NM_001195132.1:c.322G>A

CDKN2A · NP_001182061.1:p.(Asp108Asn) · NM_001195132.1

GRCh37: chr9:21971036 C>T · GRCh38: chr9:21971037 C>T

Gene: CDKN2A Transcript: NM_001195132.1

Final call

VUS

PS3 moderate PM1 supporting PM2 supporting PP4 supporting

Variant details

Gene

CDKN2A

Transcript

NM_001195132.1

Protein

NP_001182061.1:p.(Asp108Asn)

gnomAD AF

1.2452865902558815e-06 (v4.1)

ClinVar

Uncertain significance

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_001195132.1:c.322G>A (p.Asp108Asn) is a missense variant in exon 2 of CDKN2A, which encodes the p16INK4a tumor suppressor protein involved in G1 cell cycle regulation through CDK4/CDK6 inhibition.

Direct functional testing of the Asp108Asn variant by Huot et al. (2002) in patient-derived fibroblasts and recombinant expression systems demonstrated significantly reduced binding to both CDK4 and CDK6 and an intermediate reduction in cell cycle inhibitory function, classifying the variant as a partially impaired mutant (PS3_moderate).

The variant is located at a statistically significant hotspot residue within the ankyrin repeat domain of p16INK4a, a region critical for CDK4/CDK6 binding and tumor suppressor function (PM1_supporting).

The variant is absent from gnomAD v2.1 and gnomAD-Canada, and is extremely rare in gnomAD v4.1 (2/1,606,056 alleles, AF = 0.0001%), well below the 0.1% PM2 threshold (PM2_supporting).

The variant has been identified in a familial melanoma context: reported in the GEM population-based study in an individual with melanoma noted under familial melanoma, and in the Huot et al. proband who presented with melanoma in situ at age 23 with a second primary melanoma and a strong family history of cancer including melanoma in both parental lines (PP4_supporting).

Applying the ACMG/AMP 2015 generic combination rules: 1 moderate criterion (PS3) and 3 supporting criteria (PM1, PM2, PP4) are met. No benign criteria are met. The combination of 1 moderate + ≥2 supporting criteria satisfies the threshold for Likely Pathogenic classification.

Final determination: Generic ACMG/AMP 2015 fallback rules do not meet benign, likely benign, likely pathogenic, or pathogenic combination thresholds, so the variant is classified as Variant of Uncertain Significance.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	NM_001195132.1:c.322G>A is a missense variant (p.Asp108Asn) and does not fall into the generic PVS1 null-variant categories of nonsense, frameshift, or canonical ±1,2 splice consensus variants. The pvs1_variant_assessment explicitly confirms the variant bucket is 'other' and generic PVS1 framework does not apply.	pvs1_variant_assessment pvs1_gene_context pvs1_generic_framework
PS1	Not met	No alternative nucleotide change at codon 108 (Asp) resulting in the same Asp108Asn amino acid substitution has been identified as a previously established pathogenic variant. Only c.322G>A has been reported at this position.	clinvar
PS2	Not met	No de novo occurrence data for NM_001195132.1:c.322G>A was identified in the reviewed literature or ClinVar submissions. No parentage testing confirming de novo status was available.
PS3	Met	The exact variant (c.322G>A, p.Asp108Asn) was directly tested in functional assays by Huot et al. (2002, PMID:12417717). Co-immunoprecipitation experiments in patient-derived Q34 fibroblasts and recombinant expression in normal human diploid fibroblasts demonstrated significantly reduced binding of the Asp108Asn variant to both CDK4 and CDK6 (estimated at <5% of wild-type binding capacity in the compound heterozygous background, and markedly reduced relative to wild-type when expressed individually). Proliferation assays showed an intermediate reduction in cell cycle inhibitory function. The variant was classified as a 'partially impaired mutant.' Asp108 is structurally involved in stabilizing the hairpin loop between adjacent ankyrin-type repeats. A single study directly testing the exact variant with clear but partial functional impairment supports moderate-level PS3.	PMID:12417717
PS4	Not met	The variant has been observed in only 1 individual with single primary melanoma (SPM) out of 2,424 SPM cases and 0 of 1,189 multiple primary melanoma (MPM) cases in the population-based GEM study (Orlow et al. 2007). These numbers are insufficient to meet PS4 thresholds for statistically significant enrichment in affected cases versus controls.	PMID:17218939
PS5	Not met	No reputable source has recently reported this variant as definitively pathogenic with unavailable evidence. ClinVar classification is Uncertain significance (1-star review status), and the two submissions classifying as Pathogenic/Likely pathogenic are single-submitter clinical laboratories whose evidence is available for independent review.	clinvar
PM1	Met	The variant is located at a statistically significant hotspot residue (cancerhotspots.org) within the ankyrin repeat domain of p16INK4a, a functionally critical region for CDK4/CDK6 binding. The residue Asp108 is involved in stabilizing the hairpin loop between adjacent ankyrin-type repeats, and mutations in this domain are a well-established mechanism of CDKN2A pathogenicity.	PMID:12417717
PM2	Met	The variant is absent from gnomAD v2.1 and gnomAD-Canada, and is extremely rare in gnomAD v4.1 (2/1,606,056 alleles; AF = 0.000124%; highest subpopulation AF = 0.00133% in African/African American). The total allele frequency is well below the 0.1% threshold for PM2 application.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	Not met	No confirmed pathogenic or likely pathogenic variant at the same residue (Asp108) with a different amino acid change was identified. The pm5_candidates module was unable to harvest same-residue comparator variants safely.	pm5_candidates
PM6	Not met	No de novo occurrence data for this variant was identified in any reviewed publication or ClinVar submission. No confirmed de novo with maternity/paternity testing was available.
PP1	Not met	Limited segregation data is available. The variant was noted to have been observed in a familial melanoma context (Orlow et al. 2007: 1 family), and one ClinVar submitter notes cosegregation in related individuals. However, another ClinVar submission notes that in one family two of four affected members did not carry the variant, indicating incomplete cosegregation. Insufficient robust segregation data to meet PP1.	PMID:17218939 clinvar
PP2	Not met	No gene-level missense constraint data (e.g., Z-score, missense depletion metrics) was available for CDKN2A to establish a low rate of benign missense variation. While CDKN2A is a known tumor suppressor with many pathogenic missense variants reported, specific constraint metrics needed for PP2 are absent.
PP3	Not met	In silico predictions are conflicting. REVEL score of 0.676 is above the 0.5 threshold supporting a deleterious prediction. However, BayesDel score of 0.034 is strongly benign-leaning. SpliceAI delta score is 0.00 indicating no splice impact. With only one tool supporting pathogenicity and another strongly contradicting it, the 'multiple lines of computational evidence' requirement for PP3 is not satisfied.	revel bayesdel spliceai
PP4	Met	The variant has been reported in a familial melanoma context. CDKN2A is an established melanoma susceptibility gene, and the patient phenotype (melanoma) is highly specific for CDKN2A-related disease. Orlow et al. (2007) identified c.322G>A (p.Asp108Asn) in an individual with melanoma noted under 'familial melanoma.' The variant was also identified in the Huot et al. (2002) proband who presented with melanoma in situ at age 23 and a second melanoma 5 years later, with a strong family history of cancer including melanoma in both parental lines.	PMID:17218939 PMID:12417717
PP5	Not met	ClinVar classification for this variant is 'Uncertain significance' with review status 'criteria provided, single submitter' (1 star). The 3-star expert panel review threshold required for PP5 application is not met.	clinvar
BA1	Not met	The variant allele frequency in gnomAD v4.1 is 0.000124% (2/1,606,056 alleles), far below the 1% threshold for BA1. The variant is absent from gnomAD v2.1 and gnomAD-Canada.	gnomad_v2 gnomad_v4 gnomad_canada
BS1	Not met	The variant allele frequency in gnomAD v4.1 is 0.000124% (2/1,606,056 alleles), far below the 0.3% threshold for BS1.	gnomad_v2 gnomad_v4 gnomad_canada
BS2	Not met	No evidence that this variant has been observed in healthy adult individuals in the absence of disease. The 2 alleles in gnomAD v4.1 are from an exome dataset without confirmatory phenotype data sufficient to establish healthy adult status.
BS3	Not met	Functional studies by Huot et al. (2002, PMID:12417717) demonstrate a deleterious effect: significantly reduced CDK4/CDK6 binding and intermediate reduction in cell proliferation for the Asp108Asn variant. This is contradictory to BS3, which requires functional studies showing no damaging effect.	PMID:12417717
BS4	Not met	No evidence of lack of segregation was identified. While one ClinVar submitter noted that in one family two of four affected members did not carry the variant, this observation alone is insufficient to meet BS4, which requires systematic non-segregation data.
BP1	Not met	BP1 applies when a missense variant occurs in a gene where only truncating variants cause disease. CDKN2A has numerous well-established pathogenic missense variants documented in the literature and ClinVar; missense changes are a recognized mechanism of pathogenicity in this gene.	clinvar PMID:12417717
BP2	Not met	No evidence that this variant has been observed in trans with a known pathogenic dominant variant in CDKN2A or any other gene.
BP4	Not met	In silico evidence is conflicting. While BayesDel (0.034) leans benign, REVEL (0.676) predicts a deleterious effect. Multiple lines of computational evidence do not consistently support a benign interpretation.	revel bayesdel spliceai
BP5	Not met	No evidence that this variant was found in a case with an alternate molecular basis for disease. No alternative genetic diagnosis explaining the phenotype has been reported in carriers of this variant.
BP6	Not met	ClinVar does not have a benign or likely benign classification for this variant from a 3-star expert panel. The only ClinVar entries are Uncertain significance (2 laboratories), Pathogenic (1 laboratory), and Likely pathogenic (1 laboratory), all at 1-star review level.	clinvar
BP7	N/A	NM_001195132.1:c.322G>A is a missense variant (p.Asp108Asn), not a synonymous or intronic variant. BP7 applies only to synonymous variants with no predicted splice impact.
BP3	N/A	Variant is a substitution, not an in-frame indel in a non-repeat region.
PM3	N/A	Trivially not applicable: the variant is not observed in trans with a pathogenic variant for a recessive disorder (CDKN2A is associated with dominant melanoma predisposition).
PM4	N/A	The variant is a substitution, not a protein-length-altering change (in-frame deletion/insertion or stop-loss).

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