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

PANC-1 cell stably expressed with a total of 20 CDKN2A variants, 19 missense variants and 1 synonymous variant, at resides were cultured. Variant representation, as percent of reads supporting the variant sequence, before and after a period in vitro cell growth determined by next generation sequencing for 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 of 3,120 CDKN2A variants, including all possible 2,964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious or neutral based on P value. 1,182 (39.9%) of variants were classified as functionally deleterious. (B) P values for 32 benchmark pathogenic variants and 162 benign variants. All pathogenic variants were classified as functionally deleterious, and all benign variants were classified as functionally neutral, based on P values. (C) High-throughput functional assay P values for 32 CDKN2A VUSs previously reported to have functionally deleterious effects and 18 VUSs previously reported to have functionally neutral effects. (D) 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 available prediction. Number of missense variants with available prediction for each in silico model given in parentheses. 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) Missense somatic missense variants in CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT, by functional classification (deleterious – black 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.

Development and validation of high-throughput CDKN2A functional assay.

(A) Cell proliferation of PANC-1 cells stably expressing empty expression vector, one of three synonymous variants (p.L32L, p.G101G, p.V126V), or one of three pathogenic variants (p.L32P, p.G101W, p.V126D) over 14 days in culture. Cell proliferation values are given as mean of three repeats ± standard deviation normalized to PANC-1 cells that stably express empty vector. Statistically significant inhibition of cell proliferation inhibition in PANC-1 cells that stably express synonymous variants *; Student’s t test, P value < 0.001). (B) PANC-1 cells stably express codon optimized CDKN2A transduced with a CellTag lentiviral library of 20 nonfunctional barcodes were cultured and representation (percent of reads supporting each barcode) before and after a period of in vitro cell proliferation was measured determined by next generations sequencing. Percent values are given as the mean of three repeats ± standard deviation.

Functional characterization of all possible CDKN2A missense variants.

(A) Schematic representation of CDKN2A ankyrin repeats. (B) Percent of functionally deleterious (black box) and functionally neutral variants (white box) within ankyrin repeats and non-ankyrin repeat regions of CDKN2A. Ank; Ankyrin repeat. (C) Box plot showing distribution of percent functionally deleterious missense variants per residue.

Functional classification of missense somatic mutations in CDKN2A.

(A - D) Percent of missense somatic mutations in CDKN2A reported in COSMIC (A), TCGA (B), JHU (C), or MSK-IMPACT (D) that were classified as functionally deleterious (black box) or functionally neutral (white box) group by tumor type. Cancer types with 10 or more missense somatic mutations in COSMIC are presented. 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.

Aggregate functional classifications for missense somatic mutations in CDKN2A.

Percent of missense somatic mutations in CDKN2A that were classified as functionally deleterious (black box) or functionally neutral (white box) group by tumor type. Missense somatic mutations reported in COSMIC, TCGA, JHU, and MSK-IMPACT were combined. Cancer types with 10 or more missense somatic mutations in COSMIC are presented. 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.

Codon optimized CDKN2A sequence.

Assessment of in silico variant effect prediction models.