Functional, structural, and genetic evaluation of 20CDKN2Agerm line mutations identified in melanoma-prone families or patients

Abstract

Germline mutations of the CDKN2A gene are found in melanoma-prone families and individuals with multiple sporadic melanomas. The encoded protein, p16INK4A , comprises four ankyrin-type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16INK4a with its physiological targets, the cyclin-dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16 INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction.

p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

“…For example, missense mutations or deletions of the INK4/ARF locus represent the most important alterations in familial melanoma (41% of cases) (Orlow et al, 2007;Kannengiesser et al, 2009). Furthermore, in families with BRCA2, MMR genes and INK4/ARF locus mutations, there is an increased risk of cutaneous melanoma, acute lymphatic leukemia and blast leukemia in childhood (Magnusson et al, 2008;Daniotti et al, 2009).…”

Characteristics of the coexistence of melanoma and renal cell carcinoma

“…We screened for point mutations in CDKN2A (exons 1a, 2, and 3), adenosine diphosphate-ribosylation factor (ARF) (exon 1b), and CDK4 (exon 2) using denaturing high-performance liquid chromatography or direct sequencing as described previously. 28,29 Genomic deletion screening of the CDKN2A locus also was carried out as described previously. 30 …”

Familial Melanoma in Italy: A Review

“…A recent study on a series of CDKN2A missense variants, however, showed potential limits of these approaches: widely conflicting results, in fact, were obtained, with several specific variants, that were paradoxically predicted to be benign or pathogenic, depending on the software used (Kannengiesser et al, 2009). Undoubtedly, in silico approaches provide an excellent support for UVs classification, but their accuracy is apparently limited by the available evolutionary, mutational or structural databases and, in some cases, by intrinsic limitations of each individual method.…”

“…A powerful assay must be designed according to the functional properties of the encoded protein and hence each single cancer-predisposing gene requires the development of a set of specific tests. A variety of p16 missense variants were investigated by functional analysis (Kannengiesser et al, 2009;McKenzie et al, 2010;Ruas et al, 1999), since two main p16 functions can be easily measured in vitro: the CDK4/6 binding capacity and the p16 ability to arrest cellcycle. In particular, the advantage of using cell growth inhibition assays is that they evaluate a phenotype directly involved in tumorigenesis.…”

“…Interestingly, although the G101W showed some residual binding to CDK4/6 (McKenzie et al, 2010;Parry & Gordon, 1996), it was found to be clearly impaired in the ability to block cell cycle progression (Kannengiesser et al, 2009). These singular results obtained for the G101W emblematically represent the case of a mutation having discordant functional behaviours, on the basis of the particular assay used, as discussed before.…”