TSC2 c.2476C>A, p.(Leu826Met)

Reasoning

The exact TSC2 c.2476C>A variant was identified in sporadic patient S19-01 with TSC. The study states that all mutations identified in sporadic individuals were tested in the respective parents and that, apart from a different S12 mutation, none of the parents carried the mutations; it additionally states that the de novo origin of the 10 sporadic mutations was confirmed by testing both parents. This supports confirmed de novo occurrence in an affected individual and meets generic ACMG PS2 at strong strength.

Rule

Generic ACMG/AMP 2015 PS2: confirmed de novo occurrence in an affected individual with confirmed maternity and paternity; apply strong strength when the de novo event is confirmed.

Evidence considered

• PMID:9829910 reports c.2476C>A in exon 21 as a missense mutation in sporadic patient S19-01.
• PMID:9829910 states that all 11 mutations in sporadic individuals were tested in the respective parents and that none of the parents carried the TSC2 mutations except for the separate S12 familial deletion; it then states that the de novo origin of the 10 sporadic mutations was confirmed by testing both parents.
• Generic ACMG/AMP PS2 was used because no TSC2-specific VCEP/CSPEC framework was available.

Still missing

• The publication uses the protein notation Leu825Met, whereas the case normalization is p.Leu826Met; the nucleotide change c.2476C>A and exon 21 match the case variant.
• The publication does not provide a detailed individual phenotype description for S19-01 in the quoted passage.

Sources

Reasoning

BS2 is met at supporting strength because the exact p.L826M variant was reported in a clinically unaffected father, who transmitted it to both children, and the same substitution was described as a nonpathogenic change in an unaffected relative. This is evidence of the variant in clinically unaffected adults for an autosomal-dominant disorder. Human review is warranted because the reports do not provide a detailed age, examination, or penetrance assessment for the unaffected carrier, and TSC has variable expressivity and age-dependent ascertainment considerations.

Rule

generic ACMG/AMP BS2: observed in healthy adult individual(s) for a disorder expected to be highly penetrant; supporting strength

Evidence considered

• PMID:17120248 reports: 'In Family B, both children also inherited a TSC2 L826M polymorphism from their unaffected father.' The paper also states that this polymorphism has no effect on TSC2 protein function.
• PMID:15483652 identifies L826M in an unaffected relative of a TSC patient and describes it as a nonpathogenic substitution after tuberin-hamartin interaction, signaling, and rheb GTPase assays.
• The exact case variant is NM_000548.5:c.2476C>A, normalized as NP_000539.2:p.(Leu826Met); the papers use the equivalent L826M notation.

Still missing

• The cited reports do not document the unaffected father's age, formal TSC evaluation, or duration of follow-up.
• The extent to which reduced penetrance or subtle/unrecognized TSC manifestations could affect interpretation of a single unaffected carrier is not quantified.

Sources

Reasoning

The exact TSC2 p.L826M substitution demonstrated normal function in a well-established and biologically relevant assay system. Across orthogonal assays, L826M preserved tuberin-hamartin binding, mTOR pathway suppression through inhibition of S6K T389 and S6 phosphorylation, and rheb GAP activity, with results comparable to wild-type tuberin and distinct from damaging comparator variants. This supports BS3 at strong strength under the generic ACMG/AMP framework.

Rule

Generic ACMG/AMP BS3 rule: well-established functional studies showing no damaging effect support BS3; the breadth, controls, biological relevance, and concordance of the exact-variant assays justify strong strength.

Evidence considered

• PMID:15483652 identified L826M as a nonpathogenic change found in an unaffected relative and experimentally showed preserved tuberin-hamartin interaction, normal inhibition of S6K T389 and S6 phosphorylation, and retained stimulation of rheb GTPase activity.
• PMID:15483652 provides assay controls and calibration: wild-type tuberin retained activity, mock/untransfected controls established baseline signaling, pathogenic variants showed impaired readouts, L826M was tested in multiple independent experiments, and the normal-function findings were concordant across protein interaction, downstream mTOR signaling, and biochemical GAP activity assays.
• PMID:17120248 reports that both children in Family B inherited TSC2 L826M from their clinically unaffected father and states that the polymorphism had no effect on TSC2 protein function. The paper itself cites prior functional work rather than presenting a new L826M assay, so it is corroborative and not the primary assay evidence.

Sources

Reasoning

The variant shows lack of segregation with TSC: both children in Family B inherited L826M from their clinically unaffected father, and L826M was also identified in unaffected relatives of TSC patients. This is direct non-segregation evidence supporting BS4 at supporting strength under the generic ACMG/AMP framework.

Rule

Generic ACMG/AMP 2015 BS4: lack of segregation in affected members of a family; apply supporting strength when informative non-segregation is documented.

Evidence considered

• PMID:17120248 reports inheritance of L826M by both children from their unaffected father and states that the polymorphism had no effect on TSC2 protein function.
• PMID:15483652 reports L826M among nonpathogenic substitutions identified in unaffected relatives of TSC patients.
• These observations provide lack-of-segregation evidence for the disease phenotype and support BS4.

Still missing

• The reports do not provide a complete pedigree or a formal count of informative meioses for c.2476C>A.

Sources

Reasoning

No distinct nucleotide substitution producing the same amino-acid change p.Leu826Met was established. PMID:9829910 reports the exact c.2476C>A variant, not an alternate nucleotide change, and PMID:15483652 reports L826M as nonpathogenic in functional and familial context. Therefore PS1 is not met.

Rule

PS1 requires the same amino-acid change as an established pathogenic variant caused by a different nucleotide change.

Evidence considered

• PMID:15483652 explicitly studied TSC2 L826M and described it as a nonpathogenic change identified in an unaffected relative; the reported assays found no disruption of tuberin function.
• PMID:9829910 reports c.2476C>A in a sporadic TSC patient as Leu825Met in its numbering, but this is the same nucleotide substitution as the case variant rather than a different nucleotide change producing the same amino-acid substitution.

Still missing

• A distinct nucleotide change resulting in p.Leu826Met and independently established as pathogenic was not identified in the supplied evidence.

Sources

Reasoning

A well-established, biologically relevant functional study of the exact p.L826M substitution found preserved tuberin function rather than loss of function: L826M retained tuberin-hamartin interaction, inhibited S6K T389 and S6 phosphorylation comparably to wild-type tuberin, and stimulated rheb GTPase activity. The study included wild-type, mock/untransfected, and disease-associated damaging variant comparators, repeated assays, and orthogonal readouts. Therefore, the available functional evidence does not support a deleterious functional effect and PS3 is not met.

Rule

Generic ACMG/AMP functional evidence rule: PS3 requires well-established functional studies supportive of a damaging effect; no damaging effect was demonstrated for this exact variant.

Evidence considered

• PMID:15483652 directly tested the exact TSC2 L826M substitution using full-length site-directed constructs and multiple disease-relevant assays: tuberin-hamartin co-immunoprecipitation, inhibition of S6K T389 phosphorylation, inhibition of S6 phosphorylation in Tsc2-null mouse embryo fibroblasts, and in-vitro rheb GTPase activity.
• The study used appropriate functional comparators, including wild-type tuberin, mock/untransfected or protein-only controls, and pathogenic TSC2 variants; assays were repeated, with at least three replicates for the rheb GTPase experiments and five repeats for the S6 phosphorylation assay.
• The exact L826M variant retained interaction with hamartin, inhibited S6K T389 phosphorylation, inhibited S6 phosphorylation with no significant difference from wild type, and increased rheb GTPase activity. These concordant normal-function results contradict the functional-loss premise required for PS3.

Sources

Reasoning

vcep_materials.json contains no TSC2 domain_tables entry, so no authoritative VCEP domain list applies. Generic literature places residue 826 within the N-terminal TSC1-binding region (amino acids 1-900), but this region is not shown to be a PM1-qualifying critical domain lacking benign variation: L826M itself has benign functional evidence and is present in gnomAD v4.1 at AF 0.0013527. A PM1 claim is therefore not supported.

Rule

PM1 requires a variant to lie in a mutational hotspot or critical and well-established functional domain with little or no benign variation; generic domain location alone is insufficient.

Evidence considered

• vcep_materials.json reports domain_tables as an empty object and the case uses generic ACMG fallback.
• PMID:22903760 describes TSC2 amino acids 1-900 as the N-terminal region mediating interaction with TSC1; residue 826 falls within that interval.
• PMID:21309039 describes amino acids 1-900 as corresponding to the TSC1-binding domain, but provides no variant-specific p.L826M result.
• PMID:15483652 reports L826M as nonpathogenic and functionally similar to wild-type tuberin.
• gnomAD v4.1 reports the case variant at AF 0.0013527, with 2182/1613066 alleles and no homozygotes.

Still missing

• No applicable VCEP domain table or gene-specific PM1 rule was supplied.

Sources

Reasoning

PM2 is not met because the variant is not absent or sufficiently rare in population databases under the case's generic PM2 threshold of AF <0.1%. gnomAD v4.1 reports an overall AF of 0.13527% and a European non-Finnish AF of 0.17372%, both above 0.1%; gnomAD v2.1 also reports a European non-Finnish AF of 0.11274%. All queried datasets report zero homozygotes, but absence of homozygotes does not overcome the observed heterozygote frequency above the PM2 threshold.

Rule

generic_population_frequency: PM2 requires AF < 0.1% or absence in appropriate population datasets; zero homozygotes alone is insufficient

Evidence considered

• gnomAD v4.1 reports 2,182/1,613,066 alleles overall (AF 0.13527%) and 2,050/1,180,042 European non-Finnish alleles (AF 0.17372%), with zero homozygotes.
• gnomAD v2.1 reports overall AF 0.06491% and European non-Finnish AF 0.11274%, with zero homozygotes; gnomAD-Canada reports AF 0.02714%, with zero homozygotes. The modern gnomAD v4.1 and gnomAD v2.1 maximum frequencies exceed the case PM2 threshold of 0.1%.
• PMID:9829910 reports the variant-equivalent Leu825Met in a sporadic TSC patient and no observation of the six study missense mutations in at least 80 CEPH control chromosomes. This small historical control panel does not establish PM2 in the presence of the larger modern population datasets, and the paper's residue numbering differs from the normalized p.L826M notation.

Sources

Reasoning

PMID:22903760 includes a different missense substitution at the same residue, p.Leu826Pro, but the supplied extraction states that no functional result for p.L826P is provided and does not establish it as pathogenic. No established pathogenic missense comparator at residue 826 was identified; PM5 is not met.

Rule

PM5 requires a novel missense change at a residue where a different missense change is established as pathogenic.

Evidence considered

• PMID:22903760 lists p.L826P among the tested TSC2 variants, but the extracted finding explicitly states that the paper provides no functional result for p.L826M and does not establish p.L826P as pathogenic.

Still missing

• An independently established pathogenic missense variant at TSC2 residue 826 was not identified.

Sources

Reasoning

No positive co-segregation of c.2476C>A with TSC is documented. Instead, L826M was inherited from a clinically unaffected father by both children in Family B, and the variant was reported in unaffected relatives of TSC patients. These observations do not support PP1 and are inconsistent with positive disease segregation.

Rule

Generic ACMG/AMP 2015 PP1: co-segregation with disease in multiple affected family members; positive segregation is required and non-segregation does not satisfy PP1.

Evidence considered

• PMID:17120248 reports that both children in Family B inherited the TSC2 L826M polymorphism from their clinically unaffected father.
• PMID:15483652 states that L826M was identified in unaffected relatives of TSC patients.
• Because the documented observations are non-segregating rather than positively cosegregating, PP1 is not met.

Still missing

• The available reports do not provide a complete pedigree with the clinical status and genotypes of every family member carrying c.2476C>A.

Sources

Reasoning

The supplied evidence does not establish that pathogenic missense variation is common and benign missense variation is rare in TSC2 at the gene level. TSC2 has a documented missense disease mechanism, but the available studies and population data do not satisfy the generic PP2 requirement; the case variant also has direct benign functional evidence and is present in gnomAD.

Rule

PP2 is supporting evidence only when missense variation is a common disease mechanism and benign missense variation is uncommon for the gene.

Evidence considered

• PMID:15483652 documents that TSC2 missense substitutions can be pathogenic or nonpathogenic, demonstrating a missense mechanism but not the gene-level prevalence pattern required for PP2.
• PMID:21309039 evaluates TSC1/TSC2 missense variants functionally and emphasizes that missense effects require variant-specific assessment; it does not provide a gene-level PP2 calibration.
• gnomAD v4.1 reports the case variant at AF 0.0013527, providing population evidence inconsistent with treating this specific missense change as presumptively pathogenic.

Still missing

• No validated gene-level missense constraint or curated pathogenic-versus-benign missense framework for TSC2 was supplied.

Sources

Reasoning

The variant is a missense substitution (p.Leu826Met), so the generic fallback selects the REVEL missense path and does not independently apply SpliceAI. REVEL is 0.638, which is below the ClinGen SVI calibrated supporting PP3 threshold of 0.773; PP3 is not met.

Rule

Generic ACMG fallback: for a missense variant, assess one computational mechanism only using calibrated REVEL evidence; PP3 supporting requires REVEL >=0.773 (ClinGen SVI calibration, PMID:36413997).

Evidence considered

• No TSC2 VCEP/CSPEC or gene-specific PP3/BP4 lookup was available; the supplied framework therefore uses the generic missense-path selection (REVEL only) for this missense variant.
• REVEL score for NM_000548.5:c.2476C>A is 0.638. The ClinGen SVI calibrated REVEL supporting PP3 cutoff is >=0.773 (PMID:36413997); this score does not reach that threshold.
• SpliceAI max delta score is 0.005, but the generic framework explicitly does not use a missense variant's splice score to assess its protein-level PP3/BP4 computational evidence; it was not counted.
• BayesDel score was not used because no verified published BayesDel threshold is available for this pipeline.

Sources

Reasoning

BA1 is not met. Under the generic population-frequency operating threshold used for this case (allele frequency >1%), the highest observed frequency for NM_000548.5:c.2476C>A (NP_000539.2:p.(Leu826Met)) is 0.17372% in the gnomAD v4.1 European non-Finnish population, with overall frequency 0.13527%; gnomAD v2.1 and gnomAD-Canada are also below 1%. These frequencies are far below the BA1 threshold.

Rule

generic_population_frequency: BA1 requires AF > 1% in an appropriate population dataset

Evidence considered

• gnomAD v4.1 reports 2,182/1,613,066 alleles overall (AF 0.0013527; 0.13527%) and 2,050/1,180,042 European non-Finnish alleles (AF 0.00173723; 0.17372%), with zero homozygotes.
• gnomAD v2.1 reports 183/281,938 alleles overall (AF 0.00064908; 0.06491%) and a highest observed European non-Finnish AF of 0.11274%, with zero homozygotes; gnomAD-Canada reports 5/18,422 alleles (AF 0.02714%), with zero homozygotes.
• The generic ACMG/AMP framework is used because no TSC2-specific VCEP/CSPEC framework was retrieved; the case-level BA1 operating threshold is >1% and is not reached.

Sources

Reasoning

BS1 is not met under the case's generic population-frequency threshold (AF >0.3%). The highest observed frequency is 0.17372% in gnomAD v4.1 European non-Finnish individuals, below 0.3%; overall gnomAD v4.1 AF is 0.13527%. The frequency therefore does not exceed the threshold for being too common for TSC2-related tuberous sclerosis.

Rule

generic_population_frequency: BS1 requires AF > 0.3% in an appropriate population dataset

Evidence considered

• gnomAD v4.1 reports the maximum broad population AF as 0.17372% in the European non-Finnish population and overall AF as 0.13527%; both are below 0.3%.
• gnomAD v2.1 reports a highest observed European non-Finnish AF of 0.11274%, and gnomAD-Canada reports AF 0.02714%; neither exceeds 0.3%.
• The generic ACMG/AMP framework is used because no TSC2-specific VCEP/CSPEC framework was retrieved; the case-level BS1 operating threshold is >0.3% and is not reached.

Sources

Reasoning

BP1 is not applicable solely because TSC2 is also affected by truncating variants. The supplied literature documents a substantial and experimentally assessable TSC2 missense mechanism, including pathogenic missense substitutions, so TSC2 cannot be treated as a gene in which disease is caused primarily by truncating variants for this criterion.

Rule

BP1 applies to a missense variant in a gene for which primarily truncating variants cause disease; an established missense mechanism argues against BP1.

Evidence considered

• PMID:15483652 reports TSC2 missense mutations among the mutations detected and experimentally distinguishes pathogenic from nonpathogenic missense substitutions.
• PMID:22903760 functionally assessed 73 missense changes among 78 TSC2 variants and classified many functionally disruptive variants as pathogenic or probably/possibly pathogenic.
• PMID:21309039 provides a functional assay specifically for TSC1/TSC2 missense variants, supporting an established missense disease mechanism in TSC2.

Still missing

• No quantitative, gene-specific variant-spectrum framework establishing TSC2 as predominantly truncating for BP1 was supplied.

Sources

Reasoning

The variant is a missense substitution (p.Leu826Met), so the generic fallback selects the REVEL missense path and does not independently apply SpliceAI. REVEL is 0.638, which is above the ClinGen SVI calibrated supporting BP4 threshold of 0.290; BP4 is not met.

Rule

Generic ACMG fallback: for a missense variant, assess one computational mechanism only using calibrated REVEL evidence; BP4 supporting requires REVEL <=0.290 (ClinGen SVI calibration, PMID:36413997).

Evidence considered

• No TSC2 VCEP/CSPEC or gene-specific PP3/BP4 lookup was available; the supplied framework therefore uses the generic missense-path selection (REVEL only) for this missense variant.
• REVEL score for NM_000548.5:c.2476C>A is 0.638. The ClinGen SVI calibrated REVEL supporting BP4 cutoff is <=0.290 (PMID:36413997); this score does not reach that threshold.
• SpliceAI max delta score is 0.005, but the generic framework explicitly does not use a missense variant's splice score to assess its protein-level PP3/BP4 computational evidence; it was not counted.
• BayesDel score was not used because no verified published BayesDel threshold is available for this pipeline.

Sources

Reasoning

No applicable TSC2 VCEP/CSPEC was retrieved; generic ACMG/AMP is used. The exact variant was reported once among 20 sporadic tuberous sclerosis cases, but the available evidence contains neither a case-control analysis nor sufficient independent affected observations to establish statistically significant enrichment.

Rule

Generic ACMG/AMP PS4: apply only when the variant prevalence is significantly increased in affected individuals compared with controls; this threshold is not demonstrated here.

Evidence considered

• PMID:9829910 reports the variant as Leu825Met in sporadic patient S19-01 in a study of 40 unrelated tuberous sclerosis patients; this is one case observation, not an enrichment analysis.
• PMID:15483652 reports L826M in an unaffected relative of a person with tuberous sclerosis.
• PMID:17120248 reports L826M inherited from a clinically unaffected father in Family B.

Still missing

• A phenotype-defined case-control or burden study reporting exact-variant counts and controls.
• Additional independent, non-overlapping affected observations with sufficiently detailed phenotypes.

Sources

Reasoning

No applicable TSC2 VCEP/CSPEC was retrieved; generic ACMG/AMP is used. The bundle provides no phenotype for the proband and no evidence that a phenotype in this case is highly specific for TSC2-related tuberous sclerosis; PP4 cannot be evaluated.

Rule

Generic ACMG/AMP PP4: apply only when the patient's phenotype or family history is highly specific for a disease with a single genetic etiology; the required case phenotype and specificity evidence are absent.

Evidence considered

• PMID:9829910 identifies the exact variant in a sporadic tuberous sclerosis patient but the supplied extraction does not provide phenotype details that could establish PP4 specificity.
• No proband phenotype or phenotype-specificity analysis is present in the consolidated bundle.

Still missing

• Proband phenotype, including diagnostic features and the basis for the tuberous sclerosis diagnosis.
• Evidence establishing that the observed phenotype is highly specific for TSC2-related disease rather than the broader differential diagnosis.

Sources

Reasoning

ClinVar contains no exact-variant expert-panel pathogenic or likely pathogenic assertion. Therefore PP5 cannot be applied.

Rule

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

Evidence considered

• ClinVar variation 41732 is an exact match but has zero expert-panel submissions; the available assertions are ordinary laboratory submissions and include benign/likely benign and uncertain-significance classifications.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Sources

Reasoning

The available evidence does not establish the phase required for BP2. PMID:17120248 reports that L826M was inherited by two children from a clinically unaffected father, but it does not document that L826M was in trans with a known pathogenic variant in those individuals, nor does it establish a cis configuration relevant to a recessive disorder. Therefore BP2 cannot be applied or excluded from the available record.

Rule

Generic ACMG/AMP BP2: supporting benign evidence when the variant is observed in trans with a pathogenic variant for a dominant disorder, or in cis with a pathogenic variant for a recessive disorder; phase and the relevant pathogenic allele must be documented.

Evidence considered

• PMID:17120248 states: "In Family B, both children also inherited a TSC2 L826M polymorphism from their unaffected father. This polymorphism has no effect on the TSC2 protein function." The report does not identify a second pathogenic variant in the children or provide phase information.
• The exact variant is also functionally and clinically described as a nonpathogenic change identified in an unaffected relative in PMID:15483652, but that source does not provide the trans-phase observation required for BP2.
• Because BP2 requires documented phase with a pathogenic allele, the available inheritance observation is insufficient for criterion application.

Still missing

• Need family-level genotype data identifying any co-occurring pathogenic variant in the affected individuals.
• Need validated phase information showing L826M in trans with that pathogenic variant for dominant-disease BP2 application.
• The reported inheritance from an unaffected father alone is insufficient to establish BP2.

Sources

Reasoning

No proband-level evidence of an alternate molecular diagnosis that fully explains the phenotype is available in the consolidated bundle; BP5 cannot be evaluated.

Rule

Generic ACMG/AMP BP5: apply only when a variant is found in a case with an alternate molecular basis that fully explains the phenotype.

Evidence considered

• The consolidated bundle contains no proband phenotype or alternate molecular diagnosis for this case.

Still missing

• Proband phenotype and molecular testing results documenting any alternate diagnosis and whether it fully explains the presentation.

Sources

Reasoning

ClinVar contains no exact-variant expert-panel benign or likely benign assertion. Therefore BP6 cannot be applied.

Rule

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

Evidence considered

• ClinVar variation 41732 is an exact match but has zero expert-panel submissions. Benign and likely benign assertions in the record are ordinary laboratory submissions and do not qualify for BP6.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign classification.

Sources