Functional impairment of p16^INK4A due to CDKN2A p.Gly23Asp missense mutation

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• 作者：Maria Chiara Scaini ; Elisabetta Rossi ; Paula Lobao Antunes de Siqueira Torres ; Daniela Zullato ; Monia Callegaro ; Cinzia Casella ; Monica Quaggio ; Simona Agata ; S ; ro Malacrida ; Vanna Chiarion-Sileni ; An
• 关键词：Familial melanoma ; CDKN2A locus ; Functional assays ; p16^INK4A unclassified variant
• 刊名：Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
• 出版年：2009
• 出版时间：1 December 2009
• 年：2009
• 卷：671
• 期：1-2
• 页码：26-32
• 全文大小：672 K

文摘

The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16^INK4A and p14^ARF, involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16^INK4A properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16^wt, p16^23Asp, or the p16^32Pro pathogenic variant. We found that p16^23Asp was less efficient than p16^wt in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p16^23Asp in familial melanoma.