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 1a, 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 1a 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 allelespecific PCR for the recently discovered IVS2À105A4G 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 atrisk patients.

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