Structures of the ribosome bound to EF-Tu-isoleucine tRNA elucidate the mechanism of AUG avoidance.

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.

Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm2C), respectively, in the anticodon of tRNAIle to decipher AUA codon. L and agm2C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAsIle recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N6-threonylcarbamoyladenosine (ct6A), another tRNA modification, and revealed a molecular basis how ct6A contributes to efficient decoding.

Recognition of tRNAIle with a UAU anticodon by isoleucyl-tRNA synthetase in lactic acid bacteria.

In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum has a tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).

Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA.

BACKGROUND: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. METHODS: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. RESULTS: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. CONCLUSION: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.

A deafness-associated tRNA mutation caused pleiotropic effects on the m1G37 modification, processing, stability and aminoacylation of tRNAIle and mitochondrial translation.

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.

Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility.

The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.

Mitochondrial tRNAIle A4317G mutation may be associated with hearing impairment in a Han Chinese family.

Mutations in the mitochondrial genome have been identified to be associated with hearing loss. The aim of the present study was to investigate the role of mitochondrial DNA (mtDNA) variants in a Chinese family with hearing loss. Polymerase chain reaction (PCR)­Sanger sequencing was used to screen the mtDNA variants and nuclear genes [gap junction protein ß2 (GJB2) and transfer (t)RNA 5­methylaminomethyle­2­thiouridylate methyltransferase (TRMU)]; in addition, the mtDNA copy number was determined by quantitative PCR. The present study characterized the molecular features of a Chinese family with maternally­inherited hearing loss and identified mtDNA A1555G and tRNAIle A4317G mutations. The A4317G mutation was localized at the TΨC arm of tRNAIle (position 59) and created a novel base­pairing (G59­C54), which may alter the secondary structure of the tRNA. In addition, patients carrying the A4317G mutation exhibited a lower mtDNA copy number compared with the controls, suggesting that this mutation may cause mitochondrial dysfunction that is responsible for the deafness. However, no functional variants in the GJB2 and TRMU genes were detected. mtDNA A1555G and A4317G mutations may contribute to the clinical manifestation of hearing loss in this family.

Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle.

Prokaryotic toxin-antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.

First description of a novel mitochondrial mutation in the MT-TI gene associated with multiple mitochondrial DNA deletion and depletion in family with severe dilated mitochondrial cardiomyopathy.

Mitochondria are essential for early cardiac development and impaired mitochondrial function was described associated with heart diseases such as hypertrophic or dilated mitochondrial cardiomyopathy. In this study, we report a family including two individuals with severe dilated mitochondrial cardiomyopathy. The whole mitochondrial genome screening showed the presence of several variations and a novel homoplasmic mutation m.4318-4322delC in the MT-TI gene shared by the two patients and their mother and leading to a disruption of the tRNAIle secondary structure. In addition, a mitochondrial depletion was present in blood leucocyte of the two affected brother whereas a de novo heteroplasmic multiple deletion in the major arc of mtDNA was present in blood leucocyte and mucosa of only one of them. These deletions in the major arc of the mtDNA resulted to the loss of several protein-encoding genes and also some tRNA genes. The mtDNA deletion and depletion could result to an impairment of the oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patients. Our report is the first description of a family with severe lethal dilated mitochondrial cardiomyopathy and presenting several mtDNA abnormalities including punctual mutation, deletion and depletion.

Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation.

The 1555A→G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A→G mutation. Here, we report that a deafness-susceptibility allele (m.4317A→G) in the tRNAIle gene modulates the phenotype expression of m.1555A→G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A→G and m.1555A→G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A→G mutation. The m.4317A→G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNAIle We therefore hypothesized that the m.4317A→G mutation alters both structure and function of tRNAIle Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A→G and m.4317A→G mutations, three harboring only m.1555A→G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A→G mutation. We also found that the m.4317A→G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNAIle These m.4317A→G mutation-induced alterations in tRNAIle structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A→G mutation. Furthermore, mutant cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A→G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations.

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum.

In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell.

The mitochondrial calcium uniporter is involved in mitochondrial calcium cycle dysfunction: Underlying mechanism of hypertension associated with mitochondrial tRNA(Ile) A4263G mutation.

Recent studies have shown that the mitochondrial DNA mutations are involved in the pathogenesis of hypertension. Our previous study identified mitochondrial tRNA(Ile) A4263G mutation in a large Chinese Han family with maternally-inherited hypertension. This mutation may contribute to mitochondrial Ca(2+) cycling dysfuntion, but the mechanism is unclear. Lymphoblastoid cell lines were derived from hypertensive and normotensive individuals, either with or without tRNA(Ile) A4263G mutation. The mitochondrial calcium ([Ca(2+)]m) in cells from hypertensive subjects with the tRNA(Ile) A4263G mutation, was lower than in cells from normotension or hypertension without mutation, or normotension with mutation (P<0.05). Meanwhile, cytosolic calcium ([Ca(2+)]c) in hypertensive with mutation cells was higher than another three groups. After exposure to caffeine, which could increase the [Ca(2+)]c by activating ryanodine receptor on endoplasmic reticulum, [Ca(2+)]c/[Ca(2+)]m increased higher than in hypertensive with mutation cells from another three groups. Moreover, MCU expression was decreased in hypertensive with mutation cells compared with in another three groups (P<0.05). [Ca(2+)]c increased and [Ca(2+)]m decreased after treatment with Ru360 (an inhibitor of MCU) or an siRNA against MCU. In this study we found decreased MCU expression in hypertensive with mutation cells contributed to dysregulated Ca(2+) uptake into the mitochondria, and cytoplasmic Ca(2+) overload. This abnormality might be involved in the underlying mechanisms of maternally inherited hypertension in subjects carrying the mitochondrial tRNA(Ile) A4263G mutation.

The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway.

The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.

A Member of the Arabidopsis Mitochondrial Transcription Termination Factor Family Is Required for Maturation of Chloroplast Transfer RNAIle(GAU).

Plastid gene expression is crucial for organelle function, but the factors that control it are still largely unclear. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate organellar gene expression at different levels. Arabidopsis (Arabidopsis thaliana) mTERF6 is localized in chloroplasts and mitochondria, and its knockout perturbs plastid development and results in seedling lethality. In the leaky mterf6-1 mutant, a defect in photosynthesis is associated with reduced levels of photosystem subunits, although corresponding messenger RNA levels are unaffected, whereas translational capacity and maturation of chloroplast ribosomal RNAs (rRNAs) are perturbed in mterf6-1 mutants. Bacterial one-hybrid screening, electrophoretic mobility shift assays, and coimmunoprecipitation experiments reveal a specific interaction between mTERF6 and an RNA sequence in the chloroplast isoleucine transfer RNA gene (trnI.2) located in the rRNA operon. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. At present, it is unclear whether disturbed rRNA maturation is a primary or secondary defect. However, it is clear that mTERF6 is required for the maturation of trnI.2. This points to an additional function of mTERFs.

[Mitochondrial DNA mutation associated with hypertension in tRNA(Ile) and tRNA(Gln) genes].

OBJECTIVE: To study the relationship between mitochondrial DNA (mtDNA) mutations and hypertension. METHODS: Clinical data of two pedigrees with maternally transmitted hypertension was collected. Whole mtDNA sequence was analyzed. RESULTS: The family members on the maternal side presented with various levels of hypertension, with the onset age ranging from 44 to 55 years old. Analysis of the mtDNA sequence of the two families members showed all patients have carried a matrilineal 4329C> G mutation of the tRNA(Ile) and tRNA(Gln) genes. The same mutation was not found in 366 healthy controls. The 4329C site of mtDNA is highly conserved across species, and has been associated with the fidelity of amino acid accept arm of the tRNAs, as well as functionality and stability in the formation of tRNAs. CONCLUSION: The 4329C> G point mutation in tRNA(Ile) and tRNA(Gln) probably has contributed to the pathogenesis of hypertension, possibly in association with other modifying factors.

Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase.

The accurate expression of genetic information relies on the fidelity of amino acid-tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs.

Molecular characterization of a Chinese family carrying a novel C4329A mutation in mitochondrial tRNAIle and tRNAGln genes.

BACKGROUND: Hypertension is a very common cardiovascular disease influenced by multiple genetic and environmental factors. More recently, there are some studies showed that mutations in mitochondrial DNA have been involved in its pathogenesis. In this study we did further investigations on this relationship. METHODS: Epidemiological research found a Han Chinese family with probable maternally transmitted hypertension. Sequence analysis of the whole mitochondrial DNA was detected from all the family members. And evaluations of the clinical, genetic and molecular characterization were also performed. RESULTS: Matrilineal relatives within the family exhibited varying degrees of hypertension with an onset age of 48-55 years. Sequence analysis of this pedigree showed a novel homoplasmic 4329C > G mutation located at the 3' end of the tRNAIle and tRNAGln genes that was absent from 366 Chinese controls. The cytosine (C) at 4329 position was very important in the structural formation and stabilization of functional tRNAs, which was highly conserved in mitochondria of various organisms and also contributed to the high fidelity of the acceptor arm. Cells carrying this mutation were also shown to harbor mitochondrial dysfunctions. CONCLUSIONS: The C4329G point mutation in tRNAIle and tRNAGln was involved in the pathogenesis of hypertension, perhaps in association with other modifying factors.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype.

BACKGROUND/AIM: To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS: Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS: The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS: We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.