CDKN2A c.44_46dup, p.(Trp15_Leu16insArg)

Reasoning

PM2 is met at supporting strength. The exact normalized variant NM_001195132.1:c.44_46dup, corresponding to NC_000009.11:g.21974781_21974783dup and the GRCh38 allele 9-21974781-A-AGCC, is reported as absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. This supports rarity in reference populations. The bundle does not provide allele-number, coverage, or ancestry-specific quantitative data, so this is not upgraded beyond supporting strength.

Rule

Generic ACMG/AMP PM2: absent from population databases or present at an extremely low frequency, in a population appropriate to the disorder; applied at supporting strength because the variant is absent from multiple population resources but quantitative denominators are unavailable.

Evidence considered

• gnomAD v2.1 reports the exact variant as absent.
• gnomAD v4.1 reports the exact variant as absent.
• gnomAD-Canada v1.0 reports the exact variant as absent.
• The concordant absence across three population resources supports rarity, while the lack of quantitative denominator and coverage data limits threshold-level auditing.

Still missing

• Allele counts, total allele numbers, ancestry-specific counts, and coverage/quality metrics were not provided for maximum-credible-frequency or representation-artifact auditing.
• No disease-specific prevalence, penetrance, or inheritance model was supplied for a quantitative rarity threshold.

Sources

Reasoning

NM_001195132.1:c.44_46dupGGC (normalized c.44_46dup) is an in-frame 3-bp duplication in exon 1 (CDS positions 44-46) of CDKN2A transcript variant 5, predicting NP_001182061.1:p.(Trp15_Leu16insArg) - insertion of a single arginine that lengthens p16INK4A from 156 to 157 residues. No frameshift or premature termination codon is introduced, so NMD does not apply; the change is a protein length change at the protein level. Direct inspection of the reference genomic sequence (NC_000009.11, GRCh37) around chr9:21974781-21974783 shows the duplicated unit GGC (plus-strand GCC) is not embedded in a tandem repeat, homopolymer, or low-complexity array, so the insertion occurs in a non-repeat region. SpliceAI (max delta 0.014) predicts no splice impact, confirming the variant acts as a purely protein-level in-frame change rather than a splicing alteration. This satisfies generic ACMG/AMP 2015 PM4 (in-frame insertion in a non-repeat region causing protein length change). ClinVar annotates the identical consequence on the canonical transcript NM_000077.5, confirming transcript relevance of the p16INK4A isoform.

Rule

PM4 (ACMG/AMP 2015, Richards et al. PMID 25741868, generic fallback): Protein length changes as a result of in-frame deletions/insertions in a non-repeat region or stop-loss variants. Strength: Moderate.

Evidence considered

• Variant normalization (prefetch.json, Mutalyzer/VariantValidator) confirms c.44_46dupGGC is an in-frame 3-bp duplication (NM_001195132.1:c.44_46dup) predicting NP_001182061.1:p.(Trp15_Leu16insArg), a single-amino-acid insertion lengthening p16INK4A from 156 to 157 residues; no frameshift or premature stop, so NMD is not triggered.
• pvs1_variant_assessment classifies the consequence as in_frame_indel_unknown and places it in a non-null variant bucket; the variant is not a canonical splice-consensus variant, consistent with a purely protein-level in-frame change.
• Direct inspection of the reference genomic sequence (NC_000009.11, GRCh37) around chr9:21974781-21974783 shows the duplicated unit GGC (plus-strand GCC) is not part of a tandem repeat, homopolymer, or low-complexity array (no (GCC)n or (GGC)n repeat flanks the site), so the duplication lies in a non-repeat region.
• SpliceAI (spliceai source) reports max delta score 0.014 (all delta scores <= 0.014), below any splice-impact threshold, supporting a protein-level in-frame insertion rather than a splicing alteration.
• The variant lies in exon 1 (CDS c.44-46) of NM_001195132.1 (CDKN2A transcript variant 5, p16INK4A isoform); ClinVar (clinvar source) reports the identical protein consequence p.(Trp15_Leu16insArg) on the canonical transcript NM_000077.5, confirming transcript relevance.
• Generic ACMG/AMP 2015 fallback (generic_acmg_combination_rules, based on Richards et al., PMID 25741868) defines PM4 as protein length changes from in-frame deletions/insertions in a non-repeat region or stop-loss variants.

Still missing

• No variant-specific functional studies confirm the biological impact of this specific in-frame insertion (OncoKB lists no variant-specific reviewed functional evidence; functional group assesses PS3/BS3 separately).
• Residues 15-16 lie in the N-terminal region of p16INK4A upstream of the ankyrin-repeat domains; the clinical impact of this particular insertion is not independently established by functional literature.
• No ClinGen CSPEC/VCEP for CDKN2A was available, so the generic ACMG/AMP 2015 PM4 definition was applied without VCEP-specific calibration.

Sources

Reasoning

No CSPEC/VCEP framework or gene-specific lookup material governs CDKN2A PP3/BP4 for this case, so generic ACMG applies. NM_001195132.1:c.44_46dup is an in-frame insertion outside the canonical splice consensus; the only calibrated computational predictor with a named, citable threshold available for this variant type is SpliceAI. SpliceAI predicts negligible splice impact (max delta score = 0.014, i.e. all four delta scores DS_AG/AL/DG/DL <=0.014), well below the ~0.2 delta-score cutoff established in the SpliceAI publication (Jaganathan et al., Cell 2019, PMID 30661751) as the operating threshold for a predicted splice-altering effect. This low computational splice-impact score supports a benign impact on splicing, warranting BP4 at supporting strength. BayesDel and REVEL are not applicable to this non-SNV in-frame duplication and were not scored, so they contribute no additional BP4 evidence.

Rule

generic_acmg_bp4

Evidence considered

• SpliceAI Lookup query for NM_001195132.1:c.44_46dup returned max delta score = 0.014, well below the ~0.2 delta-score threshold from the SpliceAI publication (Jaganathan et al., Cell 2019, PMID 30661751) used to flag a predicted splice-altering variant.
• REVEL was explicitly skipped by the pipeline for this variant ('not snv or no coords') and BayesDel score/found are both null, so neither missense predictor contributes to BP4 for this in-frame insertion.

Still missing

• No gene-specific CDKN2A BP4 lookup spreadsheet was available to confirm gene-specific calibration for this in-frame insertion variant type.
• BayesDel score is not available for this variant (bayesdel.found=null); per pipeline policy, BayesDel is never used for PP3/BP4 in this case since no verified published threshold/table citation is available.

Sources

Reasoning

No CSPEC/VCEP framework or gene-specific PP3/BP4 lookup material is present for CDKN2A (cspec.found=false; vcep_materials.summaries and files_found are empty), so generic ACMG applies. This variant, NM_001195132.1:c.44_46dup (p.(Trp15_Leu16insArg)), is an in-frame duplication/insertion, not a missense substitution and not a canonical splice-site (±1,2) variant, so the missense in-silico path (REVEL/BayesDel/aGVGD) and the classic splice-consensus path do not apply to it. The only applicable computational evidence is SpliceAI, which predicts minimal splice impact (max delta score = 0.014; DS_AG 0.002, DS_AL 0.014, DS_DG 0.014, DS_DL 0.007), well below the ~0.2 delta threshold SpliceAI's own developers associated with a high-confidence splice-altering call. There is no computational signal supporting a damaging molecular consequence, so PP3 is not met.

Rule

generic_acmg_pp3

Evidence considered

• SpliceAI Lookup query for NM_001195132.1:c.44_46dup returned max delta score = 0.014 (DS_AG 0.002, DS_AL 0.014, DS_DG 0.014, DS_DL 0.007), indicating no predicted splice disruption.
• The variant is an in-frame 3-bp duplication (p.(Trp15_Leu16insArg)), not a missense SNV, so REVEL/BayesDel/aGVGD in-silico missense predictors are not applicable and were not run (revel skipped: 'not snv or no coords'; bayesdel score/found both null).

Still missing

• No gene-specific CDKN2A PP3/BP4 lookup spreadsheet or CSPEC was available to confirm whether this in-frame insertion falls under a different calibrated in-silico rule.
• REVEL and BayesDel are not applicable/not available for this non-missense in-frame duplication (revel.found=null, bayesdel.found=null; revel step explicitly skipped as 'not snv or no coords').

Sources

Reasoning

PP5 is not met because the exact-variant ClinVar record has no expert-panel submission. The available exact-match assertions are single-laboratory submissions, including one Pathogenic assertion, and cannot trigger PP5 under the specified rule.

Rule

Apply PP5 only when an exact-variant ClinVar expert-panel classification is Pathogenic or Likely pathogenic; single-submitter laboratory assertions do not qualify.

Evidence considered

• ClinVar variation 182408 is an exact match to NM_000077.5(CDKN2A):c.44_46dup (p.Trp15_Leu16insArg).
• The submission audit reports zero expert-panel submissions; the Pathogenic assertion is from GeneDx with review status criteria provided, single submitter, and the other submission is Uncertain significance from Labcorp with the same review status.
• Ordinary laboratory submissions and conflicting or aggregate ClinVar labels do not satisfy the required expert-panel condition for PP5.

Sources

Reasoning

BA1 is not met. The exact normalized variant NM_001195132.1:c.44_46dup (NP_001182061.1:p.(Trp15_Leu16insArg)) is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; therefore there is no population frequency at or above the stand-alone benign threshold. Quantitative allele counts and ancestry-specific denominators were not supplied, so no frequency threshold calculation is possible, but the observed evidence is incompatible with BA1.

Rule

Generic ACMG/AMP BA1: a population allele frequency sufficiently high to establish a stand-alone benign criterion; no such frequency is observed here.

Evidence considered

• gnomAD v2.1 reports the exact genomic allele 9-21974780-A-AGCC as absent.
• gnomAD v4.1 reports the exact genomic allele 9-21974781-A-AGCC as absent.
• gnomAD-Canada v1.0 reports the exact genomic allele 9-21974781-A-AGCC as absent.

Still missing

• Allele counts, total allele numbers, ancestry-specific counts, and quality/coverage metrics were not provided for quantitative threshold auditing.

Sources

Reasoning

BS1 is not met. The exact variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, so no allele frequency above the expected disease-prevalence threshold is demonstrated.

Rule

Generic ACMG/AMP BS1: allele frequency is greater than expected for the disorder; no elevated frequency is observed.

Evidence considered

• The exact normalized variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; therefore the available population evidence does not support an allele frequency exceeding a disease-specific threshold.

Still missing

• Disease prevalence, penetrance, inheritance-specific maximum credible allele frequency, and ancestry-specific population denominators were not provided for a quantitative BS1 threshold calculation.
• Allele counts and coverage/quality metrics were not provided.

Sources

Reasoning

BP3 requires an in-frame deletion/insertion located in a repeat region without known function. Although c.44_46dup is an in-frame insertion, direct inspection of the reference genomic sequence (NC_000009.11, GRCh37) at chr9:21974781-21974783 shows the duplicated unit is not within a tandem repeat, homopolymer, or low-complexity sequence region, so the repeat-region condition is not satisfied. Furthermore, CDKN2A encodes p16INK4A, a well-characterized tumor suppressor with established function, so even the 'region without known function' clause would not be met. BP3 is therefore not met.

Rule

BP3 (ACMG/AMP 2015, Richards et al. PMID 25741868, generic fallback): In-frame deletions/insertions in a repeat region without known function. Strength: Supporting (benign).

Evidence considered

• Generic ACMG/AMP 2015 BP3 (generic_acmg_combination_rules, based on Richards et al., PMID 25741868) requires an in-frame deletion/insertion in a repeat region without known function.
• Direct inspection of the reference genomic sequence (NC_000009.11, GRCh37) around chr9:21974781-21974783 shows the duplicated unit GGC (plus-strand GCC) is not embedded in a tandem repeat, homopolymer, or low-complexity array, so the repeat-region condition of BP3 is not satisfied.
• CDKN2A encodes p16INK4A, a cyclin-dependent kinase inhibitor with well-established tumor-suppressor function; even were the site repetitive, the 'region without known function' clause would not be satisfied.
• pvs1_variant_assessment confirms the variant is an in-frame non-null change (in_frame_indel_unknown), so the variant type is nominally within BP3's scope, but the location condition fails.
• SpliceAI (spliceai source) max delta 0.014 indicates no splice impact; the insertion is a pure protein-level in-frame change and does not map to any repetitive splicing element.
• The variant is absent from gnomAD v2.1/v4.1/Canada, consistent with a rare non-polymorphic insertion rather than a common repeat-region indel.

Still missing

• A formal repeat annotation (e.g., UCSC RepeatMasker track) was not present in the case bundle; the non-repeat determination rests on direct inspection of the local reference sequence context.
• No ClinGen CSPEC/VCEP for CDKN2A exists, so the generic ACMG/AMP 2015 BP3 definition was applied.

Sources

Reasoning

BP6 is not met because the exact-variant ClinVar record has no expert-panel submission with a Benign or Likely benign classification. The available submissions are a single-laboratory Pathogenic assertion and a single-laboratory Uncertain significance assertion.

Rule

Apply BP6 only when an exact-variant ClinVar expert-panel classification is Benign or Likely benign; single-submitter laboratory assertions and aggregate labels do not qualify.

Evidence considered

• ClinVar variation 182408 is an exact match to NM_000077.5(CDKN2A):c.44_46dup (p.Trp15_Leu16insArg).
• The submission audit reports zero expert-panel submissions and no Benign or Likely benign exact-variant expert-panel classification.
• The available exact-match classifications are Pathogenic from GeneDx and Uncertain significance from Labcorp, both criteria-provided single-submitter records; these cannot trigger BP6.

Sources

Reasoning

No case-specific evidence documents that the variant arose de novo in the proband, including confirmed maternity and paternity or parental genotypes. The consolidated evidence bundle contains no proband-level parental testing or de novo observation suitable for PS2.

Rule

Generic ACMG/AMP PS2 requires a confirmed de novo variant in the affected proband, with maternity and paternity confirmed; strength depends on the quality and completeness of parental confirmation.

Evidence considered

• The consolidated case-evidence bundle provides variant normalization, population data, ClinVar records, and literature triage, but no proband-specific parental testing or validated de novo observation.

Still missing

• Proband phenotype and case-level report
• Confirmed maternity and paternity
• Parental testing results for the exact variant
• De novo status and laboratory validation

Reasoning

No CDKN2A-specific VCEP/CSPEC framework was found for this case (case_summary.json cspec.found=false; framework_mode=generic_acmg), so no gene-specific PS3 functional assay calibration is available. The consolidated evidence bundle (case_summary.json compact_evidence, literature_packet, vcep_materials, source_registry) contains no functional/biochemical assay data for this variant (p16INK4a/p14ARF activity assays, cell proliferation/senescence rescue, CDK4/6 binding, etc.). OncoKB reports 'Unknown Oncogenic Effect' with no variant-specific reviewed functional evidence, and no publications in the bundle describe a validated functional assay of NM_001195132.1:c.44_46dup or the resulting p.(Trp15_Leu16insArg) in-frame insertion.

Still missing

• No validated functional assay (e.g., p16 CDK4/6 inhibition assay, cell cycle arrest/senescence rescue assay) has been identified for this specific variant
• No CDKN2A-specific VCEP functional assay specification is available to calibrate assay validity or PS3 strength

Reasoning

No case-control enrichment data, affected-case series, or sufficiently documented affected probands carrying the exact variant were provided. The available ClinVar records are single-laboratory submissions and do not establish PS4.

Rule

Generic ACMG/AMP PS4 requires statistically significant enrichment in affected individuals versus controls or an equivalent well-established case-series observation; no applicable CDKN2A VCEP specification was retrieved.

Evidence considered

• The exact ClinVar record contains one Pathogenic GeneDx submission and one Uncertain significance Labcorp submission, both criteria-provided single-submitter records; no expert-panel or case-series evidence is present.
• The retrieved literature packet contains general cancer-genetics referral guidance rather than exact-variant case-control or affected-proband evidence.

Still missing

• Case-control counts or statistically interpretable enrichment for the exact variant are unavailable.
• No adequately documented cohort of affected individuals with the exact variant and the relevant CDKN2A phenotype is available.

Sources

Reasoning

No applicable CDKN2A-specific VCEP framework was available; generic ACMG/AMP criteria were therefore considered. The case evidence bundle contains no affected-proband observations establishing a recessive disease context, no parental or family phasing, and no documented pathogenic variant in trans. The available case information is insufficient to assess PM3.

Rule

Generic ACMG/AMP PM3: apply only when a variant is observed in trans with a pathogenic variant in an affected individual or family in a recessive disease context; phase and the pathogenic status of the other allele must be established.

Evidence considered

• The case summary identifies CDKN2A and the variant NM_001195132.1:c.44_46dup, but provides no inheritance field, affected-proband genotype observations, or phase information.
• The consolidated evidence bundle reports no CDKN2A-specific VCEP/CSPEC framework, so generic ACMG/AMP PM3 logic was used only to define the missing evidence requirements.

Still missing

• Disease-specific inheritance mode for the evaluated condition is not documented in the case bundle.
• No affected-proband genotype data or independent pathogenic/likely pathogenic allele is documented.
• No parental or family phasing establishes the evaluated variant in trans with a pathogenic variant.

Reasoning

No case-specific evidence documents a presumed de novo occurrence without confirmed maternity and paternity. The available bundle does not provide a proband report, parental genotypes, or a validated de novo observation for this variant.

Rule

Generic ACMG/AMP PM6 concerns a presumed de novo variant when maternity and paternity are not confirmed; it requires a documented affected proband and credible de novo evidence.

Evidence considered

• The consolidated case-evidence bundle contains no documented presumed de novo occurrence, parental testing, or family-level clinical evidence for the exact variant.

Still missing

• Proband phenotype and case-level report
• Parental testing results for the exact variant
• Evidence establishing presumed de novo occurrence
• Assessment of maternity and paternity confirmation

Reasoning

No informative affected or unaffected relatives, meioses, phenotype concordance, or genotype results are reported for this case. Segregation cannot be assessed from the available evidence.

Rule

Generic ACMG/AMP PP1 requires cosegregation of the variant with disease in affected family members; strength may increase with multiple independent informative meioses and consistent phenotype.

Evidence considered

• The consolidated case-evidence bundle does not include family segregation data, informative meioses, relative genotypes, or phenotype concordance information for the exact variant.

Still missing

• Family pedigree and number of informative meioses
• Genotypes of affected relatives
• Genotypes of unaffected relatives
• Phenotype consistency and age-related penetrance assessment

Reasoning

The case bundle does not provide a patient phenotype, a highly specific clinical presentation, or a phenotype-to-CDKN2A syndrome match for the exact variant. General disease labels in ClinVar are insufficient to apply PP4.

Rule

Generic ACMG/AMP PP4 requires a phenotype or family history highly specific for a disease with a single-gene etiology; broad database condition labels without patient-level phenotype documentation are insufficient.

Evidence considered

• The exact ClinVar record lists broad condition labels including Familial melanoma and Hereditary cancer-predisposing syndrome, but provides no patient-level phenotype or phenotype specificity assessment.
• No CDKN2A-specific VCEP framework was retrieved; generic ACMG/AMP was therefore used.

Still missing

• Patient-level phenotype and clinical diagnostic details are unavailable.
• No evidence demonstrates that the phenotype is highly specific for a CDKN2A-related disorder.

Sources

Reasoning

BS2 cannot be assessed from the case bundle. No evidence was provided that the exact variant occurs in healthy adults, in homozygous state, or in unaffected individuals of an age and background appropriate for the relevant CDKN2A-associated disease.

Rule

Generic ACMG/AMP BS2: observed in a healthy adult in an appropriate number of individuals, or observed in a homozygous state in a disorder usually caused by heterozygous variants; no such observation is available.

Still missing

• No healthy-adult carrier or homozygote observations were provided.
• No information was provided about age, phenotype, ascertainment, penetrance, or follow-up of any putative unaffected carriers.

Reasoning

No CDKN2A-specific VCEP/CSPEC framework was found (case_summary.json cspec.found=false; framework_mode=generic_acmg). No functional assay data of any kind (showing normal/wild-type-like activity) for NM_001195132.1:c.44_46dup or p.(Trp15_Leu16insArg) is present anywhere in the consolidated evidence bundle, including compact_evidence, literature_packet, vcep_materials, or source_registry entries.

Still missing

• No functional assay evidence of any kind identified for this variant to assess a benign/wild-type functional readout

Reasoning

No unaffected relatives tested for the exact variant, non-segregation observation, phenotype information, or penetrance-adjusted family analysis is available. Absence of family data does not establish BS4.

Rule

Generic ACMG/AMP BS4 requires lack of segregation with disease in affected family members or other credible non-segregation evidence; missing family data is insufficient.

Evidence considered

• The consolidated case-evidence bundle contains no family-level non-segregation evidence or phenotype-adjusted testing results for the exact variant.

Still missing

• Genotypes of clinically unaffected relatives
• Age and phenotype assessment of tested relatives
• Number of informative non-segregating meioses
• Penetrance and phenocopy considerations

Reasoning

No applicable CDKN2A-specific VCEP framework was available; generic ACMG/AMP criteria were therefore considered. The case evidence bundle contains no affected-proband or family genotype data demonstrating the evaluated variant in trans with a pathogenic variant for a dominant disorder, or in cis with a pathogenic variant. The available evidence is insufficient to assess BP2.

Rule

Generic ACMG/AMP BP2: apply when the variant is observed in trans with a pathogenic variant in a dominant disorder, or in cis with a pathogenic variant in any disorder; the allelic relationship and pathogenic status of the other variant must be established.

Evidence considered

• The case summary and consolidated evidence bundle provide no phase, parental testing, family segregation, or compound-allelic observations for NM_001195132.1:c.44_46dup.
• The consolidated evidence bundle reports no CDKN2A-specific VCEP/CSPEC framework, so generic ACMG/AMP BP2 logic was used only to define the missing evidence requirements.

Still missing

• No affected-proband or family genotype observations establish a second pathogenic variant.
• No parental or molecular phasing establishes the evaluated variant in trans or in cis with another pathogenic variant.
• The relevant disease inheritance context is not specified sufficiently to evaluate the BP2 trans configuration.

Reasoning

No patient-level molecular workup or alternative pathogenic variant explaining the relevant phenotype was provided. The available variant and database records do not permit assessment of BP5.

Rule

Generic ACMG/AMP BP5 requires an alternate molecular finding that explains the patient's phenotype better than the assessed variant; this cannot be evaluated without patient-level data.

Evidence considered

• The case bundle contains no documented alternative molecular diagnosis or patient-level testing result that could explain a relevant phenotype independently of this variant.

Still missing

• Patient-level phenotype and molecular testing results are unavailable.
• No alternate pathogenic or likely pathogenic variant has been documented as a better explanation for the phenotype.