Denaturing high-performance liquid chromatography for the detection of mutations and polymorphisms in UBE3A

Angelman syndrome (AS) is caused by maternal deficiency of UBE3A, the gene encoding E6-AP ubiquitin-protein ligase. Our objectives were to develop conditions for denaturing high-performance liquid chromatography (dHPLC) analysis of UBE3A and to compare the sensitivity to direct genomic sequencing. Genomic DNA was obtained from 17 Angelman patients with known mutations and from 120 normal controls. DNA was amplified for the 10 coding exons and 6 upstream noncoding exons of UBE3A. Using dHPLC, the mutations previously identified in 17 Angelman patients were all easily detected using a single dHPLC condition for most exon-containing fragments. An analysis of all 16 exons in 120 normal controls identified 15 other DNA alterations of varying frequency, all of which are assumed to be benign. We conclude that dHPLC is a reliable and convenient method for detecting mutations in UBE3A causing Angelman syndrome. No disease-causing mutations were found in the noncoding exons.