Mutation analysis of the entire mitochondrial DNA using Denaturing High Performance Liquid Chromatography (DHPLC). H. Smeets¹, B. Van den Bosch¹, J. Nijland¹, H. Scholte², C. DeDie¹, R. Van den Bogaard³, M. De Visser³, I. De Coo². 1) Dept Molecular, Cell Biol & Gen, Univ Maastricht, Maastricht, Netherlands; 2) Depts Child Neurology & Biochemistry, Erasmus Univ, Rotterdam, Netherlands; 3) Depts Clin Genet & Neurol, AMC, Amsterdam, Netherlands.
   In patients with mitochondrial disease a continuously increasing number of mtDNA mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of Denaturing High Performance Liquid Chromatography (DHPLC). The entire mtDNA was amplified in 13 fragments of 1-2 kb, digested in fragments of 90-600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detectable percentage of 0.5% for the A8344G mutation. The entire muscle mtDNA from 6 patients with mitochondrial disease was screened and 3 mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA-Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy a T3271C mutation in the tRNA-Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes. Subsequently, we optimised the procedure at the single cell level to test for heteroplasmy in single oocytes and embryos to determine the natural mutation load of mtDNA and the possible presence of paternal mtDNA.