CDKN2A c.225_243del, p.Ala76CysfsTer64

Reasoning

PVS1 is met at full strength. NM_000077.5:c.225_243del is a frameshift in exon 2 of the MANE Select transcript NM_000077.5, producing NP_000068.1:p.(Ala76CysfsTer64). CDKN2A loss of function is supported as a germline disease mechanism in the gene-level assessment. The frameshift introduces a premature termination codon within exon 2, with downstream exon sequence remaining, so the predicted transcript is not an obvious terminal-exon or last-junction NMD escape. The affected region is not a documented non-critical distal tail, and the predicted protein is truncated from 157 to 139 amino acids. No named downgrade rule is supported by the available evidence; therefore the default full-strength PVS1 assignment remains appropriate.

Rule

PVS1: Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.

Evidence considered

• NM_000077.5:c.225_243del is annotated as a frameshift in exon 2 with protein consequence NP_000068.1:p.(Ala76CysfsTer64).
• The normalization record identifies NM_000077.5 as the MANE Select/refseq-select transcript for CDKN2A.
• The predicted protein sequence ends at residue 139 versus residue 157 in the reference protein, consistent with a truncating loss of 18 amino acids after the frameshifted segment.
• The gene-level PVS1 assessment marks CDKN2A loss of function as a supported germline disease mechanism and the PVS1 gene gate as eligible.
• The generic PVS1 assessment identifies frameshift variants as eligible for the generic PVS1 framework and lists NMD escape, non-critical distal location, biologically irrelevant exon, and population LoF enrichment as downgrade considerations; none is established here.

Sources

Reasoning

PS3 is met at strong strength. McKenzie et al. (PMID:20340136) directly tested the exact 19-bp deletion, c.225_243del19 (the p16-Leiden allele corresponding to NM_000077.5:c.225_243del), using orthogonal functional assays with wild-type controls. The variant showed 92.3% altered subcellular localization, a Ki67 index of 32.9% versus 9.3% for wild type, and CDK4/CDK6 binding of 1.2%/1.0% versus 100%/100% for wild type. The study used at least two independent transfection experiments for the binding and localization assays and evaluated the biologically relevant CDKN2A functions of CDK4 interaction, cellular localization, and cell-cycle inhibition. These concordant loss-of-function results directly support a damaging effect on the CDKN2A gene product; no BS3 evidence was identified.

Rule

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

Evidence considered

• McKenzie et al. 2010, Table 2, row c.225_243del19: 92.3% altered localization, Ki67 index 32.9%, CDK4 binding 1.2%, and CDK6 binding 1.0%; wild type was 2.3% altered localization, 9.3% Ki67, and 100.0% binding for both CDK4 and CDK6.
• McKenzie et al. 2010, Results: the study included the 19-bp deletion c.225_243del19, also known as p16INK4a-Leiden, among the tested CDKN2A variants.
• McKenzie et al. 2010, Methods and Figure 1 legend: mammalian two-hybrid binding assays were performed at least twice in duplicate, and localization was quantified from at least two independent transfection experiments with at least 400 cells evaluated.

Sources

Reasoning

The variant is absent from gnomAD v2.1 and is present only once among 1,604,242 alleles in gnomAD v4.1 (AF=6.23347350337418e-07; homozygotes=0), which is below the supplied PM2 supporting threshold of 0.0001. The non-cancer subsets also report the variant as absent, but no governing VCEP-specific source selection applies because this case is generic ACMG/AMP. The observed gnomAD v4.1 frequency therefore supports PM2 at supporting strength under the supplied generic SVI threshold.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v2.1 reports the variant absent.
• gnomAD v4.1 reports total AC=1, AN=1,604,242, total AF=6.23347350337418e-07, and total homozygotes=0; the highest observed ancestry frequency is European (non-Finnish) AF=8.478010583948413e-07 from 1/1,179,522 alleles, also with zero homozygotes.
• The supplied generic PM2 supporting threshold is <=0.0001; the gnomAD v4.1 total AF is below this threshold.
• The evidence record reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer; no VCEP-specific non-cancer or exome-only rule governs this generic ACMG/AMP case.

Sources

Reasoning

ClinVar contains an exact-variant Pathogenic record for NM_000077.5:c.225_243del, but the submission audit reports zero expert-panel submissions. The available records are ordinary laboratory or other non-expert assertions, and the aggregate ClinVar label cannot trigger PP5 under the specified restriction.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation. ... Generic rules for PP5/BP6: apply them only for an exact-variant ClinVar expert-panel classification: Pathogenic/Likely pathogenic supports PP5; Benign/Likely benign supports BP6. Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger either criterion.

Evidence considered

• ClinVar exact-variant record NM_000077.5(CDKN2A):c.225_243del (p.Ala76fs) is classified Pathogenic, with 9 clinical laboratories summarized.
• The ClinVar submission audit reports expert_panel_submissions: 0; no exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification is present.

Sources

Reasoning

BS3 is not met because no well-established functional study showed that the exact NM_000077.5:c.225_243del allele has no damaging effect on CDKN2A protein function or splicing. The available exact-variant study instead demonstrated marked loss of CDK4/CDK6 binding, abnormal subcellular localization, and impaired cell-cycle control, which is inconsistent with benign functional evidence. Absence of a benign assay result is not itself evidence for BS3.

Rule

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

Evidence considered

• McKenzie et al. 2010, Table 2, row c.225_243del19: CDK4 binding was 1.2% and CDK6 binding was 1.0% relative to 100.0% for wild type, with 92.3% altered localization.
• The exact-variant functional result is damaging rather than benign, so it cannot support BS3.

Still missing

• No well-established assay demonstrating preserved CDKN2A protein function or normal splicing for the exact allele was identified.

Sources

Reasoning

The exact-variant ClinVar record contains Pathogenic and Likely pathogenic laboratory assertions and reports zero expert-panel submissions; it contains no qualifying exact-variant expert-panel Benign or Likely benign classification. Ordinary laboratory submissions and aggregate labels cannot trigger BP6 under the specified restriction.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation. ... Generic rules for PP5/BP6: apply them only for an exact-variant ClinVar expert-panel classification: Pathogenic/Likely pathogenic supports PP5; Benign/Likely benign supports BP6. Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger either criterion.

Evidence considered

• ClinVar's exact-variant submission audit reports expert_panel_submissions: 0 and summarizes the available clinical-laboratory classifications as Pathogenic or Likely pathogenic.
• No qualifying exact-variant ClinVar expert-panel Benign or Likely benign classification is present.

Sources

Reasoning

The available evidence does not document a de novo occurrence of NM_000077.5:c.225_243del in a proband, confirmed maternity and paternity, disease phenotype, or absence of family history. Therefore, the PS2 requirements 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 parental testing, maternity confirmation, paternity confirmation, or de novo observation for NM_000077.5:c.225_243del was identified in the reviewed evidence.

Still missing

• Proband-level de novo observation for NM_000077.5:c.225_243del
• Confirmed maternity and paternity
• Documented disease phenotype and family-history status

Reasoning

The literature_pass packet identifies PMID:10956390 as a PS4 paper, and its abstract reports the exact p16-Leiden 19-bp deletion in 19 Dutch FAMMM families, pancreatic cancer in 15 patients from 7 mutation-positive families, and no pancreatic cancer in 8 mutation-negative families. This is a qualitative affected-versus-negative-family comparison, but it does not report the individual numbers of exact-variant carriers among affected patients and controls, nor matched control denominators, so a valid case-control OR and 95% CI cannot be computed. The family-level counts cannot be substituted for the required individual-level case-control counts. PMID:25356972 was checked in the available extraction and does not mention the exact target variant.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: 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:10956390 abstract, verified against the literature_pass abstract: a p16-Leiden 19-bp deletion was identified in 19 families; pancreatic cancer occurred in 15 patients from 7 families, while 8 p16-Leiden-negative families had no pancreatic cancer.
• The reported comparison is family-level and does not provide individual affected/control carrier counts or matched denominators; therefore no valid OR or 95% CI was computed.
• PMID:25356972 paper extraction: the study did not explicitly mention CDKN2A c.225_243del or p.(A76Cfs*64), so it supplies no exact-variant PS4 observation.

Still missing

• Individual-level exact-variant carrier counts among affected patients and unaffected or matched population controls, with denominators, are required to calculate OR and 95% CI.

Sources

Reasoning

The available evidence does not document an assumed de novo occurrence of NM_000077.5:c.225_243del or parental testing showing that the variant is absent from either parent. Therefore, PM6 cannot be assessed.

Rule

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

Evidence considered

• No case-level parental genotypes or documentation of an assumed de novo event for NM_000077.5:c.225_243del was identified in the reviewed evidence.

Still missing

• Evidence that the variant arose de novo in the proband
• Parental genotyping results, even if maternity or paternity is unconfirmed

Reasoning

ClinVar-associated material describes the variant in individuals and families with personal or family histories consistent with familial atypical multiple mole melanoma, but it does not provide an auditable count of affected relatives carrying the exact variant, tested non-carriers, or informative meioses. PMID:25356972 reports CDKN2A mutation testing in familial pancreatic cancer probands but explicitly does not mention this exact variant and provides no variant-specific segregation result. The evidence is therefore insufficient to establish PP1 or determine a stronger level.

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

• ClinVar submission-audit material contains a generic statement that the mutation was reported in numerous individuals and families with personal and/or family history consistent with familial atypical multiple mole melanoma, but it does not provide auditable segregation counts for the exact variant.
• Li et al. 2015, PMID:25356972, reports germline CDKN2A testing in familial pancreatic cancer probands; the extracted finding states that NM_000077.5:c.225_243del/p.(A76Cfs*64) was not explicitly mentioned and that no variant-specific segregation result was reported.

Still missing

• Exact-variant genotypes for affected relatives
• Exact-variant genotypes for informative unaffected relatives
• Number of informative meioses and affected carriers
• Phenotype details for each tested relative

Sources

Reasoning

No individual patient phenotype or family-history description is available in the supplied evidence. Gene-level disease associations and literature about other carriers do not establish that this patient's phenotype is highly specific for a disease with a single genetic etiology, 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 and relevant family history linked to a defined CDKN2A-associated disease are required.

Reasoning

The variant is present at total AF 6.23347350337418e-07 (1/1,604,242 alleles) in gnomAD v4.1 and absent from gnomAD v2.1, far below the generic BA1 threshold of 0.05. However, the required filtering frequency, grpmax FAF, is unavailable (null), and BA1 is a stand-alone benign criterion; therefore a definitive BA1 assessment cannot be made using the mandated FAF metric. The available population data do not support BA1, and the pathogenic ClinVar consensus further argues against ending classification on an unverified frequency threshold.

Rule

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

Evidence considered

• gnomAD v4.1 reports total AC=1, AN=1,604,242, total AF=6.23347350337418e-07, and total homozygotes=0; grpmax FAF is null.
• gnomAD v2.1 reports the variant absent.
• The generic stand-alone BA1 threshold supplied for this case is >=0.05, so the observed total AF is approximately 80,000-fold below that threshold; the mandated grpmax FAF is nevertheless unavailable.
• ClinVar reports Pathogenic with multiple submitters and no conflicts for variation 182411; this is contextual evidence against applying BA1 solely from an unverified population-frequency metric, not an independent BA1 criterion.

Still missing

• gnomAD filtering frequency (grpmax FAF) is unavailable for this variant; BA1 requires assessment with that metric and a population of at least 2000 observed alleles.

Sources

Reasoning

The variant is present at total AF 6.23347350337418e-07 (1/1,604,242 alleles) in gnomAD v4.1 and absent from gnomAD v2.1, far below the generic BS1 default threshold of 0.01. The required filtering frequency, grpmax FAF, is unavailable (null), so the mandated FAF-based BS1 assessment cannot be completed. No maximum-credible-frequency calculation is needed to explain an elevated frequency because no elevated frequency is observed, but the criterion remains not_assessed rather than met or not_met because the specified filtering metric is missing.

Rule

BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v4.1 reports total AC=1, AN=1,604,242, total AF=6.23347350337418e-07, and total homozygotes=0; grpmax FAF is null.
• gnomAD v2.1 reports the variant absent.
• The generic BS1 default supplied for this case is >=0.01; the observed total AF is approximately 16 million-fold lower, but the required grpmax FAF is unavailable.
• Whiffin et al. 2017 (PMID:28518168) is the specified publication for maximum-credible-frequency reasoning; no elevated ancestry-specific frequency requiring such a calculation is present here.

Still missing

• gnomAD filtering frequency (grpmax FAF) is unavailable; a definitive FAF-based BS1 assessment cannot be completed.

Sources

Reasoning

gnomAD v4.1 reports one heterozygous allele and zero homozygotes, but the population record does not establish that the carrier was a healthy adult or provide phenotype and age-at-assessment information. The evidence therefore does not satisfy the specific healthy-adult observation required for BS2, and no independent healthy-adult carrier report was identified in the population evidence.

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 v4.1 reports total AC=1, AN=1,604,242, total homozygotes=0, and European (non-Finnish) AC=1, AN=1,179,522, homozygotes=0.
• A gnomAD allele count alone does not document that the individual was a healthy adult or that full penetrance was expected at an early age; therefore it cannot establish BS2.

Still missing

• No documented healthy adult carrier observation with adequate phenotype and age-at-assessment information is available.

Sources

Reasoning

The available evidence does not identify affected relatives who were tested and found not to carry NM_000077.5:c.225_243del. No family-level non-segregation data are provided, and the evidence is insufficient to distinguish true lack of segregation from absent testing or phenocopy. Therefore, BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No case-level affected-relative genotypes demonstrating non-segregation for NM_000077.5:c.225_243del were identified in the reviewed evidence.

Still missing

• Affected relatives tested for the exact variant
• Genotype-negative affected relatives and their clinical diagnoses
• Family structure and phenocopy assessment

Reasoning

BP2 cannot be assessed from the available evidence. The case provides no documented pathogenic variant in trans, no documented pathogenic variant in cis, and no parent or offspring testing establishing phase for NM_000077.5:c.225_243del. The available disease context does not establish a fully penetrant dominant gene/disorder for the trans pathway, while the cis pathway still requires direct phase evidence.

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 identifies the target variant as NM_000077.5:c.225_243del (NP_000068.1:p.(Ala76CysfsTer64)) but does not document a second pathogenic variant in the same proband.
• The available case evidence contains no parent or offspring genotyping and no cis/trans phase result for this variant.
• Clinical and literature records describe CDKN2A familial melanoma/cancer predisposition, but they do not establish the fully penetrant dominant status required for the BP2 trans pathway.

Still missing

• Documented paired-variant observation in the same affected proband
• Parent or offspring testing establishing whether the paired variants are in trans or cis
• Evidence that any paired variant is pathogenic

Reasoning

The supplied evidence does not document that this variant was found in a case with an alternate molecular basis for disease. Somatic database occurrence, general CDKN2A disease literature, and unrelated molecular findings do not establish an alternate molecular diagnosis for this case, so BP5 cannot be assessed.

Rule

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

Still missing

• Case-level molecular findings establishing an alternate molecular basis for the patient's disease are required.