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CDKN 2 A Unclassified Variants in Familial Malignant Melanoma: Combining Functional and Computational Approaches for Their Assessment
Author(s) -
Scaini Maria Chiara,
Minervini Giovanni,
Elefanti Lisa,
Ghiorzo Paola,
Pastorino Lorenza,
Tognazzo Silvia,
Agata Simona,
Quaggio Monica,
Zullato Daniela,
BianchiScarrà Giovanna,
Montagna Marco,
D'Andrea Emma,
Menin Chiara,
Tosatto Silvio C.E.
Publication year - 2014
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22550
Subject(s) - in silico , biology , cdkn2a , missense mutation , computational biology , genetics , p14arf , germline , exon , mutation , melanoma , bioinformatics , gene , carcinogenesis , tumor suppressor gene
CDKN 2 A codes for two oncosuppressors by alternative splicing of two first exons: p16 INK 4a and p14 ARF . Germline mutations are found in about 40% of melanoma‐prone families, and most of them are missense mutations mainly affecting p16 INK 4a. A growing number of p16 INK 4a 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. Predicting the effect of these VUS by either a “standard” in silico approach, or functional tests alone, is rather difficult. Here, we report a protocol for the assessment of any p16 INK 4a VUS , which combines experimental and computational tools in an integrated approach. We analyzed p16 INK 4a VUS from melanoma patients as well as variants derived through permutation of conserved p16 INK 4a amino acids. Variants were expressed in a p16 INK 4a‐null cell line ( U 2‐ OS ) and tested for their ability to block proliferation. In parallel, these VUS underwent in silico prediction analysis and molecular dynamics simulations. 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.