title: Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography.
abstract: 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 tRNA(Leu(UUR)) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA(Leu(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.
authors.affiliation: Department of Molecular Cell Biology and Genetics, Maastricht University, PO Box 1475, 6201 BL Maastricht, The Netherlands.
authors.forename: B J
authors.initials: BJ
authors.lastname: van Den Bosch
authors.forename: R F
authors.initials: RF
authors.lastname: de Coo
authors.forename: H R
authors.initials: HR
authors.lastname: Scholte
authors.forename: J G
authors.initials: JG
authors.lastname: Nijland
authors.forename: R
authors.initials: R
authors.lastname: van Den Bogaard
authors.forename: M
authors.initials: M
authors.lastname: de Visser
authors.forename: C E
authors.initials: CE
authors.lastname: de Die-Smulders
authors.forename: H J
authors.initials: HJ
authors.lastname: Smeets
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics; D001483:Base Sequence; D009202:Cardiomyopathies / Q000235:genetics / Q000473:pathology; D002467:Cell Nucleus / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D016678:Genome; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D007888:Leigh Disease / Q000235:genetics / Q000473:pathology; D008931:Mitochondria, Muscle / Q000235:genetics / Q000473:pathology; D017240:Mitochondrial Myopathies / Q000235:genetics / Q000473:pathology; D025261:Mitochondrial Proton-Translocating ATPases; D009136:Muscular Dystrophies / Q000235:genetics / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D018401:Sample Size; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; C501005:MT-ATP6 protein, human; D012346:RNA, Transfer, Amino Acyl; D004262:DNA Restriction Enzymes; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases