Predicting functional significance of cancer-associated p16^INK4a mutations in CDKN2A

Inherited mutations affecting the INK4a/ARF locus (CDKN2A) are associated with melanoma susceptibility in 40% of multiple case melanoma families. Over 60 different germline INK4a/ARF mutations have been detected in more than 190 families worldwide. The majority of these alterations are missense mutations affecting p16^INK4a, and only 25% of these have been functionally assessed. There is therefore a need for an accurate and rapid assay to determine the functional significance of p16^INK4a mutations. We reviewed the performance of several in vivo functional assays that measure critical aspects of p16^INK4a function, including subcellular location, CDK binding and cell cycle inhibition. In this report the function of 28 p16^INK4a variants, many associated with melanoma susceptibility were compared. We show that assessment of CDK4 binding and subcellular localization can accurately and rapidly determine the functional significance of melanoma-ssociated p16 ^INK4a mutations. p16^INK4a-CDK6 binding affinity was unhelpful, as no disease-associated mutation showed reduced CDK6 affinity while maintaining the ability to bind CDK4. Likewise, in silico analyses did not contribute substantially, with only 12 of 25 melanoma-associated missense variants consistently predicted as deleterious. The ability to determine variant functional activity accurately would identify disease-associated mutations and facilitate effective genetic counselling of individuals at high risk of melanoma.