Abstract 4199: CDKN2A/p16INK4a 5′UTR variants in melanoma predisposition: Lost in translation, somewhere

Abstract

Abstract The CDKN2A gene is the most common high penetrance susceptibility gene identified to date in melanoma families. While functional tests for determining the pathogenicity of missense germline mutations in the CDKN2A coding region have been developed, rare polymorphisms or sequence variants at the CDKN2A/ p16INK4a 5′UTR, encountered during routine screening, are usually defined as variants with unknown significance after determining their frequency in control population and the cosegregation analysis in the family, when possible. We recently developed reporter assays to study a panel of p16INK4a 5′UTR variants identified as heterozygous changes in patients from a hospital-based series of melanoma cases (c.-21C>T; c.-25C>T&c.-180G>A; c.-56G>T; c.-67G>C). Monocistronic as well as bicistronic luciferase-based reporter vectors were developed and used to test wild type and variant p16INK4a 5′UTR activity upon transient transfection in melanoma-derived cells (WM266-4, G361 and SK-Mel-5) and in the breast cancer-derived MCF7 cells. Results revealed that the c.-21C>T variant had a strong negative impact on the reporter activity, similar to that of the known melanoma-predisposing mutation c.-34G>T, included as a control. The variants at –56 and at –25&-180 exhibited a milder impact, while results with c.-67G>C were dependent on the type of reporter vector. Quantification of the luciferase mRNA conducted in parallel with the luciferase assay indicated that the impact of the variants was mainly post-transcriptional. We also applied a polysomal profiling technique to measure allelic imbalance starting from heterozygous patient-derived cell lines and found that the c.-21C>T variant but also c.-56T>G and c.-67G>C exhibited lower association with the polysomes suggestive of reduced mRNA translation efficiency. A panel of eleven additional germline variants in the 5′UTR of p16INK4a is being investigated. In particular we are focusing on the functional interactions between wild type and variant p16INK4a 5′- and 3′-UTR sequences and on the impact the variants can have on the targeting of the p16INK4a mRNA by microRNAs (including mir-346, -636, -639, -935) or RNA binding proteins (including YB-1). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4199. doi:1538-7445.AM2012-4199