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.1 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).2 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).3 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).4 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).5 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.6
CDKN2A
Final classification
VUS
CDKN2A c.322G>A · p.Asp108Asn
CDKN2A
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.
gene-specific framework lacked a usable explicit final combination framework, so generic ACMG/AMP 2015 final-combination rules were applied as fallback; applied criteria: PS3 moderate, PM1 supporting, PM2 supporting, PP4 supporting; combination = 1 moderate + 3 supporting, which maps to VUS.
Classification rationale
PS3PM1PM2PP4
VUS
CDKN2A c.322G>A
PS3 + PM1 + PM2 + PP4
→
VUS
6
generic_acmg_combination_rules
Gene diagram
· NM_001195132.1 · variants mapped to exon structure
CDKN2A
NM_001195132.1
Fetching transcript structure from UCSC…
Applied criteria · 4 applied · 19 assessed
Applied · 4
Strength
Supporting
Moderate
Strong
Very strong
✓
PS3
moderate
Pathogenic
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.
Co-immunoprecipitation: Asp108Asn variant showed significantly reduced binding to CDK4 and CDK6 relative to wild-type p16INK4aProliferation assay: Asp108Asn produced an intermediate reduction in cell cycle inhibition (partially impaired)Structural context: Asp108 stabilizes the hairpin loop between adjacent ankyrin-type repeats
✓
PM1
supporting
Pathogenic
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.
Cancerhotspots.org: statistically significant hotspot residueAnkyrin repeat domain (ankyrin III-IV): critical for CDK4/CDK6 binding and cell cycle regulationStructural role of Asp108 in hairpin loop stabilization between ankyrin repeats
✓
PM2
supporting
Pathogenic
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 v2.1: absentgnomAD v4.1: 2/1606
✓
PP4
supporting
Pathogenic
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.
Identified in 1 familial melanoma case in GEM population-based studyHuot 2002 proband: melanoma in situ at age 23second melanoma at 28
Assessed · not applied
Pathogenic
PS1
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.
PS2
No de novo occurrence data for NM_001195132.1:c.322G>A was identified in the reviewed literature or ClinVar submissions.
PS4
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.
PM5
No confirmed pathogenic or likely pathogenic variant at the same residue (Asp108) with a different amino acid change was identified.
PM6
No de novo occurrence data for this variant was identified in any reviewed publication or ClinVar submission.
PP1
Limited segregation data is available.
PP2
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.
PP3
In silico predictions are conflicting.
PP5
ClinVar classification for this variant is 'Uncertain significance' with review status 'criteria provided, single submitter' (1 star).
Benign
BA1
The variant allele frequency in gnomAD v4.1 is 0.000124% (2/1,606,056 alleles), far below the 1% threshold for BA1.
BS1
The variant allele frequency in gnomAD v4.1 is 0.000124% (2/1,606,056 alleles), far below the 0.3% threshold for BS1.
BS2
No evidence that this variant has been observed in healthy adult individuals in the absence of disease.
BS3
Functional studies by Huot et al.
BS4
No evidence of lack of segregation was identified.
BP1
BP1 applies when a missense variant occurs in a gene where only truncating variants cause disease.
BP2
No evidence that this variant has been observed in trans with a known pathogenic dominant variant in CDKN2A or any other gene.
BP4
In silico evidence is conflicting.
BP5
No evidence that this variant was found in a case with an alternate molecular basis for disease.
BP6
ClinVar does not have a benign or likely benign classification for this variant from a 3-star expert panel.
N/A · 5
PVS1 · PM3 · PM4 · BP3 · BP7
Research & evidence
Population frequency
gnomAD v4.1
gnomAD v2.1
v4.1
This variant is present in gnomAD v4.1 (AF= 1.24529e-06; MAF= 0.00012%, 2/1606056 alleles, homozygotes = 0) and has highest observed frequency in the African/African American population (AF= 1.33298e-05; MAF= 0.00133%, 1/75020 alleles, homozygotes = 0).
v2.1
Absent from gnomAD v2.1.
🇨🇦 CA
Absent from gnomAD-Canada v1.0.
Allele frequency by ancestry
three datasets · side by side
gnomAD v4.1
0.00012%
· 2 / 1,606,056
0 hom
0 hom
African/African American 1 / 75,020 |
0.0013% |
European (non-Finnish) 1 / 1,179,392 |
8.5e-05% |
+ 8 not observed (Remaining individuals, Admixed American, European (Finnish), Amish, East Asian, Middle Eastern, South Asian, Ashkenazi Jewish)
gnomAD v2.1
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
gnomAD Canada 🇨🇦
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
ClinVar
This variant has been reported in ClinVar as Uncertain significance (2 clinical laboratories) and as Pathogenic (1 clinical laboratory) and as Likely pathogenic (1 clinical laboratory). (ClinVarID = 216275)
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). REVEL score = 0.676. BayesDel score = 0.0341573.
Functional
Likely Oncogenic
OncoKB identified variant-specific curated literature and context relevant to functional review; biological-effect context: Likely Loss-of-function; curated oncogenicity label: Likely Oncogenic.
COSMIC
Cancer hotspots
Somatic evidence
Hotspot
COSMIC
This variant lies in a statistically significant hotspot. This variant has previously been reported in somatic cancers (COSMIC; COSV58690777, n = 35 times).
Hotspots
This variant lies in a statistically significant hotspot.
Literature · how each cited paper was used
2papers cited
Each card is an audit: what was searched, what was found, whether it names the variant, which criteria it fed, and why. 6 further PMIDs triaged but not cited — see Sources & References.
Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts.
Searched
c.322G>Ap.Asp108AsnD108N322Asp108
Found
The c.322G>A (p.Asp108Asn) variant in CDKN2A was identified as one of two compound heterozygous missense mutations in the Q34 proband. Co-immunoprecipitation assays in patient-derived dermal fibroblasts and recombinant expression in normal HDFs demonstrated that the Asp108Asn variant has significantly reduced binding to both CDK4 and CDK6, with affinity markedly lower than wild-type p16INK4a. Proliferation assays showed an intermediate reduction in cell cycle inhibitory function, classifying Asp108Asn as a partially impaired mutant. Asp108 is structurally involved in stabilizing the hairpin loop between adjacent ankyrin-type repeats.
Variant
✓ Names this variant — characterised directly
Applied to
→PM1 supports · met
→PP4 supports · met
→PS3 supports · met
Why
Exact variant directly tested with clear partial loss-of-function. Referenced in PS3 (moderate), PM1 (domain hotspot), and PP4 (familial melanoma phenotype) assessments.
G to A at nucleotide 322, which changes Asp to Asn at residue 108 of p16INK4a
Location Results (pages 8136-8137): lines 208-213 describe the mutation; Figure 2C shows Cdk immunoprecipitation data; Figure 2D shows proliferation assay; Discussion (pages 8140-8141): lines 609-618 discuss Asp108Asn variant · Context Co-immunoprecipitation of endogenous p16INK4a with Cdk4/Cdk6 in Q34 patient dermal fibroblasts; retroviral expression of HA-tagged Asp108Asn in normal human diploid fibroblasts (Hs68 and 904); cell proliferation assay over 10 days · full text
CDKN2A germline mutations in individuals with cutaneous malignant melanoma.
Searched
c.322G>Ap.Asp108AsnD108N322Asp108
Found
The c.322G>A (p.Asp108Asn) variant was identified in 1 of 2,424 individuals with single primary melanoma (0.04%) and 0 of 1,189 individuals with multiple primary melanoma in the population-based GEM study. One first-degree relative with melanoma was reported. The variant was noted as previously identified in 1 familial melanoma family. Existing in vitro data at the time (Koh 1995, Ranade 1995, Reymond 1995) was classified as showing no change from wild type; however, subsequent studies (Huot 2002, PMID:12417717) demonstrated partial functional impairment.
Variant
✓ Names this variant — characterised directly
Applied to
→PP4 supports · met
Why
Variant identified in 1 familial melanoma case with low population frequency. Referenced in PS4 (not met due to insufficient case counts), PP1 (not met due to limited/incomplete segregation), and PP4 (supporting — familial melanoma context).
c.322 G>A Asp108Asn Arg163Gln No change 1 (0.04) 0 1/2 1 family
Location Table 1, rows for c.322G>A and c.306G>A+c.322G>A; Results section · Context Population-based case-control study (GEM): denaturing HPLC screening followed by Sanger sequencing of exons 1a, 2, and 3 of CDKN2A · full text
Sources & reference links
Triaged references · 6 PMIDs not cited in assessment
16896043 ↗
The prevalence of CDKN2A germ-line mutations and relative risk for cutaneous malignant melanoma: an international population-based study.
CLINVAR
17047042 ↗
High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL.
CLINVAR
25741868 ↗
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
CLINVAR
28492532 ↗
Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.
CLINVAR