TSC2 c.5335del, p.(Gln1779ArgfsTer47)

Reasoning

No TSC2-specific ClinGen VCEP CSPEC PVS1 specification is available for this case (cspec.found=false, vcep_materials empty), so generic ACMG/AMP PVS1 assessment per the ClinGen SVI PVS1 recommendations (PMC6185798) applies. Gene-level eligibility is established: pvs1_gene_context confirms germline TSC2 loss-of-function is a supported disease mechanism (lof_mechanism_supported=true, pvs1_gene_gate='eligible'), consistent with tuberous sclerosis complex pathogenesis via TSC1/TSC2-mTORC1 loss of inhibitory GAP function. NM_000548.4:c.5335del is a single-nucleotide frameshift deletion predicted to produce NP_000539.2:p.(Gln1779ArgfsTer47). Exon mapping places this variant in exon 42 of 42 (variant_exonic_positions start_exon/end_exon = 42; CDS c.1-5424 with exon 42 spanning c.5260-*110), i.e. the terminal exon of the transcript. Because the premature termination codon falls within the last exon, nonsense-mediated decay (NMD) is not predicted to occur (NMD-escape branch of the SVI PVS1 decision tree). Comparing predicted protein lengths (position_last_original=1808 [wild-type stop, 1807 aa mature protein] vs position_last_predicted=1825 for the frameshifted product) shows the frameshift does not truncate the protein at all in absolute terms - it replaces only the native C-terminal 29 residues (aa 1779-1807) with a 47-residue frameshifted tail, yielding a protein 18 residues LONGER than wild type. The region altered/lost (29 of 1807 residues, ~1.6% of the protein) is well below the ~10% threshold the SVI flowchart uses to judge whether an NMD-escaping truncation is likely functionally consequential. No domain or functional-criticality evidence for this specific C-terminal segment (aa 1779-1807) was found anywhere in the case evidence bundle (no CSPEC, no VCEP materials, no literature identifying this region as critical), so the 'truncated/altered region is critical to protein function' branch cannot be answered affirmatively. Per the SVI PVS1 decision tree, an NMD-escaping variant that removes ≤10% of the protein and lacks evidence that the affected region is functionally critical is scored PVS1_Supporting rather than full/strong/very-strong PVS1. This is applied here as PVS1 met at supporting strength only.

Rule

PVS1 (SVI generic decision tree, PMC6185798): frameshift variant in gene with confirmed LOF disease mechanism, located in the last exon (42/42) -> NMD not predicted -> altered/lost protein region (~1.6% of protein, C-terminal 29 residues, replaced by a longer frameshifted tail) is ≤10% of the protein and not established as functionally critical -> PVS1_Supporting.

Evidence considered

• case_summary.json pvs1_variant_assessment: variant classified consequence_class='frameshift', variant_bucket='frameshift', framework_source PMC6185798, apply_generic_pvs1_framework=true, suggested_default_strength='PVS1' pending downgrade checks for NMD escape and exon/region importance.
• case_summary.json pvs1_variant_assessment.downgrade_considerations explicitly flag: confirm transcript relevance, downgrade if predicted truncation escapes NMD or affects only a non-critical distal region, downgrade/withhold if the affected exon is not biologically relevant or is enriched for population LoF variation.
• prefetch.json mutalyzer selector_short exon coordinates show the transcript has 42 total exons and variant_exonic_positions (start_exon=end_exon=42 for both GRCh37/GRCh38) place c.5335del in the final exon (exon 42/42), spanning c.5260-*110.
• prefetch.json mutalyzer protein prediction: position_last_original=1808 (wild-type stop/1807 aa protein) vs position_last_predicted=1825 for the frameshifted product NP_000539.2:p.(Gln1779ArgfsTer47), showing the mutant protein is longer than wild type rather than substantially truncated, i.e. only the terminal 29 residues (1779-1807, ~1.6% of 1807 aa) are replaced by a novel 47-residue tail.
• pvs1_gene_context.json confirms germline TSC2 loss-of-function disease mechanism is supported by targeted literature review (lof_mechanism_supported=true, pvs1_gene_gate='eligible'), satisfying the gene-level LOF-mechanism gate required before any PVS1 application, generic or VCEP-based.
• case_summary.json cspec.found=false and vcep_materials.final_classification_framework.found=false confirm no TSC2 ClinGen VCEP-specific PVS1 specification exists for this case; generic ACMG/SVI PVS1 framework (PMC6185798) was used as the governing framework per instructions.
• No domain-criticality, functional, or region-importance evidence for TSC2 residues 1779-1807 (or the resulting frameshifted tail) was found in the evidence bundle, so the SVI 'critical region' branch defaults to 'not established', supporting only PVS1_Supporting rather than a higher strength for this last-exon, NMD-escaping frameshift.

Still missing

• No TSC2-specific VCEP/CSPEC PVS1 specification was available to confirm gene- and region-specific exon/NMD/critical-domain rules; generic SVI framework was used as fallback.
• No domain-level annotation (e.g., GAP domain boundaries, calmodulin-binding region, known critical C-terminal motifs) was available in the case bundle to confirm or refute functional criticality of TSC2 residues 1779-1807 or the novel frameshifted tail.
• No experimental (mRNA/NMD) data confirm last-exon NMD escape for this specific transcript position; the escape call is based on genomic exon-junction position modeling only.

Sources

Reasoning

NM_000548.4:c.5335del is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. In the absence of an applicable TSC2-specific VCEP specification, this satisfies the generic ACMG/AMP PM2 supporting criterion for absence from population databases in a disorder where the variant is expected to be rare.

Rule

generic ACMG/AMP 2015 PM2: absent from population databases; supporting strength

Evidence considered

• gnomAD v2.1 reports NM_000548.4:c.5335del as absent.
• gnomAD v4.1 reports NM_000548.4:c.5335del as absent.
• gnomAD-Canada v1.0 reports NM_000548.4:c.5335del as absent.
• No TSC2-specific VCEP/CSPEC framework was retrieved, so the generic ACMG/AMP 2015 framework was used.

Sources

Reasoning

NM_000548.4:c.5335del is a frameshift deletion (p.(Gln1779ArgfsTer47)), not a missense or synonymous variant, so the missense in-silico path (REVEL/BayesDel/aGVGD) and BP7 do not apply. The variant does not lie in the canonical splice consensus region (canonical_splice_consensus=false) and SpliceAI, the only computational splice predictor with data in this case, shows no significant splice impact (max delta score = 0.00; DS_AG/DS_AL/DS_DG/DS_DL all below the 0.2 threshold). No governing VCEP/gene-specific framework (cspec) was found for TSC2 in this case (framework_mode = generic_acmg; cspec.found = false), so generic ACMG/AMP PP3 criteria were applied. A near-zero SpliceAI delta score does not provide evidence of a deleterious splicing effect, so PP3 is not met.

Rule

generic_acmg_pp3_bp4

Evidence considered

• SpliceAI lookup for NM_000548.4:c.5335del (hg19) reports max delta score = 0.00, indicating no significant predicted splice impact.
• Variant is a frameshift deletion, not missense or synonymous, so REVEL/BayesDel/aGVGD (PP3 missense path) and BP7 (synonymous path) are not applicable.
• No TSC2 gene-specific VCEP framework or PP3/BP4 lookup spreadsheet was found in vcep_materials for this case; generic ACMG/AMP framework applied by default.

Sources

Reasoning

No population frequency at or above the stand-alone benign threshold is observed. NM_000548.4:c.5335del is reported as absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, so BA1 is not met.

Rule

generic ACMG/AMP 2015 BA1: stand-alone benign population frequency threshold; absence does not meet BA1

Evidence considered

• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports the variant as absent.
• gnomAD-Canada v1.0 reports the variant as absent.

Sources

Reasoning

The variant is absent from the available population datasets rather than present at a frequency greater than expected for TSC2-related disease. Therefore, the population-frequency requirement for BS1 is not met.

Rule

generic ACMG/AMP 2015 BS1: allele frequency greater than expected for the disorder; absent variant does not meet BS1

Evidence considered

• The available gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0 records all report absence of NM_000548.4:c.5335del; no elevated allele frequency or ancestry-specific frequency is available.

Sources

Reasoning

SpliceAI's max delta score of 0.00 is consistent with no predicted splice disruption, which would in principle support BP4 (benign computational evidence) for the splice-impact path. However, because this variant is a frameshift-causing single-base deletion producing a premature stop (p.(Gln1779ArgfsTer47)), the predicted pathogenic mechanism is loss-of-function via frameshift/PTC rather than splicing, and a benign SpliceAI score does not offset the primary LoF consequence; BP4 is not intended to counter a truncating/frameshift call. There is no missense in-silico score (REVEL/BayesDel/aGVGD) or synonymous-variant context here, so the missense BP4 path and BP7 do not apply. BP4 is therefore not met/not applicable in a way that would affect classification.

Rule

generic_acmg_pp3_bp4

Evidence considered

• SpliceAI max delta score = 0.00 for NM_000548.4:c.5335del, indicating benign-range splice prediction, but this does not translate to BP4 applicability because the variant's primary predicted mechanism is frameshift/PTC (LoF), not splicing.
• Variant is not missense or synonymous, so the BayesDel/REVEL/aGVGD BP4 path and BP7 are not applicable.

Sources

Reasoning

No proband-level de novo evidence is provided. The case bundle contains no parental genotypes, maternity/paternity confirmation, trio testing results, or documentation that the variant is absent from both biological parents. Therefore PS2 cannot be assessed.

Rule

PS2 requires the variant to be observed de novo in the affected individual with confirmed parentage; stronger application requires confirmed maternity and paternity and reliable testing showing absence of the variant in both parents.

Evidence considered

• The consolidated case-evidence bundle reports no case-specific parental testing, trio analysis, de novo observation, or family segregation data.
• No applicable TSC2-specific VCEP/ClinGen specification was available; generic ACMG/AMP criteria were therefore used as the fallback framework.

Still missing

• Proband genotype and phenotype documentation relevant to the variant
• Confirmed biological parentage
• Pathogenic-variant testing results for both biological parents
• Evidence that the variant is absent in both parents

Reasoning

No official CSPEC/VCEP framework for TSC2 was found (case_summary.json: cspec.found=false; framework_mode=generic_acmg; vcep_materials.note='no cspec url available'), so generic ACMG/AMP PS3 requirements apply: a validated functional assay demonstrating abnormal protein function for this specific variant, run alongside normal and pathogenic/benign controls. The literature packet for this case (case_summary.json literature_packet) contains only 4 PMIDs, all general TSC1/TSC2-mTOR pathway mechanistic reviews/studies (PMID:14561707, PMID:14729330, PMID:24529379, PMID:25724664) retrieved via OncoKB gene-level context. None of these describe a functional assay performed on NM_000548.4:c.5335del (p.Gln1779ArgfsTer47) or any variant-specific experimental characterization; they discuss TSC1/TSC2 complex biology and mTORC1 regulation in general terms, not this frameshift allele. No assay data, controls, or variant-specific functional results are present anywhere in the evidence bundle.

Still missing

• No variant-specific functional/complementation/rescue assay data identified for c.5335del in the retrieved literature or evidence bundle.
• No CSPEC/VCEP-specific PS3 assay framework available for TSC2 to define qualifying assay types or strength calibration.

Reasoning

No variant-specific affected-case series, case-control comparison, or statistically significant enrichment data are present in the case-evidence bundle. The available literature is functional or gene-level and does not establish PS4 for this exact variant.

Rule

Generic ACMG/AMP PS4 requires a statistically significant increase in affected individuals or affected families with the variant relative to controls; exact-variant case-control or appropriately documented case enrichment is required.

Evidence considered

• The case summary contains no affected-case or case-control evidence for NM_000548.4:c.5335del.
• The literature pass contains four gene/function-focused publications with no candidate PS4 evidence and no exact-variant clinical observations.

Still missing

• Variant-specific affected-case counts and an appropriate control comparison are unavailable.
• No exact-variant clinical case series or enrichment analysis was identified.

Reasoning

No unconfirmed de novo observation is provided. The case bundle contains no report of the variant in an affected proband with testing showing absence in the parents when parentage or parental testing is incomplete. Therefore PM6 cannot be assessed.

Rule

PM6 may be applied for a presumed de novo variant when the affected individual is reported with the variant and the variant is absent in tested parents, but parentage and/or other confirmation is insufficient for PS2.

Evidence considered

• The consolidated case-evidence bundle contains no case-specific report of an apparently de novo variant, parental absence testing, or incomplete-parentage documentation.
• No applicable TSC2-specific VCEP/ClinGen specification was available; generic ACMG/AMP criteria were therefore used as the fallback framework.

Still missing

• Affected-proband clinical and genotype documentation
• Parental variant testing or other evidence of parental absence
• Parentage information and the specific limitation preventing confirmed parentage
• Independent confirmation of the de novo observation

Reasoning

No affected relatives, informative meioses, familial genotypes, or segregation analysis are documented. The available literature in the bundle is functional or gene-level and does not report this variant in a family. PP1 therefore cannot be assessed.

Rule

PP1 supports pathogenicity when a variant cosegregates with disease in affected family members; strength depends on the number and informativeness of independent meioses and the specificity and consistency of the phenotype.

Evidence considered

• The consolidated case-evidence bundle reports no family pedigree, affected-relative observations, informative meioses, or segregation data for NM_000548.4:c.5335del.
• The four triaged publications are functional/gene-context sources and do not provide variant-specific familial segregation evidence.

Still missing

• Family pedigree and phenotype information
• Genotypes of affected and unaffected relatives
• Number of informative meioses
• Assessment of phenotype consistency among variant-positive relatives

Reasoning

No proband phenotype, clinical diagnosis, or phenotype specificity assessment is provided for this variant. Therefore, the highly specific phenotype required for PP4 cannot be evaluated.

Rule

Generic ACMG/AMP PP4 requires a patient's phenotype or family history to be highly specific for a disease with a single-gene etiology; gene-level disease descriptions alone are insufficient.

Evidence considered

• The case-evidence bundle provides variant normalization and database results but no patient phenotype or clinical diagnostic summary.
• Gene-level descriptions of tuberous sclerosis complex do not establish PP4 for an individual without documented phenotype data.

Still missing

• Clinical phenotype and diagnostic features of the proband are unavailable.
• No assessment of whether the phenotype is specific for a TSC2-related disorder is available.

Reasoning

The exact variant has no ClinVar result, no variation ID, and no expert-panel assertion in the case bundle. OncoKB is not a ClinVar expert panel and cannot trigger PP5 under the requested rule.

Rule

Apply PP5 only when an exact-variant ClinVar expert-panel assertion classifies the variant as Pathogenic or Likely pathogenic; ordinary submissions, aggregate labels, non-expert assertions, and other databases do not qualify.

Evidence considered

• ClinVar exact-variant search returned no result; therefore there is no expert-panel Pathogenic or Likely pathogenic assertion to support PP5.
• The variant-specific OncoKB label is not an exact-variant ClinVar expert-panel classification and was not used for PP5.

Still missing

• No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification is available.

Sources

Reasoning

No evidence was provided that the variant occurs in healthy adults or unaffected individuals, and no homozygote observations or phenotype/penetrance information from population cohorts are available. BS2 cannot be assessed.

Rule

generic ACMG/AMP 2015 BS2: observed in a healthy adult individual for a fully penetrant early-onset disorder, or in a healthy adult homozygote for a dominant disorder

Still missing

• No documented observations in healthy adults or unaffected individuals.
• No homozygote observations or relevant penetrance information.

Reasoning

No official CSPEC/VCEP framework for TSC2 was found (case_summary.json: cspec.found=false; framework_mode=generic_acmg), so generic ACMG/AMP BS3 requirements apply: a validated functional assay showing no damaging effect for this specific variant. The literature packet contains only general TSC1/TSC2-mTOR pathway mechanistic papers (PMID:14561707, PMID:14729330, PMID:24529379, PMID:25724664) with no assay data on this specific frameshift variant (c.5335del / p.Gln1779ArgfsTer47). No functional evidence of any kind (damaging or benign) specific to this variant is present in the evidence bundle.

Still missing

• No variant-specific functional assay data identified showing normal/benign function for c.5335del.
• No CSPEC/VCEP-specific BS3 assay framework available for TSC2.

Reasoning

No unaffected relatives known to carry the variant, no non-segregation analysis, and no phenotype-confirmed family dataset are provided. Absence of family data is not evidence of non-segregation, so BS4 cannot be assessed.

Rule

BS4 supports benignity when a variant is observed in affected families but does not segregate with disease in informative affected/unaffected relatives, accounting for penetrance, age, phenotype specificity, and possible phenocopies.

Evidence considered

• The consolidated case-evidence bundle contains no variant-positive unaffected relatives or other family data demonstrating lack of segregation with disease.
• No applicable TSC2-specific VCEP/ClinGen specification was available; generic ACMG/AMP criteria were therefore used as the fallback framework.

Still missing

• Family pedigree and phenotype assessment
• Genotypes of informative unaffected relatives
• Number of informative non-segregating meioses
• Evaluation of phenocopies, age-dependent penetrance, and variable expressivity

Reasoning

No applicable TSC2-specific VCEP framework was available, so generic ACMG/AMP rules were used. BP2 requires documented occurrence with another pathogenic or likely pathogenic variant in a relevant allelic configuration, with interpretation dependent on inheritance mode and phase. The consolidated case-evidence bundle contains no affected-proband or family-level observation documenting a second pathogenic variant in cis or trans with NM_000548.4:c.5335del, and no phase information is available. BP2 therefore cannot be assessed from the supplied evidence.

Rule

Generic ACMG/AMP BP2: consider only when the variant is observed with another pathogenic or likely pathogenic variant in a phase and inheritance context that supports a benign interpretation; phase and relevant case observation must be documented.

Evidence considered

• The governing-framework fields report no CSPEC/VCEP or gene-specific framework for TSC2 and specify generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no case-level segregation, phase, cis/trans, or affected-proband data relevant to BP2.
• The available literature records are functional or gene-level sources and do not document this variant in an allelic observation suitable for BP2.

Still missing

• Documented affected-proband or family observations involving NM_000548.4:c.5335del and a second pathogenic or likely pathogenic variant.
• Validated cis/trans phase information and inheritance context for any second variant.

Reasoning

No clinical phenotype or molecular diagnosis indicating an alternative genetic cause of the observed phenotype is available. The absence of such information prevents assessment of BP5.

Rule

Generic ACMG/AMP BP5 requires a pathogenic variant in an alternative gene that explains the phenotype; an alternative diagnosis must be documented rather than inferred from absent or nonclinical database data.

Evidence considered

• The case-evidence bundle contains no proband phenotype, family history, or alternate pathogenic variant that could support an alternative molecular explanation.

Still missing

• The patient's phenotype and indication for testing are unavailable.
• No confirmed alternate molecular etiology is documented.

Reasoning

The exact variant is absent from ClinVar and has no expert-panel Benign or Likely benign classification. No qualifying evidence for BP6 is therefore available.

Rule

Apply BP6 only when an exact-variant ClinVar expert-panel assertion classifies the variant as Benign or Likely benign; ordinary submissions, aggregate labels, and non-expert assertions do not qualify.

Evidence considered

• ClinVar exact-variant search returned no result; therefore there is no expert-panel Benign or Likely benign assertion to support BP6.
• No ordinary laboratory submission, aggregate ClinVar label, or non-expert assertion was used for BP6.

Still missing

• No exact-variant ClinVar expert-panel Benign or Likely benign classification is available.

Sources