SDHB c.178A>G, p.(Thr60Ala)

Reasoning

PM2 supporting is met. The variant is rare in population datasets: gnomAD v4.1 overall AF 0.009666%, NFE AF 0.011865%, and grpmax FAF 0.01021%, with zero homozygotes. These values are below the generic 0.1% PM2 rarity threshold used in the absence of an operational SDHB VCEP threshold.

Rule

Generic ACMG/AMP PM2 rarity fallback (<0.1%, supporting); no operational PM2 threshold was available in the retrieved SDHB VCEP ruleset.

Evidence considered

• The retrieved SDHB VCEP ruleset has no operational PM2 threshold, so generic ACMG/AMP rarity assessment was used.
• gnomAD v4.1 reports AF 0.009666% overall (156/1,613,972), 0.011865% in NFE (140/1,179,932), and grpmax FAF 0.01021%, with zero homozygotes. gnomAD v2.1 is concordant (overall AF 0.005968%; NFE AF 0.011439%; grpmax FAF 0.006668%).
• The gnomAD-Canada NFE point estimate is 0.025558% from 3/11,738 alleles, but its FAF95 is 0.006943%; this small-sample estimate remains below 0.1% and does not represent a frequency threshold artifact that would negate PM2.
• All available population estimates are below the generic 0.1% PM2 rarity threshold; PM2 is assigned at supporting strength.

Sources

Reasoning

PP3 is met at moderate strength. NM_003000.3:c.178A>G (p.Thr60Ala) is a missense substitution with REVEL = 0.845, which meets the ClinGen SVI-calibrated moderate pathogenic-evidence threshold for REVEL (>=0.773; PMID 36413997). The retrieved SDHB (ClinGen Endocrine Tumor Predisposition VCEP) specification's rule payload contains no operational, gene-specific PP3/BP4 computational-evidence thresholds or lookup table, so the generic ClinGen SVI-calibrated REVEL framework was applied as fallback. SpliceAI predicts no splice impact (max delta score 0.002, effectively null across all four splice-altering categories), so there is no separate splice-disruption signal for this exonic missense change; the missense in-silico pathway (REVEL) governs PP3/BP4 for this variant and is not double-counted against the splicing pathway. BayesDel (score 0.307627) was not used because no verified published calibration threshold for BayesDel is available to this evaluation.

Rule

Generic/ClinGen SVI-calibrated REVEL framework (PMID 36413997) applied as fallback because the SDHB VCEP specification has no operational PP3/BP4 threshold; REVEL >=0.773 triggers PP3_Moderate.

Evidence considered

• REVEL score = 0.845 for NM_003000.3:c.178A>G (p.Thr60Ala), retrieved from the local REVEL v1.3 predictor lookup.
• ClinGen SVI-calibrated REVEL thresholds (Pejaver et al. 2022, PMID 36413997): PP3_Moderate applies at REVEL >=0.773 (PP3_Supporting >=0.644, PP3_Strong >=0.932); REVEL 0.845 falls in the moderate pathogenic-evidence band.
• SpliceAI predicts no significant splice impact for this variant (max delta score = 0.002 across DS_AG/DS_AL/DS_DG/DS_DL), indicating no competing splice-disruption mechanism that would need separate PP3 consideration.
• The SDHB VCEP (ClinGen Endocrine Tumor Predisposition Expert Panel) specification was checked; its retrieved rule payload is an unstructured, incomplete ruleset with no operational PP3/BP4 computational-evidence thresholds or gene-specific lookup spreadsheet, so generic/ClinGen SVI-calibrated REVEL thresholds were used instead.
• BayesDel score (0.307627) was retrieved but not used for PP3/BP4 because no verified published calibration threshold for BayesDel is available to this evaluation.

Sources

Reasoning

PS1 requires that the exact same amino acid change (p.Thr60Ala) has been separately established as pathogenic via a different nucleotide substitution. The only ClinVar record identified for this residue/amino-acid change is the variant under evaluation itself (ClinVar 239423, NM_003000.3(SDHB):c.178A>G p.Thr60Ala), which carries conflicting submissions (Uncertain significance from 11 labs, Benign from 2, Likely benign from 1) rather than an independently confirmed pathogenic classification. No alternate codon change producing p.Thr60Ala with an established pathogenic classification was identified in the gathered evidence bundle or literature pass.

Evidence considered

• ClinVar variation 239423 for this exact variant (NM_003000.3:c.178A>G, p.Thr60Ala) shows conflicting submissions: 11 laboratories Uncertain significance, 2 Benign, 1 Likely benign — this is the variant under assessment itself, not an independent PS1 comparator record.

Still missing

• No dedicated cross-check was performed for alternate nucleotide substitutions (e.g., a different codon 60 change) that would also produce p.Thr60Ala and carry an independent pathogenic classification

Sources

Reasoning

The governing ClinGen Endocrine Tumor Predisposition VCEP specification for SDHB was located (cspec found=true) but its rule_payload is empty/unstructured (framework_complete=false), so no codified PM1 hotspot/critical-domain rule text is available to apply directly; generic ACMG reasoning is used as fallback. The published functional characterization of SDHB (PMID:41252211) maps the gene's structurally critical residues to the iron-sulfur (Fe-S) cluster-binding cysteines (e.g., C68, C93, C101, C113, C186, C189, C249, C253) that coordinate the 2Fe-2S, 4Fe-4S, and 3Fe-4S clusters essential for SDH enzymatic activity. Residue Thr60 is not among these Fe-S coordinating cysteines or any other domain explicitly flagged as structurally/functionally critical in this evidence base, and no statistically significant mutational hotspot was identified for this variant.

Evidence considered

• PMID:41252211 maps SDHB's 11 Fe-S cluster-coordinating cysteine residues (structurally critical region) via 3D structural modeling; residue 60 is not among these coordinating cysteines nor discussed as part of any other critical structural/functional domain in this paper.
• cspec (ClinGen Endocrine Tumor Predisposition Expert Panel Specifications for SDHB, v1.0) was located but its extracted rule_payload is empty, providing no codified PM1 hotspot/domain criterion text for this case.

Still missing

• The SDHB VCEP cspec document was retrieved as present but its structured rule_payload is empty, so no VCEP-specific PM1 domain/hotspot boundary definitions could be directly consulted
• No independent protein structural/domain annotation (e.g., UniProt domain boundaries) beyond the Fe-S cluster mapping in PMID:41252211 was available to confirm residue 60 falls outside all critical functional regions

Sources

Reasoning

PM5 requires a different missense change at the same residue (here, an alternate substitution at Thr60) to have been previously established as pathogenic. The functional characterization paper (PMID:41252211) directly tested SDHB p.Thr60Ile (T60I), the only alternate residue-60 missense variant identified in the evidence base, and found it was reclassified as Likely Benign after functional (succinate/fumarate ratio) testing, explicitly citing it as an example where REVEL over-predicted pathogenicity but functional evidence supported benignity. No pathogenic alternate change at residue 60 was identified. The pipeline's own same-residue candidate harvesting artifact (pm5_candidates) also returned zero same-residue candidates and flagged itself as not eligible for classic PM5 semantics.

Evidence considered

• PMID:41252211 states: 'multiple variants predicted to be damaging in silico — such as SDHB R38P, SDHB T60I, and SDHB S152F — were reclassified as LB with functional testing,' directly demonstrating that the alternate missense change at residue 60 (T60I) was determined to be likely benign rather than pathogenic.
• pm5_candidates.json artifact for this case reports zero same-residue candidates and flags the classic PM5 comparator search as not confidently applicable, consistent with no pathogenic alternate-residue-60 variant being available.

Sources

Reasoning

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

Rule

PP5 only if the exact variant has a ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Evidence considered

• For ClinVar Variation ID 239423 (NM_003000.3:c.178A>G; p.Thr60Ala), the submission audit reports zero expert-panel submissions. The record contains only ordinary laboratory assertions (Uncertain significance, Benign, and Likely benign), which are ineligible for PP5.

Sources

Reasoning

BA1 is not met. The highest reliable population estimate is gnomAD v4.1 NFE AF 0.011865% (140/1,179,932 alleles; zero homozygotes), with overall AF 0.009666% and grpmax FAF 0.01021%; this is far below the generic benign stand-alone threshold.

Rule

Generic ACMG/AMP BA1 population-frequency fallback; no operational BA1 threshold was available in the retrieved SDHB VCEP ruleset.

Evidence considered

• The SDHB ClinGen Endocrine Tumor Predisposition specification is present but its retrieved rule payload contains no operational population-frequency thresholds; generic ACMG/AMP population-frequency guidance was therefore used.
• gnomAD v4.1 reports 156/1,613,972 alleles overall (AF 0.009666%), NFE 140/1,179,932 (AF 0.011865%), grpmax FAF 0.01021%, and zero homozygotes. gnomAD v2.1 gives a concordant NFE AF of 0.011439% (13/113,648) and grpmax FAF 0.006668%; gnomAD-Canada reports NFE AF 0.025558% (3/11,738), a small-sample estimate with FAF95 0.006943%.
• These observed frequencies are orders of magnitude below the generic ACMG/AMP BA1 stand-alone benign threshold and do not create a threshold-artifact concern.

Sources

Reasoning

BS1 is not met. The highest robust population frequency is gnomAD v4.1 NFE AF 0.011865% (140/1,179,932 alleles; zero homozygotes), which is below the generic 0.3% BS1 threshold used when no gene-specific threshold is available.

Rule

Generic ACMG/AMP BS1 population-frequency fallback (0.3%); no operational BS1 threshold was available in the retrieved SDHB VCEP ruleset.

Evidence considered

• The retrieved SDHB VCEP ruleset has no operational BS1 threshold, so a generic ACMG/AMP population-frequency fallback was used.
• gnomAD v4.1 shows overall AF 0.009666%, NFE AF 0.011865% (140/1,179,932 alleles), and grpmax FAF 0.01021%, with zero homozygotes. The small gnomAD-Canada NFE sample has AF 0.025558% (3/11,738) but FAF95 0.006943%, so its point estimate does not approach the generic BS1 threshold or overturn the larger gnomAD result.
• The observed data are below the generic 0.3% BS1 threshold; no BS1 evidence is assigned.

Sources

Reasoning

BP4 is not met. REVEL = 0.845 is far above any ClinGen SVI-calibrated BP4 benign-evidence threshold (BP4_Supporting requires REVEL <=0.290; PMID 36413997), so the missense in-silico prediction supports a pathogenic rather than benign direction. SpliceAI's near-null max delta score (0.002) indicates no predicted splice disruption, but this null splicing signal is not independently applied as BP4 here because the variant's primary annotated consequence is missense and the missense predictor (REVEL) already governs the PP3/BP4 call for this variant, avoiding double counting the same variant across the splice and missense in-silico pathways. No gene-specific SDHB VCEP PP3/BP4 threshold or lookup spreadsheet was found to override this generic assessment. BayesDel (0.307627) was not used because no verified published calibration threshold is available.

Rule

Generic/ClinGen SVI-calibrated REVEL framework (PMID 36413997) applied as fallback; REVEL 0.845 is well above the BP4 benign-evidence threshold, so BP4 is not met.

Evidence considered

• REVEL score = 0.845 exceeds the ClinGen SVI-calibrated BP4_Supporting threshold of <=0.290 (Pejaver et al. 2022, PMID 36413997) in the opposite (pathogenic) direction, so BP4 cannot be met on the missense pathway.
• SpliceAI max delta score = 0.002 predicts no splice impact, but this is not independently invoked for BP4 because the variant's consequence is missense and the REVEL-based missense pathway already governs PP3/BP4 to avoid double counting across pathways for the same variant.
• The SDHB VCEP specification's retrieved rule payload contains no operational, gene-specific PP3/BP4 threshold or lookup spreadsheet that would override the generic REVEL-based assessment.
• BayesDel score (0.307627) was not applied because no verified published calibration threshold for BayesDel is available to this evaluation.

Sources

Reasoning

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

Rule

BP6 only if the exact variant has a ClinVar expert-panel Benign or Likely benign classification.

Evidence considered

• For ClinVar Variation ID 239423 (NM_003000.3:c.178A>G; p.Thr60Ala), the submission audit reports zero expert-panel submissions. The two Benign and one Likely benign entries are ordinary laboratory submissions and are ineligible for BP6.

Sources

Reasoning

No case-level de novo observation is documented. The ClinVar record includes a maternal-origin submission, but it provides no parental genotypes, identity testing, proband phenotype, or family-history data and cannot establish a de novo event.

Rule

Generic ACMG/AMP PS2 fallback: confirmed de novo occurrence requires parental testing and confirmation of both maternity and paternity.

Evidence considered

• The available SDHB CSPEC framework identifies autosomal dominant hereditary pheochromocytoma-paraganglioma but contains no retrieved PS2-specific rule.
• Generic ACMG/AMP defines PS2 as a de novo variant with maternity and paternity confirmed in an affected patient without family history (PMID:25741868, Table 3).
• ClinVar lists one submission with origin reported as maternal, but the record contains no parental genotypes, parentage confirmation, phenotype, or family-history details.

Still missing

• Obtain proband and both parental genotypes for NM_003000.3:c.178A>G.
• Confirm maternity and paternity by identity testing.
• Document the proband phenotype and family history for SDHB-related hereditary pheochromocytoma-paraganglioma.

Sources

Reasoning

No validated functional assay data (enzymatic/complex II activity, SDHB immunohistochemistry, yeast complementation, or other biologically relevant assay) for NM_003000.3:c.178A>G (p.Thr60Ala) was found in the case bundle. Two independent ClinVar submitters (Color Diagnostics/SCV006061159 and St. Jude/SCV005689294) explicitly state functional studies have not been reported for this variant. A large-scale SDHB functional characterization study (PMID:41252211, full text reviewed) profiles many SDHB missense variants but does not report data for T60A/c.178A>G specifically, so it cannot be used to support PS3 for this variant. No ClinGen Endocrine Tumor VCEP-specific PS3 assay rule/strength calibration was retrievable from the case's vcep_materials (empty/no populated rule_payload).

Evidence considered

• Two ClinVar submissions (SCV006061159, SCV005689294) state 'to our knowledge, functional studies have not been reported for this variant.'
• PMID:41252211 full text (Functional characterization of SDHB variants clarifies hereditary PPGL risk) was reviewed for a T60A/c.178A>G-specific data point; no mention of this variant or codon 60 was found in the full text.

Still missing

• No SDHB complex II/succinate dehydrogenase enzymatic activity assay data for this specific variant
• No SDHB immunohistochemistry (tumor SDHB staining) data for this specific variant
• PMID:41252211 functional dataset does not report this variant by name/position
• SDHB VCEP PS3 assay validation/strength thresholds not available in retrieved vcep_materials

Sources

Reasoning

No case-control enrichment analysis or independently documented series of unrelated affected individuals with this exact variant is available in the case evidence.

Rule

Generic ACMG/AMP PS4: significant enrichment in affected individuals versus controls; in a case-control study, OR/RR >5 with CI excluding 1.

Evidence considered

• The applicable SDHB Endocrine Tumor Predisposition Expert Panel framework was identified, but no structured PS4-specific rule was supplied in the case materials; the generic ACMG/AMP case-control standard is therefore used.
• ClinVar lists conflicting laboratory assertions for the exact variant and no expert-panel submission; these assertions do not provide carrier counts, phenotype ascertainment, or a case-control comparison.
• The generic ACMG/AMP PS4 standard requires significantly increased prevalence in affected individuals versus controls; for a case-control study, an odds ratio or relative risk greater than 5 with confidence interval excluding 1 is specified. PMID 25741868.

Still missing

• Ascertain independent carriers of NM_003000.3:c.178A>G with SDHB-related hereditary pheochromocytoma-paraganglioma and an appropriate control comparison.

Sources

Reasoning

No presumed de novo occurrence is documented. The reported maternal origin in one ClinVar submission argues against treating that submission as de novo, and no other submission supplies parental testing or family data.

Rule

Generic ACMG/AMP PM6 fallback: apply only to a documented presumed de novo event lacking confirmation of maternity and paternity.

Evidence considered

• The available SDHB CSPEC framework contains no retrieved PM6-specific rule.
• Generic ACMG/AMP defines PM6 as assumed de novo without confirmation of paternity and maternity (PMID:25741868, Table 3).
• ClinVar records one maternal-origin submission and otherwise provides germline, inherited, unknown, or unspecified origins; it does not document a proband with both parents negative.

Still missing

• Obtain parental genotypes for the proband carrying NM_003000.3:c.178A>G.
• If both parents test negative, document whether biological parentage was confirmed and the proband phenotype and family history.

Sources

Reasoning

No pedigree, affected-relative genotypes, phenotype assignments, or informative meioses are provided. The ClinVar origin fields are insufficient to establish cosegregation.

Rule

Generic ACMG/AMP PP1 fallback: require documented cosegregation in multiple affected relatives; do not infer segregation from a submission origin label.

Evidence considered

• The available SDHB CSPEC framework contains no retrieved PP1-specific rule.
• Generic ACMG/AMP defines PP1 as cosegregation with disease in multiple affected family members and notes that strength may increase with additional segregation data (PMID:25741868, Table 3).
• The ClinVar record reports origin labels for submissions but no family-level genotype-phenotype segregation data or meiosis count.

Still missing

• Provide a pedigree with affected and unaffected relatives, ages, and disease status.
• Provide variant genotypes for informative relatives and the number of independent informative meioses.
• Confirm that segregating phenotypes are consistent with SDHB-related hereditary pheochromocytoma-paraganglioma.

Sources

Reasoning

PP2 requires establishing that missense variation is a common mechanism of disease in SDHB AND that benign missense variation is uncommon in the gene. The evidence bundle does not contain gene-level missense constraint statistics (e.g., gnomAD missense Z-score/o-e ratio) or a formal benign-missense-rate calculation needed to satisfy the second prong, and the SDHB VCEP cspec framework's rule_payload is empty, so no codified VCEP-specific PP2 threshold could be consulted. While PMID:41252211 shows missense variants are a well-documented pathogenic mechanism (structural disruption of Fe-S clusters), it does not by itself establish a quantified low rate of benign missense variation across the gene.

Evidence considered

• cspec (ClinGen Endocrine Tumor Predisposition Expert Panel Specifications for SDHB, v1.0) was located but its extracted rule_payload is empty, providing no codified PP2 applicability statement or missense-constraint threshold for SDHB.

Still missing

• No gnomAD/ExAC missense constraint metric (missense Z-score or observed/expected ratio) was available in the evidence bundle
• No formal calculation of the proportion of benign/likely benign missense variants in SDHB was available
• cspec rule_payload is empty; no VCEP-specific PP2 applicability statement could be retrieved

Sources

Reasoning

No proband phenotype or family-history information is supplied, so phenotype specificity for an SDHB-related disorder cannot be evaluated.

Rule

Generic ACMG/AMP PP4: patient phenotype or family history highly specific for a disease with a single genetic etiology.

Evidence considered

• The applicable framework identifies hereditary pheochromocytoma-paraganglioma as the SDHB-associated disease, inherited autosomal dominantly.
• No individual-level phenotype or family-history data are present in the consolidated case evidence.
• Generic ACMG/AMP PP4 requires a patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. PMID 25741868.

Still missing

• Provide the proband's phenotype, tumor type and location, biochemical findings, age at diagnosis, and relevant family history.

Sources

Reasoning

BS2 is not assessed. Population databases report zero homozygotes, and the bundle provides no phenotype-verified unaffected adult carriers or other observations in healthy adults for this adult-onset, incompletely penetrant autosomal-dominant SDHB-associated condition.

Rule

BS2 requires observation in healthy adults; population frequency or unphenotyped database carriers alone are insufficient, particularly for an incompletely penetrant adult-onset dominant condition.

Evidence considered

• gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada each report zero homozygotes for this variant; absence of homozygotes is not evidence for BS2.
• The SDHB VCEP material identifies hereditary pheochromocytoma-paraganglioma as autosomal dominant. The supplied evidence contains no phenotype-verified unaffected adult carriers, so population allele counts alone cannot establish BS2.

Still missing

• Phenotype-verified observations of sufficiently aged unaffected heterozygous carriers, interpreted with SDHB penetrance and surveillance status, are required to assess BS2.

Sources

Reasoning

No functional assay evidence of normal/neutral SDHB activity for NM_003000.3:c.178A>G (p.Thr60Ala) was identified. No enzymatic complex II activity assay, SDHB IHC data, or other validated benign-supporting functional study for this specific variant was found in the case bundle, ClinVar submission text, or the full-text SDHB functional characterization paper reviewed (PMID:41252211), which does not report data for this variant.

Evidence considered

• PMID:41252211 full text reviewed for T60A/c.178A>G-specific functional data; variant not found in the dataset.

Still missing

• No SDHB functional/enzymatic assay data of any kind (pathogenic or benign direction) available for this specific variant
• PMID:41252211 large-scale functional dataset does not include this variant

Sources

Reasoning

No affected family member is documented to lack NM_003000.3:c.178A>G. A maternal-origin ClinVar submission does not establish non-segregation because the mother's phenotype and genotype context, as well as affected-relative testing, are not provided.

Rule

Generic ACMG/AMP BS4 fallback: require documented lack of segregation in affected family members after considering phenocopies and other molecular explanations.

Evidence considered

• The available SDHB CSPEC framework contains no retrieved BS4-specific rule.
• Generic ACMG/AMP defines BS4 as lack of segregation in affected members of a family and cautions that phenocopies and multiple pathogenic variants can confound apparent non-segregation (PMID:25741868, Table 4).
• The ClinVar record provides no affected-relative genotype-phenotype data for NM_003000.3:c.178A>G.

Still missing

• Provide genotype and phenotype data for affected relatives who do and do not carry the variant.
• Assess phenocopies and alternative molecular causes before interpreting any apparent non-segregation.

Sources

Reasoning

No qualifying co-occurrence observation is provided. The record does not identify an established pathogenic/likely pathogenic SDHB variant in the same individual, does not establish cis or trans phase, and does not document an unaffected carrier context that could support BP2. The governing SDHB condition is autosomal dominant, and the supplied framework does not provide a BP2-specific modification.

Rule

Generic ACMG/AMP BP2: observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern; no qualifying co-occurrence was documented.

Evidence considered

• The governing SDHB Endocrine Tumor Predisposition Specification identifies hereditary pheochromocytoma-paraganglioma as autosomal dominant and contains no BP2-specific rule in the supplied material.
• The exact-variant ClinVar record has conflicting classifications and submission origin labels only; its supplied submission data do not report a co-occurring pathogenic/likely pathogenic SDHB variant or phase.

Still missing

• A case-level co-occurrence report naming the comparator variant and its pathogenic/likely pathogenic evidence.
• Reliable phase evidence showing cis co-occurrence, or a disease-context-qualified trans co-occurrence observation, together with phenotype and penetrance-relevant information.

Sources

Reasoning

No case-level molecular testing result documents an alternate molecular basis for the proband's disease.

Rule

Generic ACMG/AMP BP5: variant found in a case with an alternate molecular basis for disease.

Evidence considered

• No proband-level alternate molecular diagnosis or co-occurrence evidence is included in the consolidated case evidence.
• Generic ACMG/AMP BP5 applies when a variant is found in a case with an alternate molecular basis for disease. PMID 25741868.

Still missing

• Provide case-level testing results establishing a separate pathogenic or likely pathogenic molecular diagnosis that explains the presenting phenotype.

Sources