on referral indications only.
• PMID:27748099 (GeneReviews BAP1-TPDS chapter): no abstract or full text available in this case; metadata only; no variant-specific content retrievable.
• PMID:28492532 (Sherloc methods paper): background review of ACMG/AMP criteria refinement; no variant-specific or case-level segregation content.

Still missing

• No proband clinical phenotype is provided for this variant, so it cannot be established that the proband has the relevant BAP1-TPDS phenotype.
• No parental genotype testing (confirmed or unconfirmed parentage) is reported for any carrier of c.126T>C; a confirmed de novo occurrence would be required to consider PS2.

Sources

Reasoning

PS3 (well-established functional studies show a deleterious effect) cannot be assessed: no functional assay evidence for BAP1 c.126T>C (p.Pro42=) exists in any bundled source. The variant is synonymous (exon 4, NP_004647.1:p.(Pro42=)); no in-vitro/in-vivo assay, RNA/splicing assay, or BAP1 deubiquitinase activity study of this variant was retrieved. The only splice-relevant data point is SpliceAI (max delta score = 0.01), an in-silico prediction that does not constitute a well-established functional study and cannot support PS3 in either direction. No VCEP/CSPEC specification exists for BAP1, so generic ACMG/AMP 2015 (PMID 25741868) applies; under that framework PS3 requires established functional assay evidence of a deleterious effect, which is absent. Status set to not_assessed (evidence insufficient), not not_met, because no functional evidence was available to evaluate rather than evidence being present but negative.

Rule

Generic ACMG/AMP 2015 (PMID 25741868) PS3: Well-established functional studies show a deleterious effect. Not assessable - no functional assay evidence.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'): max delta score 0.01 (DS_DG 0.01) - in-silico splice prediction only, not a functional assay; consulted to confirm no splice-impact signal but insufficient for PS3
• Generic ACMG/AMP 2015 rules (source_registry key 'generic_acmg_combination_rules', source_pmid 25741868): PS3 requires well-established functional studies showing a deleterious effect; no such studies present for this variant
• Case evidence bundle: no functional assay records for c.126T>C in evidence.json, prefetch.json, or literature_pass.json (criterion_screened empty; no functional/splicing assay results)
• OncoKB entry is non-variant-specific (Unknown Oncogenic Effect, is_variant_specific=false) and provides no functional assay data for this variant

Still missing

• No functional assay (e.g., BAP1 deubiquitinase activity, protein stability, RNA splicing/minigene assay, or patient-derived cell-based assay) has been performed for c.126T>C
• No published functional study of this exact variant was found in the literature packet or retrieved publications
• SpliceAI in-silico prediction (max delta 0.01) is not a substitute for functional assay evidence and was not used to determine PS3

Sources

Reasoning

PS4 requires a statistically significant increase in the prevalence of the variant in affected individuals versus controls (case-control enrichment or equivalent cohort evidence). No case-control study, cohort prevalence estimate, or variant-specific affected-vs-control comparison exists for BAP1 c.126T>C in the evidence bundle. The only population-level data are gnomAD v4.1 frequencies (total AF 3.10757e-06, 5/1,608,972 alleles, 0 homozygotes; absent from gnomAD v2.1), which are population/control frequencies and do not constitute PS4-type enrichment evidence. The available literature (PMID:25394175 referral guideline; PMID:27748099 GeneReviews chapter; PMID:28492532 Sherloc methods paper) contains no case-control or prevalence-in-affected data for this variant. Because the required evidence class is entirely absent, PS4 is not assessed rather than affirmatively excluded.

Rule

ACMG/AMP 2015 PS4 (PMID 25741868): prevalence of the variant in affected individuals is significantly increased compared with controls; requires case-control or equivalent cohort enrichment data. Applied under generic ACMG/AMP fallback (no CSPEC/VCEP for BAP1).

Evidence considered

• gnomAD v4.1 reports total AF 3.10757e-06 (5/1,608,972 alleles, 0 homozygotes; grpmax FAF 4.43e-06); this is population allele frequency, not case-control or affected-prevalence data, and cannot establish PS4.
• gnomAD v2.1 reports the variant absent; absence from population databases does not provide affected-vs-control enrichment evidence.
• No publication in the source registry reports this variant in affected individuals with a control comparison.

Still missing

• No case-control or affected-cohort prevalence study for BAP1 c.126T>C identified; no variant-specific enrichment odds ratio or p-value available.
• No proband-level or cohort-level case data accompany this variant in the evidence bundle.

Sources

Reasoning

No de novo occurrence of c.126T>C is reported anywhere, with or without confirmed parentage. ClinVar submissions for VCV000515124 do not assert de novo origin (origins reported as germline x3 and unknown x1), and no literature in this case describes a de novo observation of this variant. Generic ACMG/AMP 2015 PM6 (assumed de novo without confirmation of paternity and maternity) therefore cannot be evaluated; the gap is absence of any de novo occurrence report, not a judgment on parentage confirmation.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) PM6: assumed de novo, but without confirmation of paternity and maternity. Applied because no BAP1 CSPEC/VCEP specification exists (framework_mode: generic_acmg).

Evidence considered

• ClinVar VCV000515124 submission audit: SCV000727119 (germline), SCV001170879 (germline), SCV005406131 (origin unknown), SCV005692622 (germline); no submission records a de novo origin or parental testing.
• PMID:25394175: full-text review found no variant mention and no de novo/parental-testing content.
• PMID:27748099 and PMID:28492532: no variant-specific or case-level data available (no abstract/full text; methods paper respectively).

Still missing

• No report of a de novo occurrence of c.126T>C in any proband; without a documented de novo event, PM6 cannot be met.
• No parental testing results (even with unconfirmed parentage) are available for any carrier.

Sources

Reasoning

No co-segregation data for c.126T>C exists in this case. None of the four ClinVar submissions report segregation testing in affected family members, and no publication available here describes the variant segregating with BAP1-TPDS-related disease. Generic ACMG/AMP 2015 PP1 (co-segregation with disease in multiple affected family members) cannot be applied: the absence of any affected-family segregation data is an evidence gap, not evidence against segregation.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) PP1: co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Applied because no BAP1 CSPEC/VCEP specification exists (framework_mode: generic_acmg).

Evidence considered

• ClinVar VCV000515124: no submission contains a PP1/segregation signal (criteria signals found: PP3 only in SCV000727119; others none).
• PMID:25394175: full-text review found no segregation or variant-specific content (searched: segregation, c.126T>C, p.Pro42= - zero matches).
• PMID:27748099, PMID:28492532: no case-level segregation data available in this case.

Still missing

• No segregation testing of c.126T>C in affected relatives is reported; PP1 requires segregation in multiple affected family members.
• No family-history or pedigree data for any carrier of this variant is present in the bundle (ClinVar submissions carry no segregation assertions).

Sources

Reasoning

PP4 requires a phenotype or family history that is highly specific for a disease caused by a single genetic etiology. The evidence bundle contains no proband phenotype description, clinical history, or family history data whatsoever for this case, so the phenotype-specificity of the presentation cannot be evaluated. In addition, even at the gene level, BAP1 tumor predisposition syndrome manifests as a spectrum of multiple cancer types (uveal/cutaneous melanoma, mesothelioma, renal cell carcinoma, meningioma) that overlaps other hereditary cancer syndromes and would not ordinarily satisfy the 'highly specific, single genetic etiology' requirement of PP4. With no case-level phenotype data, PP4 is not assessed.

Rule

ACMG/AMP 2015 PP4 (PMID 25741868): phenotype or family history highly specific for a disease with a single genetic etiology. Applied under generic ACMG/AMP fallback (no CSPEC/VCEP for BAP1).

Evidence considered

• No case-level phenotype or family-history data present in review_manifest.json, case_summary.json, or prefetch.json.
• Gene-level sources (PMID:27748099 GeneReviews BAP1-TPDS chapter) describe the syndrome spectrum but provide no variant-specific phenotype evidence for this case and do not establish phenotype specificity for this proband.

Still missing

• No proband phenotype or family history information provided in the case evidence bundle.
• No clinical description (age of onset, tumor types, syndromic features) is available to judge phenotype specificity for BAP1-TPDS.

Sources

Reasoning

BS3 (well-established functional studies show no damaging effect on protein function or splicing) cannot be met: no well-established functional study of BAP1 c.126T>C (p.Pro42=) was retrieved. SpliceAI predicts no significant splice impact (max delta score = 0.01), but this is an in-silico splicing prediction, not a functional assay; under ACMG/AMP 2015 BS3 requires well-established functional studies (e.g., RNA/minigene splicing assays, protein function assays), which are absent. The variant's synonymous nature and benign ClinVar submissions (3x Likely benign, 1x Benign; ClinVar ID 515124) were NOT used to infer BS3 per instruction not to infer a criterion from another source's final classification. Status set to not_assessed (evidence insufficient) rather than not_met because the required functional-assay evidence class was never available to evaluate.

Rule

Generic ACMG/AMP 2015 (PMID 25741868) BS3: Well-established functional studies show no damaging effect on protein function or splicing. Not assessable - no functional assay evidence.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'): max delta score 0.01 - in-silico splice prediction; noted as consistent with no splice impact but does not meet BS3's well-established-functional-study requirement
• Generic ACMG/AMP 2015 rules (source_registry key 'generic_acmg_combination_rules', source_pmid 25741868): BS3 requires well-established functional studies showing no damaging effect; none available for this variant
• ClinVar classification (source_registry key 'clinvar') deliberately NOT used as BS3 evidence - final classifications from other submitters are not independent functional evidence
• Case evidence bundle: no functional assay records (RNA, protein, or cellular) for c.126T>C in evidence.json, prefetch.json, or literature_pass.json

Still missing

• No well-established functional study demonstrating normal splicing or normal BAP1 protein function for c.126T>C (e.g., RNA-seq/minigene assay, BAP1 deubiquitinase activity assay)
• SpliceAI max delta 0.01 is in-silico only; a validated RNA/splicing assay would be required to support BS3
• No BAP1-specific VCEP/CSPEC functional assay calibration exists (no cspec available; generic ACMG/AMP 2015 fallback)

Sources

Reasoning

BS4 (lack of segregation in affected members of a family) requires documented non-segregation of the variant with disease. No family segregation data of any kind (positive or negative) is available for c.126T>C in this case: ClinVar submissions report no segregation results and the available literature contains no variant-specific segregation analyses. Absence of data cannot be scored as BS4. Note: the gnomAD v4.1 population frequency (5/1,608,972 alleles, grpmax FAF 4.43e-06) is population-frequency evidence for BS1/BA1 adjudication (handled by another group), not BS4, and was not used here.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) BS4: lack of segregation in affected members of a family. Applied because no BAP1 CSPEC/VCEP specification exists (framework_mode: generic_acmg).

Evidence considered

• ClinVar VCV000515124: no submission reports segregation or non-segregation results.
• PMID:25394175: full-text review found no segregation/non-segregation or variant-specific content.
• PMID:27748099, PMID:28492532: no family segregation data available in this case.

Still missing

• No affected-family segregation study for c.126T>C is available; BS4 needs documented non-segregation (e.g., affected relative(s) who do not carry the variant).
• No pedigree/segregation assertions are provided in any ClinVar submission for VCV000515124.

Sources

Reasoning

BP5 applies when a variant is found in a case with an alternate molecular basis for the disease. The evidence bundle contains no case-level data: there is no proband, no reported additional pathogenic variant or alternate molecular explanation, and no segregation/testing context. Without any case-level information indicating an alternate molecular basis of disease, BP5 cannot be assessed.

Rule

ACMG/AMP 2015 BP5 (PMID 25741868): variant found in a case with an alternate molecular basis for disease. Applied under generic ACMG/AMP fallback (no CSPEC/VCEP for BAP1).

Evidence considered

• No source in the bundle reports an alternate molecular basis of disease for any case carrying this variant.

Still missing

• No case-level data in the evidence bundle; results of proband testing for alternate molecular causes (e.g., a second pathogenic variant or another explanatory mechanism) are unavailable.
• No proband is described, so the presence or absence of an alternate molecular basis cannot be determined.

Reasoning

BP7 (synonymous variant with no predicted splice impact AND nucleotide not highly conserved) cannot be affirmatively met: two of its three conditions are satisfied, but the conservation condition is unverifiable from the case bundle. (1) The variant is synonymous: NM_004656.4:c.126T>C => NP_004647.1:p.(Pro42=), confirmed by Mutalyzer/VariantValidator normalization and by pvs1_variant_assessment (consequence_class='synonymous'; variant is in exon 4, not in the canonical splice consensus, canonical_splice_consensus=false). (2) Splicing prediction algorithms predict no impact: SpliceAI max delta score = 0.01 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.0), far below the recommended 0.2 delta threshold (Jaganathan et al. 2019, PMID 30661751); no alternate splice-site creation or consensus disruption is predicted. (3) The required condition that the nucleotide is NOT highly conserved cannot be established: no nucleotide conservation data (e.g., phyloP, GERP, or equivalent) is present anywhere in the evidence bundle. Because BP7's conservation sub-condition is a genuine requirement under generic ACMG/AMP 2015 (PMID 25741868) and no data exists to satisfy it, the criterion is returned not_assessed with this explicit gap rather than met. The same SpliceAI prediction was not additionally used to meet BP4 (single line, see BP4) to avoid double-counting. ClinVar classifications were not used per the no-inference rule.

Rule

Generic ACMG/AMP 2015 (PMID 25741868) BP7: A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new alternate splice site AND the nucleotide is not highly conserved. Not assessable - nucleotide conservation data missing.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'): max delta score 0.01 (all delta scores <= 0.01), below the recommended 0.2 delta threshold (Jaganathan et al., Nature Genetics 2019, PMID 30661751) - satisfies BP7's no-splice-impact condition
• Variant consequence (source_registry key 'pvs1_variant_assessment' and prefetch normalization): NM_004656.4:c.126T>C is synonymous, NP_004647.1:p.(Pro42=), exon 4, not in canonical splice consensus (canonical_splice_consensus=false) - satisfies BP7's synonymous condition
• Generic ACMG/AMP 2015 rules (source_registry key 'generic_acmg_combination_rules', source_pmid 25741868): BP7 requires (a) synonymous variant, (b) splicing prediction algorithms predict no impact to splice consensus or new alternate splice site, AND (c) nucleotide is not highly conserved; condition (c) unverifiable from available evidence
• Case evidence bundle: no phyloP/GERP/conservation score for this position found in prefetch.json, evidence.json, or case_summary.json - conservation condition cannot be established

Still missing

• Nucleotide conservation data (phyloP/GERP/UCSC conservation track or equivalent) for the c.126 position (genomic chr3:g.52442619A>G GRCh37 / g.52408603A>G GRCh38) is absent from the case bundle; BP7 requires the nucleotide to be NOT highly conserved and this cannot be affirmed
• Human review is requested: a conservation lookup would allow BP7 to be finalized (met if the position is not highly conserved; the splice-impact and synonymous conditions are already satisfied)
• No BAP1 VCEP/CSPEC variant of this type exists; generic ACMG/AMP 2015 BP7 wording applied

Sources