Denaturing High Performance Liquid Chromatography (DHPLC) used in the detection of mutations and polymorphisms in the UBE3A gene. D. Bercovich, E. Lev-Lehman, A.L Beaudet. Human and Molecular Genetics, Baylor college of medicine, Houston, TX.
   One of the most sensitive method for detecting point mutation causing human disease is PCR followed by direct sequencing. However, sequencing of those genes is technically demanding, costly, and time consuming; in addition, heterozygous mutations may go undetected. There are reports that Denaturing High Performance Liquid Chromatography (DHPLC) is as sensitive as direct sequencing for detection of point mutations, and we have evaluated the ability of DHPLC to detect known mutation in the UBE3A locus, which encodes E6-AP ubiquitin-protein ligase. Large deletions, imprinting defects or loss-of-function mutation in the maternal allele for UBE3A cause Angelman syndrome (AS), which is characterized by mental retardation, absence of speech, seizures and motor dysfunction. We studied 17 mutations in the UBE3A gene that was identified by Fang et al (Hum. Mol. Genet. Vol 8, 129-135, 1999), two new mutations and two polymorphic sites. The UBE3A gene is a very AT rich locus so we compared the use of primers designed for sequencing analysis and not for the DHPLC to primers with GC clamps. In some of the fragments, the use of GC clamp was necessary to detect the DNA alteration (exon 13/14, 16), and in some fragments (exons 8,9, 15) the primers designed for sequencing where adequate. Because the fragments are AT rich, the gradient temperature was always above the Tm of the fragments (+ 1 to 5 degrees). DHPLC detected 100% of the DNA alterations, and 30 controls samples were scored correctly. In addition, DHPLC allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. In some fragments, 10-bp GC clamp showed DNA alteration in controls, probably due to Taq polymerase mismatch in amplification, and the GC clamp was reduce to 5-bp. We conclude that DHPLC is a highly sensitive, efficient and economical method to screen for point mutation in Angelman syndrome.