SDHB c.158G>A, p.(Gly53Glu)

Reasoning

The allele is consistently enriched in the Ashkenazi Jewish population above the applicable generic BA1 threshold of 1.0%: 1.09768% in gnomAD v4.1 (325/29,608 alleles) and 1.08004% in gnomAD v2.1 (112/10,370 alleles). The v4.1 observation includes three homozygotes. This large, ancestry-specific frequency is incompatible with a pathogenic SDHB allele for autosomal-dominant hereditary pheochromocytoma-paraganglioma under the generic population framework.

Rule

Generic ACMG/AMP population-frequency fallback: BA1 when the highest reliable ancestry-specific allele frequency exceeds 1.0%; applied after confirming the SDHB CSpec contains no population criterion specification.

Evidence considered

• The SDHB ClinGen Endocrine Tumor Predisposition CSpec record was reviewed first. Its rules table is empty and the record is in Pilot Rules In Prep, so it provides no applicable population-frequency thresholds; generic ACMG/AMP population thresholds were used.
• gnomAD v4.1 reports Ashkenazi Jewish AF 1.09768% (325/29,608 alleles) with 3 homozygotes; gnomAD v2.1 independently reports Ashkenazi Jewish AF 1.08004% (112/10,370 alleles) with 1 homozygote.
• The generic case framework specifies BA1 for population frequency greater than 1.0%.

Sources

Reasoning

PS1 requires that the exact same amino acid change (p.Gly53Glu) has already been established as pathogenic by an independent nucleotide change. Here the amino acid change in question IS this variant, and ClinVar (VCV000184148) classifies NM_003000.3:c.158G>A / p.(Gly53Glu) as Benign (7 labs Benign, 6 Likely benign, no P/LP submissions). A recent functional complementation study (succinate/fumarate ratio and SDH activity assay) independently reclassified SDHB G53E as benign, concordant with ClinVar. There is no established pathogenic precedent for the Gly53Glu amino acid change, so PS1 is not met.

Rule

PS1_generic

Evidence considered

• ClinVar VCV000184148 for NM_003000.3(SDHB):c.158G>A (p.Gly53Glu) shows classification Benign/Likely benign with 15 submissions, 0 pathogenic or likely pathogenic, criteria provided multiple submitters no conflicts.
• PMID:41252211 (functional complementation assay paper) states: 'SDHB C68Y and SDHB G53E (indicated with asterisks) were recently reclassified in ClinVar as pathogenic and benign, respectively (Figure 4A), aligning with the results of our functional assay,' confirming the same p.Gly53Glu change is benign, not pathogenic.

Sources

Reasoning

PM1 requires the variant to lie in a well-established mutational hotspot or critical/well-characterized functional domain without benign variation. cancerhotspots.org returned no statistically significant hotspot for SDHB codon 53. The functional characterization study (PMID:41252211) mapped SDHB's three Fe-S clusters and their 11 coordinating cysteine residues (explicitly named cysteines include C93, C101, C113, C186, C189, C249, C253); residue Gly53 is not among the Fe-S cluster-binding cysteines or any other domain flagged as functionally critical in that study. No CSPEC/VCEP-specific PM1 hotspot/domain rule payload is available for SDHB (rule_payload is empty in the ClinGen Endocrine Tumor Predisposition EP specification). Absent hotspot or critical-domain evidence, PM1 is not met.

Rule

PM1_generic

Evidence considered

• cancerhotspots.org search for 'SDHB G53' returned no_result / 'This variant does not lie in a statistically significant hotspot.'
• PMID:41252211 structurally mapped SDHB's Fe-S cluster-binding cysteines (2Fe-2S, 4Fe-4S, 3Fe-4S clusters) as the functionally critical/conserved residues; G53 is not among the 11 Fe-S-coordinating cysteines or other domains highlighted as structurally essential (e.g., C93, C101, C113, C186, C189, C249, C253).
• The SDHB ClinGen Endocrine Tumor Predisposition EP specification (cspec) was consulted but its extracted rule_payload is empty/unstructured, providing no codified PM1 hotspot/domain criteria to apply.

Still missing

• No structured CSPEC/VCEP PM1 hotspot/domain table was available for SDHB to confirm domain boundaries definitively.

Sources

Reasoning

PM2 is not met because the allele is not absent or rare in population databases. Its Ashkenazi Jewish allele frequency is approximately 1.1% in both gnomAD v4.1 and v2.1, far above the generic PM2 rarity threshold of less than 0.1%.

Rule

Generic ACMG/AMP population-frequency fallback: PM2 requires absence or extreme rarity, operationalized in this case framework as an ancestry-specific allele frequency below 0.1%.

Evidence considered

• gnomAD v4.1: Ashkenazi Jewish AF 1.09768% (325/29,608 alleles); total AF 0.02838% (458/1,614,034 alleles).
• gnomAD v2.1: Ashkenazi Jewish AF 1.08004% (112/10,370 alleles); total AF 0.04915% (139/282,780 alleles).
• The generic case framework specifies PM2 only for an allele frequency below 0.1%; ancestry-stratified frequency is used to avoid dilution of a founder-enriched allele in the global aggregate.

Sources

Reasoning

NM_003000.3:c.158G>A (p.Gly53Glu) is a missense variant. The governing ClinGen Endocrine Tumor Predisposition VCEP specification for SDHB (cspec doc 101200173, v1.0) contains no populated PP3/BP4 rule payload or gene-specific in-silico lookup table for this variant (criteria/rule_payload fields empty), so generic ACMG/AMP calibrated in-silico evidence applies. REVEL score = 0.608, below the ClinGen SVI-recommended supporting threshold for pathogenic evidence (REVEL>=0.644-0.773 range for PP3_Supporting per Pejaver et al. calibration, PMID 36413997) and above the BP4 supporting threshold (REVEL<=~0.29-0.30), placing it in the indeterminate zone where REVEL crosses neither PP3 nor BP4 calibrated cutoffs. BayesDel score (0.345884) was also computed but is not used: no verified published calibration/threshold publication for BayesDel is available to this pipeline, so it is treated as not_available for PP3/BP4 regardless of the raw score. SpliceAI predicts no splice impact (max delta score = 0.00), which is consistent with a missense-only mechanism and does not itself support PP3 for a splicing mechanism. On the missense in-silico path, no calibrated predictor crosses a pathogenic-supporting threshold, so PP3 is not met.

Rule

generic_ACMG_calibrated_insilico

Evidence considered

• REVEL score = 0.608 for NM_003000.3:c.158G>A (p.Gly53Glu), from local REVEL v1.3 predictor lookup; this falls short of the ClinGen SVI/Pejaver et al. (PMID 36413997) calibrated PP3_Supporting threshold (~0.644 and above).
• SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00; pangolin_SG=0.001, pangolin_SL=-0.001), so no splicing mechanism supports PP3.
• The SDHB cspec (ClinGen Endocrine Tumor Predisposition EP, v1.0, doc_id 101200173) supporting materials retrieved for this case contain an empty rule_payload and criteria list, with no gene-specific PP3/BP4 lookup spreadsheet or pre-assigned code found for this variant.
• BayesDel score = 0.345884 was computed but is not used for PP3/BP4 because no verified published BayesDel calibration threshold/publication is available to this pipeline.

Still missing

• No SDHB VCEP-specific PP3/BP4 in-silico lookup table or pre-assigned code is available for this variant in the cspec materials retrieved for this case.
• BayesDel score is present but cannot be applied without a verified published calibration threshold.

Sources

Reasoning

No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion is present; PP5 is not met.

Rule

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

Evidence considered

• The exact variant ClinVar audit identifies 15 submissions and zero expert-panel submissions. The observed benign and likely benign laboratory assertions are not usable for PP5.

Sources

Reasoning

BP1 applies to missense variants in genes where primarily truncating variants are known to cause disease (i.e., missense is not a plausible disease mechanism). SDHB does not fit this profile: PMID:41252211 functionally validated 27 distinct pathogenic missense SDHB variants (elevated succinate/fumarate ratio, markedly reduced SDH activity, meeting PS3 strong criteria) alongside 9 benign missense variants, and reclassified 14 additional missense VUS as likely pathogenic based on loss-of-function biochemistry, demonstrating that missense variation is a well-established, common mechanism of SDHB-related disease (not merely a rare or tolerated event). Because missense pathogenicity is clearly established for this gene, BP1 (which presumes missense is not expected to be pathogenic) does not apply / is not met for this variant class.

Rule

BP1_generic

Evidence considered

• PMID:41252211 reports characterizing 36 ClinVar-classified SDHB missense variants (9 benign, 27 pathogenic) via a succinate/fumarate functional assay: 'all 27 pathogenic variants showed elevated succinate/fumarate ratios, consistent with amorphic or severely hypomorphic allele activity,' and separately reclassified 14 of 39 tested VUS missense alleles to Likely Pathogenic using PS3-strong functional evidence, establishing missense variation as a well-documented, common SDHB disease mechanism (not one where truncating variants are uniquely causal).

Sources

Reasoning

Same underlying computational data as PP3. REVEL = 0.608 does not fall at or below the calibrated BP4_Supporting threshold (~0.29-0.30 per Pejaver et al., PMID 36413997), so REVEL does not support a benign call either. SpliceAI predicts no splice impact (max delta = 0.00), which is consistent with absence of a splicing mechanism but this variant's primary consequence is missense, and a negative SpliceAI score alone is not used to invoke BP4 for a missense variant under generic ACMG guidance (SpliceAI absence of signal is expected default for most missense variants and is not itself calibrated evidence of benign impact on protein function). BayesDel (0.345884) is not applied per lack of a verified published calibration threshold. No SDHB VCEP-specific PP3/BP4 lookup spreadsheet pre-assigns a code here. Therefore BP4 is not met.

Rule

generic_ACMG_calibrated_insilico

Evidence considered

• REVEL score = 0.608 for NM_003000.3:c.158G>A (p.Gly53Glu); this is above the ClinGen SVI/Pejaver et al. (PMID 36413997) calibrated BP4_Supporting threshold (~0.29-0.30), so REVEL does not support benign in-silico evidence.
• SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00); absence of a predicted splice effect on a missense variant does not itself constitute calibrated BP4 evidence.
• The SDHB cspec (ClinGen Endocrine Tumor Predisposition EP, v1.0, doc_id 101200173) supporting materials retrieved for this case contain an empty rule_payload and criteria list, with no gene-specific PP3/BP4 lookup spreadsheet or pre-assigned code found for this variant.
• BayesDel score = 0.345884 was computed but is not used for PP3/BP4 because no verified published BayesDel calibration threshold/publication is available to this pipeline.

Still missing

• No SDHB VCEP-specific PP3/BP4 in-silico lookup table or pre-assigned code is available for this variant in the cspec materials retrieved for this case.
• BayesDel score is present but cannot be applied without a verified published calibration threshold.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign assertion is present; BP6 is not met.

Rule

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

Evidence considered

• The exact variant ClinVar audit identifies 15 submissions and zero expert-panel submissions. The observed benign and likely benign laboratory assertions are not usable for BP6.

Sources

Reasoning

No case-level proband phenotype, parental genotypes, parentage confirmation, or de novo testing result is provided. PS2 cannot be assessed.

Rule

Generic ACMG/AMP de novo evidence definition used because the available SDHB specification has no criterion-specific PS2 rule: PS2 requires a confirmed de novo occurrence in an affected individual with confirmed maternity and paternity.

Evidence considered

• The available SDHB Endocrine Tumor Predisposition Specification v1.0 identifies autosomal-dominant hereditary pheochromocytoma-paraganglioma as the disease context but contains no extracted PS2-specific rule payload.
• The consolidated case evidence contains no proband-parent trio result, parentage result, or reported de novo observation for NM_003000.3:c.158G>A.

Still missing

• Proband phenotype and age at diagnosis.
• Orthogonal parental testing for c.158G>A in both biological parents.
• Confirmation of maternity and paternity, and assessment for parental mosaicism if the variant is absent from blood.

Sources

Reasoning

No functional/biochemical assay data (e.g., complex II enzymatic activity, SDHB immunohistochemistry, yeast/cell-based complementation) for NM_003000.3:c.158G>A (p.Gly53Glu) was found in the case evidence bundle or in the fetched full-text publications. PMID:41252211 is a functional characterization study of SDHB missense variants (IHC/enzymatic assays), but exhaustive search of its available text found no mention of this specific variant (G53E/p.Gly53Glu/c.158G>A); it is therefore not usable to support PS3 for this variant. The ClinGen Endocrine Tumor Predisposition VCEP SDHB specification (v1.0) is the governing framework but its rule_payload/PS3-BS3 code-specific text was not populated in vcep_materials.json for this case, so no VCEP-specific PS3 strength calibration could be applied either.

Evidence considered

• Searched full-text of PMID:41252211 (Functional characterization of SDHB variants clarifies hereditary pheochromocytoma and paraganglioma risk and genotype-phenotype relationships) for 'G53E', 'Gly53Glu', 'c.158G', '158G>A', 'p.G53', '53Glu' - no matches found in the available fetched text
• Searched entire criteria_evidence_bundle.txt for assay/activity/immunohistochemistry/functional-study terms combined with this variant - no PS3/BS3-supporting content found

Still missing

• No validated functional assay (enzymatic activity, IHC, complementation) reported for this exact variant in any reviewed source
• PMID:41252211 (SDHB functional characterization study) does not mention this variant in its available text; unable to confirm whether the variant was included in that study's cohort without further full-text access
• VCEP SDHB PS3/BS3 code-specific point-strength rules (e.g. required assay validation criteria) were not available in vcep_materials.json for this case

Sources

Reasoning

The available exact-variant papers document c.158G>A (p.Gly53Glu) in SDHB mutation cohorts but do not provide an exact-variant affected-case count and an appropriate control comparison from which case-control enrichment can be calculated. PS4 is therefore not assessed.

Rule

No applicable quantitative PS4 threshold was retrieved from the SDHB CSPEC material; use generic ACMG/AMP PS4 only when statistically supported case-control enrichment is available.

Evidence considered

• PMID:19802898 lists c.158G>A (p.Gly53Glu) in its SDHB mutation table and reports 0/314 control chromosomes, but the table does not state the number of affected carriers for this exact variant.
• PMID:23072324 lists c.158G>A (p.Gly53Glu) among SDHB variants in a 501-proband PPGL/HNPGL referral cohort, without an exact-variant carrier count or control comparison.

Still missing

• An exact-variant case count and ancestry-matched control data, or a published odds ratio with confidence interval, are needed to assess enrichment.

Sources

Reasoning

PM5 requires a different missense change at the same residue (Gly53) that has been established as pathogenic. The pipeline's PM5 candidate harvesting step explicitly did not run a same-residue comparator search for this variant ('Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting'), returning zero candidates. No other alternate amino acid substitutions at codon 53 were identified anywhere in the case bundle or the reviewed full-text SDHB functional paper (which examined a broad set of named SDHB missense variants but did not report any other p.Gly53X substitution). Given the absence of any comparator data, PM5 cannot be reliably assessed and is flagged for human review rather than defaulted to not_met.

Rule

PM5_generic

Evidence considered

• pm5_candidates.json records eligible_for_classic_pm5_search=false and candidate_summary with 0 total_candidates_seen, 0 same_residue_candidates, explicitly instructing that PM5 should not be applied from this artifact alone.

Still missing

• No same-residue (codon 53) comparator variant search was performed by the pipeline; a manual ClinVar/literature search for other Gly53 substitutions (e.g., p.Gly53Ala, p.Gly53Val, p.Gly53Arg) is needed to confirm or rule out PM5.

Sources

Reasoning

No reported de novo occurrence or parental testing result is available; therefore presumed de novo evidence cannot be assessed.

Rule

Generic ACMG/AMP de novo evidence definition used because the available SDHB specification has no criterion-specific PM6 rule: PM6 requires a presumed de novo occurrence without confirmation of maternity and paternity.

Evidence considered

• The available SDHB Endocrine Tumor Predisposition Specification v1.0 contains no extracted PM6-specific rule payload.
• The consolidated case evidence contains no parental genotype information or de novo report for NM_003000.3:c.158G>A.

Still missing

• A reported affected proband carrying c.158G>A.
• Parental testing showing absence of the variant when parentage confirmation is unavailable.

Sources

Reasoning

No informative relatives, affected-status data, genotypes, or meioses are reported for c.158G>A; segregation cannot be evaluated.

Rule

Generic ACMG/AMP segregation evidence definition used because the available SDHB specification has no criterion-specific PP1 rule: PP1 requires cosegregation with disease in multiple affected family members, with strength determined by the available segregation evidence.

Evidence considered

• The available SDHB Endocrine Tumor Predisposition Specification v1.0 contains no extracted PP1-specific rule payload.
• The consolidated case evidence contains no pedigree, relative genotypes, phenotype segregation data, or informative meiosis count for NM_003000.3:c.158G>A.

Still missing

• Pedigree with phenotype status, ages, and diagnoses for tested relatives.
• Genotypes for informative affected and unaffected relatives and the number of independent informative meioses.
• Phenotype definition appropriate to SDHB-related hereditary pheochromocytoma-paraganglioma and consideration of age-dependent penetrance.

Sources

Reasoning

PP2 requires that the gene has a low rate of benign missense variation and missense variants are a common mechanism of disease. The ClinGen Endocrine Tumor Predisposition EP specification for SDHB was located (cspec) but its extracted rule payload is empty/unstructured and does not codify a PP2 recommendation for SDHB (nor does it flag PP2 as not applicable). No gene-level missense constraint metric (e.g., a missense Z-score or benign-missense-rate figure) is present anywhere in the case bundle to independently evaluate this criterion. While PMID:41252211 shows both pathogenic and benign missense variants occur throughout SDHB (consistent with missense being a real, non-rare disease mechanism), that alone is insufficient to confirm a 'low rate of benign missense variation' as PP2 requires, and no explicit VCEP position on PP2 applicability was found. This is flagged for human review rather than guessed.

Rule

PP2_generic

Evidence considered

• vcep_materials.json shows the ClinGen Endocrine Tumor Predisposition EP SDHB specification (cspec) was found but framework_complete=false and rule_payload={} with no PP2-specific guidance codified.

Still missing

• No structured VCEP guidance on PP2 applicability/thresholds for SDHB is present in vcep_materials.json (rule_payload empty).
• No gene-level missense constraint statistic (e.g., ExAC/gnomAD missense Z-score) was retrieved for SDHB in this case bundle.

Sources

Reasoning

No case-level phenotype is supplied for the individual carrying this variant. Consequently, phenotype specificity for SDHB-related hereditary pheochromocytoma-paraganglioma cannot be evaluated.

Rule

No applicable SDHB-specific PP4 rule was retrieved; generic ACMG/AMP PP4 requires a phenotype highly specific for disease caused by the gene.

Evidence considered

• The SDHB CSPEC framework identifies hereditary pheochromocytoma-paraganglioma as the disease context, but no individual phenotype is present in the case-evidence bundle.

Still missing

• Case-level phenotype, age at onset, tumor site(s), biochemical findings, and differential diagnostic evaluation are needed.

Sources

Reasoning

Population databases document homozygotes but do not provide the phenotyped, unaffected adult observations required for BS2. The three gnomAD v4.1 homozygotes therefore cannot be treated as BS2 evidence.

Rule

BS2 requires well-phenotyped unaffected adult individual(s) for a disorder expected to be fully penetrant at an early age; unphenotyped population database homozygotes do not satisfy this requirement.

Evidence considered

• gnomAD v4.1 reports 3 homozygotes and gnomAD v2.1 reports 1 homozygote, but neither source supplies disease phenotype or longitudinal ascertainment for those individuals.
• Because hereditary pheochromocytoma-paraganglioma is autosomal dominant and incompletely penetrant, an unphenotyped population-database observation does not establish that a carrier is unaffected.

Still missing

• Phenotyped adult carrier data demonstrating absence of SDHB-related hereditary pheochromocytoma-paraganglioma in a number of individuals adequate for BS2 are not available in the reviewed evidence bundle.

Sources

Reasoning

No functional/biochemical assay data demonstrating normal/wild-type activity for NM_003000.3:c.158G>A (p.Gly53Glu) was found in the case evidence bundle or fetched full-text publications. As with PS3, PMID:41252211 is a relevant SDHB functional characterization study but does not mention this specific variant in its available text, so no benign functional evidence could be extracted to support BS3.

Evidence considered

• Same search as PS3: no functional assay results (normal or abnormal) for this variant found in the bundle or PMID:41252211 full text

Still missing

• No functional assay data (of any result direction) identified for this exact variant
• PMID:41252211 does not mention this variant in the available fetched text

Sources

Reasoning

No informative non-segregation observations are reported; BS4 cannot be assessed.

Rule

Generic ACMG/AMP non-segregation evidence definition used because the available SDHB specification has no criterion-specific BS4 rule: BS4 requires lack of segregation with disease in affected family members; unaffected carriers alone require careful interpretation in an age-dependent predisposition condition.

Evidence considered

• The available SDHB Endocrine Tumor Predisposition Specification v1.0 identifies an autosomal-dominant hereditary pheochromocytoma-paraganglioma context but contains no extracted BS4-specific rule payload.
• The consolidated case evidence contains no family phenotype-genotype discordance data for NM_003000.3:c.158G>A.

Still missing

• Documented informative affected relatives who lack c.158G>A, with verified phenotype and relationship information.
• Documented unaffected carriers with age and surveillance information sufficient to interpret non-segregation in this age-dependent hereditary cancer-predisposition disorder.

Sources

Reasoning

No eligible variant-level co-occurrence was provided: there is no affected proband reported with a pathogenic/likely pathogenic variant in trans, no confirmed cis observation with such a variant, and no phase data. Therefore BP2 cannot be applied.

Rule

Generic ACMG/AMP BP2 requires a qualifying observation of the variant in cis with a pathogenic variant for a dominant disorder, or in trans with a pathogenic variant for a fully penetrant dominant disorder; neither observation is available.

Evidence considered

• The consolidated evidence bundle contains no PM3/BP2 candidate evidence and no reported phase or co-occurring pathogenic/likely pathogenic allele for c.158G>A.

Still missing

• Documented co-occurrence of c.158G>A with a pathogenic/likely pathogenic variant in an appropriate individual, including confirmed phase and phenotype, is required to assess BP2.

Reasoning

No affected-individual clinical record or independently established alternative molecular diagnosis is supplied, so BP5 cannot be evaluated.

Rule

Generic ACMG/AMP BP5 requires an alternate molecular basis for disease in an affected individual.

Still missing

• Clinical phenotype and testing results demonstrating an alternative molecular diagnosis that fully explains the phenotype are needed.