TSC1 c.2865C>T, p.(Thr955=)

Reasoning

BS1 (Richards et al. 2015, PMID:25741868) is met when the allele frequency in a control/general population is greater than expected for the disorder. TSC is an autosomal dominant disorder with near-complete penetrance and an estimated prevalence of ~1/6,000-1/10,000; TSC1 accounts for roughly one-third of TSC cases, so the maximum credible population allele frequency for a single disease-causing TSC1 allele is on the order of 0.006% or less (maximum credible AF approach, Whiffin et al. 2017, Genet Med 19:1151-1158, PMID:28518168). The observed frequency of this variant is ~40-50x higher than that expectation in the largest populations: NFE AF 0.316% (gnomAD v4.1), grpmax FAF 0.308% (gnomAD v4.1), NFE AF 0.285% (gnomAD v2.1), grpmax FAF 0.299% (gnomAD v2.1). A single synonymous allele at this frequency would imply far more TSC carriers than the disorder's prevalence allows, so the frequency is clearly greater than expected for a disease-causing allele. Note: against the pipeline's numeric BS1 calibration of 0.3%, gnomAD v4.1 NFE AF (0.316%) and grpmax FAF (0.308%) exceed the threshold while gnomAD v2.1 NFE AF (0.285%) falls marginally below it; however, both datasets exceed the biologically derived maximum credible disease-allele frequency by ~40-50x, so BS1 is met regardless of dataset choice. BS1 is applied at strong strength per ACMG/AMP 2015 weighting.

Rule

BS1 (Richards et al. 2015, PMID:25741868): allele frequency greater than expected for the disorder (strong benign)

Evidence considered

• gnomAD v4.1 (source: gnomad_v4): NFE AF 0.003161 (3730/1180034 alleles), grpmax FAF 0.003076 - exceeds maximum credible disease-allele AF for TSC1 (~0.00006, i.e. ~0.006%) by ~50x
• gnomAD v2.1 (source: gnomad_v2): NFE AF 0.002849 (368/129158), grpmax FAF 0.002993 - exceeds maximum credible TSC1 disease-allele AF by ~45x
• Maximum credible allele frequency approach (Whiffin et al. 2017, Genet Med 19(10):1151-1158, PMID:28518168): AF_max for a single TSC1 pathogenic allele estimated from TSC prevalence ~1/6,000-1/10,000 (autosomal dominant, near-full penetrance) and TSC1 causal fraction ~1/3, giving AF_max ~0.006% or less
• BS1 rule (Richards et al. 2015, PMID:25741868, full text): 'an allele frequency in a control population that is greater than expected for the disorder (Table 6) is considered strong support for a benign interpretation for a rare Mendelian disorder (BS1)'

Still missing

• No VCEP-specified BS1 frequency threshold exists for TSC1; adjudication relies on the qualitative ACMG/AMP 2015 rule plus maximum credible AF reasoning (Whiffin et al. 2017, PMID:28518168)

Sources

Reasoning

BS2 (Richards et al. 2015, PMID:25741868) is met when the variant is observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, or in a healthy adult for a disorder with full penetrance at an early age. TSC is an autosomal dominant disorder that is essentially fully penetrant and manifests in early childhood (multi-organ hamartomas, epilepsy, intellectual disability). This variant is observed in the homozygous state in 10 individuals in gnomAD v4.1 (9 European non-Finnish, 1 Admixed American; 8 exome + 2 genome homozygotes among 1,614,154 alleles) and in 1 homozygote in gnomAD v2.1 (NFE exome). Homozygosity for a truly pathogenic TSC1 allele would be expected to produce severe, early-onset disease and would not be anticipated in a general-population cohort; the observation of 10 homozygotes is strong evidence of benignity. BS2 is applied at strong strength per ACMG/AMP 2015 weighting. gnomAD is not a strictly healthy cohort, but the homozygote count far exceeds any plausible expectation for a pathogenic dominant allele; ClinVar's 2-star Benign/Likely benign consensus (25 submissions, criteria provided, no conflicts) is consistent with this assessment but was not used to establish the criterion.

Rule

BS2 (Richards et al. 2015, PMID:25741868): observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, or in a healthy adult for a disorder with full penetrance at an early age (strong benign)

Evidence considered

• gnomAD v4.1 (source: gnomad_v4): total homozygotes = 10 (9 NFE, 1 AMR; exome 8, genome 2) among 1,614,154 alleles - homozygous observation in a general population cohort for a fully penetrant dominant disorder
• gnomAD v2.1 (source: gnomad_v2): total homozygotes = 1 (NFE exome) among 282,842 alleles
• gnomAD-Canada v1.0 (source: gnomad_canada): 0 homozygotes (24/18420 alleles) - no contradiction
• BS2 rule (Richards et al. 2015, PMID:25741868, full text): 'if the disease under investigation is fully penetrant at an early age and the variant is observed in a well-documented healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) condition then this is considered strong evidence for a benign interpretation (BS2)'

Still missing

• gnomAD v4.1 includes some affected individuals and is not a strictly healthy cohort; however, 10 homozygotes in a general-population cohort is accepted practice for BS2 in fully penetrant dominant disorders

Sources

Reasoning

No case-control enrichment evidence exists for NM_000368.5:c.2865C>T (p.Thr955=). The only case-series source that mentions this exact variant, PMID:10533067 (germline TSC1/TSC2 analysis of 126 unrelated TSC patients), lists it under 'TSC1 Polymorphisms' (older nomenclature 3086C>T, codon T955, synonymous) at 0.009 (2/112 chromosomes) with no odds ratio or case-control statistics; the authors explicitly classify it as a polymorphism. The large TSC diagnostic cohort PMID:15798777 (490 patients) does not report this variant as disease-associated at all. gnomAD v2.1 shows total AF 0.16688% (472/282842 alleles, 1 homozygote) and gnomAD v4.1 shows total AF 0.25252% (4076/1614154 alleles, 10 homozygotes), i.e. the variant is common in general populations, which is incompatible with a statistically significant increase in prevalence among TSC cases versus controls. PS4 therefore not met.

Rule

ACMG/AMP 2015 (PMID:25741868), PS4 (strong): prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls (case-control enrichment).

Evidence considered

• PMID:10533067: TSC1/TSC2 mutational analysis of 126 unrelated TSC patients lists 3086C>T (T955, silent change) under 'TSC1 Polymorphisms', frequency 0.009 (2/112 chromosomes); no case-control enrichment analysis or OR reported; variant treated as a polymorphism by the authors.
• PMID:15798777: diagnostic TSC cohort of 490 patients; full text contains no occurrence of c.2865C>T / p.Thr955= / T955, i.e., the variant is not reported as disease-associated in this large case series.
• gnomad_v2: exact variant 9-135772681-G-A total AF 0.00166878 (0.16688%, 472/282842 alleles), 1 homozygote, grpmax FAF 0.00299349.
• gnomad_v4: exact variant chr9-132897294-G-A total AF 0.00252516 (0.25252%, 4076/1614154 alleles), 10 homozygotes, grpmax FAF 0.00307583.

Still missing

• No formal case-control study (affected vs. control cohorts) reporting allele counts and an odds ratio/confidence interval for c.2865C>T is available in the case bundle.

Sources

Reasoning

PM2 (Richards et al. 2015, PMID:25741868) requires the variant to be absent from (or below the expected carrier frequency if recessive) a large race-matched general population or control cohort. For this non-VCEP case the pipeline calibration threshold is AF <= 0.1% (0.001) in gnomAD. The variant is present at AF 0.2525% overall (gnomAD v4.1), 0.1669% overall (gnomAD v2.1), with a maximum subpopulation frequency of 0.3161% (NFE, gnomAD v4.1) and 10 homozygotes - i.e. it is common rather than absent or extremely rare. The population-frequency evidence does not support the rarity required for PM2; instead it positively supports benign population criteria (BS1, BS2). PM2 is not met.

Rule

PM2 (Richards et al. 2015, PMID:25741868): absent (or below expected carrier frequency if recessive) in a large race-matched general population or control cohort (pathogenic; commonly applied at supporting strength)

Evidence considered

• gnomAD v4.1 (source: gnomad_v4): total AF 0.002525 (0.253%) > 0.1% PM2 calibration threshold; NFE AF 0.003161 (0.316%)
• gnomAD v2.1 (source: gnomad_v2): total AF 0.001669 (0.167%) > 0.1% PM2 calibration threshold
• gnomAD-Canada v1.0 (source: gnomad_canada): AF 0.001303 (0.130%) > 0.1%
• PM2 rule (Richards et al. 2015, PMID:25741868, full text): 'If a variant is absent from (or below the expected carrier frequency if recessive) a large general population or a control cohort (>1000 individuals) and the population is race-matched to the patient harboring the identified variant, then this observation can be considered a moderate piece of evidence for pathogenicity (PM2)' - variant is present at population frequency far above the rarity required
• PM2 non-VCEP gnomAD calibration threshold AF <= 0.1% (pipeline standard for absence/extreme rarity)

Sources

Reasoning

PP3 requires multiple lines of computational evidence supporting a deleterious effect on the gene or gene product. The only applicable computational evidence for this synonymous variant (VEP most severe consequence = synonymous_variant; p.(Thr955=)) is the splice-impact path: SpliceAI (SpliceAI Lookup, Broad Institute) reports max delta score 0.17 (DS_AG=0.0, DS_DG=0.0, DS_AL=0.17, DS_DL=0.1), below the published 0.20 'possibly splice-altering' threshold (Jaganathan et al. 2019, PMID 30661751), so no splice impact is predicted. The missense in-silico path (REVEL, BayesDel, aGVGD) is both unavailable in this case (REVEL found:false; BayesDel found:false and lacking any verified published threshold; aGVGD absent) and inapplicable to a synonymous variant. No computational line supports a deleterious effect; PP3 is not met.

Rule

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.) - Richards et al. 2015 (PMID:25741868).

Sources

Reasoning

PP5 is applied only for an exact-variant ClinVar expert-panel classification of Pathogenic/Likely pathogenic. ClinVar record VCV000049008 for NM_000368.5(TSC1):c.2865C>T (p.Thr955=) contains 27 submissions, all from ordinary clinical laboratories/research programs (review status 'criteria provided, single submitter' or 'no assertion criteria provided'); the submission audit reports expert_panel_submissions = 0 and the ClinVar expert_panels object is empty. No expert-panel Pathogenic/Likely pathogenic classification exists for this variant (the aggregate classification is Benign/Likely benign, 2 stars). PP5 therefore not met.

Rule

ACMG/AMP 2015 (PMID:25741868), PP5 (supporting): reputable source recently reports variant as pathogenic; restricted per governing instruction to exact-variant ClinVar expert-panel Pathogenic/Likely pathogenic classifications only.

Evidence considered

• clinvar (VCV000049008): aggregate classification 'Benign/Likely benign'; review status 'criteria provided, multiple submitters, no conflicts' (2 stars); 27 submissions from 26 submitters, 0 expert-panel submissions (expert_panels: {}); no Pathogenic/Likely pathogenic expert-panel assertion exists for this exact variant.

Sources

Reasoning

BA1 (Richards et al. 2015, PMID:25741868) requires an allele frequency >5% in a large general/control population database for stand-alone benign evidence. The highest observed frequency for NM_000368.5:c.2865C>T (TSC1, p.Thr955=, rs45468995) is in European (non-Finnish) gnomAD v4.1 at AF 0.00316 (0.316%), with grpmax FAF 0.00308 (0.308%); gnomAD v2.1 NFE AF is 0.00285 (0.285%) with grpmax FAF 0.00299 (0.299%); gnomAD-Canada AF is 0.00130 (0.130%). Every observed frequency is at least an order of magnitude below the 5% BA1 threshold (and below the pipeline's 1% BA1 calibration). BA1 is not met.

Rule

BA1 (Richards et al. 2015, PMID:25741868): allele frequency >5% in a large general/control population database (stand-alone benign)

Evidence considered

• gnomAD v4.1 (source: gnomad_v4): total AF 0.002525 (4076/1614154 alleles), NFE AF 0.003161 (3730/1180034), grpmax FAF 0.003076 - all far below 5% BA1 threshold
• gnomAD v2.1 (source: gnomad_v2): total AF 0.001669 (472/282842), NFE AF 0.002849, grpmax FAF 0.002993 - far below 5%
• gnomAD-Canada v1.0 (source: gnomad_canada): AF 0.001303 (24/18420) - far below 5%
• BA1 threshold >5% per Richards et al. 2015 ACMG/AMP guidelines (PMID:25741868, full text: 'if over 5% then it is considered as stand-alone support (BA1)')

Sources

Reasoning

BP4 requires MULTIPLE lines of computational evidence suggesting no impact on the gene or gene product. The only no-impact computational line available for this synonymous variant is SpliceAI (max delta 0.17 < 0.20, PMID 30661751). REVEL and BayesDel were not found in the case bundle (found:false), BayesDel additionally lacks a verified published threshold, and aGVGD is absent; moreover these missense tools do not apply to a synonymous variant. Per this group's no-double-count rule, the single SpliceAI no-impact prediction is allocated to BP7 (the synonymous-specific criterion) and cannot simultaneously satisfy BP4's multiple-line requirement. With only one independent line available, BP4 is not met.

Rule

BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.) - Richards et al. 2015 (PMID:25741868).

Still missing

• Only one independent computational no-impact line (SpliceAI) is available; REVEL/BayesDel/aGVGD are absent or inapplicable for this synonymous variant, so the 'multiple lines' requirement of BP4 cannot be satisfied from the current case bundle.

Sources

Reasoning

BP6 is applied only for an exact-variant ClinVar expert-panel classification of Benign/Likely benign. Although ClinVar VCV000049008 does carry an aggregate 'Benign/Likely benign' label (18 Benign + 7 Likely benign among 27 submissions, 2 stars, no conflicts), the submission audit shows expert_panel_submissions = 0 and expert_panels: {} — every submission is an ordinary laboratory/clinical-testing assertion (e.g., Athena Diagnostics, Illumina, GeneDx, Invitae/Labcorp, Mayo Clinic, Ambry, Myriad, ARUP, PreventionGenetics) or a no-criteria submission. Under the governing instruction, ordinary laboratory submissions and aggregate ClinVar labels must never trigger BP6. BP6 therefore not met despite the benign reputation; the aggregate benign label is documented here but not counted as a criterion.

Rule

ACMG/AMP 2015 (PMID:25741868), BP6 (supporting): reputable source recently reports variant as benign; restricted per governing instruction to exact-variant ClinVar expert-panel Benign/Likely benign classifications only.

Evidence considered

• clinvar (VCV000049008): aggregate 'Benign/Likely benign' (2 stars, 27 submissions, 26 submitters, no conflicts); all 27 submissions are non-expert-panel laboratory assertions (is_expert_panel = false for every submission in clinvar_submission_audit.json); no ClinGen or other expert-panel classification exists for this exact variant.

Sources

Reasoning

No proband-level de novo observation of TSC1 c.2865C>T is reported anywhere in the case evidence bundle. The ClinVar record (VCV000049008, 27 submissions, all Benign/Likely benign or no-assertion) contains no de novo or parental-testing assertion for this variant. The only de novo-themed literature source in the bundle (PMID:15798777, Sancak et al. 2005, 490-patient TSC diagnostic cohort) was reviewed in full text and does not mention c.2865C>T / p.Thr955=, so no variant-specific de novo occurrence with confirmed paternity/maternity exists. Without a documented de novo event, PS2 cannot be applied.

Rule

ACMG/AMP 2015 PS2: De novo (paternity and maternity confirmed) in a patient with the disease and no family history (Richards et al., PMID 25741868). Applied via generic ACMG fallback because no TSC1 ClinGen CSPEC/VCEP exists.

Evidence considered

• PMID:15798777 (Sancak et al. 2005, EJHG; TSC1/TSC2 diagnostic cohort of 490 patients, de novo-themed; full text searched for c.2865C>T/p.Thr955= — variant not mentioned, no variant-specific de novo data)
• clinvar (VCV000049008, TSC1 c.2865C>T; 27 submissions Benign/Likely benign; no de novo or parental-testing assertion recorded)

Still missing

• No de novo observation of c.2865C>T in any proband with confirmed paternity and maternity (no parental testing data in any source in the bundle)

Sources

Reasoning

PS3 requires well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (ACMG/AMP 2015, PMID 25741868). No functional assay evidence for TSC1 c.2865C>T (p.Thr955=) exists anywhere in the case evidence bundle or the available full texts. The variant is synonymous and located mid-exon 21 (c.2843-3030), away from canonical splice consensus; no expression, splicing (RT-PCR/minigene), immunoblot, or other wet-lab study of this variant was identified. The papers triaged as 'functional' in literature_pass.json were reviewed and contain no variant-specific assay: PMID 24033266 and PMID 26467025 are generic variant-scoring-system papers that do not mention TSC1 or this variant; PMID 15798777 (TSC diagnostic cohort) does not mention this variant or any assay of it. SpliceAI (max delta score 0.17) is an in silico splice predictor, not a functional assay, and cannot support PS3. With no well-established functional study available, PS3 is not assessable.

Rule

PS3 - Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (ACMG/AMP 2015, Richards et al., PMID 25741868)

Evidence considered

• PMID:25741868 (ACMG/AMP 2015 standards; defines PS3 as requiring well-established functional studies supportive of a damaging effect — no such study exists for this variant)
• spliceai (SpliceAI max delta score 0.17 for NM_000368.5:c.2865C>T; in silico splice prediction only, not functional assay evidence for PS3)

Still missing

• No well-established in vitro/in vivo functional study of TSC1 c.2865C>T (p.Thr955=) identified; a variant-specific functional assay (e.g., splicing/minigene assay or a TSC1 function assay such as mTORC1/S6K readout) would be required to assess PS3.

Sources

Reasoning

No de novo occurrence of TSC1 c.2865C>T is reported in any proband in the bundle, with or without confirmation of parental identity. The ClinVar record contains no de novo assertions, and the de novo-themed full-text source reviewed (PMID:15798777) does not mention this variant. In the absence of any reported de novo event, PM6 (assumed de novo without confirmation of parentage) cannot be applied.

Rule

ACMG/AMP 2015 PM6: Assumed de novo, but without confirmation of paternity and maternity (Richards et al., PMID 25741868). Applied via generic ACMG fallback because no TSC1 ClinGen CSPEC/VCEP exists.

Evidence considered

• PMID:15798777 (Sancak et al. 2005; reviewed in full text for any de novo report of c.2865C>T — variant not mentioned)
• clinvar (VCV000049008; 27 submissions, no de novo assertion recorded)

Still missing

• No de novo observation of c.2865C>T reported in any proband (even without paternity/maternity confirmation)

Sources

Reasoning

No co-segregation data exist for TSC1 c.2865C>T. The only source mentioning the variant, PMID:10533067 (Niida et al.), reports it in Table 4 as a silent TSC1 polymorphism (their numbering 3086C>T -> T955, 2/112 chromosomes) among 126 unrelated TSC patients, but performs no segregation analysis for this variant and reports no affected-family-member testing. PMID:15798777 does not mention the variant. Without segregation data in multiple affected family members, PP1 cannot be applied; the variant's status as a common synonymous polymorphism (gnomAD AF ~0.17-0.25%) makes pathogenic co-segregation unlikely, but that is not PP1 evidence.

Rule

ACMG/AMP 2015 PP1: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease (Richards et al., PMID 25741868). Applied via generic ACMG fallback because no TSC1 ClinGen CSPEC/VCEP exists.

Evidence considered

• PMID:10533067 (Niida et al., Hum Mutat 2001; Table 4 lists c.3086C>T -> T955 as a silent TSC1 polymorphism, 2/112 chromosomes; no segregation testing reported for this variant)
• PMID:15798777 (Sancak et al. 2005; full text searched — variant not mentioned, no segregation data)
• clinvar (VCV000049008; no segregation assertion recorded in any submission)

Still missing

• No segregation analysis of c.2865C>T in affected family members in any source (no affected-relative genotype data in the bundle)

Sources

Reasoning

PP4 requires a phenotype or family history highly specific for a disease with a single genetic etiology (TSC). No proband-level phenotype or family-history data for this exact variant is present anywhere in the case bundle: the case is variant-centric with no clinical case description, and the ClinVar submissions (VCV000049008) carry only testing indications (e.g., 'Tuberous sclerosis 1', 'Hereditary cancer-predisposing syndrome', 'not provided', 'not specified') rather than documented phenotypes of carriers. There is therefore no phenotypic evidence on which to evaluate PP4; the criterion is not assessed and the gap is identified.

Rule

ACMG/AMP 2015 (PMID:25741868), PP4 (supporting): phenotype or family history highly specific for a disease with a single genetic etiology.

Still missing

• No proband phenotype or family-history data for this exact variant (c.2865C>T / p.Thr955=) is available in the case bundle; a clinical case description with a TSC-specific phenotype would be required to evaluate PP4.

Reasoning

BS3 requires well-established functional studies showing no damaging effect on protein function or splicing (ACMG/AMP 2015, PMID 25741868). No well-established functional study of TSC1 c.2865C>T (p.Thr955=) exists in the evidence bundle or the available full texts, so the criterion cannot be applied. The following do NOT qualify as functional assay evidence for BS3: (1) the synonymous consequence itself — a synonymous call is not a functional study; (2) SpliceAI max delta score 0.17 — in silico prediction, and per adjudication rules an in silico score without a named published threshold is treated as not_available, and in any case in silico prediction falls under BP4/BP7 (other groups), not BS3; (3) ClinVar Benign/Likely benign classifications from 24 submissions — final classifications from other sources are never used to infer criteria; (4) population frequency (gnomAD AF ~0.25%) — this is BA1/BS1 territory, not functional evidence; (5) OncoKB 'Unknown Oncogenic Effect' with is_variant_specific=false. With no variant-specific functional study available, BS3 is not assessable.

Rule

BS3 - Well-established functional studies show no damaging effect on protein function or splicing (ACMG/AMP 2015, Richards et al., PMID 25741868)

Evidence considered

• PMID:25741868 (ACMG/AMP 2015 standards; defines BS3 as requiring well-established functional studies showing no damaging effect — no such study exists for this variant)
• spliceai (SpliceAI max delta score 0.17; consulted to check the splicing angle but in silico prediction is not a well-established functional study and cannot satisfy BS3)

Still missing

• No well-established functional study demonstrating normal protein function or splicing for TSC1 c.2865C>T; a variant-specific assay (e.g., splicing/minigene assay or TSC1 functional readout) would be required to apply BS3.

Sources

Reasoning

BS4 requires demonstrated lack of segregation, i.e., affected family members who do not carry the variant. No such data exist for c.2865C>T in the bundle. PMID:10533067 classifies the variant as a benign silent TSC1 polymorphism found in affected TSC patients (2/112 chromosomes), which is case-observation/population-frequency evidence relevant to other criteria (e.g., BA1/BS1/BP7), but the paper reports no testing of affected relatives for this variant, so it does not constitute a segregation (non-segregation) test. The ClinVar record carries no segregation assertions. BS4 therefore cannot be met or refuted from available evidence.

Rule

ACMG/AMP 2015 BS4: Lack of segregation in affected family members (Richards et al., PMID 25741868). Applied via generic ACMG fallback because no TSC1 ClinGen CSPEC/VCEP exists.

Evidence considered

• PMID:10533067 (Niida et al., Hum Mutat 2001; variant reported as TSC1 polymorphism in affected TSC patients, but no affected-relative segregation testing reported — cannot support BS4)
• clinvar (VCV000049008; no segregation/non-segregation assertion recorded in any submission)

Still missing

• No affected-family-member genotype data for c.2865C>T (no demonstration that an affected relative lacks the variant)

Sources

Reasoning

BP2 (ACMG/AMP 2015) is applicable in principle: TSC1 is a fully dominant disease gene, so 'observed in trans with a pathogenic variant for a fully dominant disease/mechanism; or observed in cis with a pathogenic variant in any inheritance pattern' would constitute benign evidence. However, the consolidated case-evidence bundle contains no phase or co-occurrence data whatsoever: no proband observation with a second (pathogenic) TSC1 or TSC2 variant, no parental segregation/phase testing, no cis/trans determination in any ClinVar submission (all 27 submissions record only the single-variant classification), and no such information in the retrieved literature (PMID:10533067 Table 4 reports the T955 synonymous polymorphism in a TSC diagnostic cohort with allele counts only, no phase; PMID:15798777 does not report the variant). Population homozygosity in gnomAD (1 homozygote in v2.1; 10 in v4.1) is not phase-with-a-pathogenic-variant evidence and belongs to frequency-based criteria, not BP2. With no documented trans/cis observation against a pathogenic variant, BP2 cannot be evaluated; it is therefore not_assessed pending proband-level phase data.

Rule

BP2 (ACMG/AMP 2015, PMID 25741868): Observed in trans with a pathogenic variant for a fully dominant disease/mechanism; or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• PMID:25741868: ACMG/AMP 2015 BP2 definition consulted to define the evidence required (trans with pathogenic variant in fully dominant disease, or cis with pathogenic variant in any inheritance pattern).
• clinvar: all 27 ClinVar submissions for VCV000049008 reviewed; none report a second variant, phase, or co-occurrence information.
• PMID:10533067: full text reviewed; Table 4 reports the T955 synonymous polymorphism (legacy cDNA numbering c.3086C>T) at frequency 0.009 (2/112) with no phase or co-occurrence information.

Still missing

• No proband-level co-occurrence or phase data: need documentation of the variant in trans with a pathogenic TSC1/TSC2 variant in an affected individual (dominant mechanism), or in cis with any pathogenic variant, with confirmed phase (e.g., parental testing or long-read/allele-specific data).
• ClinVar submissions reviewed do not report phase or a second variant for this variant; a dedicated co-occurrence query of diagnostic laboratory data (e.g., trans observation with a known pathogenic TSC1 variant) is required.

Sources

Reasoning

BP5 requires the variant to have been found in a case with an alternative molecular basis for disease (relevant to a dominant disorder such as TSC). No case-level data for this exact variant is available in the case bundle: there are no proband descriptions, no co-occurrence observations, and no reports of an alternate cause identified in a carrier. Evidence is insufficient, so BP5 is not assessed and the gap is identified.

Rule

ACMG/AMP 2015 (PMID:25741868), BP5 (supporting): variant found in a case with an alternate molecular basis for disease.

Still missing

• No proband/case-level data demonstrating an alternative molecular basis of disease (e.g., a pathogenic variant in TSC2 or another gene) in a carrier of c.2865C>T; co-occurrence evidence would be required to evaluate BP5.

Reasoning

BP7 requires: (1) a synonymous (silent) variant, (2) splicing prediction algorithms predicting no impact to the splice consensus sequence nor creation of a new alternate splice site, AND (3) the nucleotide is not highly conserved. Conjuncts 1 and 2 are met: VEP most severe consequence = synonymous_variant (p.(Thr955=)); SpliceAI max delta 0.17 < 0.20 (threshold PMID 30661751) with DS_AG=0.0 and DS_DG=0.0, and the variant sits mid-exon 22 (c.2865; exon spans c.2814-2975; DP_AL=51 nt from the upstream acceptor, DP_DL=-110 nt from the downstream donor), i.e., outside any splice consensus region. Conjunct 3 is unverifiable from the case bundle: no conservation data (phyloP, GERP, or any other conservation score) for the c.2865 position is present in prefetch.json, evidence.json, or the retrieved publications, and no publication in the case mentions the variant. Because one required conjunct of BP7 cannot be verified, BP7 is recorded as not_assessed pending conservation data (or an applicable VCEP BP7 specification); flagged for human review.

Rule

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 - Richards et al. 2015 (PMID:25741868).

Still missing

• No conservation score (e.g., phyloP/GERP) for the c.2865 position (exon 22) is present in the case bundle; BP7's 'nucleotide is not highly conserved' conjunct is unverifiable. Resolution requires a conservation track (phyloP/GERP) or an applicable VCEP/gene-specific BP7 specification.

Sources