Functional analysis of CDKN2A/p16INK4a 5′-UTR variants predisposing to melanoma

Germline CDKN2A mutations are observed in 20 -50% of melanoma-prone families.We identified melanoma patients that were heterozygous for non-coding germline variants in the 5 0 -UTR of) and examined their impact on the p16 INK4a 5 0 -UTR activity using two luciferase-based reporter vectors that differ in basal transcription level and that were transfected into the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells.The wild-type 5 0 -UTR sequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G > T), was included as controls.Results revealed that the variants at -21 and -34 severely reduced the reporter activity.The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C were dependent on the plasmid type.Quantification of the luciferase mRNA indicated that the effects of the variants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmed that c.-21C > T and c.-34G > T had a severe negative impact in both cell lines.We also applied a polysomal profiling technique to samples heterozygous for the 5 0 -UTR variants, including patient-derived lymphoblasts.Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > C variants also reduced mRNA translation efficiency.Overall, our results suggest that the c.-21C > T sequence variant is a melanoma-predisposing mutation.The c.-25C > T&c.-180G > A and particularly the c.-56G > T variants showed a range of intermediate functional defects in the different assays, and were not observed in the control population.We propose that these variants should be considered as potential mutations.