title: Functional analysis of CDKN2A/p16INK4a 5'-UTR variants predisposing to melanoma.
abstract: 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'-UTR of CDKN2A (c.-21C > T; c.-25C > T&c.-180G > A; c.-56G > T; c.-67G > C) and examined their impact on the p16(INK4a) 5'-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'-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'-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.
authors.affiliation: Unit of Molecular Mutagenesis and DNA Repair, National Institute for Cancer Research IST, 16132 Genoa, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bisio
authors.forename: Sabina
authors.initials: S
authors.lastname: Nasti
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Jordan
authors.forename: Sara
authors.initials: S
authors.lastname: Gargiulo
authors.forename: Lorenza
authors.initials: L
authors.lastname: Pastorino
authors.forename: Alessandro
authors.initials: A
authors.lastname: Provenzani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Quattrone
authors.forename: Paola
authors.initials: P
authors.lastname: Queirolo
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bianchi-Scarrà
authors.forename: Paola
authors.initials: P
authors.lastname: Ghiorzo
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
mesh_terms: D020121:5' Untranslated Regions; D045744:Cell Line, Tumor; D015331:Cohort Studies; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation; D006801:Humans; D007558:Italy; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide
chemical_list: D020121:5' Untranslated Regions; D019941:Cyclin-Dependent Kinase Inhibitor p16