Case Report

Recurrent strokes in a 34-year-old man

The phenotypic expression of disorders caused by point mutations, deletions or depletions within the mitochondrial genome (mtDNA) is heterogeneous. This relates to the phenomena of heteroplasmy, tissue threshold as well as the distribution of mutant DNA among tissues. Hence, the diagnostics of these disorders demands highly specific, sensitive and quantitative methods.

We have developed an allele-specific quantitative real-time PCR method for the detection of two of the most prevalent disease causing mitochondrial mutations, m.3243A > G (MELAS) and m.8993T > G (NARP). Locked Nucleic Acid (LNA) modified primers were used to obtain high allele specificity. In order to monitor mtDNA depletion a real-time method for mtDNA/nuclear DNA copy number ratio determination was developed.

Rapid and sensitive detection and quantification of MELAS and NARP mtDNA alleles were achieved. Heteroplasmy levels as low as 0.01% could be detected, and the mtDNA/nuclear DNA ratio could be determined.

The present method that allows simultaneous determination of heteroplasmy levels and mtDNA/nuclear DNA copy number ratio, will provide a useful tool in molecular diagnostics and in future epidemiological studies of mitochondrial diseases.

This chapter presents the investigation of metabolic myopathies. Glycogen storage disorders (the glycogenoses) are a group of rare inherited metabolic diseases due to abnormal synthesis or breakdown of glycogen. Most affect single cytoplasmic enzymes, with the exception of α-glucosidase (acid maltase) deficiency which involves the lysosomal glycogen degradation pathway. The glycogen storage disorders generally present clinically in one of two ways: exercise intolerance, muscle cramps and intermittent rhabdomyolysis, or progressive proximal weakness. Defects of mitochondrial oxidative phosphorylation are an important cause of muscle disease and are often described as mitochondrial myopathies. The biochemistry and genetics of these disorders are much more complex than either glycogen storage disorders or fatty acid oxidation, predominantly due to the involvement of the mitochondrial genome (mtDNA). In addition, mitochondrial oxidative phosphorylation disorders — all of which are characterized by the inability of the cell to produce enough ATP on account of respiratory chain dysfunction—may present with a vast array of different clinical features, which makes them enter the differential diagnosis of many different neurological conditions.

Mitochondrial respiratory chain abnormalities are an important cause of neuromuscular disease and may be due to defects of either the mitochondrial or nuclear genome. On account of the clinical and genetic heterogeneity exhibited by the mitochondrial myopathies, their investigation and diagnosis remains a challenge, requiring a combination of techniques including muscle histochemistry, biochemical assessment of respiratory chain function and molecular genetic studies. Here, we describe a step-by-step approach to the clinical and laboratory diagnosis of mitochondrial muscle disease, highlighting the many potential problems that can hinder reaching the correct diagnosis.

Mitochondrial DNA (mtDNA) mutations are an important cause of neurological disease. The identification of causative mtDNA mutations may be particularly troublesome in blood where there are often low levels of mutant mtDNA. This is evident from a recent study in which heteroplasmic mtDNA mutations in cytochromecoxidase genes were incorrectly thought to be linked to Alzheimer's disease. We wished to explore whether analysis of blood mtDNA, prepared by a number of DNA extraction procedures, influenced the diagnosis of mtDNA disease. DNA was extracted by different procedures from 4 patients with heteroplasmic mtDNA mutations, and the level of heteroplasmy investigated by radioactive PCR-RFLP analysis. Whilst there was no consistent decrease in the level of mtDNA heteroplasmy, we observed the coamplification of a novel mtDNA pseudogene from DNA samples extracted by a simple ‘boiling’ procedure using primers designed to screen for the tRNA^Leu(UUR)A3243G mutation. This pseudogene was readily amplified from DNA extracted from ρ° (mtDNA-less) cells, confirming its nuclear location. We believe that mtDNA pseudogenes may therefore present significant difficulties in the accurate identification of pathogenic heteroplasmic mtDNA mutations in blood.