MTO1 mutations are associated with hypertrophic cardiomyopathy and lactic acidosis and cause respiratory chain deficiency in humans and yeast

Abstract

We report three families presenting with hypertrophic cardiomyopathy, lactic acidosis, and multiple defects of mitochondrial respiratory chain (MRC) activities. By direct sequencing of the candidate gene MTO1, encoding the mitochondrial-tRNA modifier 1, or whole exome sequencing analysis, we identified novel missense mutations. All MTO1 mutations were predicted to be deleterious on MTO1 function. Their pathogenic role was experimentally validated in a recombinant yeast model, by assessing oxidative growth, respiratory activity, mitochondrial protein synthesis, and complex IV activity. In one case, we also demonstrated that expression of wt MTO1 could rescue the respiratory defect in mutant fibroblasts. The severity of the yeast respiratory phenotypes partly correlated with the different clinical presentations observed in MTO1 mutant patients, although the clinical outcome was highly variable in patients with the same mutation and seemed also to depend on timely start of pharmacological treatment, centered on the control of lactic acidosis by dichloroacetate. Our results indicate that MTO1 mutations are commonly associated with a presentation of hypertrophic cardiomyopathy, lactic acidosis, and MRC deficiency, and that ad hoc recombinant yeast models represent a useful system to test the pathogenic potential of uncommon variants, and provide insight into their effects on the expression of a biochemical phenotype.

Keywords: MTO1; hypertrophic cardiomyopathy; lactic acidosis; mitochondrial disorder; yeast.

Figure 1

Pedigrees and radiological features. A: Pedigrees and electropherograms of the MTO1 genomic region encompassing the nucleotide substitutions in patients and available parents. Black symbols designate affected subjects. B: Brain MRI of Pt1. Transverse FLAIR image showing abnormal hyperintensity in the region of the claustrum and surrounding capsulae (arrows). C: Brain MRI of Pt2. Coronal T2-weighted sequence showing abnormal hyperintense signals of the thalami and diffusely abnormal signal in the subcortical white matter. Lesions are also present in the brainstem. The cerebellar folia are normal.

Figure 2

In silico structural analysis. A: Alignment of Mto1 proteins from animals, yeasts, plants, and eubacteria around mutated amino acids. In yellow the amino acids corresponding to mutations hThr411Ile and hArg477His, in green the amino acid corresponding to mutation Ala428Thr [Ghezzi et al., 2012]. The corresponding secondary structure elements for the C. tepidum GidA structure are indicated and colored according to Meyer et al. [2008]. B: Structure of the C. tepidum GidA region around the FAD moiety. Amino acids of the motif 2 of GidA which bind the FAD group are indicated. For simplicity, the numbers refer to the equivalent position in yeast Mto1. C: Structure of the C. tepidum GidA region around the FAD moiety superimposed to the model structure of GidA. The wt structure of the amino acids equivalent to threonines 414 (T414) and 415 (T415) in yeast Mto1 is in red. The predicted structure of the amino acids equivalent to yeast mutant isoleucine 414 (I414, corresponding to the human mutation Thr411Ile) and adiacent threonine 415 (T415) is in yellow. For simplicity, the numbers refer to the position in yeast Mto1. D: Overall structure of the C. tepidum GidA with basic amino acids (in red), which form a pocket who is predicted to bind the D-stem of the incoming tRNA. The bacterial Arg436 residue (R436), equivalent to hArg477 and yArg481, is in magenta.

Figure 3

Yeast studies. A: Growth of Δmto1 strain transformed with MTO1 wt allele, mto1^R481H, and mto1^T414I mutant alleles or empty plasmid on YP medium supplemented with 2% glucose (left panel) or 2% glycerol (left panel). Cells were pregrown on YP+glucose and plated after serial dilutions to obtain spots of 5 × 10⁴, 5 × 10³, 5 × 10², and 5 × 10¹ cells/spot. Pictures were taken after 2 days of growth. B: Respiratory activity of Δmto1 strains transformed with MTO1 wt allele, mto1^R481H, and mto1^T414I mutant alleles or empty plasmid. Respiratory rates were normalized to the strain transformed with wt MTO1, for which the respiratory rate was 34.7 nmol min⁻¹ mg⁻¹. Values are the mean of three independent experiments, each with an independent clone. Two-tail, paired t-test was applied for statistical significance. ^***P < 0.001. C: In vivo mitochondrial translation of Δmto1 strain transformed with MTO1 wt allele, mto1^R481H, and mto1^T414I mutant alleles or empty plasmid. Mitochondrial gene products were labeled with [³⁵S]-methionine in whole cells in the presence of cycloheximide for 10 min at 28°C. Cox: cytochrome c oxidase; Cob: cytochrome b; Atp: ATP synthase; Var1: small mitochondrial ribosome subunit. D: Cytochrome c oxidase (CIV) activity of Δmto1 strain transformed with MTO1 wt allele, mto1^T414I, mto1^A431T, mto1^R481H, and mto1^P622X mutant alleles or empty plasmid. Cytochrome c oxidase activities were normalized to the strain transformed with wt MTO1, for which the activity was 368.8 units per mg of mitochondrial proteins. Values are the mean of three independent experiments, each with an independent clone. Two-tail, unpaired t-test was applied for statistical significance. ^**P < 0.01.