title: Germline splicing mutations of CDKN2A predispose to melanoma.
abstract: Coding mutations of the CDKN2A gene on chromosome 9p21 cosegregate with 25-60% of familial melanoma cases, but there remains a number of 9p21-linked kindreds that lack germline coding mutations of CDKN2A. We sequenced CDKN2A exons 1alpha, 2, 3, and the adjacent intronic regions in 167 melanoma-prone families (at least two affected first-degree relatives), and detected four splice site variations, three of which cosegregate with the disease. RT-PCR experiments verified that these three variants, including an AGgt to ATgt mutation that demonstrates a founder effect, do affect splicing. While an exon 1alpha splice donor site mutation incompletely abolishes splicing, the correctly spliced mRNA yields a protein (Q50P) that cannot effectively interact with CDK4. We also performed RT-PCR on mRNA from 16 melanoma-prone kindreds to search for cryptic splice sites deep within introns, but identified no splice variants. Meanwhile, we screened 139 affected families using allele-specific PCR for the recently discovered IVS2-105A>G mutation, but found only one family that possesses this alteration. We conclude that splice site mutations do predispose to disease in a subset of melanoma-prone kindreds. Characterization of additional splice site variants and other noncoding alterations of CDKN2A should allow us to detect a wider range of mutations in at-risk patients.
mesh_terms: D017398:Alternative Splicing; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010375:Pedigree; D022821:RNA Splice Sites; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D022821:RNA Splice Sites