CDKN2A unclassified variants in familial malignant melanoma: combining functional and computational approaches for their assessment

Hum Mutat. 2014 Jul;35(7):828-40. doi: 10.1002/humu.22550. Epub 2014 May 21.


CDKN2A codes for two oncosuppressors by alternative splicing of two first exons: p16INK4a and p14ARF. Germline mutations are found in about 40% of melanoma-prone families, and most of them are missense mutations mainly affecting p16INK4a. A growing number of p16INK4a 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 p16INK4a VUS, which combines experimental and computational tools in an integrated approach. We analyzed p16INK4a VUS from melanoma patients as well as variants derived through permutation of conserved p16INK4a amino acids. Variants were expressed in a p16INK4a-null cell line (U2-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.

Keywords: CDKN2A; VUS; familial melanoma; p16INK4a; variants of uncertain significance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Substitution
  • Cell Line, Tumor
  • Cell Proliferation
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism
  • Genetic Variation*
  • Humans
  • Melanoma / diagnosis
  • Melanoma / genetics*
  • Models, Molecular
  • Mutation, Missense
  • Protein Conformation
  • Skin Neoplasms


  • Cyclin-Dependent Kinase Inhibitor p16

Supplementary concepts

  • Melanoma, Cutaneous Malignant