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

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.

Dried blood spot sampling for detection of monoclonal immunoglobulin gene rearrangement.

Molecular methods are important tools for diagnosis and monitoring of many lymphoproliferative disorders. The reliability of lymphoma diagnoses is strikingly different between developed and developing countries, partly due to lack of access to these advanced molecular analyses. To overcome these problems, we propose a new application of dried blood spots (DBS) for detecting clonal B-cell populations in peripheral blood (PB). We ensured that the DBS contained sufficient lymphocytes to perform a PCR-based clonality assay without producing false positives. Using the Namalwa B-cell line, we established that the assay is sensitive enough to detect 200 clonal cells in the analyzed sample. Very similar clonal results were obtained between DNA from DBS and fresh whole blood from patients with B-cell chronic lymphocytic leukemia. B-cell clonality can also be detected in DBS from African children with EBV-associated diseases. This is the first study demonstrating that clonality testing can be performed on DBS samples, thus improving the diagnostic and monitoring options for lymphoproliferative diseases in resource-limited settings.

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

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.

Association between MDM2-SNP309 and age at colorectal cancer diagnosis according to p53 mutation status.

A single-nucleotide polymorphism (SNP) in the promoter of the MDM2 gene, SNP309 (a T-->G change), was recently implicated in the early onset of cancer in individuals with Li-Fraumeni syndrome and of sporadic soft-tissue sarcoma. SNP309 induces an increase in the level of Mdm2 protein, which causes attenuation of the p53 pathway. To investigate the effect of this polymorphism in colorectal cancer pathogenesis, we genotyped 153 colorectal cancer patients who were randomly selected from among 330 consecutive patients stratified according to p53 mutation status and age at diagnosis, for alleles of MDM2-SNP309. Among the 77 patients with p53 wild-type tumors, the median age at colorectal cancer diagnosis was 71.5 years for patients with the T/T genotype and 61.0 years for patients with SNP309 (T/G or G/G genotype) (estimated difference between medians [Hodges-Lehmann method] = 8.0 years, 95% confidence interval = 1.0 to 16.0 years; P = .03 [two-sided Wilcoxon rank sum test]). Our data indicate that MDM2-SNP309 is a modifier of the age at colorectal cancer onset for patients whose tumors have a wild-type p53 gene.