CYP2C19 c.681G>A, p.Pro227=

Reasoning

PMID:8195181 directly studied CYP2C19 c.681G>A in poor metabolizers and reported a G>A mutation in exon 5 that creates an aberrant splice site, shifts the reading frame from amino acid 215, introduces a premature stop codon 20 amino acids later, and produces a truncated non-functional protein. The genotype-phenotype relationship was observed in 7/10 Caucasian and 10/17 Japanese poor metabolizers who were homozygous for the defect. This is variant-specific functional evidence for a damaging effect on the gene product and supports PS3. The study is not documented as a validated, reproducible clinical diagnostic assay with explicitly described assay controls, so the evidence is calibrated below strong as moderate.

Rule

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

Evidence considered

• The abstract of de Morais et al. (1994), PMID:8195181, reports that the exon 5 G>A mutation creates an aberrant splice site, alters the reading frame beginning at amino acid 215, creates a premature stop codon 20 amino acids downstream, and produces a truncated non-functional protein.
• The same report states that 7/10 Caucasian and 10/17 Japanese poor metabolizers were homozygous for the defect, linking the variant to the poor-metabolizer phenotype.
• Because explicit clinical-laboratory validation, reproducibility, and detailed controls are not documented in the available report, the functional evidence is calibrated as moderate rather than strong.

Still missing

• The available report does not document validation and reproducibility in a clinical diagnostic laboratory or detailed assay-control performance sufficient for strong strength.

Sources

Reasoning

The variant is synonymous (NM_000769.4:c.681G>A; NP_000760.1:p.(Pro227=)), so the applicable generic computational path is SpliceAI only; REVEL is not applicable. SpliceAI max delta is 0.994, which meets the supplied generic PP3 supporting threshold of >=0.2 from the SpliceAI recommended cutoff (Jaganathan et al. 2019, PMID:30661751). The same SpliceAI prediction is not counted again as independent evidence for BP4 or BP7.

Rule

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

Evidence considered

• SpliceAI Lookup for NM_000769.4:c.681G>A reports DS_AG 0.994, DS_AL 0.889, DS_DG 0.001, DS_DL 0.000, and max delta 0.994.
• The variant is annotated as synonymous_variant, so SpliceAI is the sole applicable PP3/BP4 predictor path and REVEL is not applicable.
• The supplied generic calibration assigns PP3 supporting at SpliceAI max delta >=0.2, using the SpliceAI recommended cutoff from Jaganathan et al. 2019 (PMID:30661751).

Sources

Reasoning

NM_000769.4:c.681G>A (CYP2C19*2; rs4244285) is common in population databases. The default all-comers gnomAD v2.1 frequency is 0.174893 (33,738/192,906 alleles), and gnomAD v4.1 is 0.160234 (234,686/1,464,646 alleles); both exceed the generic BA1 threshold of 0.05. The non-cancer exome-only gnomAD v2.1 frequency is also high at 0.178200 (26,226/147,172 alleles).

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 all-comers: AF=0.174893, 33,738/192,906 alleles, 3,460 homozygotes; South Asian AF=0.323955.
• gnomAD v4.1 all-comers: AF=0.160234, 234,686/1,464,646 alleles, 20,742 homozygotes; South Asian AF=0.320310.
• gnomAD v2.1 non-cancer exomes: AF=0.178200, 26,226/147,172 alleles, 2,776 homozygotes; this corroborates the high frequency but is not needed for the default generic assessment.
• Generic calibration supplied for BA1: allele frequency >=0.05.

Sources

Reasoning

The variant is not absent or extremely rare in controls. It has AF=0.174893 in gnomAD v2.1 and AF=0.160234 in gnomAD v4.1, with 3,460 and 20,742 homozygotes, respectively. The non-cancer subsets also show high frequencies: 0.178200 in v2.1 exomes and 0.168221 in v3.1 genomes.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 all-comers: AF=0.174893, 33,738/192,906 alleles, 3,460 homozygotes.
• gnomAD v4.1 all-comers: AF=0.160234, 234,686/1,464,646 alleles, 20,742 homozygotes.
• gnomAD v2.1 non-cancer exomes: AF=0.178200, 26,226/147,172 alleles, 2,776 homozygotes.
• gnomAD v3.1 non-cancer genomes: AF=0.168221, 24,749/147,122 alleles, 2,294 homozygotes.
• Generic calibration supplied for PM2: allele frequency <=0.0001.

Sources

Reasoning

The governing rule requires a reputable source to report the variant as pathogenic when the evidence is unavailable for independent evaluation. The exact-variant ClinVar audit reports zero expert-panel submissions; the available exact-match submissions include a drug-response practice-guideline entry and a likely-benign clinical-laboratory entry, with no pathogenic or likely pathogenic expert-panel assertion. PMID:21716271 reports CYP2C19*2 (c.681G>A; rs4244285) as a loss-of-function allele associated with adverse outcomes during clopidogrel therapy, not as a pathogenic classification for a Mendelian disease. PP5 is therefore not met.

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 audit: expert_panel_submissions=0; recorded exact-match submissions include a CPIC drug-response practice-guideline entry and a Likely benign clinical-laboratory entry, with no pathogenic or likely pathogenic expert-panel assertion.
• PMID:21716271 abstract and literature-packet entry: CPIC reports CYP2C19*2 (c.681G>A; rs4244285) as a common loss-of-function allele associated with adverse cardiovascular outcomes during clopidogrel therapy, not as a pathogenic Mendelian-disease classification.

Sources

Reasoning

The variant is synonymous, so the applicable generic computational path is SpliceAI only; REVEL is not applicable. SpliceAI max delta is 0.994, which does not meet the supplied generic BP4 supporting threshold of <=0.1 from the SpliceAI recommended cutoff (Jaganathan et al. 2019, PMID:30661751); it instead predicts splice impact. The SpliceAI prediction is not counted again as independent evidence for PP3 or BP7.

Rule

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

Evidence considered

• SpliceAI Lookup for NM_000769.4:c.681G>A reports max delta 0.994.
• The variant is annotated as synonymous_variant, so SpliceAI is the sole applicable PP3/BP4 predictor path and REVEL is not applicable.
• The supplied generic calibration assigns BP4 supporting at SpliceAI max delta <=0.1, using the SpliceAI recommended cutoff from Jaganathan et al. 2019 (PMID:30661751).

Sources

Reasoning

The governing rule requires a reputable source to report the variant as benign when the evidence is unavailable for independent evaluation. The exact-variant ClinVar audit records zero expert-panel submissions; the available likely-benign assertion is from a clinical laboratory, and the overall record also contains a drug-response classification. Under the required restriction, ordinary laboratory submissions, aggregate labels, and non-expert assertions cannot trigger BP6. BP6 is therefore not met.

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 audit: expert_panel_submissions=0; the submission summary includes one Likely benign clinical-laboratory assertion and one drug-response assertion, not an expert-panel benign or likely-benign classification.
• The ClinVar record's aggregate review status does not convert the non-expert laboratory assertion into an expert-panel classification.

Sources

Reasoning

The variant is synonymous, satisfying the variant-type requirement, but SpliceAI max delta is 0.994 and therefore predicts splice impact rather than no impact. Because the BP7 rule requires no predicted splice impact and a non-highly-conserved nucleotide, the criterion is not met; the conservation condition need not be resolved after the splice condition fails.

Rule

A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.

Evidence considered

• The variant is annotated as synonymous_variant (NM_000769.4:c.681G>A; NP_000760.1:p.(Pro227=)).
• SpliceAI Lookup reports max delta 0.994, so the required prediction of no impact to the splice consensus sequence or creation of a new splice site is not satisfied.

Sources

Reasoning

No case-level evidence documents a patient with a relevant disease, absence of family history, or confirmed maternity and paternity for NM_000769.4:c.681G>A. The available literature concerns common pharmacogenomic CYP2C19 variation and does not report a de novo family investigation; therefore PS2 cannot be assessed.

Rule

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

Evidence considered

• No case-level proband, parental-testing, or family-history data are reported for NM_000769.4:c.681G>A.
• The available variant-specific literature describes CYP2C19 pharmacogenomic effects rather than a de novo disease case or parent-child investigation.

Still missing

• Affected-proband phenotype and disease diagnosis are not documented.
• Maternity and paternity confirmation are not documented.
• Family-history information is not documented.

Reasoning

The governing ACMG/AMP definition requires that the prevalence of the variant in affected individuals be significantly increased compared with controls. Its specific note requires RR or OR >5.0 with a confidence interval that does not include 1.0. The literature-pass PS4 leads were checked against the available abstract or full-text extracts: PMID:27026020 does not mention CYP2C19 or c.681G>A, and PMID:27981572 identifies c.681G>A as CYP2C19*2 but reports no variant-specific affected/control cohort, carrier counts, OR, RR, or confidence interval. No qualifying counts were available, so no OR or 95% CI could be computed. Pharmacogenomic drug-response associations and high population frequency are not PS4 case-control evidence.

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

• PMID:27026020 full-text extraction: the article discusses clopidogrel and P2Y12 inhibitors but does not mention CYP2C19 c.681G>A and provides no relevant variant observation or case-control data.
• PMID:27981572 full-text extraction: the guideline states that the most common no-function allele is *2 (c.681G>A; rs4244285), but provides no variant-specific affected/control cohort, carrier counts, OR, RR, or confidence interval.
• The literature-pass PS4-indexed material was reviewed; no verified qualifying case-control counts or effect estimate for this exact variant were available.
• No qualifying numeric comparison was available; consequently no OR or 95% CI was computed.

Still missing

• A variant-specific affected cohort and matched unaffected or population control cohort with carrier counts or frequencies is required.
• An odds ratio or relative risk with a 95% confidence interval, or sufficient counts to calculate both, is required.

Sources

Reasoning

PM3 requires a recessive-disorder context and evidence that NM_000769.4:c.681G>A is in trans with a pathogenic variant, with parental or offspring testing establishing phase. The available case evidence documents no affected proband, no co-occurring pathogenic variant, and no parental, offspring, or other phase testing; therefore PM3 cannot be assessed.

Rule

For recessive disorders, detected in trans with a pathogenic variant ... This requires testing of parents (or offspring) to determine phase.

Evidence considered

• ClinVar variation 16897 identifies the variant record but provides no affected-proband observation, co-occurring pathogenic allele, or parental/offspring phase result applicable to PM3.
• No source in the case evidence documents a recessive disorder, an affected proband carrying this variant with a pathogenic variant in trans, or testing that establishes phase.

Still missing

• A documented affected proband with a recessive disorder relevant to CYP2C19.
• A co-occurring pathogenic variant in CYP2C19 in the same proband.
• Parental, offspring, or equivalent testing establishing that c.681G>A and the pathogenic variant are in trans.

Sources

Reasoning

No case-level evidence documents an assumed de novo occurrence of NM_000769.4:c.681G>A in an affected patient. Neither parental genotypes nor a clinical report establishing de novo status without maternity and paternity confirmation is available, so PM6 cannot be assessed.

Rule

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

Evidence considered

• No case-level report establishes NM_000769.4:c.681G>A as de novo or presumed de novo.
• No parental genotypes or parent-child transmission data are available.

Still missing

• An affected proband with a relevant disease diagnosis is not documented.
• A de novo occurrence is not documented.
• Parental testing information is not documented.

Reasoning

No affected family members, informative meioses, genotype-phenotype co-segregation, or disease-specific family study is documented for NM_000769.4:c.681G>A. The available pharmacogenomic studies do not provide segregation data in a disease family, so PP1 cannot be assessed.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... Note: May be used as stronger evidence with increasing segregation data.

Evidence considered

• No family pedigree, affected-relative genotypes, or disease-specific segregation analysis is available for NM_000769.4:c.681G>A.
• The available studies address population pharmacogenomics or treatment response and do not report co-segregation with a disease phenotype.

Still missing

• Affected relatives and their phenotypes are not documented.
• Genotypes of affected and unaffected relatives are not documented.
• Informative meioses and disease-specific segregation analysis are not documented.

Reasoning

The available case materials provide no patient phenotype or family history for the carrier. The reviewed literature concerns CYP2C19 pharmacogenomic drug response and population variation rather than a phenotype highly specific for a disease with a single genetic etiology. Therefore the criterion cannot be assessed from the available clinical information.

Rule

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

Evidence considered

• The case summary identifies the variant and gene but supplies no patient phenotype or family history.
• The reviewed literature describes CYP2C19 pharmacogenomic drug response and allele frequency, not a disease-specific phenotype for this patient.

Still missing

• Patient phenotype and family-history details are required.
• A defined disease phenotype with evidence of high specificity and a single genetic etiology is required.

Reasoning

The allele frequency is far above the supplied generic 0.01 BS1 default (0.174893 in gnomAD v2.1 and 0.160234 in gnomAD v4.1), but BS1 requires that frequency to be greater than expected for a specific disorder. This case identifies no Mendelian CYP2C19 disorder and provides no disease prevalence, penetrance, allelic heterogeneity, or maximum-credible-frequency calculation. The allele is a common pharmacogenomic allele, so the default alone is insufficient for a disorder-based BS1 call.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v2.1 all-comers: AF=0.174893, with South Asian AF=0.323955 and 3,460 homozygotes.
• gnomAD v4.1 all-comers: AF=0.160234, with South Asian AF=0.320310 and 20,742 homozygotes.
• The supplied generic BS1 default is >=0.01, but the governing disorder-specific inputs needed to establish 'greater than expected for the disorder' are not available.
• The case evidence describes CYP2C19*2 as a pharmacogenomic allele and does not establish a Mendelian CYP2C19 disease model for this assessment.

Still missing

• A defined Mendelian disorder, disease prevalence, penetrance, allelic heterogeneity, and maximum credible allele-frequency calculation are unavailable.

Sources

Reasoning

gnomAD reports many homozygotes (3,460 in v2.1; 20,742 in v4.1), but the database counts do not document that these individuals are healthy adults, their ages, or absence of a fully penetrant early-onset disorder. The case also provides no established Mendelian CYP2C19 disorder to which the BS2 applicability conditions can be applied.

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

• gnomAD v2.1 reports 3,460 homozygotes overall and 999 homozygotes in the South Asian population.
• gnomAD v4.1 reports 20,742 homozygotes overall and 3,593 homozygotes in the South Asian population.
• Population-database homozygote counts alone do not establish healthy-adult status or the absence of a fully penetrant early-onset disorder.

Still missing

• Individual-level healthy-adult status, age, phenotype, and a defined fully penetrant early-onset Mendelian disorder are unavailable.

Sources

Reasoning

No variant-specific functional study in the reviewed evidence demonstrates that CYP2C19 c.681G>A has no damaging effect on protein function or splicing. The available functional report instead supports a damaging effect, which cannot be used as BS3 evidence.

Rule

BS3

Still missing

• A well-established variant-specific functional study showing no damaging effect on protein function or splicing is absent.

Reasoning

No affected family members with documented absence of NM_000769.4:c.681G>A are reported. Because there is no disease-family pedigree or genotype data showing non-segregation, BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-relative genotype data or family-level non-segregation analysis is available for NM_000769.4:c.681G>A.

Still missing

• Affected family members are not documented.
• Genotypes of affected relatives are not documented.
• A family-level non-segregation analysis is not documented.

Reasoning

BP2 requires either observation in trans with a pathogenic variant in a fully penetrant dominant gene/disorder or observation in cis with a pathogenic variant in any inheritance pattern. The available case evidence contains no affected-proband or other individual-level cis/trans observation and no documented pathogenic variant in the relevant phase; therefore BP2 cannot be assessed.

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

• ClinVar variation 16897 identifies the variant record but provides no individual-level cis/trans observation with a documented pathogenic variant.
• No source in the case evidence establishes either the trans configuration required for a fully penetrant dominant gene/disorder or the cis configuration required for BP2 in any inheritance pattern.

Still missing

• An individual-level observation containing c.681G>A and a documented pathogenic variant in the same gene.
• Reliable phase determination showing trans status for a fully penetrant dominant disorder or cis status for any inheritance pattern.
• Affected-proband and inheritance information sufficient to establish the applicable BP2 pathway.

Sources

Reasoning

The governing rule requires the variant to be found in a case with an alternate molecular basis for disease. The available materials do not identify a disease case carrying NM_000769.4:c.681G>A together with an independently established alternate molecular cause. Pharmacogenomic treatment-response associations and population presence do not satisfy BP5.

Rule

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

Evidence considered

• No case-level report in the reviewed materials documents NM_000769.4:c.681G>A with an alternate molecular diagnosis.
• Population frequency and pharmacogenomic drug-response data are not alternate molecular bases for a disease case.

Still missing

• A documented disease case carrying the variant is required.
• An independently established alternate molecular basis for that case's disease is required.