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INVITED REVIEW ABSTRACT: The mitochondrial encodes 13 . All are subunits of the respiratory chain complexes involved in energy metab- olism. These proteins are translated by a set of 22 mitochondrial transfer (tRNAs) that are required for codon reading. Human mitochondrial tRNA are hotspots for pathogenic 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 . 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 machinery and cause disease. Muscle Nerve 37: 150–171, 2008

HUMAN MITOCHONDRIAL TRANSFER RNAs: ROLE OF PATHOGENIC IN DISEASE

FERNANDO SCAGLIA, MD, and LEE-JUN C. WONG, PhD

Department of Molecular and Human , 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 . It encodes 13 of The adaptation of the bacterial 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 cess of oxidative phosphorylation. However, the pres- . These include two rRNA genes and 22 ence in mitochondria of an 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 (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, ; C, ; COX, of ROS in most tissues. MtDNA is located in the mito- ; CPEO, chronic progressive external ophthalmoplegia; chondrial matrix and its close proximity to the respi- DHU, ; G, ; 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 , causing ROS, ; rRNA, ribosomal ribonucleic acid; SNHL, sen- its high (10–17-fold higher rate of mu- sorineural ; 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 . 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 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 correlates with clin- volved recognize a specific structure of a tRNA.196 ical severity and 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 to deplete affected with the mutation, the position of the their mitochondrial DNA.81 mutation within the structure of the tRNA , the However, some pathogenic mtDNA mutations efficiency in the utilization of different codons fail to meet these established canonical criteria. In for the same , 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 in the the pathogenic mechanism of disease of known and offspring of a homoplasmic and mildly affected well-established mutations, the description of new ,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 can be divided into three classes: identification because of poor evolutionary conser- pathogenic, adaptive (advantageous in certain envi- vation in the region or because segregation with ronments), and neutral (accumulated by chance). disease cannot be demonstrated in small families.

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 151 In order to take into consideration these known maximum of 8 points. The sequences exceptions to the canonical criteria, newer classifica- from (), sea urchin tion systems have been proposed by different (Strongylocentrotus purpuratus), fruit fly ( groups. Mitchell et al.108 designed a scoring system melanogaster), frog (Xenopus laevis), mouse (Mus mus- to assess the pathogenicity of a given sequence vari- culus), cattle (Bos taurus), and human (Homo sapiens) ant in the mitochondrial encoded complex I were compared. One point was subtracted for each (MTND) subunit genes. Another scoring system spe- nucleotide variation occurring in the species listed cific for the mitochondrial tRNA gene mutations above. One additional point was subtracted for vari- listed on MITOMAP was devised by McFarland et ations that occurred in each of the four adjacent al.101 with specific scoring criteria that gave higher . For nucleotides in the stem region, scores to transmitochondrial cybrid experiments, their complementary nucleotides rather than the measurements of steady-state levels, and segregation adjacent nucleotides were considered. The existence of the mutation with a biochemical defect at the level of more than one independent report and the pres- of single cell. ence of heteroplasmy were given 5 points each. Three points were given for segregation of the mu- CRITERIA FOR ASSESSMENT OF PATHOGENICITY OF tation with the disease (Table 1). For each mutation, mtDNA ALTERATIONS AND CLASSIFICATION OF references were evaluated according to the criteria MUTATIONS described above and a score was given. If an exper- iment was not performed or the data were not pro- We evaluated the mitochondrial tRNA mutations vided for any reason, a score of 0 was given to that listed on MITOMAP, which is the largest publicly criterion. Based on our scoring system, each muta- available compendium of mtDNA mutations and tion could receive a maximum score of 40. polymorphisms (MITOMAP: A Human Mitochon- Overall, the variations in the mitochondrial drial Genome Database; http://www.mitomap.org tRNA genes that were classified according to these accessed on January 31, 2007). In addition, we new criteria were assigned to four distinct categories: searched PubMed (http://www.ncbi.nlm.nih.gov/ (1) definite pathogenicity (Ն30 points), (2) proba- entrez accessed January 31, 2007) in order to analyze ble pathogenicity (20–29 points), (3) possible patho- a thorough and complete list of mitochondrial tRNA genicity (10–19 points), and (4) unlikely pathoge- gene mutations. nicity (Յ9 points). The scores listed in Table 2 Since the determination of the pathological sig- represent the minimal scores since not all the rele- nificance of these mitochondrial tRNA variants is vant experiments to confirm pathogenicity were per- required in order to provide accurate genetic ser- formed in each case. vices, we evaluated the mutations in the mitochon- drial tRNA genes using a modified scoring system by taking earlier scoring systems101,108 into consider- ation. Functional studies including steady-state levels Table 1. Scoring criteria applied to mitochondrial tRNA mutations of mutated mitochondrial tRNA, evidence of patho- listed on MITOMAP and PubMed. genicity from cybrid cells or single fiber studies, were Score given the highest scores. We did not score the pres- Pathogenic criteria (points) ence of positive single fibers additively since the Evolutionary conservation of the base No change 8 levels of the mutant tRNA would be higher in COX- Each change Ϫ1 negative fibers than in adjacent COX-positive fibers More than one independent report Yes 5 if there is a decline in steady-state mRNA level or No 0 Presence of heteroplasmy Yes 5 evidence of pathogenicity from transmitochondrial No 0 cybrid studies. Positive evidence from any of these Histochemical evidence Strong evidence 5 experiments was scored a maximum of 9 points. The Weak evidence 3 presence of strong histochemical evidence including No evidence 0 COX-negative ragged-red fibers or cristae abnormal- RC defects (in tissues) Strong 5 Moderate 3 ities by electron microscopy was assigned 5 points. No 0 The respiratory chain (RC) defects were classified Segregation of mutation with the Yes 3 according to the modified Walker criteria8 and a disease No 0 score of 5 or 3 points was given for fitting major or Single fiber studies, or steady-state Yes 9 levels, or cybrid studies No 0 minor criteria, respectively. Evolutionary conserva- tion weighed second to functional evidence, with a RC, respiratory chain.

152 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 Table 2. Structural distribution of tRNA mutations and clinical . Nucleotide Clinical features of related tRNA gene change Region Het/Hom Score Inheritance References MTTF 582 TϾC Acceptor stem M.M., EI, tachycardia Het 35 S (female) 112 MTTF 583 GϾA Acceptor stem MELAS, EI, RP Het 38 D 30,57 MTTF 608 AϾG AC stem TIN Homo 11 I 184 MTTF 611 GϾA AC wobble base MERRF Het 35 D 94 MTTF 618 TϾC AC stem MM Het 26 D 85 MTTF 622 GϾA Variable loop EI Het 31 D/I 33 MTTV 1606 GϾA Acceptor stem Ataxia, PEM, SNHL, Sz, RP, Het 27 I, D 130,178 cataracts, hypotonia MTTV 1624 CϾT DHU stem Adult LS Homo 27 I 100 MTTV 1642 GϾA AC stem MELAS Het 26 D 31 MTTV 1644 GϾT Variable region LS Het 21 I 21 MTTV 1659 TϾCT␺C stem Movement disorder, Het 16 I 12 hemiplegia MTTL (UUR) 3243 AϾG DHU loop MELAS, DM, SNHL Het 40 I 25 MTTL (UUR) 3243 AϾT DHU loop PEM, MM Het 21 ND 150 MTTL (UUR) 3249 GϾA DHU loop KSS Het 26 I 147 MTTL (UUR) 3250 TϾC DHU loop MM Het 18 I 49 MTTL (UUR) 3251 AϾG DHU loop MM Het 25 I 168 MTTL (UUR) 3252 AϾG DHU loop MELAS Het 28 I 111 MTTL (UUR) 3254 CϾG DHU stem MM Het 13 I 78 MTTL (UUR) 3255 GϾA DHU stem MERRF/KSS overlap Het 35 I 115 MTTL (UUR) 3256 CϾT DHU stem MERRF, MELAS Het 40 I, D 75,110 MTTL (UUR) 3258 TϾC AC stem MM Het 39 I 18,163 MTTL (UUR) 3260 AϾG AC stem MM, HCM Het 40 I 98 MTTL (UUR) 3264 TϾC AC loop DM Het 10 I 166 MTTL (UUR) 3271 TϾC AC stem MELAS, DM Het 27 I 132 MTTL (UUR) 3271 delT AC stem PEM Het 21 D 152 MTTL (UUR) 3273 TϾC AC stem PEO, EI Het 35 I 17 MTTL (UUR) 3274 AϾG AC stem Depression, cataracts Het 33 D 73 MTTL (UUR) 3280 AϾGT␺C stem MM Het 40 I 18,163 MTTL (UUR) 3287CϾAT␺C loop PEM, RP, SNHL Het 33 ND 3 MTTL (UUR) 3288 AϾGT␺C loop MM Het 21 I 54 MTTL (UUR) 3291 TϾCT␺C loop MELAS Het 21 D 50 MTTL (UUR) 3302 AϾG AC stem MM Het 40 I 11,187 MTTL (UUR) 3303 CϾT Acceptor stem HCM Het/homo 29 I 48,155 MTTI 4267 AϾG Acceptor stem MM, ataxia, SNHL Het 30 D 172 MTTI 4269 AϾG Acceptor stem DCM, Sz, SNHL, FSGS Het 27 I 170 MTTI 4274 TϾC DHU stem CPEO, ALS Het 37 ND 13,23 MTTI 4284 GϾA DHU/AC stem CPEO, HCM Het 22 I 26 MTTI 4285 TϾC AC stem PEO Het 26 ND 156 MTTI 4291 TϾC AC loop HTN, dsylipidemia Homo 21 I 195 MTTI 4295 AϾG AC stem HCM Het 21 I 105 MTTI 4298 GϾA AC stem CPEO, MS, MM Het 38 S (female) 29,171 MTTI 4300 AϾG AC stem HCM Homo 34 I 20,173 MTTI 4309 GϾAT␺C stem CPEO Het 27 ND 43 MTTI 4317 AϾGT␺C loop DCM Het 33 ND 169 MTTI 4320 CϾTT␺C stem PEM, HCM Het 13 ND 134 MTTQ 4332GϾA Acceptor stem Atypical MELAS, DM Het 30 D 6 MTTQ 4336 TϾC Acceptor stem Alzheimer Homo 15 ND 39 disease/Parkinson disease MM Het 30 S (male) 34 MTTM 4409 TϾC DHU stem EI, muscle atrophy, Het 30 D 190 hypotonia, short stature MTTM 4450 GϾAT␺C stem Splenic lymphoma Het 27 ND 92 MTTW 5521 GϾA DHU stem M M Het 35 I 157 MTTW 5532 GϾA DHU stem MNGIE-like syndrome Het 34 I 96 MTTW 5537 InsT AC stem MILS Het 38 I 137, 182 MTTW 5540 GϾA AC stem PEM Het 32 ND 158

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 153 Table 2. Continued Nucleotide Clinical features of related tRNA gene change Region pathologies Het/Hom Score Inheritance References MTTW 5543 TϾC AC loop MM Het 35 D 3 MTTW 5549 GϾA AC stem Dementia, chorea, DM Het 23 ND 113 MTTA 5591 GϾA Acceptor stem MM, myalgia, high CK Het 34 I 167 MTTA 5628 TϾC AC stem CPEO, weakness, Het 33 I 160 dysphagia MTTA 5650 GϾA Acceptor stem MM, myotonic dystrophy- Het 32 I 40,66 like phenotype MTTN 5692 TϾC AC loop CPEO Het 36 ND 143 MTTN 5693 TϾC AC loop PEM, hypotonia, liver Homo 28 ND 27 dysfunction MTTN 5698 GϾA AC loop CPEO, MM Het 35 D 161,163 MTTN 5703 GϾA AC stem CPEO, MM Het 32 ND 61,192 MTTN 5728 TϾC Acceptor stem FSGS, GH deficiency, PEM Het 34 D 106 MTTC 5783 GϾAT␺C stem MM, HCM, TIN, SNHL Het 26 I 38 MTTC 5814 TϾC DHU stem MELAS, Sz, PEO, ataxia, Het 39 I 76,95,136 HCM MTTY 5843 AϾGT␺C loop FSGS Homo 16 I 138 MTTY 5874 TϾC DHU stem EI, weakness Het 35 S (female) 123 MTTY 5877 CϾT DHU loop CPEO Het 26 I 131 MTTY 5885 DelT Acceptor stem CPEO Het 35 S (female) 126 MTSS1 7443 AϾG Acceptor stem SNHL Homo 20 I 119 (UCN) MTSS1 7444 GϾA Acceptor stem SNHL Homo 20 I 119 (UCN) MTSS1 7445 AϾC Acceptor stem SNHL Homo 15 I 119 (UCN) MTTS1(UCN) 7445 AϾG Acceptor stem SNHL, palmoplantar Het/homo 32 I 127, 149 keratoderma MTTS1(UCN) 7472insA Variable region Sz Homo 40 I 72 MTSS1 7472insC Variable region SNHL, MERRF, EI Het 40 I 124,177 (UCN) MTSS1 7480AϾG AC loop MM, SNHL, dementia, Het 35 D 9 (UCN) ataxia MTTS1(UCN) 7497GϾA DHU stem MM, PEM, EI Het/homo 29 I 51,72 MTTS1(UCN) 7510 TϾC Acceptor stem SNHL Het 15 I 68 MTTS1(UCN) 7511 TϾC Acceptor stem SNHL Homo 40 I 22,164 MTTS1 7512 TϾC Acceptor stem PEM Homo 23 I 72 (UCN) MTTD 7526 AϾG connector region MM Het 26 S (female) 148 (between acceptor/D stem) MTTD 7543 AϾG AC stem Sz, DD Het 22 I 154 MTTK 8296 AϾG Acceptor stem DM, SNHL, MERRF, Het 21 ND 133 MELAS MTTK 8300 TϾC Acceptor stem EI Het 22 I 46 MTTK 8313 GϾA DHU stem MNGIE-like syndrome Het 27 D 189 MTTK 8326 AϾG AC stem Movement disorder Het 16 I 198 MTTK 8328 GϾA AC stem EM Het 35 D 67 MTTK 8342 GϾAT␺C stem PEO, movement disorder Het 27 S (female) 179 MTTK 8344 AϾGT␺C loop MERRF Het 38 I 7 MTTK 8348 AϾGT␺C loop HCM Het 8 I 175 MTTK 8355 TϾCT␺C stem CPEO, MM, hypoventilation Het 32 ND 163 MTTK 8356 TϾCT␺C stem MERRF, MELAS, SNHL Het 38 I 99 MTTK 8361 GϾA Acceptor stem MERRF Het 33 I 129 MTTK 8362 TϾG Acceptor stem MM Het 32 ND 163 MTTK 8363 GϾA Acceptor stem MERRF Het 35 I 118 MTTG 9997 TϾC Acceptor stem HCM Het 21 I 104 MTTG 10006 AϾG DHU loop CIP, CPEO Nd 5 ND 89

154 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 Table 2. Continued Nucleotide Clinical features of related tRNA gene change Region pathologies Het/Hom Score Inheritance References MTTG 10010 TϾC DHU stem EM, MM, polyendocrine Het 40 ND 10,29,114 dysfunction MTTG 10044 AϾGT␺C loop EM Het 13 I 135 MTTR 10438 AϾG AC loop EM Het 21 I 185 MTTH 12147 GϾA DHU stem MELAS, MERRF, cerebral Het 40 D 103, 174 edema MTTH 12183 GϾAT␺C stem RP, SNHL, muscle atrophy, Het 30 I 28 ataxia MTTH 12192 GϾAT␺C stem HCM, DCM Homo 11 ND 151 MTTS2 12246 CϾGT␺C loop CIP, CPEO Homo 5 ND 89 (AGY) MTTS2 12258 CϾA Acceptor stem DM, SNHL Het 35 I 93, 97 (AGY) MTTL (CUN) 12276 GϾA DHU stem CPEO Het 16 ND 19 MTTL(CUN) 12297 TϾC AC loop DCM Het 26 I 53,176 MTTL (CUN) 12301 GϾA AC loop AISA Het 15 S (male) 47 MTTL (CUN) 12311 TϾC Variable loop CPEO Het 18 ND 63 MTTL (CUN) 12315 GϾAT␺C stem MM, PEO Het 38 D 44, 77 MTTL (CUN) 12320 AϾGT␺C loop MM Het 35 ND 194 MTTL (CUN) 12334 GϾA Acceptor stem M.M. Het 35 S (female) 191 MTTE 14687 TϾCT␺C loop MM, HCM, RP Het 31 S (male) 16 MTTE 14696 AϾG DHU stem EM Het 21 I 185 MTTE 14709 TϾC AC loop DM, SNHL, FSHD, hydrops Homo 40 I 56, 59, fetalis 102, 107 MTTE 14710GϾAAC(EϾK) CPEO, fatigue, weakness, Het 35 D 3 EI MTTT 15915 GϾA AC stem Sz, SNHL, MR, movement Het 21 D 116,144 disorder MTTT 15923 AϾG AC loop LIMM Homo 0 I 14 MTTT 15940delT T␺C loop MM Homo 0 I 145 MTTT 15950 GϾA Acceptor stem Alzheimer homo 0 ND 52 disease/Parkinson disease MTTP 15990 CϾTAC(PϾS) MM, CPEO Het 35 S (female) 71 MTTP 15995 GϾA AC stem Movement disorder Het 15 D 198 MTTP 16002 TϾC DHU stem Ptosis, CPEO Het 19 S (male) 146

AC, anticodon; AISA, acquired idiopathic sideroblastic anemia; ALS, amyotrophic lateral sclerosis; CIP, chronic intestinal pseudoobstruction; CPEO, chronic progressive external ophthalmoplegia; D, de novo; DCM, dilated cardiomyopathy; DD, developmental delay; DHU, dihydrouridine; DM, diabetes mellitus; EI, exercise intolerance; EM, encephalomyopathy; FSGS, focal segmental glomerulosclerosis; FSHD, facioscapulohumeral muscular dystrophy; GH, deficiency growth hormone deficiency; HCM, hypertrophic cardiomyopathy; Het, heteroplasmy; Homo, homoplasmy; I, inherited; KSS, Kearns Sayre syndrome; LIMM, lethal infantile mitochondrial myopathy; LS, Leigh syndrome; MELAS, mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes syndrome; MERRF, myoclonic epilepsy associated with ragged-red fibers; MILS, maternally inherited Leigh syndrome; MM, mitochondrial myopathy; MNGIE, mitochondrial neurogastrointestinal syndrome; MR, mental retardation; MS, multiple sclerosis; ND, not determined; PEM, progressive encephalomyopathy; PEO, progressive external ophthalmoplegia; RP, ; S, sporadic; SNHL, sensorineural hearing loss; Sz, seizure disorder; TIN, tubulointerstitial nephritis. Note: tRNAs for pro, glu, ser(UCN), tyr, cys, asn, ala, and gln are transcribed from the light strand. The nucleotide changes listed in Table 2 refer to the nucleotide and position in the mitochondrial DNA sequence as shown in http://www.mitomap.org

We also listed the nucleotide changes, the re- remainder of the mutations were classified as possibly gions in the tRNA cloverleaf structure where these (18 of 124, 14.5%), probably (40 of 124, 32.3%), and mutations were present, and the assigned clinical definitely (60 of 124, 48.4%) pathogenic (Table 3). phenotype for these mutations (Table 2). Only the mutations with definite and probable patho- genicity (scoring 20 points or above) were further re- PATHOGENIC MUTATIONS AND BENIGN VARIANTS viewed for the purpose of this study. Based on the newly adopted classification, among all the tRNA mutations analyzed in this study there was a Mitochondrial tRNAPhe Mutations. Mutations in this small percentage of mutations that were classified as gene have presented with a myopathic phenotype neutral polymorphisms (6 of 124, 4.8%), whereas the in the affected subjects. Four heteroplasmic muta-

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 155 Table 3. Distribution of pathogenic mutations and benign variants among mitochondrial tRNA genes. Benign tRNA Definite Probable Possible variation Definite ϩ probable Predominant gene (Ն 30) (20–29) (10–19) (Յ 9) Total N (% total) Phenotype F 4 1 1 0 6 5 (83%) MM V 0 4 1 0 5 4 (80%) Multisystemic/LS L(UUR) 10 9 3 0 22 19 (86%) MELAS/DM/SNHL/LS I 5 6 1 0 12 11 (92%) CM, PEO Q 2 0 1 0 3 2 (67%) EM, MM M 1 1 0 0 2 2 (100%) MM W 5 1 0 0 6 6 (100%) MM/MNGIE/LS/EM A 3 0 0 0 3 3 (100%) MM N 4 1 0 0 5 5 (100%) PEO/MM C 1 1 0 0 2 2 (100%) Multisystemic Y 2 1 1 0 4 3 (75%) MM/PEO S(UCN) 5 4 2 0 11 9 (82%) SNHL D 0 2 0 0 2 2 (100%) MM K 7 4 1 1 13 11 (85%) MERRF G 1 1 1 1 4 2 (50%) EM R 0 1 0 0 1 1 (100%) EM H 2 0 1 0 3 2 (67%) Multisystemic S(AGY) 1 0 0 1 2 1 (50%) DM/SNHL L(CUN) 3 1 3 0 7 4 (57%) PEO/MM/CM/AISA PEO/MM/DM/SNHL/ E 3 1 0 0 4 4 (100%) EM/HCM T 0 1 0 3 4 1 (25%) EM P 1 0 2 0 3 1 (33%) MM/PEO Total 60 40 18 6 124 100 (82%)

See Table 2 for definition of abbreviations. tions: 582 TϾC, 583 GϾA, 618 TϾC, and 622 GϾA CNS, and skeletal muscle involvement with reduced were identified in four unrelated patients with RC enzymes in skeletal muscle. mitochondrial myopathy112 and exercise intoler- McFarland et al.100 described a family with a ho- ance.30 The 583 GϾA mutation has also been re- moplasmic 1624 CϾT mutation that resulted in several ported in a patient with a MELAS-like pheno- neonatal deaths and one surviving child with Leigh type.57 syndrome. The was minimally symptomatic, In a patient with typical clinical, histological, and but a severe biochemical defect was present in a de- biochemical features of MERRF, Mancuso et al.94 ceased infant and the mother. The 1624 CϾT change reported a novel GϾA transition in nucleotide posi- affected a highly conserved basepair at the stem region tion 611, affecting the wobble base. The finding of of the dihydrouridine (DHU) arm. In addition, there this mutation revealed that MERRF syndrome is not was a striking reduction in the steady-state mitochon- always associated with tRNALys mutations. drial tRNAVal in different tissues obtained from some of these patients. Northern blot analysis did not reveal Mitocondrial tRNAVal Mutations. Patients carrying evidence of unprocessed intermediates from a large mutations in this gene have had either multisystemic polycistronic RNA unit pointing toward a fast degrada- involvement or a clinical picture consistent with tion of the mutant tRNA, which would have a major Leigh syndrome. These include a 1642 GϾAina impact on mitochondrial protein synthesis. The MELAS patient, a 1644 GϾT in a patient with Leigh marked difference in phenotypic expression between syndrome, a 1606 GϾA in patients with multisys- the mother and her children could not be explained temic disorders, and a homoplasmic 1624 CϾTmu- by the mtDNA mutation alone since it is homoplasmic tation in a family with Leigh syndrome and incom- in every tissue of all matrilineal family members. Epi- plete penetrance. The GϾA substitution at genetics or nuclear-encoded elements might act as nucleotide position 1606 was found on two occasions modifiers. by two groups confirming the pathogenicity of this mutation.130,178 These two patients exhibited similar Mitochondrial tRNALeu(UUR) Mutations. Mutations in clinical phenotype: hearing loss, followed by ocular, this gene are associated with diverse phenotypes in-

156 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 cluding MELAS syndrome, myopathies, maternally tion (5-taurinomethyluridine) at the anticodon wob- inherited , and sensorineural ble position, substantially decreasing UUG(Leu)– hearing loss (SNHL). More than 22 point muta- dependent protein synthesis.83 This mechanism tions have been identified in this 77 nucleotide could explain the reduced translation of UUG-rich tRNALeu(UUR) gene. At least 19 of them are of prob- genes such as ND6. Most other mitochondrial tran- able or definite pathogenicity. The majority are lo- scripts preferentially use the UUA(Leu) codon, cated in the DHU and anticodon arms. is hence their translation is not substantially affected.84 the most frequently used amino acid in human mi- Deficient wobble modification may be one of the tochondrial-encoded proteins. The usage of UUR many factors responsible for the phenotypic features and CUN codons is ϳ17%–83%. The common 3243 of MELAS syndrome. AϾG mutation alters a highly conserved nucleotide The morbidity and mortality of stroke-like epi- and this mutation is predicted to cause a pro- sodes observed in MELAS syndrome could also be L nounced change in the structure of the tRNA - associated with the presence of vessels strongly pos- eu(UUR). The impact of this structural change on itive for (SSVs). These aminoacylation has been studied.120 Studies using SSVs positive for cytochrome c oxidase activity may cybrid cell lines that are generated by transferring play an important pathogenic role.62,117 Although mitochondria isolated from patients harboring there is decreased cytochrome c oxidase activity for pathogenic mutations into cultured human cells each individual , there is a high in- have detected decreased respiratory chain enzyme crease in mitochondrial mass in these SSVs that leads activities.141 Moreover, additional studies have re- to an increase in total cytochrome c oxidase activity. vealed defects in aminoacylation,74 protein synthe- Nitric oxide (NO) binds the active sites of cyto- sis,24 RNA processing,86 and levels of tRNA modifi- chrome c oxidase and displaces the heme-bound cation.202 Mitochondrial processing intermediates, oxygen.193 This could lead to oxygen depletion in consisting of RNA species referred to as RNA19, the surrounding tissue and decreased unbound NO, could play a significant role in this syndrome, as with an ensuing microvascular stroke-like pattern. pointed out in a recent review.139 These mitochon- Thus, the stroke-like episodes observed in MELAS drial processing intermediates corresponding to the syndrome could be caused by alterations in NO ho- 16S ribosomal RNA (rRNA), tRNALeu(UUR), and meostasis resulting in microvascular damage. the nicotinamide adenine dinucleotide dehydroge- Another common mutation in tRNALeu(UUR) is nase subunit 1 (ND1) genes were observed in cybrid the 3271 TϾC mutation at the anticodon stem re- cell lines expressing the 3243 AϾG.82 Since 16S gion, which is also associated with MELAS syndrome rRNA is contained in precursor RNA19, this tran- and diabetes mellitus. In transmitochondrial cybrid script could be incorporated into the , lines generated with this mutation, a lack of post- leading to stalling. The percentage corre- transcriptional modification,202 decreased steady- sponding to the 3243 AϾG mutation in the process- state levels,202 and abnormal intermediates of pro- ing intermediates is higher than the percentage ob- tein synthesis64 have been observed. The anticodon served in mtDNA, suggesting that the processing stem of the tRNALeu(UUR) is a loosely structured unit intermediates that contain mutations may be more formed by four AU basepairs within the wildtype difficult to process than their cellular counter- tRNA.197 The high AU content of the anticodon parts.86,87 stem causes weakness in this domain and susceptibil- In addition, this tRNA molecule has unstable ity to rupture when mismatch mutations such as the structural domains, and one of the effects of the 3271 TϾC is present. Chemical studies revealed that 3243 AϾG mutation might be the formation of a the anticodon stem was denatured in the mutant.197 dimeric complex that interferes with the function of This mutation caused an aminoacylation the tRNA within the context of protein-RNA assem- defect connected to Km abnormalities.197 bly.196 Since the DHU stem of human mitochondrial tRNALeu(UUR) contains only two Watson–Crick pairs, Mitochondrial tRNAIle Mutations. The mitochondrial the dimerization of this tRNA caused by this muta- tRNAIle gene is the third most commonly mutated tion may lead to its disruption. among the mitochondrial tRNA genes. Among the The observed reduction in complex I associated 12 reported mutations, 11 of them score above 20. with MELAS syndrome could be linked to a skewing The predominant clinical phenotypes associated of the tRNALeu anticodon availability. The mitochon- with mutations in this mitochondrial tRNA are iso- drial tRNALeu(UUR) gene harboring the common lated cardiomyopathy (4295 AϾG, and 4300 MELAS mutation lacks the normal modifica- AϾG),20,105 and progressive external ophthalmople-

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 157 gia (4274 TϾC, 4285 TϾC, 4298 GϾA, and 4309 cise intolerance and dystrophic changes on skeletal GϾA).13,43,156,171 Other phenotypes such as com- muscle.190 Because of the role of tRNAMet in trans- bined cardiomyopathy and PEO (4284 GϾA),26 mul- lation initiation, the high level of the mutation ob- tisystemic (4269 AϾG),170 encephalomyopathic served in this patient’s skeletal muscle is likely to (4267 AϾG),172 and hypertension and dyslipidemia cause a deficiency of mtDNA-encoded subunits, po- (4291 TϾC)195 have been described. Studies of dis- tentially affecting the capacity for ATP production. ease-related tRNAIle mutations using cell lines re- In addition, a computerized analysis revealed a vealed changes in tRNA stability in the presence of markedly altered structure induced by this muta- the 4269 AϾG mutation that affects an AU pair in tion.190 the acceptor stem of the human mitochondrial tR- Another mutation, a GϾA transition at position NA.Ile An accelerated tRNA degradation related to 4450 in this gene, was initially described by Lombes the loss of structural integrity caused by the mutation et al.92 in a patient with splenic lymphoma. The was observed.201 patient’s lymphocytes revealed abnormal mitochon- The 4285 TϾC and the 4298 GϾA mutations dria and multiple RC defects. Transfer of the mutant (located in the anticodon stem) diminished the ef- mitochondria to Rho0 cells demonstrated a severe ficiency of aminoacylation by as much as 1,000- respiratory defect. However the link of this mutation fold.79 It was also found that mutant tRNAs were able to the underlying disease remains unclear. to inhibit the charging of the wildtype tRNAIle80. Two studies have revealed that the cardiomyop- Mitochondrial tRNATrp Mutations. Six pathogenic athy-associated mutation 4317 AϾG induces an ab- mutations have been reported in this tRNA gene and errant secondary structure in the T-stem due to the cause a diverse clinical picture. A novel pathogenic instability introduced by the native CA pair.32,180 mutation 5521 GϾA was found to be associated with This aberrant structure may decrease aminoacyla- late-onset mitochondrial myopathy.157 Histochemi- tion. It seems that for this class of mutations in this cal and biochemical studies documented COX-neg- particular tRNA, a weak structure may amplify the ative ragged-red fibers. This mutation located in the effect of the mutation. D-stem could affect the tRNA-ribosome interaction, resulting in reduced mitochondrial protein synthe- Mitochondrial tRNAGln Mutations. Mutations in this sis. Alternatively, since this mutation is located close gene have been found in patients with either an to the origin of mtDNA light-strand replication, it encephalomyopathic or myopathic presentation. A could interfere with the regulation of the mtDNA heteroplasmic GϾA transition at nucleotide 4332 in light strand, altering the L-strand secondary struc- the tRNAGln gene was identified in a subject present- ture formed after the H-strand displacement. ing with stroke-like episodes, deafness, leukoenceph- The combination of Leigh syndrome, muscle alopathy, COX-negative ragged-red fibers, and basal COX deficiency, complex I deficiency, and a hetero- ganglia calcifications with late-onset findings that plasmic 5537 insertion T has been described on two were atypical for MELAS syndrome.6 occasions in different families by two independent Dey et al.34 reported a 12-year-old boy with a groups.137,182 This mutation affects the structure and myopathy in association with a single nucleotide in- stability of the anticodon stem region. Insertions in sertion, 4370insA (TϾAT), in the mitochondrial the tRNA genes are a rare cause of mitochondrial tRNAGln gene. The muscle biopsy was remarkable, a disorders, but these cases suggested that this muta- majority of the fibers were COX-deficient, and there tion should be screened in cases of Leigh syndrome were decreased steady-state levels of subunit II of and COX deficiency when mutations in SURF1 have COX and subunit 1 of complex I. Addition of the been excluded. extra nucleotide in the anticodon loop of this gene Other heteroplasmic point mutations in this changes seven evolutionary-conserved bases in the tRNA gene includes 5532 GϾA in a patient with a anticodon loop to eight. In many cases, tRNAs with neurogastrointestinal syndrome,96 a novel patho- eight or nine base anticodon loops have been asso- genic 5540 GϾA transition in a patient with sporadic ciated with frameshifting.183 Occasional frameshift encephalomyopathy characterized by spinocerebel- during translation may lead to a defect in mitochon- lar ataxia,158 a 5543 TϾC causing mitochondrial my- drial protein synthesis or misincorporation of amino opathy, and a 5549 GϾA mutation associated with acids. dementia, chorea, and diabetes. Complex IV defi- ciency may be the most common RC defect found in Mitochondrial tRNAMet Mutations. A 4409 TϾCmu- patients affected with mutations in the mitochon- tation was found in a 30-year-old woman with exer- drial tRNATrp gene. This finding may be explained

158 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 by the higher relative content of in COX tion could affect the anticodon loop structure of the subunits COI and COIII, 3.1% and 4.6%, respec- tRNAAsn since it is located next to the anticodon tively, compared to an average of 2.5% in mtDNA GUU. encoded complex I subunits.157 Another mutation at nucleotide position 5728 in the same tRNA gene was reported as the cause of Mitochondrial tRNAAla. Mutations in this gene have multiorgan failure.106 been associated with a myopathic phenotype. The Ͼ Cys T C transition at nucleotide position 5628 was iden- Mitochondrial tRNA Mutations. The 5783 GϾA tified in a patient with late-onset chronic progressive was found in a patient with myopathy, cardiomyop- external ophthalmoplegia (CPEO), dysphagia, and athy, renal failure, and sensorineural hearing loss mild proximal myopathy.160 Two mutations were (SNHL).38 Another pathogenic mutation is the 5814 found at the amino acid acceptor stem region: the TϾC mutation reported independently by three Ͼ Ͼ 5591 G A and the 5650 G A; both cause mitochon- groups to be associated with MELAS syndrome and 66,40,167 drial myopathy. Both mutations create a mis- with progressive external ophthalmoplegia in the matched basepair that can be designated as G4:U69 first two cases.76,95,136 This mutation shares similar and U6:G67 for 5650 GϾA and 5591 GϾA, respec- L features with the 3243 AϾG mutation in the tRNA - tively, according to the canonical structure. These eu(UUR)76 in that both mutations are located at the mismatches lie next to a G5:U68 wobble in the Ala D-stem/D-loop boundary (positions 14 and 13 at the amino acid acceptor stem of the tRNA molecule. cloverleaf). These nucleotides seem to provide sta- One role of the G5:U68 pair is to provide the tRNA bility to the tertiary structure of mitochondrial conformation that is most favorable for binding and tRNAs and regulate the efficiency of aminoacyla- positioning the tRNA acceptor end at the enzyme tion.55 catalytic site.45 The gain of an additional wobble may distort the conformation of the tRNA and render Mitochondrial tRNATyr. Patients with mutations in that structure unsuitable for aminoacylation. this gene predominantly exhibit a myopathic pheno- type, but more cases are required in order to estab- Mitochondrial tRNAAsn Mutations. Mutations in this lish a genotype–phenotype correlation. gene have been associated either with a CPEO phe- The first reported pathogenic mutation in this notype or with a myopathic phenotype. The first 123 Ͼ mutation associated with an A to G substitution in gene was a somatic 5874 T C transition in a nucleotide position 5692143 was found in a patient woman with exercise intolerance and bilateral ptosis. presenting with CPEO. The mutation could cause a The transition changed a highly evolutionarily con- possible reorganization of the anticodon loop lead- served nucleotide and may alter the transcriptional ing to inhibition of assembly of the ribosome-mRNA- processing or translation of mitochondrial proteins. tRNA complex required for translation. This mutation may preferentially affect complex III A de novo 5698 GϾA mutation has been de- and IV. This finding could be explained by the fact scribed in two patients presenting with a phenotype that the percentage of residues is among the consisting of isolated mitochondrial myopathy with highest in , COXI, and COX III sub- chronic progressive ophthalmoplegia.163,161 units. Two patients with early onset of progressive ex- Two additional cases with CPEO, myopathy, and ternal ophthalmoplegia and fatigability were de- exercise intolerance associated with mutations in Ͼ 126,131 scribed to have the 5703 GϾA mutation in the this tRNA gene were 5877 C T and 5885delT. tRNAAsn.61,192 Studies using transmitochondrial cy- The 5885delT shortened the aminoacyl-acceptor brid cell lines containing this mutation revealed im- stem.126 Since this 7-basepair stem is strictly con- Tyr paired oxidative phosphorylation and protein syn- served among mammalian mitochondrial tRNA thesis with severe reduction in steady-state levels of and is a canonical feature of tRNA in general, a the tRNAAsn pool.61 This was accompanied by a con- deletion may impair the formation of a proper sec- formational change in the tRNAAsn that may impair ondary and tertiary structure. The 5877 CϾT muta- aminoacylation. tion occurs at the DHU loop of this gene.131 This In contrast, the patient described by Coulbault et mutation seems to alter an essential residue for the al.27 presented with an encephalomyopathy and did configuration of the L-shaped tertiary structure of not have features of CPEO. A TϾC substitution at tRNA. Studies using transmitochondrial cybrids re- position 5693 in the tRNAAsn gene was found in vealed decreased oxygen consumption and compro- blood and muscle. It was suggested that this muta- mised cell viability.

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 159 Mitochondrial tRNASer(UCN). Patients carrying muta- ragged-red fibers, lactic acidosis, and respiratory tions in this gene usually present with SNHL. The chain complexes deficiency.51,72 mutations associated with SNHL include the 7443 AϾG, 7444 GϾA, 7445 AϾG, 7472 insC, the 7480 Mitochondrial tRNAAsp Mutations. Two probable AϾG, and the T to C changes at nucleotide positions pathogenic mutations in the tRNAAsp gene have 7510, 7511, and 7512. The pathogenic mutations been reported. The first heteroplasmic pathogenic located at the boundary between the termination mutation in this gene was reported154 in a patient codon of COI and the 3Ј end of the tRNASer(UCN) with myoclonic epilepsy and psychomotor regression gene (7443 AϾG, 7444 GϾA, and 7445 AϾG) affect and other affected family members. The mutation the processing and stabilization of the precursor abolished a highly conserved pairing in the antico- Ser(UCN) tRNA rather than affecting the termination don domain of the tRNAAsp gene that could affect codon of CO1.199 These mutations affect the second- the double helix, resulting in an alteration of the Ј ary structure at the 3 tRNA cleavage site for the secondary structure of this tRNA. In addition, an- L processing by tRNaseZ , leading to decreased steady- other pathogenic mutation in this gene was report- state levels of tRNA.90,200 ed148 in a young girl who presented with isolated The 7472 insertion C mutation was found in a progressive exercise intolerance. family who exhibited SNHL and a MERRF-like phe- 177 notype and in another patient with isolated myop- Mitochondrial tRNALys Mutations. The tRNALys is an athy, expanding the clinical phenotype of the muta- example of an oversimplified structure with a small 124 tion. The insertion adds a seventh cytosine to a DHU loop. The small DHU loop may limit the num- ⌿ six-cytosine run in the T C loop, potentially alter- ber of contacts necessary to stabilize the tertiary fold ing the secondary structure of the mitochondrial Ser(UCN) and the intrinsically weak structure of the human tRNA gene. Analysis of cybrids containing mitochondrial tRNALys depends on the existence of the 7472 insertion C revealed a decrease in the modified bases to maintain tRNA integrity. The ex- abundance of this particular tRNA, suggesting that it istence of posttranscriptional modifications plays an 181 could be the result of altered processing. important role in stabilizing the structure of mito- In addition, the 7480 AϾG mutation was de- chondrial tRNALys. Proper folding and aminoacyla- scribed in a woman who presented with a progressive tion require a 1-methyladenine residue at nucleotide mitochondrial myopathy and later developed SNHL, 9 to prevent formation of an alternative structure dementia, and ataxia.9 This mutation could with an extended acceptor stem.65 lengthen the anticodon stem by additional pairing, The 8344 AϾG mutation located in this gene is drastically reducing the size of the anticodon loop, present in 80%–90% of patients with myoclonic ep- and compromising anticodon function by the dis- ilepsy associated with ragged-red fibers (MERRF).36 ruption of codon matching. These SNHL-related tRNASer(UCN) mutations oc- Cybrid cells carrying this mutation demonstrated curred multiple times on different genetic back- reduced protein synthesis as a consequence of di- grounds at either heteroplasmic or homoplasmic minished tRNA stability and concomitant reduction in the aminoacylation level for the mutant states, and the clinical severity does not appear to Lys37 correlate with the proportion of mutant hetero- tRNA. plasmy.22,68,69,186,188 In addition to hearing impair- It has also been found that the levels of posttran- ment, there are inter- and intrafamilial differences scriptional modification in the wobble position of Lys in the penetrance of the neurological dysfunction. the anticodon tRNA were decreased, leading to These results and the lack of correlation between the disruptive translation.203 The disruption in transla- mutant load and the phenotype suggest that nuclear tion was related to the defective binding of the mu- Ͼ genes may play a role in the clinical expression of tant 8344 A G to the mRNA-ribosome complex and these mutations. Furthermore, the reason that these defective translation of both AAA and AAG codons mutations preferentially affect the function of the due to weak codon–anticodon interaction in this inner ear is currently not known. mutant tRNA. Further work revealed lack of taurine Interestingly, a heteroplasmic mutation in this modification at the wobble of mitochondrial same tRNA, 7497 GϾA, does not cause SNHL but tRNALys from pathogenic cells of MERRF patients rather a mitochondrial myopathy.51,72 This mutation carrying 8344 AϾG mutation.165 The 2-thio group of has been reported in a 13-year-old girl with exercise the wobble uridine is involved in mediating a stable intolerance and lactic acidosis, and in another two codon–anticodon interaction,4 hence the loss of the unrelated families with progressive myopathy, 2-thio group associated with this mutation is thought

160 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 to be one of the main reasons for the weak binding tional modification, which suggests functional rele- to cognate codons. vance.15 Two other pathogenic mutations in this same gene, 8356 TϾC and 8363 GϾA, seem to account for Mitochondrial tRNAHis. The heteroplasmic 12147 most of the remaining MERRF cases36 and more GϾA mutation was found in a patient with a MELAS recently the 8361 GϾA has been added to the ex- phenotype that eventually evolved into a MELAS/ panding mutational spectrum of MERRF.129 How- MERRF phenotype.103 The change most likely would ever, the clinical phenotype associated with muta- perturb secondary and tertiary structure.103 As pre- tions in this gene can be broad. The 8361 GϾA viously described,60 mutations in the dihydrouridine mutation has been associated with cardiomyopa- arm might cause mitochondrial dysfunction by im- thy129 and other mutations in the same gene are pairing mitochondrial protein synthesis. This may be associated with exercise intolerance (8300 TϾC),46 pertinent for COIII, the richest polypeptide in histi- atypical MELAS (8296 AϾG),133 and a phenotype dine residues,103 which may account for the associ- evocative of mitochondrial neurogastrointestinal en- ated COX deficiency. cephalomyopathy (MNGIE) syndrome (8313 Another pathogenic heteroplasmic mutation in GϾA).189 In addition, other clinical presentations tRNAHis is the 12183 GϾA at the T stem region include encephalomyopathy (8328 GϾA),67 myop- identified in a patient with pigmentary retinopathy, athy (8362 TϾG),163 PEO and myopathy (8355 SNHL, muscle hypotrophy, and ataxia.28 TϾC),163 and PEO with movement disorder (8342 GϾA).179 The potential pathomechanistic effects of Mitochondrial tRNASer(AGY) Mutations. Two families the 8344 AϾG mutation were studied in vitro and with novel 12258 CϾA mutation in the tRNASer(AGY) the mutant transmitochondrial cybrids exhibited gene have been reported. In the first case the patient poor respiration and defective mitochondrial pro- had a history of cataracts, SNHL, cerebellar ataxia, tein synthesis and respiratory chain enzyme activity and diabetes.93 The second time the mutation was in addition to a marked decrease in tRNALys steady- reported in a large pedigree of five generations with state levels and aminoacylation.5 24 affected matrilineal family members presenting with retinitis pigmentosa and progressive SNHL.97 Mitochondrial tRNAGly Mutations. Two pathogenic The 12258 CϾA substitution altered a highly con- mutations in this tRNA gene have been reported. served basepair in the amino acid acceptor stem that The 9997 TϾC mutation caused hypertrophic car- potentially could affect the secondary and tertiary diomyopathy.104 The 10010 TϾC mutation has been structures and therefore the function of this associated with diverse clinical phenotypes that in- tRNA.162 It is interesting to note that the only re- clude an encephalomyopathic presentation with sei- ported pathogenic mutation in the tRNASer(AGY) zures, choreoathetosis, ataxia, and spastic tetrapare- gene, 12258 CϾA change, causes SNHL, similar to sis10; a myopathic presentation with fatigue and mutations in tRNASer(UCN). It is not clear why muta- elevated serum creatine kinase114; and a multisys- tions in the tRNASer genes predominantly cause temic presentation with exercise intolerance, poly- SNHL. neuropathy, and hypothyroidism.29 This mutation is located within a highly conserved stem region of the Mitochondrial tRNALeu(CUN) Mutations. The myo- DHU arm, which underlies its pathogenicity. pathic phenotype is predominantly associated with the mutations in this tRNALeu(CUN). The 12297 TϾC Mitochondrial tRNAArg Mutations. The heteroplas- mutation, located right next to the anticodon, mic 10438 AϾG in the tRNAArg gene was found in a caused dilated cardiomyopathy. The heteroplasmic boy affected with mild cognitive impairment, ataxia, 12315 GϾA mutation was diagnosed in a young nystagmus, and muscle weakness.185 This mutation woman affected with exercise intolerance, bilateral changed the nucleotide flanking the anticodon. ptosis, and PEO.77 The mutation disrupts a highly Point mutations flanking the anticodon triplet may conserved G-C basepair in the T⌿C stem of the affect codon matching and potentially tRNA syn- molecule. This mutation had been previously re- thetase recognition, ultimately leading to a decrease ported in a middle-aged man who had developed in the accuracy and rate of translation.204 The posi- ptosis, CPEO, weakness, retinitis pigmentosa, hear- tion 10438 in mitochondrial DNA corresponds to ing loss, and peripheral neuropathy.44 position 37 in the tRNA molecule. Through se- Another heteroplasmic mutation 12320 AϾG af- quencing of human mitochondrial tRNAs, it is fecting the T⌿C loop was identified in a patient with known that this nucleotide undergoes posttranscrip- an isolated skeletal myopathy.194 In vivo, this muta-

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 161 tion increased over time from 70%–90%. Concur- ria for probable pathogenicity is the 15915 GϾA. rently, the patient’s myopathy progressed from mild This mutation was found in a patient with mitochon- to profound. drial encephalomyopathy, myoclonus, ragged-red fi- The somatic 12334 GϾA mutation affecting the bers, and progressive calcification in the basal gan- acceptor stem was identified in a subject who exhib- glia and cerebral atrophy.116,144 ited an isolated skeletal myopathy.191 The patient presented with exercise intolerance, ragged-red fi- Mitochondrial tRNAPro Mutations. The 15990 CϾT bers, and deficiencies of complex III and IV in skel- mutation in the tRNAPro gene has been associated etal muscle. with myopathy and CPEO.109 This mutation would convert the anticodon into a antico- Mitochondrial tRNAGlu Mutations. A 14687 TϾCso- don resulting in altered charging properties and matic mutation affecting the T⌿C loop of the mito- impairment of aminoacylation. The tRNA anticodon chondrial tRNAGlu was associated with mitochon- swap could affect protein synthesis by enhancing drial myopathy, respiratory failure, COX-negative misprocessing of polycistronic primary transcripts, ragged-red fibers, and complex I and IV deficien- amino acid misincorporation, and reduced or defec- cies.16 tive charging of the mitochondrial tRNAPro gene. The 14696 AϾG mutation was detected in a pa- This mutation could also have effects on the tient with psychomotor retardation, hypotonia, dys- posttranscriptional modification of other nucleo- phasia, combined complex I and III deficiencies, tides and potentially lead to hypomethylation or no and ragged-red fibers. This mutation changes a nu- in nucleotide 37 in the mitochondrial cleotide in the DHU stem, creating a novel basepair tRNAPro gene.15 This hypomodification may attenu- and reducing the wobble.185 ate the severity of disease and act as a negative The heteroplasmic 14709 TϾC mutation in the determinant interfering with the recognition of a tRNAGlu gene has been reported several times, pre- tRNA by a noncognate aminoacyl-tRNA syn- senting with a very heterogeneous clinical pheno- thetase,125 leading to mischarging of the mutant type including diabetes, myopathy, ataxia, congeni- tRNA instead of a complete lack of charging. tal encephalopathy, and a presentation suggestive of facioscapulohumeral muscular dystrophy.56,59,102 ASCERTAINMENT BIAS Some of the clinical diversity could be accounted for Mitochondrial tRNA mutations have been tradition- by the intratissue variability of the mutant load, but ally characterized by the focal accumulation of large the involvement of nuclear factors cannot be dis- numbers of morphologically and biochemically ab- counted. It has been documented that the mutation normal mitochondrial that present as ragged-red has attained homoplasmy within a family and in a fibers on skeletal muscle histology.153 This finding variety of tissues.102 Cybrid studies revealed that this may have led to an ascertainment bias since screen- mutation alone was not sufficient to lead to impair- ing for mutations in mitochondrial tRNA genes is ment of mitochondrial function in these homoplas- usually done when ragged-red fibers are found, per- mic cybrid clones.121 Reduced levels of mutant haps contributing to the disproportionately large tRNAs were observed. This nucleotide located imme- number of reported mitochondrial tRNA mutations. diately next to the anticodon may be important to maintain the stability of the tRNA for codon-bind- ing. PATHOGENICITY AND LOCATION OF MUTATIONS IN THE THREE-LEAVES CLOVER STRUCTURE OF tRNA The 14710 GϾA mutation3 was associated with a phenotype consisting of retinopathy, PEO, ptosis, The different distribution patterns of pathogenic and mitochondrial myopathy. This mutation re- and neutral variants in the tRNA structure may prove sulted in an anticodon swap from Glu (CUU) to Lys useful in devising an algorithm to predict the patho- (UUU) in this particular gene, potentially having an genic effects of novel mitochondrial tRNA changes. effect on tRNA aminoacylation. This is one of the After reviewing and scoring the published muta- few mitochondrial tRNA mutations that changes an tions for pathogenicity based on our scoring system, anticodon amino acid. The other one is the 15990 it is apparent that a significant percentage of muta- CϾT in the tRNAPro gene, that changes to serine tions are not supported by sufficient evidence to be (see below).109 considered pathogenic. These are variations that score below 20 points and are considered to be of Mitochondrial tRNAThr Mutations. The only mutation benign or possible pathogenicity. Of the listed mu- in the mitochondrial tRNAThr gene that meets crite- tations on MITOMAP and PubMed, 4.8% are con-

162 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 sidered neutral variants. If we add the mutations with Among the three genes the tRNALeu(UUR) gene is possible pathogenicity (14.5%), it is found that a hotspot for pathogenic mutations consistent with about 19% of the surveyed mutations either consti- the findings of a previous study.141 Moreover, we tute benign variants or have weak evidence for observed the highest sequence variability (22 vari- pathogenicity (Tables 3, 4). Some of these published ants) and pathogenic mutation (definite plus prob- mutations were reported before the canonical crite- able) to nonpathogenic variant (possible plus un- ria35 were established. It is interesting to note that in likely) ratio (19:3 ϭ 6.3) occurred in the the article published by McFarland et al.101 26% of mitochondrial tRNALeu(UUR) gene. However, these the reviewed mutations listed as pathogenic on observations may also reflect an ascertainment bias MITOMAP did not have enough evidence to be since, in some studies of mitochondrial patients with considered pathogenic. The same group also stated defined clinical syndromes (MERRF, MELAS, mito- that only 16% of the analyzed mutations were patho- chondrial myopathy, PEO, and Leigh syndrome), genic beyond doubt,101 in contrast to our review, only the mitochondrial tRNALeu(UUR) and tRNALys where around 48% of the analyzed mutations have genes were targeted to look for known mutations definite pathogenicity (Tables 3, 4). previously associated with these particular clinical The difference in results could be accounted by phenotypes, thus ignoring the potential contribu- the different scoring criteria used in both studies tion of other mitochondrial tRNA genes to these and by the fact that their analysis only included phenotypes. The distribution of pathogenic muta- mutations listed on MITOMAP as pathogenic as of tions encompasses all of the 22 mitochondrial tRNA December 1, 2003, whereas our review includes mu- genes (http://www.mitomap/org) (Table 3). tations listed on the same database as pathogenic as Our review revealed that although the distribu- of January 31, 2007. In addition, mutations retrieved tion of mitochondrial tRNA mutations includes any from PubMed and not contained in MITOMAP were domain of the cloverleaf structure, most of the also included. The reports of newly described muta- pathogenic mutations occur in stem structures since tions have included a more careful functional anal- among the 100 mutations (definite plus probable) ysis of their pathogenicity. found in mitochondrial tRNA genes, 68% occurred A small number of common mutations are re- in stem structures. There was also a predominance of sponsible for the majority of mitochondrial disor- possible pathogenic mutations in stem structures (12 ders due to mitochondrial DNA mutations. Three of 18; 66.6%). In the case of benign polymorphisms, mitochondrial tRNAs (tRNAIle, tRNALeu(UUR), and mutations in loop structures (5 of 6; 83.3%) predom- tRNALys) concentrate almost half (41%) of the inated over mutations in stem regions (1 of 6; known mutations associated with definite and prob- 16.6%). able pathogenicity (Table 3). This finding has been McFarland et al.101 also commented on the fact noted before,58 and the percentage found in our that the majority of pathogenic mutations in tRNAs review is in agreement with previous reviews196 are located in stem structures (73%), with mutation where mutations in those three genes accounted for hotspots in the anticodon and the aminoacyl accep- almost half of the mutations deemed to be associated tor stems.101 According to our review, 45 definite with pathogenicity. and probable mutations (45 of 100; 45%) occur in

Table 4. Distributions of tRNA mutations according to affected region of cloverleaf structure. Definite Probable Possible Benign (Ն 30) (20–29) (10–19) (Յ 9) Total Acceptor stem 15 9 3 1 28 AC stem 12 9 3 0 24 AC loop 9 4 2 1 16 T␺C stem 5 4 3 0 12 T␺C loop 5 2 2 3 12 DHU stem 10 4 3 0 176 DHU loop 1 5 1 1 8 Variable 3 1 1 0 5 Connector region (Acceptor/DHU stem) 0 1 0 0 1 DHU/AC stem 0 1 0 0 1 Total 60 40 18 6 124

See Table 2 for abbreviations.

Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 163 the acceptor and anticodon stems, edging closer to tent with previous studies,41,142,163 the known patho- 50% of the total number of pathogenic mutations. genic 8344 AϾG transition in the mitochondrial Alterations in these regions may exert significant tRNALys gene affects a nonconserved nucleotide. impact on codon binding and amino acid charging This implies that the presence of a variant at a con- thus impairing mitochondrial protein synthesis. served site is not an absolute requirement to predict Few mitochondrial tRNA genes harbor mutations its pathogenicity.41 in the loop structures. However, an exception occurs At the level of the primary sequence the changes in the mitochondrial tRNALeu(UUR) gene, where five introduced by the mutations in mitochondrial tRNA pathogenic mutations (one definite and four prob- genes are known to be mainly transitions (replace- able) are located in the dihydrouridine loop (DHU ment of a by a purine or a by a loop) (see Table 2). Of interest, the structure of this pyrimidine) rather than transversions (replacement particular mitochondrial tRNA gene might explain of a purine by a pyrimidine or vice versa). Upon the why mutations occurring in the loop may signifi- analysis of our data we found that an overwhelming cantly affect the function. The tertiary structure of majority of pathogenic mutations were transitions this mitochondrial tRNA gene is different from the (89%), with the remainder transversions (5%), and structure of the cytosolic tRNAs and this difference is deletions/insertions (6%). These findings support achieved by a process of posttranscriptional modifi- the notion that transversions may be extremely det- cations.159 This tRNA has the largest DHU loop and rimental from a cellular standpoint and that a large T⌿C loop. An alteration in the base compo- “milder” changes are better tolerated.41 It is plausi- sition of these large loops may render this structure ble to speculate that it might be easier to achieve a vulnerable by altering the tertiary hydrogen bonds in transition than a transversion due to the structural these loops that are critical in maintaining the sta- similarity. The predominance of chemically “mild” bility of this tRNA molecule. Structural anomalies mutations is a concept that was first introduced by caused by the 3243 AϾG mutation were noted in the Florentz and Sissler.41 In addition, among the tran- folding of the DHU-arm of the mitochondrial sitions there is a predominance of AG changes (57 of Leu(UUR) tRNA , but structural anomalies were not ob- 89; 64%) when compared to TC changes (36%). served when the mutation was in the stem structure One of the most common oxidation products of as in the case of the 3271 TϾC.159 Another example mtDNA, 8-oxoguanine, has been found at high levels of mutations that occur in large loop structures is the in human cells under conditions of oxidative stress,2 Ͼ Lys 8344 A G mutation in the tRNA gene. This mi- suggesting a possible role of oxidative stress in mito- ⌿ tochondrial tRNA has the largest T C loop, and a chondrial DNA damage and generation of muta- mutation in this structure may also affect the integ- tions. However, it is not known whether this could be rity of the tRNA. Thus, when pathogenic mutations one of the mechanisms responsible for the predom- occur in loop structures, they tend to occur in loops inance of AG changes. that are unusual in size and influence the tertiary Although most of the mutations analyzed in this folding or function of the tRNA. review appeared to be inherited, eight patients were It is interesting to note that substitutions in the found to exhibit somatic mutations that probably anticodon triplet are extremely rare. Only three arose during early . In this Ͼ Phe pathogenic mutations, the 611 G A in the tRNA , small cohort there was a predominance of females (8 Ͼ Glu Ͼ the 14710 G A in the tRNA , and the 15990 C T of 11; 72.7%). However, more patients with somatic Pro in the tRNA have been determined to affect any of mutations in mitochondrial tRNA genes will need to 3,94,109 the three bases of the anticodon. The 611 be analyzed in order to know whether there is a clear Ͼ G A involves a wobble base change and it does not gender trend due to a biological mechanism that is change the amino acid to be charged. The other two still elusive to us. anticodon mutations indeed alter the amino acid to be charged as specified by the tRNA. Due to the HOMOPLASMY OF PATHOGENIC MUTATIONS fundamental role played by the anticodon in select- ing tRNA identity, a high of mutations is Among the 100 pathogenic mutations described in not expected in these preserved residues, as they this review, 11 occurred in the homoplasmic state, in would lead to lethal cellular consequences incom- which clinical expression and disease severity do not patible with . correlate with mutant load in affected tissues. Appar- Although most of the pathogenic mitochondrial ently, nuclear genes play a role in the disease expres- tRNA mutations listed in Table 3 (definite plus prob- sion of these homoplasmic mutations. Homoplasmic able) were found in highly conserved sites, consis- pathogenic mutations have also been reported in

164 Human Mitochondrial tRNAs MUSCLE & NERVE February 2008 mitochondrial genes encoding for mRNAs. The in- mutations per kb for mRNA and tRNA genes, respec- creasing frequency of reports of homoplasmic muta- tively. There is almost 10-fold higher frequency in tions of definite and probable pathogenicity weakens generating pathogenic mutations in tRNA genes a rigid concept of heteroplasmy as sine qua non of than in mRNA genes. If it is assumed that the muta- pathogenicity, requiring a more comprehensive tion events occur randomly regardless of the nucle- functional approach to clearly determine pathoge- otide location, these results suggest that nucleotide nicity in these cases. substitutions in tRNA genes are more detrimental from a structural and functional standpoint than CORRELATION BETWEEN CLINICAL PHENOTYPE mutations in mRNA genes. Nucleotide substitutions AND NATURE OF tRNA MUTATION in tRNA genes may affect their fragile structure, unlike nucleotide substitutions in mRNA genes that There is a wide range of clinical manifestations that do not lead to a disruption in mitochondrial protein are characteristic of disorders caused by mtDNA mu- synthesis. tations, and in particular of patients harboring mu- tations in the mitochondrial tRNA genes. As de- CONCLUSIONS scribed earlier, there appears to be little correlation between the clinical manifestations observed in a Further analysis of an expanded series of tRNA mu- particular patient and the specific mitochondrial tants will be required in order to generate a consen- tRNA harboring that mutation. However, mutations sus or framework that would allow the description of at the same position in the cloverleaf structure of the cellular and phenotypic effects of disease-related different tRNA genes may be associated with similar tRNA mutations. It is important to consider the ef- clinical phenotypes.142 Mutations at nucleotide posi- fects of pathogenic mutations on the structure of tion 4284 in the tRNAIle and at nucleotide position mitochondrial tRNA genes because the structural 5703 in the tRNAAsn, both at position 27 of the tRNA distortions probably underlie the biochemical and structure, are associated with PEO. In addition, mu- clinical phenotypes observed in these patients. The L tations at nucleotide position 3303 of the tRNA - human mitochondrial tRNAs represent a class of Lys eu(UUR) and nucleotide position 8363 of the tRNA , tRNAs with intrinsically weak structures that may be both at position 72, are associated with cardiomyop- the result of expedited and streamlining of athy. Similarly, mutations at nucleotide position the human mitochondrial genome. The inherent 4269 of the tRNAIle and at nucleotide position 9997 conformational fragility and instability of these of the tRNAGly, both at position 7, are associated with tRNAs may amplify the effects of single-base substi- cardiomyopathy. Encephalomyopathy has been ob- tutions, by disrupting a native conformation of struc- served in association with mutations at nucleotide tural elements or by promoting the formation of positions 3271 of the tRNALeu(UUR) and 5549 of the intermediate, unstable, non-native structures. tRNATrp, which correspond to position 39. Lastly, multisystemic involvement was observed with muta- tions at nucleotide positions 1606 of tRNAVal and REFERENCES nucleotide position 7512 of tRNASer(UCN) that corre- 1. Allen JF, Raven JA. Free-radical-induced mutation vs redox sponded to position 5 of the cloverleaf structure. regulation: costs and benefits of genes in . J Mol Evol 1996;42:482–492. These findings may add further evidence to support 2. Ames BN. 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