Functional evaluation of tumour-specific variants of p16(INK4a)/CDKN2A: correlation with protein structure information

Inherited mutations in the CDKN2A/INK4a/MTS1 tumour suppressor gene on chro mosome 9p21 are associated with familial predisposition to melanoma and oth er tumour types. Nonsense and missense mutations are also found in a variet y of sporadic cancers, and over 140 sequence variants have already been rec orded in the literature. In assessing the relevance of these variants and f or counselling members of affected families, it is important to distinguish inactivating mutations from harmless polymorphisms. Existing functional as says have frequently reached conflicting conclusions and no single test app ears adequate. Here we evaluate a number of alternatives including a novel assay based on retroviral delivery of p16(INK4a) cDNAs into human diploid f ibroblasts. Among the 17 sequence variants analysed, three distinct categor ies can be distinguished: those that abrogate the binding of p161(INK4a) to CDK4 and CDK6, those that alter the properties of the protein without prev enting it from interacting with CDKs, and those that have no discernible ef fect on protein function. These distinctions can be rationalized by conside ring the impact of the amino acid changes on the three-dimensional structur e of the protein.