Antonio L Andreu Ramon Martf and Michio Hirano 1 Introduction

Mitochondria are the powerhouses of eukaryotic cells. These organelles generate energy in the form of adenosine triphosphate (ATP) from carbohydrates, fats, and proteins, via oxidative phosphorylation. By virtue of possessing their own genetic material—mitochondrial DNA (mtDNA)—mitochondria are unique mammalian organelles. Normal human mtDNA is a 16,569 base-pair (bp), double-stranded, circular molecule (1). The molecules contain tightly compacted genes for 22 transfer (tRNAs), 13 polypeptides, and two ribosomal RNAs (rRNAs) (Fig. 1). All 13 polypeptides are sub-units of the oxidative phosphorylation system: seven belong to Complex I (NADH-CoQ oxidoreductase), one to Complex III (CoQ-cytochrome c oxidoreductase), three to Complex IV (cytochrome c oxidase or COX), and two to Complex V (ATP syn-thase). These subunits are synthesized within the mitochondrion, where they are assembled together with a larger number of subunits encoded by the nuclear DNA (nDNA), that are synthesized in the cytoplasm and are transported into the mitochondrion (2). Approximately 1,000 mitochondrial polypeptides are encoded in nDNA. Complex II (succinate dehydrogenase-CoQ oxidoreductase), of which succinate dehy-drogenase (SDH) is a component, is encoded entirely by nuclear genes; SDH thus serves as a marker for mitochondrial number and activity, independent of the mtDNA.

Since mitochondria are inherited only from the mother (3), defects in mtDNA genes result in pedigrees exhibiting a pattern of solely maternal inheritance. Moreover, because there are hundreds or even thousands of mitochondria in each cell, with an average of 5 mtDNAs per organelle (4,5), mutation in mtDNA may result in two populations of mtDNAs—mutated and wild-type—a condition known as heteroplasmy. The phenotypic expression of a mtDNA mutation is regulated by the threshold effect, that is, the mutant phenotype is expressed in the heteroplasmic cells only when the relative proportion of mutant mtDNAs reaches a certain value (6). A respiratory chain deficiency may become manifest is some tissues, but not in others, if a number of mutant mtDNA exceeds a certain critical threshold. The threshold varies among tissues, depending on the oxidative energy requirements of that tissue: brain, heart, and skeletal muscle have extremely high energy requirements, and therefore it is no surprise that mitochondrial disorders frequently affect brain and muscle (i.e., mitochondrial encephalomyopathies and mitochondrial cardiomyopathies). Because both mitochon-

From: Methods in Molecular Biology, vol. 217: Neurogenetics: Methods and Protocols Edited by: N. T. Potter © Humana Press Inc., Totowa, NJ

Fig. 1. Map of the human mitochondrial genome. The structural genes for the mtDNA-encoded 12S and 16S ribosomal RNAs, the subunits of NADH-coenzyme Q oxidoreductase (ND), cytochrome c oxidase (COX), cytochrome b (Cyt b), and ATP synthase (A), and 22 tRNAs (1-letter amino acid nomenclature), are shown. The origins of light-strand (OL) and heavy-strand (OH) DNA replication, and of the promoters for initiation of transcription from the light-strand (LSP) and heavy-strand (HSP), are shown by arrows. The "common" deletion, a mtDNA species often found in sporadic KSS/PEO is shown, as are common point mutations associated with maternally inherited encephalomyopathies (boxed).

Fig. 1. Map of the human mitochondrial genome. The structural genes for the mtDNA-encoded 12S and 16S ribosomal RNAs, the subunits of NADH-coenzyme Q oxidoreductase (ND), cytochrome c oxidase (COX), cytochrome b (Cyt b), and ATP synthase (A), and 22 tRNAs (1-letter amino acid nomenclature), are shown. The origins of light-strand (OL) and heavy-strand (OH) DNA replication, and of the promoters for initiation of transcription from the light-strand (LSP) and heavy-strand (HSP), are shown by arrows. The "common" deletion, a mtDNA species often found in sporadic KSS/PEO is shown, as are common point mutations associated with maternally inherited encephalomyopathies (boxed).

drial division and mtDNA replication are random processes unrelated to cell division, a dividing cell will donate variable numbers of mitochondria and mtDNAs to its progeny. This process, known as mitotic segregation, can be important clinically if a patient harbors heteroplasmic populations or normal and mutated mtDNAs in his/her tissues. The phenotypic expression of a mutation may then vary among tissues or may change within a tissue over time.

From a practical point of view, the fact that mtDNA is polyplasmic presents serious problems when looking for point mutation with variable heteroplasmy in different tissues; therefore, it is best to analyze affected tissue. To date, the majority of mtDNA

mutations have been identified in genes encoding tRNA, most notably, mutations associated with the clinical syndromes: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and myoclonus-epilepsy and ragged-red fibers (MERRF) (7-18). Our strategy for identifying mutations is based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using, as a template, total DNA from patients. Because most of the mtDNA mutations present clinically as encephalomyopathies, skeletal muscle tissue typically harbors a high mutation load, thereby accounting for the greater likelihood of successfully identifying a pathogenic mutation in this tissue.

Another practical issue when performing mutational analysis of mtDNA is how to quantitate properly the percentage of mutated genomes. A PCR-based approach has a limitation when an accurate result is required. Typically, mutated and wild-type molecules co-exist and form heteroduplexes during PCR annealing steps. When the RFLP analysis is based on digestion of the mutant molecules, heteroduplex structures lead to underestimation of the amount of mutated mtDNA molecules. To avoid this artifact, a simple strategy is to add a radiolabeled nucleotide (i.e., 32P-dATP) prior to the last PCR cycle, so that the percentage of mutated genomes will accurate represent the level in the tissue.

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