Pathogenic variants in glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial cardiomyopathy disorder

Mitochondrial protein synthesis requires charging a mitochondrial tRNA with its amino acid. Here, the authors describe pathogenic variants in the GatCAB protein complex genes required for the generation of glutaminyl-mt-tRNAGln, that impairs mitochondrial translation and presents with cardiomyopathy.

inotropes, and persistent pulmonary hypertension treated with nitric oxide. Echocardiogram showed bradycardia, thickened myocardium primarily in the right atrium and atrial septum and moderately decreased contractility of both ventricles, and signs of pulmonary hypertension.
Adrenal function was normal with cortisol 245 nmol.L -1 (normal 150-500 nmol.L -1 ). Prominent metabolic acidosis with lactate 22.2 mmol.L -1 evolved. Despite intensive care treatment, the boy died on the second day of life. Autopsy revealed massively increased myelopoiesis and erythropoiesis, pulmonary hypoplasia and cardiomegaly (Figure 1h). Lysosomal storage disease testing in fibroblasts was negative.
Patient 1B (II-3): After a healthy daughter had been born for these same parents, this third pregnancy was complicated by intrauterine growth retardation, cardiomegaly and pericardial effusion. From 30 weeks gestational age on there was relative bradycardia of 110.min -1 , and from 33 weeks gestational age a pericardial effusion was present without signs of cardiac decompensation or other signs of hydrops. Investigations for TORCHES serology and SS-A and SS-B antibodies were negative. After a Caesarean section at a gestational age of 37 weeks, a girl was born with Apgar scores 3, 3, 7 after 1, 5, and 10 minutes, respectively. Respiratory insufficiency rapidly necessitated artificial ventilation. There was an asymmetric growth restriction with a birth weight of 1,525 g (-5.6 SD), length of 43 cm , and a head circumference of 29 cm (-3.8 SD). The infant had no dysmorphic features, and only mild peripheral edema. Heart rate was low at 105.min -1 and cardiac ultrasound revealed biventricular impaired cardiac contractility with a shortening fraction of 27% but no dilation (left ventricular diameters at the 90th percentile), and mild pericardial effusion. There was anemia with a hemoglobin concentration of 10.6 g.dL -1 . Initially there was hypoglycemia 19.8 mg.dL -1 at 2 hours after birth, which normalized within 4 hours after birth with 7.6 mg.kg -1 .min -1 intravenous glucose administration. Blood lactate levels increased from 21.6 mmol.L -1 to 33.6 mmol.L -1 . In subsequent hours vital functions could not be stabilized despite intensive ventilatory and inotropic support, and carnitine supplementation, and she died within 24 hours after birth. Metabolic investigations of plasma acylcarnitines and sialotransferrin profile were normal, and urine organic acid analysis showed only massive lactate excretion. A microarray was normal. A respiratory chain defect was clinically suspected. Clinical sequencing did not identify pathogenic variants in mtDNA, or in the TUFM, TSFM,GFM1,GFM2,SCO2,or POLG gene. An autopsy was declined.

Family 2
Patient 2A (II-2): The proband was the second child to non-consanguineous parents of Caucasian and Japanese descent (Figure 1c). Their previous child was a healthy male. The proband was born by normal delivery after an uneventful pregnancy, and only had a brief admission for transient hypothermia with normal glycemia. He had normal newborn screening but failed his hearing screen and had bilateral profound sensorineural hearing loss. At 3 months of age, he presented acutely unwell with fever followed by encephalopathy, hypotonia, and poor perfusion. He was hypoglycemic with metabolic lactic acidosis pH 7.23, bicarbonate 6.3 mM, anion gap 25.3 mmol.L -1 , BE -20 mEq.L -1 , and lactate 14.9 mM. There was hepatomegaly confirmed on ultrasound, with mildly elevated transaminases AST 89 IU.L -1 , γ-GT 304, and abnormal clotting with an INR 1.4 (normal 0.9-1.3), aPTT 57 sec (normal 28-41), fibrinogen 0.88 g.L -1 (1.80-4.0) without D-dimers. Hemoglobin was 10 g.dL -1 (normal 10-14). After normalization of the glycemia, fluid status and cardiovascular status, the lactic acidosis persisted, albeit improved (lactate 6.4 mM), and he remained lethargic. Multiple viral studies, ammonia, lipid, and α1-antitrypsin were normal. Urine organic acids showed ketonuria, lactate and pyruvate, Krebs cycle intermediates, and p-hydroxyphenyllactate and p-hydroxyphenylpyruvate. Amino acids in plasma showed elevated alanine and glutamine. His general condition improved clinically sufficient to begin feeding. After 48 hours, he suffered a sudden cardiac arrest and could not be resuscitated. Autopsy showed cardiomegaly, pericardial and pleural effusions, and hepatic steatosis. On electron microscopy, there were megamitochondria with tightly packed cristae.

Family 3
Patient 3A (II-3): The proband was the product of the third pregnancy to non-consanguineous Caucasian parents (Figure 1d). A previous pregnancy resulted in intrauterine demise at 25 weeks with a severely hydropic fetus of unknown etiology but with a normally sized heart.
Maternal hypothyroidism was adequately treated throughout pregnancy. The pregnancy of the proband was complicated at 19 weeks with the fetus showing a dilated heart with ventricular wall thickening, decreased biventricular function, and pericardial effusion. Infectious causes were excluded including using pericardiocentesis; karyotype and microarray were normal, as was Pompe disease enzyme activity. The mother was treated with intravenous immunoglobulin. Severe fetal anemia was identified and treated with fetal red blood cell transfusions given at 28, 29, and 30 weeks gestation. There were no hemoglobin abnormalities and no signs of immune-mediated hemolysis. Due to worsening fetal biventricular cardiac dysfunction, ascites and pericardial effusion, this girl was born at 31 weeks gestation by urgent Caesarean section. She was intubated and ventilated shortly after birth for poor respiratory effort. Birth weight was 1.68 kg (+0.59 SD), length 39 cm (+1.44 SD), and head circumference 30 cm (-0.33 SD), all within normal range. At birth, she had a hematocrit of 28% hemoglobin 8.9 g.dL -1 (normal 11.6-16.6), platelets 84,000.mm3 -1 (normal 150-500), and she received a red blood cell transfusion. She developed rapidly worsening lactic acidosis from lactate 14 mM at 2 h of life progressing to 24 mM, and with a lactate/pyruvate ratio elevated at 77 increasing to 99, and with pH remaining below 7.0 despite treatment. She had ascites which was drained, and increased PT (INR 3.1), prolonged PTT 129 sec (normal 29-48 sec), and D-dimers present, elevated troponin and increased creatine kinase (CK) levels 413. On echocardiogram, she had a noncompacted myocardium, biventricular hypertrophy, and pericardial effusion, and on X-ray cardiomegaly. Initially ventricular function was preserved with high inotropic support for low blood pressures. Cortisol was 1.2 µg.dL -1 (normal 1.7-14.1), and hydrocortisone was given. p.Val375Met), with no second variant found and no exonic copy number change such as a deletion identified. A rapid autopsy was done with samples obtained within one hour after her death. The autopsy showed a very marked cardiomegaly (heart weight 39.2 g, normal value for gestational age 9 ± 2.8 g) with biventricular thickening of both atria (right more than left) and ventricles. On microscopic examination, there was no cardiomyocyte hypertrophy but cardiomyocyte perinuclear clearing ( Figure 1g). On electron microscopy, there was extensive mitochondrial proliferation with swelling and poor inner mitochondrial membrane, and decreased contractile elements ( Figure 1j). Skeletal muscle showed rare hypertrophied fibers and more pronounced mitochondrial staining, and electron microscopy showed ballooned rarefied mitochondria without inclusions. The lungs were small 23.8 g (normal 28.5 g) with hyaline membranes and modest intima thickening of arterioles. There were no sideroblasts either in the bone marrow or in the extramedullary hematopoiesis present in the liver.

Family 4
Three infants, two males and a female, were born to parents who were first cousins of Druze origin following full term pregnancies ( Figure 1e). Pregnancies were described as uneventful and initial development was normal. All three children presented at ages 2-3 months with cyanosis and respiratory deterioration.
Skeletal muscle biopsy was taken for mitochondrial studies.
Patient 4B (II-3) -The boy had reportedly been healthy, with no symptoms of cardiac disease or failure to thrive, until at age 2 months, when during routine echocardiography he developed cyanosis and respiratory distress necessitating mechanical ventilation. Echocardiography demonstrated concentric hypertrophy with an echo-bright endocardium and reduced global function. Laboratory findings showed severe acidosis, with intermittently high serum lactate levels up to 14 mmol.L -1 , although cerebrospinal fluid lactate was normal. He also had elevated creatine phosphokinase 938 U.L -1 , and elevated liver enzymes AST, ALT and LDH. His alanine was elevated 850 µmol.L -1 with branched amino acids slightly above the normal range, and urine organic acids showed high lactate and ketones. No defects in β-oxidation of fatty acids were observed in lymphocytes. The child was stabilized and treated with captopril, digoxin, carnitine and thiamine. Physical examination revealed a systolic 2/6 murmur at the left sternal border, he had normal muscle tone and no hepatomegaly or splenomegaly. Following repeat episodes of cyanosis and respiratory failure, the child died. Pyruvate dehydrogenase activity was normal.
Patient 4C (II-5) -The girl was born following full-term pregnancy; prenatal echocardiography was normal. She was reportedly healthy until her first cyanosis event during feeding at age 3 months. Chest X-ray was normal. At age 3.5 months, a recurrent cyanotic event left the child unconscious with severe lactic acidosis with lactate 6.1 to 15.7 mmol.L -1 , creatine phosphokinase 998 to 1877 U.L -1 , troponin 4.29 ng.mL -1 (normal <0.28), and elevated liver enzymes LDH, AST, and GGT. Echocardiography revealed concentric hypertrophy of the left ventricle with reduced left ventricular function. The child died after several days of intensive care treatment.

Family 5
The parents are healthy first-degree cousins of Druze origin (Figure 1f), with no known relation to Family 4, but residing in an adjacent village. The family history revealed a paternal aunt (I-2) who had died at the age of 4 months from progressive cardiomyopathy and pleural effusion.
She, reportedly, had reduced respiratory complexes I and IV on muscle biopsy. Patient 5A (II-2) -The proband was born at week 33 as part of a twin gestation, with his twin succumbing within one hour after birth from a congenital abdominal wall defect. He was born with a low Apgar score, but quickly recovered and had normal development thereafter. At age 5.5 months he exhibited reduced eating and general deterioration; several days later he was hospitalized due to cardiomyopathy and pericardial effusion, and required mechanical ventilation. Echocardiography revealed severe concentric hypertrophic cardiomyopathy.
Laboratory workup revealed metabolic acidosis with pH 7.27, elevated blood lactate 8 mmol.L -1 which later normalized, elevated creatine phosphokinase 660-1146 U.L -1 , elevated troponin and anemia with hemoglobin 9.2 g.dL -1 (normal 11-14). He had mildly elevated alanine 649 µmol.L -1 (normal 170-600), and on urine organic acid high lactate, succinate, fumarate, 3methylglutaconate and ketones. A mitochondrial disorder was suspected. He was treated with pericardiocentesis, and inotropes, which resulted in mild improvement. He was discharged on captopril and diuretics. He returned to the hospital after one month with cough, oxygen desaturation and fever. Repeat echocardiography revealed combined dilated and hypertrophic cardiomyopathy. During this hospitalization, his respiratory and metabolic state deteriorated and he eventually died at age 6.5 months. Skeletal muscle biopsy was obtained postmortem for mitochondrial studies.
Patient 5B (II-3) -In a subsequent pregnancy, the parents chose not to pursue prenatal diagnosis, and were followed up with fetal echocardiograms, which were normal. A girl was born by Caesarean section. Development and echocardiography testing remained normal until age 3 months, when asymptomatic mild concentric cardiomyopathy, more prominent in the left ventricle, was first detected; no signs of heart failure were observed. At age 5 months she was asymptomatic, with normal development and weight gain, but echocardiography revealed considerable left ventricle and septum hypertrophy with normal systolic function and mild pericardial effusion. Treatment with furosemide was started. She had elevated lactate 18 mmol.L -1 ; but normal creatine phosphokinase and transaminases. During a genetics evaluation at 6 months, she was tachypneic with perioral cyanosis, but no other signs of heart failure.
Within 10 days, she stopped feeding and showed breathing difficulties requiring hospitalization; she died suddenly the next day despite attempted resuscitation.

Supplementary Figure 2: mRNA levels of subunits of the GatCAB complex.
Legend: The amount of the mRNA is determined by qPCR in exponentially growing fibroblasts. RNA was extracted with Trizol reagent (Sigma), and cDNA synthesized using the QuantiTect Reverse Transcription Kit (Qiagen), followed by SYBR green RTqPCR using an Applied Biosystems Step-One Plus real-time thermocycler (Applied Biosystems, ThermoFisher), consisting of an initial 10 min denaturation step at 95°C followed by 40 cycles of denaturation (15 sec at 95°C) and annealing/extension (1min at 60°C). The mRNA relative copy numbers were determined by qPCR normalizing to HPRT levels, using for each PCR target two primer pairs (see Supplementary methods). Mean and standard deviation are shown.

Supplementary Figure 3: Cellular localization of QARS.
Legend: (a) Cellular localization of the QARS-GFP construct (green) analyzed by confocal microscopy. Cells were counterstained for the mitochondrial import protein TOM20 (red). CMAC and DAPI were used to stain the cytoplasm and cell nuclei, respectively. Scale bar -10 micrometers. (b) Cellular localization of the mitochondrially targeted GFP (MTS-GFP, green), used here as a control, analyzed by confocal microscopy. Cells were counterstained for the mitochondrial import protein TOM20 (red). CMAC and DAPI were used to stain the cytoplasm and cell nuclei, respectively. Scale bar -10 micrometers. (c) HeLa cells were fractionated into cytosol ("C", lane 2) and mitochondria ("M", lanes 3-5). The mitochondrial fraction was treated with proteinase K (lane 4) or proteinase K and Triton X (lane 5). "T" (lane 1) depicts total cell extraction. Sub-cellular fractions were analyzed by western blotting with antibodies against QARS, NDUFB8 (mitochondrial complex I) and GAPDH (cytosol).

Supplementary Figure 4: mRNA levels of nuclear encoded OXPHOS subunits.
Legend: RT-qPCR analysis of NDUFB8, UCQCR2 and COX5A mRNA levels in indicated samples, normalized to HPRT levels. Shown are the fold changes in relative mRNA levels as mean ± standard deviation for triplicate samples, compared to the control set to 1.0. *, p value < 0.05; ***, p value < 0.001; ns = not significant.

Supplementary Figure 5: pet112 phenotypes.
Legend: a) Oxidative growth: the pet112Δ strains harboring PET112 wild-type allele, pet112 F103L mutant allele, or the empty vector were serially diluted and spotted on synthetic complete agar plates supplemented with 2% glucose or 2% ethanol and incubated at 28°C. (b) Oxygen consumption rate: cells were grown at 28°C in SC medium supplemented with 0.6% glucose. Values were normalized to the wild-type strain. The data are the results of at least three measurements, and the error bars indicate the standard deviation. Statistical analysis was performed by paired, two-tail Student's t test comparing mutant strains to wild type strain: ***p<0.001, NS = not significant. Legend: The pet112Δ strains harboring PET112 wild-type allele, pet112 F103L mutant allele, or the empty vector were grown at 37°C in SC medium supplemented with 0.6% glucose. Values were normalized to the wild-type strain. The data are the results of at least three measurements, and the error bars indicate the standard deviation. Statistical analysis was performed by paired, two-tail Student's t test comparing mutant strains to wild type strain: ***p<0.001, *p<0.05.  Figure 3 panel a with molecular weight markers. Please note that the same blot was used for both stainings: first by using an anti-GatB antibody (left image), thereafter the same blot (without stripping of anti-GatB and secondary antibodies) was incubated with anti-porin (right image)*. Therefore, on the right image there is some GatB signal present.

Anti-GatB
Anti Legend: Uncropped images of Western blot shown in Figure 3 panel b with molecular weight markers. * Please note that the complex II blot (right image) was also stained with antibodies against complex III and complex V. § non-specific band only observed in control C3 but not in other controls. Legend: Uncropped images of Western blot shown in Figure 3 panel e with molecular weight marker indicated. The portion of the blots presented in Figure 3 panel e are indicated by a red box. Please, note that for GatA the same blot was used for both stainings: first by using an anti-GatA antibody (left image), thereafter the same blot (without stripping of anti-GatA and secondary antibodies) was incubated with anti-porin (right image). For GatB and GatC twin gels were carried out for problem and control antibodies.