CDKN2A c.76G>C, p.(Glu26Gln)

Reasoning

The variant is extremely rare in population data: gnomAD v4.1 total AF is 0.000124% (2/1,611,012 alleles), with maximum observed ancestry AF 0.00220% (South Asian, 2/91,020); the older gnomAD v2.1 maximum is 0.00657% (2/30,456). These rare frequencies support PM2 at supporting strength and do not constitute absence.

Rule

Generic ACMG/AMP PM2 at supporting strength: variant is absent or extremely rare in population databases; the observed maximum frequency remains in the extremely rare range.

Evidence considered

• gnomAD v4.1: total AC/AN 2/1,611,012 (AF 0.000124%), South Asian AC/AN 2/91,020 (AF 0.00220%), zero homozygotes, and grpmax FAF 0.000365%.
• gnomAD v2.1: total AC/AN 2/238,850 (AF 0.00084%), South Asian AC/AN 2/30,456 (AF 0.00657%), zero homozygotes, and grpmax FAF 0.001087%.
• gnomAD-Canada v1.0 reported the variant absent.

Sources

Reasoning

No CSPEC/VCEP-specific PP3/BP4 lookup exists for CDKN2A (framework_mode = generic_acmg), so generic calibrated computational evidence applies. SpliceAI predicts no splicing impact (max delta score = 0.006, far below any splice-altering threshold), consistent with a benign computational splicing prediction. REVEL for the resulting missense change p.(Glu26Gln) scores 0.155, below calibrated REVEL benign-supporting thresholds (ClinGen SVI REVEL calibration, PMID 36413997), consistent with a benign missense in silico prediction. Both the splice-impact path and the missense in silico path converge on a benign-leaning prediction, supporting BP4 at supporting strength. BayesDel is not used because no verified published calibration threshold is available to this pipeline.

Rule

generic_acmg_fallback

Evidence considered

• SpliceAI Lookup for NM_000077.4:c.76G>C reports max delta score = 0.006 (DS_AG=0.001, DS_AL=0.0, DS_DG=0.006, DS_DL=0.0), indicating no predicted disruption of splicing, consistent with a benign in silico prediction.
• Local REVEL v1.3 lookup for this variant returns a score of 0.155, below calibrated REVEL benign-supporting thresholds (ClinGen SVI REVEL calibration, PMID 36413997), consistent with a benign missense in silico prediction.

Still missing

• BayesDel score is available (-0.34194) but cannot be applied due to lack of a verified published calibration threshold for this pipeline.
• No CSPEC/gene-specific PP3-BP4 lookup table exists for CDKN2A.

Sources

Reasoning

No governing VCEP/CSPEC was found for CDKN2A (framework_mode = generic_acmg; vcep_materials.json note: 'no cspec url available'), so generic calibrated computational predictors were used. SpliceAI predicts negligible splice impact (max delta score = 0.006, well below any pathogenic-supporting splicing threshold), and REVEL for this missense change (NP_000068.1:p.(Glu26Gln)) scores 0.155, well below any calibrated pathogenic-supporting REVEL threshold. No computational evidence supports a deleterious effect for this variant, so PP3 is not met.

Rule

generic_acmg_fallback

Evidence considered

• SpliceAI Lookup (spliceailookup.broadinstitute.org) for NM_000077.4:c.76G>C reports DS_AG=0.001, DS_AL=0.0, DS_DG=0.006, DS_DL=0.0; max delta score = 0.006, indicating no predicted splicing impact.
• Local REVEL v1.3 lookup for this variant returns a score of 0.155, far below calibrated pathogenic-supporting REVEL thresholds (e.g., ClinGen SVI-recommended REVEL calibration, PMID 36413997), so REVEL does not support a deleterious in silico prediction.

Still missing

• BayesDel score is available (-0.34194) but this pipeline has no verified published calibration/threshold publication for BayesDel, so it cannot be used to support or refute PP3/BP4.
• No CSPEC/gene-specific PP3-BP4 lookup table exists for CDKN2A to confirm/override the generic calibration.

Sources

Reasoning

The exact-variant ClinVar record has no expert-panel submission and all three laboratory submissions classify the variant as uncertain significance; it does not meet the required expert-panel pathogenic/likely pathogenic assertion.

Rule

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

Evidence considered

• ClinVar variation 919572 is an exact match for CDKN2A c.76G>C (p.Glu26Gln). Its submission audit reports zero expert-panel submissions; the three submissions are single-submitter uncertain-significance assertions from Color Health, Labcorp, and Ambry Genetics.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion.

Sources

Reasoning

The highest observed population allele frequency is 0.00657% in gnomAD v2.1 South Asian samples (2/30,456 alleles); gnomAD v4.1 reports 0.00220% in South Asian samples (2/91,020 alleles). These frequencies are far below a stand-alone benign frequency level.

Rule

Generic ACMG/AMP BA1: apply only when allele frequency alone is sufficiently high to establish benignity; this variant's observed frequencies do not meet that standard.

Evidence considered

• gnomAD v2.1: total AC/AN 2/238,850 (AF 0.00084%), South Asian AC/AN 2/30,456 (AF 0.00657%), no homozygotes, and grpmax FAF 0.001087%.
• gnomAD v4.1: total AC/AN 2/1,611,012 (AF 0.000124%), South Asian AC/AN 2/91,020 (AF 0.00220%), no homozygotes, and grpmax FAF 0.000365%.

Sources

Reasoning

Observed frequencies are too low to be greater than expected for CDKN2A-associated hereditary cancer susceptibility. The highest observed value is 0.00657% (gnomAD v2.1 South Asian population), with only two alleles observed and no homozygotes in either gnomAD release.

Rule

Generic ACMG/AMP BS1: apply only when allele frequency is greater than expected for the disorder; the available frequencies are not.

Evidence considered

• gnomAD v2.1 maximum observed population frequency is 2/30,456 South Asian alleles (0.00657%); total frequency is 2/238,850 (0.00084%), with zero homozygotes.
• gnomAD v4.1 maximum observed population frequency is 2/91,020 South Asian alleles (0.00220%); total frequency is 2/1,611,012 (0.000124%), with zero homozygotes.

Still missing

• No CDKN2A-specific BS1 frequency threshold was retrieved; assessment therefore uses the generic ACMG/AMP requirement that frequency be greater than expected for the disorder.

Sources

Reasoning

BP1 applies when a missense variant occurs in a gene for which primarily truncating variants are known to cause disease. CDKN2A is a well-established tumor suppressor in which germline missense substitutions (not only truncating/null variants) are a recognized, common disease mechanism for familial melanoma predisposition, so the BP1 premise (truncating-only gene mechanism) does not hold for this gene; BP1 is therefore not met.

Evidence considered

• pvs1_variant_assessment.json confirms consequence_class='missense'; CDKN2A is broadly established as a gene in which missense variants (not solely truncating variants) are a recognized pathogenic mechanism, precluding a truncating-only BP1 gene profile.

Still missing

• No gene-specific missense-constraint metric was retrieved in this case to formally quantify the missense disease-mechanism rate; this conclusion rests on the well-established CDKN2A missense disease mechanism rather than a case-specific constraint statistic.

Reasoning

The exact-variant ClinVar record has no expert-panel submission and no expert-panel Benign or Likely benign assertion; ordinary laboratory uncertain-significance submissions cannot trigger BP6.

Rule

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

Evidence considered

• ClinVar variation 919572 is an exact match for CDKN2A c.76G>C (p.Glu26Gln). Its submission audit reports zero expert-panel submissions; the three available single-submitter classifications are uncertain significance.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Sources

Reasoning

PS1 requires an established pathogenic variant that produces the identical amino-acid change (p.Glu26Gln, NP_000068.1) via a different nucleotide substitution. No CDKN2A CSPEC/VCEP is available for this case (cspec.found=false, vcep_materials empty), and no source in this case's evidence bundle identifies any other reported variant resulting in p.(Glu26Gln). ClinVar record 919572 for this exact variant is itself Uncertain significance with no PS1-relevant same-amino-acid comparator surfaced.

Evidence considered

• case_summary.json compact_evidence.clinvar shows only the exact-match variant (ClinVar 919572, Uncertain significance); no alternate-codon same-amino-acid comparator was surfaced by the pipeline's PS1/PM5 search.

Still missing

• No same-amino-acid-change (p.Glu26Gln) comparator variant with an independent pathogenic/likely pathogenic classification was retrieved in this case bundle.

Reasoning

No case-level parental testing establishes the variant as de novo in an affected individual.

Rule

Generic ACMG/AMP 2015 fallback applies because no CDKN2A VCEP/CSPEC or local gene-specific framework was retrieved; PS2 requires a confirmed de novo occurrence in an affected individual.

Evidence considered

• The consolidated case-evidence bundle contains no proband-level phenotype, parental genotypes, parentage confirmation, or documented de novo observation for NM_000077.4:c.76G>C.

Still missing

• Obtain maternal and paternal targeted test results for NM_000077.4:c.76G>C, including confirmation of biological parentage when available.
• Document the proband phenotype and its consistency with a CDKN2A-associated germline condition.

Reasoning

No CDKN2A ClinGen VCEP-specific functional assay framework is available for this case (cspec.found = false; vcep_materials note states 'no cspec url available'), so generic ACMG/AMP PS3 requirements apply. No validated functional assay (e.g., cell cycle/CDK4-6 inhibition, yeast complementation, RB-binding, thermal stability) targeting NM_000077.4:c.76G>C (p.Glu26Gln) was identified in the evidence bundle. OncoKB found no variant-specific reviewed functional evidence, COSMIC has no record of this variant in somatic cancers, and it does not lie in a mutational hotspot. The ClinVar submitter (Color Diagnostics, SCV001342084) explicitly states in their evidence signals: 'to our knowledge, functional studies have not been reported for this variant.' No publication in the literature packet reports a functional/biochemical assay of this specific variant; PMID:25741868 is the ACMG/AMP 2015 methods standards paper (not a functional study), and PMID:25394175/26389258/26389333 are practice guidelines/PDQ summaries unrelated to variant-level functional data. Without a validated assay for this specific variant, PS3 cannot be assessed.

Evidence considered

• ClinVar submitter (SCV001342084, Color Diagnostics) evidence_signals for PS3 states: 'to our knowledge, functional studies have not been reported for this variant.'
• OncoKB found no variant-specific reviewed functional evidence for CDKN2A E26Q; only gene-level curated context is available.
• COSMIC has no record of this variant in somatic cancers, and it does not lie in a statistically significant mutational hotspot per the hotspots screenshot data.
• No cspec (ClinGen CDKN2A VCEP specification) was found for this case (cspec.found = false; vcep_materials note: 'no cspec url available').

Still missing

• No published functional/biochemical assay (e.g., CDK4/CDK6 kinase inhibition, RB-pathway reporter, cell proliferation/senescence, thermal stability) specific to NM_000077.4:c.76G>C / p.(Glu26Gln) was found in the case evidence.
• No CDKN2A-specific VCEP functional assay calibration framework was available in this case to define an approved assay/strength mapping.

Reasoning

No case-control or other statistically valid enrichment data for the exact CDKN2A c.76G>C (p.Glu26Gln) variant were provided.

Rule

Generic ACMG/AMP 2015 PS4: prevalence in affected individuals must be significantly increased compared with controls.

Evidence considered

• No CDKN2A VCEP/ClinGen gene-specific specification was retrieved; the case framework therefore specifies generic ACMG/AMP fallback.
• The ClinVar record provides only three single-submitter uncertain-significance assertions and no affected-versus-control counts or enrichment analysis.

Still missing

• A phenotype-defined case series or case-control study reporting exact-variant counts in affected individuals and appropriate controls, with an analyzable enrichment statistic.

Sources

Reasoning

PM1 requires the variant to lie in a mutational hotspot or well-established critical functional domain lacking benign variation. The CancerHotSpots screen returned found=false with evidence_sentence 'This variant does not lie in a statistically significant hotspot,' which argues against a somatic mutational hotspot basis for PM1. However, this case bundle contains no protein-domain/structural annotation source (no UniProt/Pfam/InterPro entry in source_registry, no CDKN2A CSPEC domain table) to evaluate whether residue 26 of p16INK4A falls within a critical functional domain lacking benign missense variation, so a domain-based PM1 call cannot be made from the available sources.

Evidence considered

• CancerHotSpots.org screen: 'This variant does not lie in a statistically significant hotspot' (found=false), which weighs against PM1 on hotspot grounds but does not by itself rule out a critical-domain basis, which was not otherwise investigated in this case's evidence bundle.

Still missing

• No domain/structural annotation source (UniProt, Pfam, or CDKN2A-specific VCEP domain table) was retrieved to assess whether p.Glu26 lies in a critical functional region.
• CancerHotSpots.org (a somatic hotspot resource) found no statistically significant hotspot at this position, but this does not directly address germline critical-domain criteria.

Reasoning

PM5 requires a different pathogenic missense change at the identical residue (position 26) from the variant under review. The pipeline's own PM5 candidate-harvesting step explicitly declined to search: pm5_candidates.json reports pm5_mode='unknown', eligible_for_classic_pm5_search=false, with the explicit instruction 'Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting' and 'do not apply PM5 from this artifact alone.' No same-residue comparator variants (candidate_summary.same_residue_candidates=0) were identified anywhere else in the bundle.

Evidence considered

• pm5_candidates.json: eligible_for_classic_pm5_search=false, same_residue_candidates=0, recommendation.level='not_applicable', explicitly instructing not to apply PM5 from this artifact alone.

Still missing

• Same-residue (codon 26) pathogenic/likely pathogenic missense comparator search was not performed for this case; a manual ClinVar/literature search restricted to CDKN2A p.Glu26X changes is needed.

Sources

Reasoning

No reported de novo occurrence with unconfirmed parentage is available for this variant.

Rule

Generic ACMG/AMP 2015 fallback applies because no CDKN2A VCEP/CSPEC or local gene-specific framework was retrieved; PM6 requires a de novo occurrence when parentage is not confirmed.

Evidence considered

• The consolidated case-evidence bundle contains no parental testing data or de novo case observation for NM_000077.4:c.76G>C.

Still missing

• Obtain targeted parental testing or a published case report documenting de novo occurrence of NM_000077.4:c.76G>C.
• Document the affected individual's phenotype and whether biological parentage was confirmed.

Reasoning

No informative family pedigree, relative genotypes, affected-relative phenotypes, or meiosis count is available to assess co-segregation.

Rule

Generic ACMG/AMP 2015 fallback applies because no CDKN2A VCEP/CSPEC or local gene-specific framework was retrieved; PP1 requires co-segregation with disease in multiple affected family members.

Evidence considered

• The consolidated case-evidence bundle contains no family segregation data, pedigree, or informative meioses for NM_000077.4:c.76G>C.

Still missing

• Obtain a pedigree with affected and unaffected relatives, their targeted genotypes for NM_000077.4:c.76G>C, relevant ages, and phenotype/cancer diagnoses.
• Determine the number of informative segregations and account for age-dependent penetrance.

Reasoning

PP2 requires the variant to be missense in a gene with a low rate of benign missense variation and where missense variants are a common mechanism of disease. Consequence class is confirmed missense (pvs1_variant_assessment.consequence_class='missense', p.(Glu26Gln)). CDKN2A/p16INK4A is well known in the melanoma-predisposition literature as a gene where germline missense substitutions are a recognized disease mechanism (disrupting CDK4/6 binding), but this case's evidence bundle contains no gnomAD gene-level missense constraint metric (missense Z-score / O:E ratio) or CDKN2A-specific VCEP guidance on PP2 applicability (cspec.found=false; vcep_materials empty), so the quantitative low-benign-missense-rate threshold cannot be verified from sourced case data.

Evidence considered

• pvs1_variant_assessment.json confirms consequence_class='missense' for NP_000068.1:p.(Glu26Gln).

Still missing

• No gnomAD gene constraint metric (missense Z-score / observed:expected ratio) for CDKN2A was retrieved in this case to quantitatively support or refute a low rate of benign missense variation.
• No CDKN2A-specific VCEP/CSPEC guidance on PP2 applicability was available for this case.

Reasoning

No proband phenotype, pedigree, or phenotype-to-genotype differential assessment was provided for this variant; phenotype specificity cannot be evaluated.

Rule

Generic ACMG/AMP 2015 PP4: the patient's phenotype or family history must be highly specific for a disease with a single genetic etiology.

Evidence considered

• No CDKN2A VCEP/ClinGen gene-specific specification was retrieved; the case framework therefore specifies generic ACMG/AMP fallback.
• ClinVar lists familial melanoma and hereditary cancer-predisposing syndrome as record-level conditions, but supplies no individual-level phenotype attributable to this variant.

Still missing

• Detailed proband phenotype and family history, including melanoma/cancer diagnoses, ages at onset, and assessment for alternative genetic or non-genetic explanations.

Sources

Reasoning

The available population records report allele and homozygote counts but do not document unaffected adult carriers or age/phenotype data. Absence of homozygotes is not BS2 evidence for a heterozygous cancer-predisposition condition.

Rule

Generic ACMG/AMP BS2: requires observation in healthy adult individual(s) for a disorder expected to be fully penetrant at an early age; population database carrier counts without phenotype data are insufficient.

Evidence considered

• gnomAD v2.1 and v4.1 both report zero homozygotes, but neither dataset supplies the unaffected-adult phenotype evidence required for BS2.

Still missing

• Well-phenotyped, unaffected adult heterozygote observations with age and relevant cancer-history information are required to assess BS2.

Sources

Reasoning

No validated functional assay data (variant-specific, with appropriate wild-type/null controls) showing normal/wild-type function for NM_000077.4:c.76G>C (p.Glu26Gln) was identified. As with PS3, the ClinVar submitter explicitly notes functional studies have not been reported for this variant, and no CDKN2A VCEP-specific assay calibration framework is present in this case (cspec.found = false). Without any reported functional study of this variant (showing normal or abnormal function), BS3 cannot be assessed.

Evidence considered

• ClinVar submitter (SCV001342084, Color Diagnostics) evidence_signals for PS3 states: 'to our knowledge, functional studies have not been reported for this variant' — the same absence of functional data applies to BS3.
• No cspec (ClinGen CDKN2A VCEP specification) was found for this case (cspec.found = false; vcep_materials note: 'no cspec url available').

Still missing

• No published functional assay data (of any outcome) specific to NM_000077.4:c.76G>C / p.(Glu26Gln) was found, precluding assessment of normal function for BS3.
• No CDKN2A-specific VCEP functional assay calibration framework was available in this case.

Reasoning

No informative non-segregation is documented; in particular, no affected non-carrier or convincingly unaffected carrier data are available.

Rule

Generic ACMG/AMP 2015 fallback applies because no CDKN2A VCEP/CSPEC or local gene-specific framework was retrieved; BS4 requires evidence that the variant does not segregate with disease.

Evidence considered

• The consolidated case-evidence bundle contains no informative affected non-carriers, unaffected carriers, or other family non-segregation observations for NM_000077.4:c.76G>C.

Still missing

• Obtain pedigree-linked genotypes and phenotype/cancer histories for affected and unaffected relatives.
• Evaluate any apparent non-segregation in the context of CDKN2A-associated condition definition, age-dependent penetrance, and phenocopies.

Reasoning

No CDKN2A gene-specific specification was retrieved. The consolidated evidence contains no observation of c.76G>C (p.Glu26Gln) in cis or trans with a pathogenic/likely pathogenic variant in the same individual, and no phase data. BP2 cannot be assessed.

Rule

Generic ACMG/AMP BP2: observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• The ClinVar material in the consolidated bundle contains only germline submissions for the exact variant and no co-occurrence or phasing information.
• No publication in the consolidated literature set was identified as providing allelic, phase, or affected-proband evidence for this variant.

Still missing

• Provide an individual-level co-occurrence report identifying the companion variant, its independently established pathogenic/likely pathogenic classification, the relevant phenotype and inheritance context, and confirmed cis/trans phase.

Sources

Reasoning

No affected individual and no alternate molecular diagnosis explaining a reported phenotype were provided, so BP5 cannot be assessed.

Rule

Generic ACMG/AMP 2015 BP5: a variant is found in a case with an alternate molecular basis for disease.

Evidence considered

• No CDKN2A VCEP/ClinGen gene-specific specification was retrieved; the case framework therefore specifies generic ACMG/AMP fallback.
• The available ClinVar record contains no individual-level phenotype or alternate molecular diagnosis.

Still missing

• An affected individual's phenotype plus a confirmed alternative molecular diagnosis that fully explains that phenotype.

Sources