SDHB c.137G>A, p.Arg46Gln

Reasoning

PS3 is met at strong strength. PMID:22835832 directly studied the exact SDHB p.R46Q variant in transfected HeLa cells using cycloheximide and [35S]-methionine pulse-chase protein-stability assays. R46Q showed markedly accelerated degradation, with a half-life of 1.73 hours versus 11.23 hours for wild-type SDHB by radiolabeled pulse-chase analysis and 0.74 hours in the cycloheximide assay. This variant-specific quantitative loss of SDHB protein is a functional result supportive of a damaging effect. The study did not establish clinical diagnostic-laboratory validation or independent replication, so no stronger-than-strong interpretation is made; such limitations do not negate the ACMG/AMP strong PS3 category.

Rule

PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Yang et al. 2012, PMID:22835832, Results, Fig. 2C-D: exact SDHB R46Q was tested in transfected HeLa cells with cycloheximide and [35S]-methionine pulse-chase assays.
• Yang et al. 2012, PMID:22835832: R46Q protein half-life was 1.73 hours by radiolabeled pulse-chase analysis versus 11.23 hours for wild-type SDHB; the cycloheximide assay estimated 0.74 hours for R46Q.
• Yang et al. 2012, PMID:22835832: the direct quote supporting the assay result is "We identified a marked reduction in the [35S]-labeled SDHB protein in the R46Q mutant over the course of 12 h".

Still missing

• Independent replication and validation in a clinical diagnostic laboratory were not documented in the reviewed source.

Sources

Reasoning

PM2 is met at supporting strength. No governing VCEP criterion specifies a non-cancer or exome-only source, so the default all-comers gnomAD entries govern, while the available non-cancer subsets are concordant. gnomAD v4.1 reports AF=1.67302e-05 from 27/1613850 alleles, zero homozygotes, and grpmax FAF=1.298e-05; gnomAD v2.1 exome reports 1/251358 alleles, zero homozygotes; gnomAD v2.1 non-cancer is absent and gnomAD v3.1 non-cancer reports AF=6.76535e-06, 1/147812 alleles, zero homozygotes, grpmax FAF=4.88e-06. The all-comers filtering frequency is below the generic PM2 supporting threshold of <=0.0001.

Rule

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers: AF=1.67302e-05, 27/1613850 alleles, homozygotes=0, grpmax FAF=1.298e-05.
• gnomAD v2.1 exome: AF=3.97839e-06, 1/251358 alleles, homozygotes=0.
• gnomAD v2.1 non-cancer: absent.
• gnomAD v3.1 non-cancer genomes: AF=6.76535e-06, 1/147812 alleles, homozygotes=0, grpmax FAF=4.88e-06; this independently supports an extremely low population frequency.
• No released SDHB VCEP criteria specify a non-cancer or exome-only source, so all-comers gnomAD v4.1 is the default governing source.
• Applied published generic calibration: PM2 (supporting) <=0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868).

Sources

Reasoning

PP1 is met at supporting strength. In Benn et al., the exact c.137G>A (R46Q) variant occurred in family F04: the proband had carotid body and adrenal paragangliomas, additional retroperitoneal paragangliomas were reported in family members, and the inheritance was paternal. This is compatible with segregation of the variant with disease in more than one affected family member, but the report does not provide a complete individual-level genotype/phenotype table for every affected relative; human review is retained for pedigree-level confirmation. The Japanese family report adds transmission to an asymptomatic carrier, so it does not strengthen affected-member cosegregation.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• Benn et al. 2003, Table 1, family F04: proband with carotid body and adrenal paragangliomas; additional retroperitoneal paragangliomas in family; paternal inheritance of the SDHB mutation.
• Benn et al. 2003, Results: the exact c.137G>A (R46Q) variant was identified in family F04.
• Takekoshi et al. 2008: the third son carrying R46Q was asymptomatic with normal catecholamines and no tumor on CT or MIBG, so this report is not additional affected-member cosegregation.

Still missing

• Confirm the exact genotype and phenotype of each affected F04 relative from the original pedigree before considering any strength above supporting.

Sources

Reasoning

The variant is a missense change, NP_002991.2:p.(Arg46Gln), so the governing generic rule selects the REVEL path only; SpliceAI is not used for PP3/BP4 in a missense variant. REVEL is 0.911, meeting the published generic PP3 supporting threshold of >= 0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997). BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... Missense variant -> REVEL only. ... PP3 (supporting): >= 0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• The normalized consequence is missense, NP_002991.2:p.(Arg46Gln); therefore the applicable computational path is REVEL only, and the SpliceAI score is not counted for PP3/BP4.
• Local REVEL predictor lookup reports a score of 0.911 for NM_003000.3:c.137G>A.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997) sets generic PP3 supporting at >= 0.644; REVEL 0.911 meets this threshold.
• BayesDel score 0.581872 is not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The variant is the same c.137G>A nucleotide change in the available reports of SDHB p.Arg46Gln, including family-based and cohort reports. PS1 requires the same amino-acid change to have been established as pathogenic regardless of nucleotide change, so an independently reported alternate nucleotide change producing Arg46Gln is not demonstrated here. The available evidence therefore supports prior observation of this exact variant but does not satisfy PS1's synonymous-protein-change requirement.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• Benn et al. 2003, Results paragraph 2 and Table 1 family F04: reports SDHB c.137G>A; R46Q.
• Kuroda et al. 2008, Results and Figure 2 caption: reports a heterozygous exon 2 G>A transition changing arginine 46 to glutamine.
• van der Harst et al. 2012, Results: lists c.137G>A p.Arg46Gln among SDHB missense mutations; no alternate nucleotide encoding Arg46Gln is reported in the extracted evidence.

Still missing

• A verified pathogenic alternate-nucleotide variant producing p.Arg46Gln is not documented.

Sources

Reasoning

PS2 is not met. The variant was reported as inherited rather than de novo: in family F04, SDHB c.137G>A (R46Q) was associated with paternal inheritance, and the Japanese family report identified the variant in the proband and his third son. The available reports do not document confirmed maternity and paternity for a de novo occurrence, and the familial observations directly contradict a de novo interpretation.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• Benn et al. 2003, Results/Table 1, family F04: SDHB c.137G>A (R46Q) occurred in a family with the proband's carotid body and adrenal paragangliomas, additional retroperitoneal paragangliomas in the family, and paternal inheritance.
• Takekoshi et al. 2008, Results: germline R46Q was found in the proband and his third son, while the other two sons were negative; the third son was an asymptomatic carrier.
• No report documents a de novo occurrence with both maternity and paternity confirmed.

Sources

Reasoning

Generic ACMG/AMP PS4 requires a statistically significant increase in affected individuals, with a relative risk or odds ratio >5.0 and a 95% confidence interval that does not include 1. PMID:12618761 reports the exact c.137G>A (R46Q) variant in 1 of 7 studied probands and in 0 of 100 normal alleles. I treated this as a case-control comparison and computed the odds ratio from those counts: the uncorrected OR is infinite because the control cell is zero; the exact conditional 95% CI has a lower bound of approximately 0.366 and an infinite upper bound, so it includes 1. Therefore the reported comparison does not satisfy the PS4 threshold. The other reviewed exact-variant reports (PMID:18362451 and PMID:23083876) are family/cohort observations without affected-versus-unaffected or population control counts sufficient to establish the required OR and confidence interval.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• Benn et al. 2003, Results and Table 1: SDHB c.137G>A (R46Q) occurred in family F04, one of seven probands studied; the paper states that R46Q was not identified in 100 normal alleles.
• Computed case-control comparison from the reported counts: 1/7 affected probands versus 0/100 controls; uncorrected OR = infinity because the control carrier count is zero, and the exact conditional 95% CI lower bound is approximately 0.366 with no finite upper bound, so the interval includes 1.
• PMID:18362451 reports a Japanese family with an affected proband and an asymptomatic carrier, not a case-control prevalence comparison.
• PMID:23083876 lists p.Arg46Gln among germline SDHB mutations in 14 patients from 12 renal-cell-carcinoma families but gives no variant-specific control count or case-control estimate.

Sources

Reasoning

The governing SDHB VCEP is listed but has no released criteria, and vcep_materials.json contains no SDHB domain-table entry. The available Cancer Hotspots result reports no statistically significant hotspot for SDHB R46. No complete, well-established critical-domain or hotspot evidence with absence of benign variation places residue 46 within the generic PM1 definition.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• The SDHB CSPEC is present but has no released criterion specifications; vcep_materials.json has domain_tables={}.
• Cancer Hotspots query SDHB R46 returned no result and states that the variant does not lie in a statistically significant hotspot.

Sources

Reasoning

PM6 is not met. The available evidence does not describe an assumed de novo variant; instead, the variant was reported with paternal inheritance in family F04 and in both a Japanese proband and his carrier son. Therefore, the evidence does not support a de novo interpretation even without parental confirmation.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• Benn et al. 2003, Table 1 and inheritance results: family F04 had paternal SDHB mutation inheritance.
• Takekoshi et al. 2008, Results: R46Q was present in the proband and his third son, with the son classified as an asymptomatic carrier.
• No evidence supports an assumed de novo event.

Sources

Reasoning

The exact variant has Pathogenic and Likely pathogenic ClinVar submissions, but the ClinVar audit reports zero expert-panel submissions; the available assertions are ordinary laboratory submissions or aggregate labels. Under the specified generic rule, those assertions do not qualify for PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-match record NM_003000.3(SDHB):c.137G>A (p.Arg46Gln): Pathogenic in 21 clinical laboratories and Likely pathogenic in 2, with 0 expert-panel submissions.
• The available laboratory submissions and aggregate ClinVar label are excluded by the specified PP5 rule.

Sources

Reasoning

BA1 is not met. The default generic stand-alone threshold is >=0.05. The gnomAD v4.1 filtering frequency is grpmax FAF=1.298e-05, far below 0.05; the highest observed subpopulation point estimate, 3.20123e-05 in Remaining individuals from 2/62476 alleles, is not used for BA1 and does not meet the required population-frequency evidence. gnomAD v2.1 also showed only 1/251358 alleles, with no homozygotes. No governing SDHB criterion specifies a different population source or threshold.

Rule

BA1: >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v4.1: total AF=1.67302e-05, 27/1613850 alleles, homozygotes=0; filtering grpmax FAF=1.298e-05.
• gnomAD v4.1 highest observed subpopulation: Remaining individuals AF=3.20123e-05, 2/62476 alleles; this point estimate was not used for BA1.
• gnomAD v2.1 exome: AF=3.97839e-06, 1/251358 alleles, homozygotes=0; grpmax FAF was unavailable.
• The CSPEC record lists SDHB criteria but provides no released criterion specifications, so generic ACMG/AMP rules govern and no alternate population source is specified.
• Applied published generic calibration: BA1 (stand-alone) >=0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

BS1 is not met. The generic default threshold is >=0.01, and the required filtering frequency is gnomAD v4.1 grpmax FAF=1.298e-05, not the popmax point estimate. This is far below 0.01, and the highest observed subpopulation point estimate (3.20123e-05 from 2/62476 alleles) cannot override the lower FAF. The allele frequency therefore is not greater than expected for SDHB-related disease on the available evidence; no maximum-credible-frequency calculation is needed because the observed filtering frequency is orders of magnitude below the default.

Rule

Allele frequency is greater than expected for the disorder. ... BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v4.1 filtering frequency: grpmax FAF=1.298e-05; total AF=1.67302e-05 from 27/1613850 alleles and no homozygotes.
• gnomAD v4.1 highest observed subpopulation point estimate: 3.20123e-05 in Remaining individuals from 2/62476 alleles; this was not used for BS1.
• gnomAD v2.1 exome: AF=3.97839e-06 from 1/251358 alleles and no homozygotes.
• The CSPEC record has no released SDHB criteria, so generic ACMG/AMP rules apply.
• Applied the generic BS1 default of >=0.01 supplied in the case instructions; the disorder-specific maximum-credible-frequency approach is described by Whiffin et al. 2017 (PMID:28518168).

Sources

Reasoning

BS3 is not met. The exact-variant study in PMID:22835832 did not show absence of a damaging effect: although the authors evaluated intrinsic enzymatic function, the R46Q protein underwent markedly accelerated degradation, with a 1.73-hour half-life versus 11.23 hours for wild-type SDHB in the radiolabeled pulse-chase assay and 0.74 hours in the cycloheximide assay. Thus, the available functional evidence is directionally damaging rather than benign and cannot support BS3.

Rule

BS3: Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• Yang et al. 2012, PMID:22835832, Results, Fig. 2C-D: exact SDHB R46Q was evaluated in transfected HeLa cells using protein-stability assays.
• Yang et al. 2012, PMID:22835832: R46Q showed accelerated degradation, with a 1.73-hour half-life versus 11.23 hours for wild-type SDHB by radiolabeled pulse-chase analysis and 0.74 hours in the cycloheximide assay.

Sources

Reasoning

BS4 is not met. The available reports do not identify an affected family member who lacks the variant. The Japanese family report instead documents an unaffected carrier, which is not lack of segregation in an affected member and may reflect age-dependent or incomplete penetrance. The F04 report describes additional affected family members and paternal inheritance rather than a documented affected non-carrier.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• Takekoshi et al. 2008, Results: R46Q was found in the proband and his third son, while the other two sons were negative; the carrier son had no tumor detected by CT or MIBG and was classified as asymptomatic.
• Benn et al. 2003, Table 1, family F04: additional retroperitoneal paragangliomas were reported in the family and paternal inheritance was recorded; no affected non-carrier is documented in the extracted evidence.

Still missing

• The available reports do not provide a complete affected-relative genotype table.

Sources

Reasoning

BP1 is not supported because SDHB disease-associated missense variation is documented rather than the gene being characterized primarily by truncating disease variants. The supplied evidence reports p.Arg46Gln in affected families and lists four missense variants alongside one nonsense, splice-site variants, and exon deletions in an SDHB-associated RCC cohort. This evidence does not support applying a truncating-predominant missense-benign rule to this variant.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• Benn et al. 2003, Results and Table 1: reports SDHB p.Arg46Gln in a disease-associated family.
• van der Harst et al. 2012, Results: reports four missense mutations, one nonsense mutation, two splice-site mutations, and exon 1 deletions in SDHB-associated RCC families.
• Yang et al. 2012, Results: studies multiple SDHB missense mutations, including R46Q, in protein-stability experiments.

Sources

Reasoning

The variant is a missense change, so the governing generic rule selects the REVEL path only; the SpliceAI result cannot provide BP4 support for this missense variant. REVEL is 0.911, which is above the generic BP4 supporting cutoff of <= 0.29 and therefore does not suggest no impact. BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) ... Missense variant -> REVEL only. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• The normalized consequence is missense, NP_002991.2:p.(Arg46Gln); therefore the applicable computational path is REVEL only, and the SpliceAI score is not counted for PP3/BP4.
• Local REVEL predictor lookup reports a score of 0.911 for NM_003000.3:c.137G>A.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997) sets generic BP4 supporting at <= 0.29; REVEL 0.911 does not meet this threshold.
• BayesDel score 0.581872 is not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The exact-variant ClinVar record contains no expert-panel classification and no Benign or Likely benign expert-panel assertion. The available ClinVar record is Pathogenic/Likely pathogenic from non-expert laboratory submissions, which cannot trigger BP6 under the specified generic rule.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-match record reports Pathogenic/Likely pathogenic laboratory submissions and 0 expert-panel submissions; no qualifying Benign or Likely benign expert-panel assertion is present.

Sources

Reasoning

The variant is a missense change at SDHB Arg46, so PM5 is applicable in principle. The available comparator artifact contains zero candidates and explicitly states that classic same-residue PM5 semantics could not be confirmed safely. No independently verified pathogenic alternate missense change at Arg46 is available in the supplied evidence, so PM5 cannot be assigned or excluded conclusively.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• pm5_candidates.json reports total_candidates_seen=0, same_residue_candidates=0, and recommends review rather than applying PM5 from the artifact alone.

Still missing

• A systematic, verified search for pathogenic SDHB alternate missense substitutions at residue Arg46 is required.

Sources

Reasoning

The supplied evidence documents pathogenic SDHB missense observations, including p.Arg46Gln, but does not provide a gene-level benign-missense rate or a sufficiently comprehensive demonstration that missense variants are a common disease mechanism under the PP2 definition. The exact variant's very low population frequency does not establish either gene-level PP2 condition.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• Benn et al. 2003 reports the R46Q missense variant in a disease-associated family and states it was absent from 100 normal alleles.
• van der Harst et al. 2012 reports four SDHB missense mutations in an RCC cohort, including p.Arg46Gln, but does not establish the gene-level benign-missense rate or the required PP2 mechanism threshold.

Still missing

• Gene-level analysis of benign missense variation and the proportion of SDHB disease-causing variants that are missense is needed.

Sources

Reasoning

PP4 requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available evidence reports the variant in pheochromocytoma/paraganglioma families, but it does not provide the current case's phenotype or family history in a form that permits assessment of specificity, and hereditary pheochromocytoma-paraganglioma has multiple possible genetic etiologies. A patient-specific PP4 determination therefore cannot be made from the available evidence.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• PMID:12618761 reports R46Q in a family with carotid-body, adrenal, and retroperitoneal paragangliomas/phaeochromocytomas.
• PMID:18362451 reports R46Q in a family with abdominal and thoracic paragangliomas and an asymptomatic carrier.
• These reports do not substitute for the current patient's phenotype-specific assessment.

Still missing

• Patient-specific phenotype and family-history details, including whether the presentation is highly specific for SDHB-related disease, are required.

Sources

Reasoning

BS2 is not assessed. PMID:18362451 reports an asymptomatic familial SDHB R46Q carrier whose CT and MIBG evaluations found no tumor, but the extracted report does not establish that this was a healthy adult or provide the age and penetrance context required to apply BS2. The generic rule requires an observation in a healthy adult with full penetrance expected at an early age; those prerequisites cannot be confirmed from the available record.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• PMID:18362451, Results, SDHB mutation analysis: the heterozygous R46Q variant was identified in the proband and his third son; the son had no tumor detected by CT or MIBG and was described as an asymptomatic carrier.
• The available paper extraction does not document the son's age at examination, establish that he was a healthy adult, or establish full penetrance at an early age.
• The CSPEC record lists autosomal dominant inheritance for hereditary pheochromocytoma-paraganglioma but contains no released SDHB criterion specifications.

Still missing

• Confirm the carrier's age and clinical status at observation.
• Establish whether full penetrance at an early age is expected for the specific SDHB-associated disorder.
• Obtain an appropriate unaffected-adult cohort or family observation if available.

Sources

Reasoning

BP2 requires either observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observation in cis with a pathogenic variant in any inheritance pattern. SDHB is recorded in the CSPEC material as autosomal dominant, but the available reports of p.Arg46Gln describe a heterozygous variant and do not document a pathogenic partner variant, cis or trans, in the same individual. The family reports therefore do not establish BP2; targeted parental, offspring, or allele-specific testing would be needed to determine phase and identify a qualifying pathogenic partner.

Rule

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.

Evidence considered

• CSPEC raw rule origin: SDHB hereditary pheochromocytoma-paraganglioma is specified as autosomal dominant inheritance.
• Benn et al. 2003, PMID:12618761, Table 1 family F04 and Results: the case report identifies SDHB c.137G>A (R46Q) in the family but does not report a second pathogenic variant or cis/trans phase with one.
• Nakamura et al. 2008, PMID:18362451, family-based germline analysis: the proband and one son carried heterozygous R46Q, but no pathogenic partner variant or cis/trans phase was reported.

Still missing

• No documented pathogenic partner variant with experimentally established cis or trans phase.
• No evidence establishes that the relevant dominant SDHB disorder is fully penetrant for the BP2 trans path.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The reviewed reports describe disease in carriers of SDHB R46Q, but they do not establish an alternate molecular diagnosis for the current case or for the reported R46Q case that would satisfy BP5. The current case's phenotype and molecular workup are not supplied.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• PMID:12618761 reports R46Q-associated pheochromocytoma/paraganglioma and does not document an alternate molecular basis in family F04.
• PMID:18362451 reports R46Q in an affected proband and an asymptomatic carrier, without an alternate molecular diagnosis.

Still missing

• A documented alternate molecular diagnosis in the same affected case is required.

Sources