MTO1 Mutations are Associated with Hypertrophic Cardiomyopathy and Lactic Acidosis and Cause Respiratory Chain Deficiency in Humans and Yeast

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.

The clinical course during the following years was complicated by feeding difficulties, failure to thrive and neurological symptoms, e.g. myoclonic seizures for the first years of life. Nowadays, aged 14 years, her weight is at the 10 th percentile, neurological development is moderately delayed, with hypotonia, dystonia and poor speech. Several brain MRIs showed abnormal bilateral hyperintensities in the capsulae surrounding the claustra ( Figure 1B). From the age of 8 years she has suffered of hypertrophic cardiomyopathy, particularly in the posterior wall of the left ventricle (6 mm, n.v. 4) with reduced systolic fraction (40%). At age 7 years, DCA treatment was stopped because of abnormalities of visual and brainstem evoked potentials and nerve conduction velocities. Subsequent metabolic follow-up revealed mildly elevated blood lactate but no further episodes of metabolic acidosis. A second muscle biopsy at 8 years again showed severe reduction of CI (14%) and CIV (27%) activities, whereas the other MRC activities were normal. Oxygen consumption, assessed through micro-oxygraphy in cultured fibroblasts, displayed significant reduction of MRR, SRC and OCR/ECAR whereas RCR, an index of mitochondrial OXPHOS coupling, was normal (Supp . Table S2).

Patient 2
Patient 2 (Pt2) was born at 37 weeks of gestation (birth weight of 2.38kg; 4 th percentile) as the male first child of 1 st cousin consanguineous Pakistani parents (Family 1; Figure 1A). On the first day of life, he developed severe poor feeding and mild hypoglycaemia and was admitted to the Special Care Baby Unit. Over the first 3 months of life, he developed hypotonia, his weight gain was poor and an echocardiography (performed at 3 months of age because of the detection of a cardiac murmur) demonstrated severe left ventricular hypertrophy with posterior wall thickness (8mm). Lactic acidemia was noted, with blood lactate varying between 9.5 and 14.6 mM. Metabolic work-up showed high plasma alanine (690μM, n.v. <400), and increased urinary lactate, 3-methylglutaconic acid and accumulation of Krebs cycle intermediates. A muscle biopsy, taken at 6 months of age, showed decreased staining for cytochrome c oxidase (COX) and severe deficiency in both CI and CIV activities (<10% residual activities). Brain MRI showed symmetrical, bilateral abnormal signals in fornices, globus pallidus, thalamus, subthalamic nucleus, substantia nigra, dorsal mesencephalon, pons and to a lesser extent dentate nuclei of the cerebellum ( Figure 1C). A lactate peak was detected on [H + ]-MR Spectroscopy.
The clinical course during the following months was complicated by persistent hypotonia and failure to thrive despite nasogastric feeding. At 12 months of age the child developed pneumonia associated with worsening metabolic acidosis and died of irreversible cardiorespiratory arrest.
Oxygen consumption assessed by micro-oxygraphy in cultured fibroblasts displayed significant reduction of MRR, SRC and OCR/ECAR with normal RCR (Supp. Table S2). The mtDNA sequence was normal.

Patient 3
Patient 3 (Pt3), the younger brother of Patient 2, was born at 34 weeks gestation (birth weight of 2.17kg; 25 th percentile). This child was born with hypospadias and an accessory digit at the base of the palmar aspect of the left thumb. In view of the family history, plasma lactate was monitored in the neonatal period and found to be elevated (7-10 mM). An echocardiogram at 1 month of age showed mild left ventricular hypertrophy with a posterior wall thickness of 7mm.
His subsequent clinical course was complicated by feeding difficulties and failure to thrive. He was admitted for nasogastric tube feeding and no further invasive investigations were performed at the family's wishes. He received palliative care and died suddenly at home at the age of 3 months.

Patient 4
Patient 4 (Pt4) was born at term as the first female child of 1 st cousin consanguineous Pakistani parents (Family 2; Figure 1A). No feeding or respiratory difficulties occurred in the perinatal period. At three months of age, she developed severe metabolic acidosis with lactic acidemia associated with bronchiolitis-like illness. Metabolic work-up showed increased urinary lactate. A muscle biopsy, taken at 3 months of age, revealed decreased histochemical reactivity for COX and a severe CIV deficiency (<10% of controls), with CI activity reported as normal.
Echocardiography demonstrated mild biventricular hypertrophic cardiomyopathy, which improved on serial scans over a number of years and did not require medication. The clinical course during the following years was complicated by speech and language delay, failure to thrive and recurrent hospital admissions with lactic acidosis associated with intercurrent infections with an admission to intensive care at age 2 years due to generalised seizures and encephalopathy. Nowadays, aged 19 years, her weight is <3 rd percentile, her psychomotor development is mildly delayed and she is in special secondary education. Her menarche occurred normally, at 13 years of age. The frequency and severity of admissions has reduced gradually with age and her last acute admission was aged 8 years. She has persistent fatigue with chronic lactic acidosis (5.0-8.0mM) and Vitamin D deficiency for which she takes regular ergocalciferol and sodium bicarbonate supplements. DCA was used regularly from the age of 16 years with a reduction in resting plasma lactate levels to 4.0 mM but has now been stopped.

Patient 5
Patient 5 (Pt5), the younger sister of Patient 4 was born at term (birth weight of 2890g; 9 th -25 th percentile), with no perinatal respiratory or feeding difficulties. At five months of age the patient developed severe metabolic acidosis and lactic acidemia (>22.0mM), associated with an upper respiratory illness. Echocardiography demonstrated dilated cardiomyopathy with left ventricular hypertrophy and an electrocardiogram demonstrated a Wolff-Parkinson-White syndrome.
Metabolic work-up showed high plasma alanine (695μM), and increased urinary lactate with ketonuria and dicarboxylic aciduria. A muscle biopsy, taken at 7 months of age, showed similar findings to her sister, i.e. severe COX defect. She had a further severe decompensation aged 9 months associated with bronchiolitis and was ventilated for 4 weeks with repeated attempts at extubation failing due to rising lactic acidosis and worsening cardiomyopathy (fractional shortening 15%), with pericardial effusion. She required drug treatment for cardiac failure and her lactic acidosis was successfully treated with DCA at 50mg/kg/day with plasma lactate levels falling to 3.0 mM. The clinical course during the following years was complicated by psychomotor delay, increasing lower limb spasticity, failure to thrive (needing nasogastric feeding) and recurrent hospital admissions with lactic acidosis associated with intercurrent infections. Nowadays, aged 12 years of age, her weight and height are at <3 rd percentile and psychomotor development is severely delayed. She is able to walk independently but has no speech and limited non-verbal communication; she is also in special education. The frequency and severity of admissions has reduced gradually with age and her last acute admission was at aged 11 years. Her cardiomyopathy has gradually improved with a fractional shortening of 30% and she takes only digoxin and lisinopril. Chronic DCA has been discontinued. Full mitochondrial DNA sequencing was negative in this family, as was the diagnostic screening of several COX assembly factor genes including COX10 and COX15.