The evaluation of mutation analysis using DHPLC in CMT patients with MPZ, PMP22, GJB1, EGR2 mutations. H. Takashima¹, C.F. Boerkoel¹, J.R. Lupski^1,2. 1) Mole Human Gen/TX Med Ctr, Baylor Col Medicine, Houston, TX; 2) Department of Pediatrics, Texas Childrens Hospital, Houston, TX.
   Mutation detection occupies an important position in the clinical diagnosis of CMT patients. As disease-causing mutations have been identified in more and more genes, the labor and expense of screening for such mutations by nucleotide sequencing has increased substantially. Although techniques such as SSCP, DGGE are more economical, the sensitivity of these techniques is under 90% and a new method of screening for mutations is needed. Based on recent reports, Denaturing High-Performance Liquid Chromatography (DHPLC) appears to be an economical and highly sensitive method for mutation detection. We report optimal conditions for and the results of using DHPLC to identify mutations within some genes associated with CMT. We performed DHPLC on 50 patients with known mutations (8 with PMP22 mutations, 23 with MPZ mutations, 9 with GJB1 mutations, and 10 with EGR2 mutations) and controls using the Transgenomic Wave system. PCR reactions were performed using conventional hot start methods. Each coding exon was scanned (PMP22, 4 exons; MPZ, 6 exons; GJB1, 1 large exon split into 2 amplicons and EGR2, 2 exons split into 6 amplicons). The sequence alterations identified by DHPLC were confirmed by Dye Primer sequencing using an ABI Prism 377 sequencer. The PCR amplicons for DHPLC were prepared by slowly cooling from 95C to 25C over 45 min in order to form the heteroduplex. For males with GJB1 mutations, the heteroduplexes were formed as above after combining the PCR products from the patient with those from a normal control. We could identify the heterozygous mutations in each patient. The most important condition was the temperature of separation. Identification of mutations in patients with homozygous mutations is not possible and requires screening of the parents. In summary, DHPLC is an effective method for screening for heterozygous mutations in patients with CMT.