TM6SF2 c.499G>A, p.Glu167Lys

Reasoning

BA1 is met. The variant's filtering frequency exceeds the generic stand-alone threshold in adequately sized populations: gnomAD v2.1 non-cancer exomes report grpmax FAF=0.08671611, and gnomAD v4.1 reports grpmax FAF=0.08901678. These FAF values are above 0.05, and the relevant South Asian population has 28,920 observed alleles in v2.1 non-cancer exomes and 89,404 observed alleles in v4.1, exceeding the 2,000-observed-allele minimum. The high frequency is not confined to a small isolate, and no pathogenic ClinVar consensus or established fully penetrant Mendelian disease association for this variant is documented in the available evidence.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v2.1 non-cancer exomes: AF=0.0652861, 14,587/223,432 alleles, 544 homozygotes, grpmax FAF=0.08671611; South Asian subgroup=2,591/28,920 alleles, AF=0.089592.
• gnomAD v4.1 all-comers: AF=0.0708821, 113,660/1,603,508 alleles, 4,135 homozygotes, grpmax FAF=0.08901678; South Asian subgroup=8,106/89,404 alleles, AF=0.090667.
• The generic BA1 calibration supplied for this case is >=0.05, identified as the ACMG/AMP 2015 stand-alone benign allele-frequency threshold (PMID:25741868).
• No ClinVar result or variant-specific pathogenic consensus is documented for NM_001001524.3:c.499G>A in the case evidence.

Sources

Reasoning

The variant is a missense substitution, NM_001001524.3:c.499G>A (p.Glu167Lys), so the applicable generic PP3/BP4 path is REVEL only; SpliceAI is not used for a missense variant, and BayesDel has no established generic ACMG-strength calibration. REVEL is 0.075, which is at or below the generic BP4 supporting threshold of <= 0.29 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997), so BP4 is met at supporting strength.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• NM_001001524.3:c.499G>A is a missense variant, NP_001001524.2:p.(Glu167Lys), so REVEL is the sole applicable computational path; SpliceAI is not counted.
• Local REVEL lookup reports score 0.075 for NC_000019.10:g.19268740C>T.
• The published generic BP4 supporting cutoff is REVEL <= 0.29, from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997); 0.075 meets it.
• BayesDel score -0.480376 is not used because no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

The variant is NP_001001524.2:p.(Glu167Lys), but the available evidence documents no established pathogenic variant producing the same amino-acid change at residue 167. The exact variant is absent from ClinVar, no relevant PMID was identified, and no same-amino-acid pathogenic comparator is documented. The missense consequence is supported and SpliceAI predicts a low maximum delta score of 0.074, so there is no evidence here that the comparison is confounded by a splice effect.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• The normalized consequence is NP_001001524.2:p.(Glu167Lys) / p.(E167K), a missense substitution at residue 167.
• ClinVar returned no exact result for NM_001001524.3:c.499G>A, and the literature packet contained zero PMIDs; no established pathogenic same-amino-acid comparator was identified.
• SpliceAI maximum delta score is 0.074, with the source describing no significant splice impact.

Sources

Reasoning

Residue Glu167 is not supported by a statistically significant cancer hotspot: the Cancer Hotspots search returned no result. COSMIC reports the exact variant six times somatically, but the generic rule requires a mutational hotspot or critical, well-established functional domain and does not make an isolated COSMIC occurrence sufficient. No VCEP covers TM6SF2, vcep_materials.json has no domain-table entry, and no authoritative critical-domain evidence is present in the case materials.

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

• Cancer Hotspots returned no statistically significant hotspot result for TM6SF2 E167.
• COSMIC records the exact variant as COSV66985100 with a somatic count of 6; this is not itself a statistically significant hotspot or established functional-domain designation.
• No VCEP-specific domain table or approved critical-domain list is present for TM6SF2.

Sources

Reasoning

PM2 is not met. The variant is common in the specified control datasets rather than absent or extremely rare: gnomAD v2.1 non-cancer exomes show AF=0.0652861 (14,587/223,432 alleles), gnomAD v3.1 non-cancer genomes show AF=0.059017 (8,722/147,788 alleles), and gnomAD v4.1 shows AF=0.0708821 (113,660/1,603,508 alleles). These frequencies are far above the supplied 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 v2.1 non-cancer exomes: AF=0.0652861, 14,587/223,432 alleles, 544 homozygotes.
• gnomAD v3.1 non-cancer genomes: AF=0.059017, 8,722/147,788 alleles, 278 homozygotes.
• gnomAD v4.1 all-comers: AF=0.0708821, 113,660/1,603,508 alleles, 4,135 homozygotes.
• The generic PM2 calibration supplied for this case is <=0.0001, identified as the ClinGen SVI recommendation to downgrade PM2 to supporting (PMID:25741868).

Sources

Reasoning

The variant is a novel missense substitution at Glu167Lys, but no different missense change at residue 167 that was determined to be pathogenic is documented in the case evidence. ClinVar returned no exact variant record, the literature packet contained no PMIDs, and the same-residue comparator collection contains zero candidates.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• The observed change is NP_001001524.2:p.(Glu167Lys) / p.(E167K).
• No exact ClinVar result or relevant PMID was available, and pm5_candidates.json contains zero same-residue candidates.
• No pathogenic comparator at residue 167 was identified, so the required alternate-missense comparison is not met.

Sources

Reasoning

The variant is a missense substitution, NM_001001524.3:c.499G>A (p.Glu167Lys), so the applicable generic PP3/BP4 path is REVEL only; SpliceAI is not used for a missense variant, and BayesDel has no established generic ACMG-strength calibration. REVEL is 0.075, below the generic PP3 supporting threshold of >= 0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997), so PP3 is not met.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• NM_001001524.3:c.499G>A is a missense variant, NP_001001524.2:p.(Glu167Lys), so REVEL is the sole applicable computational path; SpliceAI is not counted.
• Local REVEL lookup reports score 0.075 for NC_000019.10:g.19268740C>T.
• The published generic PP3 supporting cutoff is REVEL >= 0.644, from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997); 0.075 does not meet it.
• BayesDel score -0.480376 is not used because no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

The governing rule concerns a reputable source recently reporting the exact variant as pathogenic when the underlying evidence is unavailable for independent evaluation. The exact variant is reported absent from ClinVar, with no expert-panel pathogenic or likely pathogenic classification and no other qualifying reputable-source classification in the available evidence.

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-variant search reports no result and no review status or classification; therefore there is no expert-panel Pathogenic or Likely pathogenic assertion to apply.

Sources

Reasoning

The governing rule concerns a reputable source recently reporting the exact variant as benign when the underlying evidence is unavailable for independent evaluation. The exact variant is reported absent from ClinVar, with no expert-panel benign or likely benign classification and no other qualifying reputable-source classification in the available evidence.

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-variant search reports no result and no review status or classification; therefore there is no expert-panel Benign or Likely benign assertion to apply.

Sources

Reasoning

The available case evidence does not document a proband with a defined disease phenotype, absence of family history, or confirmed maternity and paternity with the variant absent in both parents. Therefore, the required de novo observation cannot be assessed.

Rule

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

Evidence considered

• The case evidence contains no pedigree, parental testing results, proband phenotype description, or de novo assessment for NM_001001524.3:c.499G>A (TM6SF2 p.Glu167Lys).

Still missing

• Clinical diagnosis and phenotype consistency for the proband are not documented.
• Family-history status is not documented.
• Parental genotypes and confirmation of both maternity and paternity are not documented.

Reasoning

No variant-specific in vitro or in vivo functional study, assay result, assay control set, or validated clinical-laboratory functional evidence was identified for TM6SF2 c.499G>A (p.Glu167Lys). Therefore, the evidence is insufficient to determine whether the variant has a damaging effect under PS3.

Rule

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

Evidence considered

• The case evidence contains no variant-specific functional assay results, functional-study publication, or validated clinical-laboratory assay evidence for TM6SF2 c.499G>A (p.Glu167Lys).
• Computational predictions and population-frequency observations were not used to assess PS3 because they do not satisfy the functional-study definition.

Still missing

• A variant-specific functional assay with appropriate wild-type, benign, and damaging controls is needed.
• Reproducibility and biological relevance of any assay result remain unestablished.

Reasoning

The governing rule requires significantly increased prevalence in affected individuals compared with controls, with OR or RR >5.0 and a confidence interval excluding 1.0. The case packet contains no variant-specific affected-case carrier counts, unaffected-control counts, or case-control estimate. The variant is common in gnomAD v4.1 (AF 0.0708821; 113660/1603508 alleles), but that population frequency alone is not a case-control comparison and cannot establish PS4. literature_pass.json reports zero PMIDs and packet.ai_classified_papers contains no papers to verify.

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

• gnomAD v4.1 reports AF 0.0708821 from 113660/1603508 alleles; this is population frequency evidence, not a case-control comparison.
• No papers were listed in literature_pass.json, and no PS4 paper findings were available for abstract or full-text verification.

Still missing

• Variant-specific affected-case and control carrier counts or a reported OR/RR with 95% CI are needed.

Sources

Reasoning

PM3 cannot be assessed because the available case evidence does not document an affected proband carrying NM_001001524.3:c.499G>A with a pathogenic variant in trans, parental or offspring testing establishing phase, or an established recessive TM6SF2 disorder context. Population observations of this common variant do not establish allelic phase or satisfy the PM3 observation requirement.

Rule

For recessive disorders, detected in trans with a pathogenic variant

Evidence considered

• The case evidence identifies TM6SF2 NM_001001524.3:c.499G>A (p.Glu167Lys) but provides no affected-proband observations, second pathogenic allele, phase testing, or inheritance information relevant to PM3.

Still missing

• Affected-proband genotype and phenotype information
• A pathogenic or likely pathogenic allele identified in the same proband
• Parental or offspring testing establishing trans phase
• Established recessive TM6SF2 disorder and inheritance context

Reasoning

The available case evidence does not document an assumed de novo occurrence in a proband with the disease, nor does it provide parental genotypes or a family-history assessment. The assumed de novo criterion therefore cannot be assessed.

Rule

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

Evidence considered

• The case evidence contains no parental testing, pedigree, proband phenotype description, or documented assumption that NM_001001524.3:c.499G>A arose de novo.

Still missing

• Clinical diagnosis and phenotype consistency for the proband are not documented.
• Parental genotypes are not documented.
• The basis for assuming de novo status and the family-history status are not documented.

Reasoning

The available case evidence provides no affected family members, informative meioses, genotype results, or segregation analysis. It also does not establish a definitive disease relationship for TM6SF2 in the reported clinical context, so co-segregation cannot be assessed.

Rule

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

Evidence considered

• The case evidence contains no family pedigree, affected relatives, genotype-phenotype cosegregation data, meioses, or segregation statistics for NM_001001524.3:c.499G>A.

Still missing

• Affected relatives and their clinical phenotypes are not documented.
• Genotypes of affected and unaffected relatives are not documented.
• Informative meioses and segregation results are not documented.
• The relevant definitive TM6SF2 disease relationship is not documented in the case evidence.

Reasoning

The variant is missense, but the case evidence does not establish both gene-level requirements for PP2: a low rate of benign missense variation and missense variants as a common disease mechanism. The observed variant is common in gnomAD v2.1 (AF 0.0645174) and v4.1 (AF 0.0708821), but those variant-level frequencies do not establish the required gene-wide missense constraint or disease mechanism.

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

• The consequence is missense, p.(Glu167Lys).
• The variant has gnomAD v2.1 allele frequency 0.0645174 and gnomAD v4.1 allele frequency 0.0708821, demonstrating that this individual variant is common in population data.
• No source in the case establishes the gene-wide low rate of benign missense variation and common missense disease mechanism required by PP2.

Still missing

• A validated gene-level assessment of benign missense variation and missense disease mechanism for TM6SF2 is required.

Sources

Reasoning

The governing rule requires a patient phenotype or family history highly specific for a disease with a single genetic etiology. No patient phenotype, family history, or disease-specific clinical presentation is provided for this variant case, so PP4 cannot be assessed.

Rule

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

Still missing

• A documented patient phenotype or family history and the relevant single-gene disease context are needed.

Reasoning

BS1 is not assessed rather than applied from the generic 1% default alone. The variant has high filtering frequencies (gnomAD v2.1 non-cancer exome grpmax FAF=0.08671611; gnomAD v4.1 grpmax FAF=0.08901678), but the available evidence does not define a specific fully penetrant disorder, disease prevalence, penetrance, or allelic heterogeneity for which a maximum credible allele frequency can be calculated. TM6SF2 is represented here in common metabolic and liver-disease susceptibility contexts, so the generic 1% default cannot establish that this frequency is greater than expected for a defined disorder without disorder-specific inputs.

Rule

BS1

Evidence considered

• gnomAD v2.1 non-cancer exomes report grpmax FAF=0.08671611; gnomAD v4.1 reports grpmax FAF=0.08901678.
• The supplied generic BS1 calibration is >=0.01, but the prompt requires disorder-specific prevalence, penetrance, allelic heterogeneity, and maximum-credible-frequency consideration before applying the default.
• Whiffin et al. 2017 (PMID:28518168) is the specified source for maximum credible allele-frequency modeling.

Still missing

• A defined disorder and its inheritance model are needed.
• Disease prevalence, penetrance, allelic heterogeneity, and a maximum credible allele frequency calculation are unavailable.

Sources

Reasoning

BS2 is not assessed. gnomAD reports many homozygotes, including 544 in gnomAD v2.1 non-cancer exomes, 598 in the corresponding v2.1 non-cancer combined data, 278 in gnomAD v3.1 non-cancer genomes, and 4,135 in gnomAD v4.1 all-comers. However, these population records do not establish that the individuals were healthy adults, nor do they establish an early-onset, fully penetrant dominant, recessive, or X-linked disorder against which the criterion can be applied.

Rule

BS2

Evidence considered

• gnomAD v2.1 non-cancer exomes report 544 homozygotes; the corresponding non-cancer combined v2.1 data report 598 homozygotes.
• gnomAD v3.1 non-cancer genomes report 278 homozygotes.
• gnomAD v4.1 all-comers report 4,135 homozygotes.
• These aggregate population datasets provide no individual-level healthy-adult phenotype, age, inheritance-model, or penetrance evidence for BS2.

Still missing

• Phenotypic and age-at-evaluation data for homozygous carriers are unavailable.
• A defined inheritance model and evidence that full penetrance is expected at an early age are unavailable.

Sources

Reasoning

No variant-specific in vitro or in vivo functional study, assay result, assay control set, or validated clinical-laboratory functional evidence was identified for TM6SF2 c.499G>A (p.Glu167Lys). Therefore, the evidence is insufficient to determine whether the variant shows no damaging effect on protein function or splicing under BS3.

Rule

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

Evidence considered

• The case evidence contains no variant-specific functional assay results, functional-study publication, or validated clinical-laboratory assay evidence for TM6SF2 c.499G>A (p.Glu167Lys).
• Computational predictions and population-frequency observations were not used to assess BS3 because they do not satisfy the functional-study definition.

Still missing

• A variant-specific functional assay with appropriate wild-type, benign, and damaging controls is needed.
• A validated result showing no damaging effect on protein function or splicing is unavailable.

Reasoning

The available case evidence does not identify affected family members who were tested for the variant, so there is no documented non-segregation. BS4 cannot be assessed, and lack of reported family data is not evidence that the variant fails to segregate.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The case evidence contains no family segregation table, affected-relative testing, or documented genotype-phenotype discordance for NM_001001524.3:c.499G>A.

Still missing

• Affected family members are not identified.
• Genotypes and phenotypes of affected relatives are not documented.
• Potential phenocopies and alternative pathogenic variants cannot be evaluated without family data.

Reasoning

The variant is missense, but the case evidence does not establish that TM6SF2 is a gene for which primarily truncating variants are known to cause disease. The available population frequency data show this variant is common, but do not determine the gene's established disease mechanism or the relative contribution of truncating versus missense variants.

Rule

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

Evidence considered

• The observed variant is a missense substitution, p.(Glu167Lys).
• No authoritative gene-level source in the case establishes a primarily truncating disease mechanism for TM6SF2.

Still missing

• A validated gene-level disease-mechanism assessment comparing truncating and missense causes is required.

Reasoning

BP2 cannot be assessed because the available case evidence does not document NM_001001524.3:c.499G>A with a pathogenic variant in trans in an established fully penetrant dominant disorder, or with a pathogenic variant in cis in any inheritance pattern. No individual-level genotype or phase data are available, and the available TM6SF2 context does not establish a qualifying dominant disorder.

Rule

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

• The case evidence provides no affected-proband or family genotype data, no pathogenic partner variant, and no cis/trans phase information for NM_001001524.3:c.499G>A.

Still missing

• Individual-level genotype data for a pathogenic partner variant
• Testing establishing cis or trans phase
• Evidence that any dominant TM6SF2 disorder is fully penetrant when the trans pathway is considered

Reasoning

The governing rule requires the variant to be found in a case with an alternate molecular basis for disease. The available case evidence contains no documented alternate molecular diagnosis or case-level evidence linking this variant to a patient with such an alternative cause, so BP5 cannot be assessed.

Rule

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

Still missing

• A documented variant-positive case with an established alternate molecular basis for the disease is needed.