DHPLC as a Method for High Throughput, Cost Effective Mutation Screening: Application to Rett Syndrome. J.M. Devaney¹, K. Hoffbuhr², S.N. Siranni², S. Naidu³, M. Marino¹, E. Hoffman². 1) Transgenomic, Inc., Gaithersburg, MD; 2) Research Center for Genetic Medicine, Children's National Medical Center, Washington DC; 3) Neurogenetics Unit, Kennedy Krieger Institute, Johns Hopkins University, Balitmore, MD.
   Rett syndrome is a neurodevelopmental disorder affecting females and is characterized by developmental regression, severe mental retardation and stereotypic hand movements. Recent studies have shown that 60-80% of girls with Rett syndrome have mutations within the X-linked gene encoding the methyl-CpG-binding protein 2 (MeCP2). We have received more than 270 samples for diagnostic mutation testing for Rett syndrome in a short period of time. Mutation screening was complicated by most cases representing de novo mutations and expression of the disease as a heterozygote. To efficiently screen this large number of samples, we have developed a "targeted sequencing" approach using a combination of denaturing high performance liquid chromatography (DHPLC), a mutation screening method that relies on heteroduplex detection, and direct sequencing.
   We examined the sensitivity of DHPLC for detection of MeCP2 mutations by performing DHPLC analysis on 18 mutation positive samples, representing 10 different MeCP2 mutations, and 22 mutation negative samples as determined by direct sequencing. Heteroduplex peaks were detected for every mutation positive sample. These results indicated a high level of sensitivity, approaching 100%, for mutations within the MeCP2 gene. DHPLC has many advantages over conventional mutation screening methods, including semi-automated analysis of 96 PCR samples in less than 12 hours, the absence of fluorescent or radioactive labeling, and low relative cost per sample approximately $0.40. Targeting samples for direct sequencing after DHPLC analysis proved to be a more efficient method for MeCP2 mutation detection by reducing the amount of sequencing performed on each sample and by localizing the mutation to a certain region of the MeCP2 gene.