Pooled analysis of CDKN2A variants at two residues with previously reported pathogenic and benign variants.

PANC-1 cell stably expressing one of 20 CDKN2A variants, 19 missense variants and 1 synonymous variant, at reside p.V126 or p.R144 were cultured. Variant representation, as the percent of reads supporting the variant sequence, before and after a period in vitro cell growth determined by next generation sequencing for the two residues, p.V126 (A) or p.R144 (B). CDKN2A variant p.V126D (*) was previously reported as pathogenic and increased representation during in vitro growth. CDKN2A variant p.R144C (**) was previously reported as benign variant and maintained representation during in vitro growth.

Functional characterization of all possible CDKN2A missense variants.

(A) Functional classifications for 3,120 CDKN2A variants, including 2,964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious, indeterminate function, or neutral based on P value using gamma GLM. 525 (17.7%) variants were classified as functionally deleterious. (B) Log2 P values for 32 benchmark pathogenic variants, 6 benign variants, 31 VUSs previously reported to have functionally deleterious effects, and 18 VUSs previously reported to have functionally neutral effects. (C) Heat map with P values for all 3,120 CDKN2A variants assayed.

Comparison of functional classifications and in silico variant effect predictions for all possible CDKN2A missense variants.

Variant effect predictions for CDKN2A missense variants using CADD, PolyPhen-2, SIFT, VEST, AlphaMissense, ESM1b, and PrimateAI-3D. Predicted deleterious, damaging, or pathogenic effects (black box) and predicted neutral, tolerated, benign, or ambiguous effects (white box) presented as percent of missense variants with an available prediction. Number of missense variants with an available prediction for each in silico model given in parentheses. Accuracy shown as a red line. CADD; Combined Annotation Dependent Depletion, PolyPhen-2; Polymorphism Phenotyping v2, SIFT; Sorting Intolerant From Tolerant, VEST; Variant Effect Scoring Tool score.

Functional classification of missense somatic mutations in CDKN2A.

(A) Somatic missense variants in CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box). (B) Distribution of functionally deleterious missense somatic mutations CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT by ankyrin (ANK) repeat. (C) Percent of missense somatic mutations in CDKN2A that were classified as functionally deleterious (black box), indeterminate function (gray box), or functionally neutral (white box) group by tumor type. Missense somatic mutations reported in COSMIC, TCGA, JHU, and MSK-IMPACT were combined. The number of missense somatic mutations for each tumor type given in parentheses. COSMIC; the Catalogue Of Somatic Mutations In Cancer, TCGA; The Cancer Genome Atlas, JHU; The Johns Hopkins University School of Medicine, MSK-IMPACT; Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets.

CDKN2A synonymous and missense variants reported in gnomAD and Clinvar.

(A) Synonymous and missense variants in CDKN2A reported in gnomAD. (B) 287 CDKN2A missense variants reported in gnomAD, by ACMG guideline classification. (C) 264 missense variants in CDKN2A reported in gnomAD, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box). (D) 395 missense variants in CDKN2A reported in Clinvar, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box).