Germline splicing mutations of CDKN2A predispose to melanoma

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 1 α , 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 A G gt to A T gt mutation that demonstrates a founder effect, do affect splicing. While an exon 1 α 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.