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Nucleic Acids Research, 2000, Vol. 28, No. 20 e89
© 2000 Oxford University Press

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

Bianca J. C. van den Bosch, René F. M. de Coo1, Hans R. Scholte2, Jeroen G. Nijland, Ruud van den Bogaard3, Marianne de Visser4, Christine E. M. de Die-Smulders and Hubert J. M. Smeets*

Department of Molecular Cell Biology and Genetics, Maastricht University, PO Box 1475, 6201 BL Maastricht, The Netherlands, 1Department of Child Neurology, University Hospital Rotterdam, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands, 2Department of Biochemistry, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands, 3Department of Clinical Genetics, Academic Medical Center and 4Department of Neurology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (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 complete mitochondrial genome 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 detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNALeu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNALeu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried 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.

* To whom correspondence should be addressed. Tel: +31 43 3875843; Fax: +31 43 3877877; Email: bert.smeets@molcelb.unimaas.nl The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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