INVITED REVIEW ABSTRACT: The human mitochondrial genome encodes 13 proteins. All are subunits of the respiratory chain complexes involved in energy metab- olism. These proteins are translated by a set of 22 mitochondrial transfer RNAs (tRNAs) that are required for codon reading. Human mitochondrial tRNA genes are hotspots for pathogenic mutations and have attracted interest over the last two decades with the rapid discovery of point mutations associated with a vast array of neuromuscular disorders and diverse clinical phenotypes. In this review, we use a scoring system to determine the pathogenicity of the mutations and summarize the current knowledge of structure–function relationships of these mutant tRNAs. We also provide readers with an overview of a large variety of mechanisms by which muta- tions may affect the mitochondrial translation machinery and cause disease. Muscle Nerve 37: 150–171, 2008 HUMAN MITOCHONDRIAL TRANSFER RNAs: ROLE OF PATHOGENIC MUTATION IN DISEASE FERNANDO SCAGLIA, MD, and LEE-JUN C. WONG, PhD Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA Accepted 17 September 2007 Normal mitochondrial function depends on both tion and termination factors, ribosomal proteins, the expression of the mitochondrial genome and the and enzymes involved in maturation and posttran- expression of at least 1,300 nuclear genes whose scriptional modification of tRNAs are nuclear en- products are imported into the mitochondria. Hu- coded. However, the complete set of 22 tRNAs and man mitochondrial DNA (mtDNA) is a 16,569-kb two major rRNAs are of mitochondrial origin. circular, double-stranded molecule. It encodes 13 of The adaptation of the bacterial ancestors of mito- more than 80 polypeptide subunits of the mitochon- chondria to the new environment of eukaryotic cells drial respiratory chain complexes and contains 24 brought the advantage of ATP production via the pro- additional genes required for mitochondrial protein cess of oxidative phosphorylation. However, the pres- biosynthesis. These include two rRNA genes and 22 ence in mitochondria of an electron transport chain tRNA genes, one for each of 20 amino acids, and two that continuously reduces oxygen to build up an elec- each for tRNALeu (UUR and CUN codons) and trochemical gradient required for ATP synthesis has a tRNASer (UCN and AGY codons).140 All partners of potentially deleterious side effect for these eukaryotic the mitochondrial protein synthetic machinery such cells. This side effect consists of the constant genera- as aminoacyl-tRNA synthetases, initiation, elonga- tion of reactive oxygen species (ROS). Due to the continuous function of the electron transport chain, the minimal electron leakage is sufficient to make mi- Available for Category 1 CME credit through the AANEM at www.aanem.org. tochondrial O2.- generation the major cellular source Abbreviations: A, adenine; ATP, adenosine triphosphate; C, cytosine; COX, of ROS in most tissues. MtDNA is located in the mito- cytochrome c oxidase; CPEO, chronic progressive external ophthalmoplegia; chondrial matrix and its close proximity to the respi- DHU, dihydrouridine; G, guanine; mtDNA, mitochondrial deoxyribonucleic acid; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke- ratory chain complexes embedded in the inner mem- like episodes; MERRF, myoclonic epilepsy with ragged-red fibers; NO, nitric oxide; PEO, progressive external ophthalmoplegia; RC, respiratory chain; brane renders it susceptible to oxidative stress, causing ROS, reactive oxygen species; rRNA, ribosomal ribonucleic acid; SNHL, sen- its high mutation rate (10–17-fold higher rate of mu- sorineural hearing loss; T, thymine; tRNA, transfer ribonucleic acid; U, uracil 1,122 Key words: cardiomyopathy; chronic progressive external ophthalmoplegia; tations than the nuclear genome). diabetes mellitus; encephalomyopathy; focal segmental glomerulosclerosis; Since the first description of pathogenic muta- human mitochondrial tRNA genes; mitochondrial cytopathies; mitochondrial myopathy; myoclonic epilepsy; pathogenic mutations; ragged-red fibers; ret- tions in the mitochondrial genome, over 200 disease- initis pigmentosa; sensorineural hearing loss; stroke-like episodes correlated point mutations and rearrangements Correspondence to: L. J. C. Wong; e-mail: [email protected] have been found in association with a variety of © 2007 Wiley Periodicals, Inc. 88 Published online 9 November 2007 in Wiley InterScience (www.interscience. mitochondrial cytopathies. More than half of these wiley.com). DOI 10.1002/mus.20917 mutations have been located in tRNA genes that 150 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 constitute ϳ9% of the entire mitochondrial ge- Most of the observed mtDNA changes represent neu- nome.91 Thus, mitochondrial tRNA genes are hot- tral polymorphisms and have been used to track spots for mitochondrial pathogenesis and contribute human migrations.70 The large prevalence of varia- in a disproportionate way to the etiology of disorders tions in tRNA genes calls for the elucidation of their caused by mitochondrial DNA mutations, which is pathogenicity. In addition, clinical misattribution of conceivable due to their central role in mitochon- pathogenicity is an important issue due to the con- drial protein synthesis. In comparison, a little less sequences for genetic counseling that is provided to than half of the mitochondrial mutations affect pro- individuals and families with mitochondrial disease. tein coding genes, which comprises 68% of the en- Benign variants can be mistaken as bona fide patho- tire mitochondrial genome.42 genic mutations when found in one or a limited To achieve their goal of carrying amino acids for number of individuals, especially if present in the protein synthesis, these tRNA molecules have to un- heteroplasmic state, which has been regarded as di- dergo different steps of processing and modifica- rect evidence for pathogenicity. tion, and interact with many protein factors.128 Dis- A set of rules had to be established to confirm the ease-related mutations could potentially affect the pathogenic nature of novel mtDNA mutations. Di primary, secondary, and tertiary structures of a given Mauro and Schon35 have previously described the mitochondrial tRNA. Misfolding and instability of canonical pathogenic criteria for mtDNA point mu- the mutant tRNA result in increased degradation tations. These rules specified that the mutation and ultimately lead to decreased steady-state levels of should (1) be absent in healthy subjects, (2) alter an a mitochondrial tRNA. In addition, the efficiency of evolutionary conserved residue, (3) be heteroplas- the aminoacylation process will be particularly sen- mic and segregate with disease, (4) be such that the sitive to point mutations because the enzymes in- degree of mutant heteroplasmy correlates with clin- volved recognize a specific structure of a tRNA.196 ical severity and cell pathology as documented by There are still many unanswered questions re- single-fiber polymerase chain reaction (PCR), and garding the phenotypic effect of these mutations. (5) cause defects of mitochondrial protein synthesis Perhaps one of the most intriguing is the incredible and respiratory chain deficiencies in single or mul- diversity of clinical phenotypes associated with mito- tiple affected tissues of patients and demonstrable in chondrial tRNA mutations. This is a phenomenon cybrid cell lines. Cybrid cell lines are generated by that has been observed even among individuals har- transferring mitochondria isolated from patients boring the same mutation. Several factors including harboring pathogenic mutations into cultured hu- the percentage of heteroplasmy, the different tissues man cells treated with ethidium bromide to deplete affected with the mutation, the position of the their mitochondrial DNA.81 mutation within the structure of the tRNA gene, the However, some pathogenic mtDNA mutations efficiency in the utilization of different codons fail to meet these established canonical criteria. In for the same amino acid, and the different amino particular, the homoplasmic mutations often do not acid composition among the mitochondrial DNA- demonstrate the required segregation with either encoded protein subunits may account for this vari- biochemical deficiency or clinical disease. It is now ability. In addition, nuclear genes, different mito- known that homoplasmic mitochondrial tRNA mu- chondrial genetic backgrounds, and environmental tations can be pathogenic, as demonstrated with factors may also exert a modifier effect. several mutations in the tRNASer(UCN) gene,72 the The aim of this review is to focus on the assess- 1624 CϾT mutation in the tRNAVal that caused mul- ment of pathogenic mitochondrial tRNA mutations, tiple neonatal deaths and Leigh syndrome in the the pathogenic mechanism of disease of known and offspring of a homoplasmic and mildly affected well-established mutations, the description of new woman,100 the 4291 TϾC in the tRNAIle gene asso- pathogenic mutations, and the elucidation of their ciated with hypertension and dyslipidemia,195 the clinical significance. 4300 AϾG in the tRNAIle gene responsible for ma- ternally inherited cardiomyopathy in two families,173 CANONICAL CRITERIA FOR ASSESSING and the 5693 TϾC change in the mitochondrial PATHOGENICITY OF MITOCHONDRIAL tRNA tRNAAsn gene that caused encephalomyopathy.27 In MUTATIONS addition, true pathogenic mutations may escape MtDNA alterations