All rights reservedĪ germline polymorphism of the MITF gene encoding a SUMOylation deficient E318K mutated protein has recently been described as a medium-penetrance melanoma gene. Evaluation of in silico and functional data disclosed a high agreement for 15/16 missense mutations, suggesting that this approach could represent a pilot study for the definition of a protocol applicable to VUS in general, involved in other diseases, as well.This article is protected by copyright. In parallel, these VUS underwent in silico prediction analysis and molecular dynamics simulations. Variants were expressed in a p16INK4a-null cell line (U2-OS) and tested for their ability to block proliferation. We analyzed p16INK4a VUS from melanoma patients as well as variants derived through permutation of conserved p16INK4a amino acids. Here we report a protocol for the assessment of any p16INK4a VUS, which combines experimental and computational tools in an integrated approach. Predicting the effect of these VUS by either a “standard” in silico approach, or functional tests alone, is rather difficult. A growing number of p16INK4a Variants of Uncertain Significance (VUS) are being identified but, unless their pathogenic role can be demonstrated, they cannot be used for identification of carriers at risk. Germline mutations are found in about 40% of melanoma-prone families, and most of them are missense mutations mainly affecting p16INK4a. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.ĬDKN2A codes for two oncosuppressors by alternative splicing of two first exons: p16INK4a and p14ARF. We observed no interaction between CDKN2A status and the presence of MC1R variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C) the novel 5'UTR -21C > T variant was not detected in controls. One novel mutation, T77A, was identified. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. The G101W and E27X founder mutations were the most common.
Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases.
The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27 95% CI 2.43-7.53). We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95).
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