Mitochondrial alanyl-tRNA Synthetase Research Articles

Reassessment of Gene-Elusive Familial Dilated Cardiomyopathy Leading to the Discovery of a Homozygous AARS2 Variant—The Importance of Regular Reassessment of Genetic Findings

Background: AARS2 encodes the mitochondrial protein alanyl-tRNA synthetase 2 (MT-AlaRS), an important enzyme in oxidative phosphorylation. Variants in AARS2 have previously been associated with infantile cardiomyopathy. Case summary: A 4-year-old girl died of infantile-onset dilated cardiomyopathy (DCM) in 1996. Fifteen years later, her 21-year-old brother was diagnosed with DCM and ultimately underwent heart transplantation. Initial sequencing of 15 genes discovered no pathogenic variants in the brother at the time of his diagnosis. However, 9 years later re-screening in an updated screening panel of 129 genes identified a homozygous AARS2 (c.1774C > T) variant. Sanger sequencing of the deceased girl confirmed her to be homozygous for the AARS2 variant, while both parents and a third sibling were all found to be unaffected heterozygous carriers of the AARS2 variant. Discussion: This report underlines the importance of repeated and extended genetic screening of elusive families with suspected hereditary cardiomyopathies, as our knowledge of disease-causing mutations continuously grows. Although identification of the genetic etiology in the reported family would not have changed the clinical management, the genetic finding allows genetic counselling and holds substantial value in identifying at-risk relatives.

Generation of three human induced pluripotent stem cell lines, LUMCi024-A, LUMCi025-A, and LUMCi026-A, from two patients with combined oxidative phosphorylation deficiency 8 and a related control

Combined Oxidative Phosphorylation Deficiency 8 (COXPD8) is an autosomal recessive disorder causing lethal childhood-onset hypertrophic cardiomyopathy. Homozygous or compound heterozygous mutations in the nuclear-encoded mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene underly the pathology. We generated induced pluripotent stem cells (hiPSCs) from two patients carrying the heterozygous compound c.1774 C>T, c.2188 G>A and c.2872 C>T AARS2 mutations, as well as a related healthy control carrying the c.2872 C>T AARS2 mutation. All hiPSC-lines expressed pluripotency markers, maintained a normal karyotype, and differentiated towards the three germ layer derivatives in vitro. These lines can be used to model COXPD8 or mitochondrial dysfunction.

Novel alanyl-tRNA synthetase 2 (AARS2) homozygous mutation in a consanguineous Chinese family with premature ovarian insufficiency

To explore the candidate pathogenic gene in a premature ovarian insufficiency (POI) proband from a consanguineous marriage and detect the potential effects of mutation on cellular energy metabolism. Genetic and functional studies. Reproductive medicine center. A patient with POI, from a consanguineous family, and her family members were recruited from the Reproductive Center of the First Affiliated Hospital of Anhui Medical University. Whole exome sequencing (WES) was performed for the proband. Variation revealed by WES sequencing was validated by Sanger sequencing in her family. Sequencing data were combined with those of other sporadic cases listed in public databases to identify the causative gene. Rare homozygous nonsynonymous variants were identified and included in subsequent analysis. Metabolic analyzes were performed using Seahorse XFe96 analyzers to measure oxygen consumption and then obtain further results of ATP production and extracellular acidification rate. The differences in energy metabolism measurements between wild type and mutation were analyzed and compared. A novel alanyl-tRNA synthetase 2 (AARS2) homozygous mutation (NM_020745: exon2: c.337G>C: p. G113R) was identified in the aminoacylation region using WES. The mutation was highly conserved among species and predicted to be disease causing. AARS2 encodes mitochondrial alanyl-tRNA synthetase, which attaches alanine onto tRNA-ala. AARS2 mutations were previously reported in female leukodystrophy patients with POI. In mitochondrial stress tests, the ATP productions of the mutation group (3.58 ± 0.46fmol/min/cell) was significantly lower than that of the wild type group (6.96 ± 1.56fmol/min/cell). This is the first report of a homozygous pathogenic AARS2 mutation in POI. This mutation may lead to incorrect aminoacylation of tRNA, affect mitochondrial translation, and cause oxidative phosphorylation defects, which may be responsible for POI.

Expansion of the clinical spectrum associated with AARS2‐related disorders

Biallelic pathogenic variants in AARS2, a gene encoding the mitochondrial alanyl-tRNA synthetase, result in a spectrum of findings ranging from infantile cardiomyopathy to adult-onset progressive leukoencephalopathy. In this article, we present three unrelated individuals with novel compound heterozygous pathogenic AARS2 variants underlying diverse clinical presentations. Patient 1 is a 51-year-old man with adult-onset progressive cognitive, psychiatric, and motor decline and leukodystrophy. Patient 2 is a 34-year-old man with childhood-onset progressive tremor followed by the development of polyneuropathy, ataxia, and mild cognitive and psychiatric decline without leukodystrophy on imaging. Patient 3 is a 57-year-old woman with childhood-onset tremor and nystagmus which preceded dystonia, chorea, ataxia, depression, and cognitive decline marked by cerebellar atrophy and white matter disease. These cases expand the clinical heterogeneity of AARS2-related disorders, given that the first and third case represent some of the oldest known survivors of this disease, the second is adult-onset AARS2-related neurological decline without leukodystrophy, and the third is biallelic AARS2-related disorder involving a partial gene deletion.

The G3-U70-independent tRNA recognition by human mitochondrial alanyl-tRNA synthetase.

Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNAAla as a major determinant. However, this base pair is divergent in human mitochondrial tRNAAla, but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNAAla, in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNAAla in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg663, likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.

Novel <i>AARS</i>2 Gene Mutation Producing Leukodystrophy: A Patient with Peripheral Demyelinating Polyneuropathy

Novel AARS2 gene mutations encoding mitochondrial alanyl-tRNA synthetase are important in the spectrum of different phenotypes expressed in the nervous system. Leukodystrophy and ovarian failure in females are common phenotypes. Peripheral demyelination is not a recognized aspect of the AARS2 phenotype. A patient with preceding Lyme neuroborreliosis developed progressive leukodystrophy and peripheral demyelinating motor polyneuropathy. Serial magnetic resonance imaging showed progressive inflammatory demyelination extending to the corticospinal tracts. Treatment with a standard of care of antibiotics and immune-modulatory therapy employing intravenous immune globulin was employed. The contribution of neuroborreliosis is not well understood in the expression of the AARS2 phenotype.

Overexpression of human mitochondrial alanyl-tRNA synthetase suppresses biochemical defects of the mt-tRNAAla mutation in cybrids.

Mutations of mitochondrial transfer RNAs (mt-tRNAs) play a major role in a wide range of mitochondrial diseases because of the vital role of these molecules in mitochondrial translation. It has previously been reported that the overexpression of mitochondrial aminoacyl tRNA synthetases is effective at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs in cells. Here we report a detailed analysis of the suppressive activities of mitochondrial alanyl-tRNA synthetase (AARS2) on mt-tRNAAla 5655 A>G mutant. Mitochondrial defects in respiration, activity of oxidative phosphorylation complexes, ATP production, mitochondrial superoxide, and membrane potential were consistently rescued in m.5655A>G cybrids upon AARS2 expression. However, AARS2 overexpression did not result in a detectable increase in mutated mt-tRNAAla but caused an increase incharged mt-tRNAAla in mutant cybrids, leading to enhanced mitochondrial translation. This indicated that AARS2 improved the aminoacylation activity in the case of m.5655A>G, rather than having a stabilizing effect on the tRNA structure. The data presented in this paper deepen our understanding of the pathogenesis of mt-tRNA diseases.

Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2) defects predicts differential effects on aminoacylation.

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

Novel (ovario) leukodystrophy related to AARS2 mutations.

The study was focused on leukoencephalopathies of unknown cause in order to define a novel, homogeneous phenotype suggestive of a common genetic defect, based on clinical and MRI findings, and to identify the causal genetic defect shared by patients with this phenotype. Independent next-generation exome-sequencing studies were performed in 2 unrelated patients with a leukoencephalopathy. MRI findings in these patients were compared with available MRIs in a database of unclassified leukoencephalopathies; 11 patients with similar MRI abnormalities were selected. Clinical and MRI findings were investigated. Next-generation sequencing revealed compound heterozygous mutations in AARS2 encoding mitochondrial alanyl-tRNA synthetase in both patients. Functional studies in yeast confirmed the pathogenicity of the mutations in one patient. Sanger sequencing revealed AARS2 mutations in 4 of the 11 selected patients. The 6 patients with AARS2 mutations had childhood- to adulthood-onset signs of neurologic deterioration consisting of ataxia, spasticity, and cognitive decline with features of frontal lobe dysfunction. MRIs showed a leukoencephalopathy with striking involvement of left-right connections, descending tracts, and cerebellar atrophy. All female patients had ovarian failure. None of the patients had signs of a cardiomyopathy. Mutations in AARS2 have been found in a severe form of infantile cardiomyopathy in 2 families. We present 6 patients with a new phenotype caused by AARS2 mutations, characterized by leukoencephalopathy and, in female patients, ovarian failure, indicating that the phenotypic spectrum associated with AARS2 variants is much wider than previously reported.

Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNAAla; a G3A change in Ashbya abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.

Exome Sequencing Identifies Mitochondrial Alanyl-tRNA Synthetase Mutations in Infantile Mitochondrial Cardiomyopathy

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.

Expression of Arabidopsis thaliana Mitochondrial Alanyl-tRNA Synthetase Is Not Sufficient to Trigger Mitochondrial Import of tRNAAla in Yeast

It has often been suggested that precursors to mitochondrial aminoacyl-tRNA synthetases are likely carriers for mitochondrial import of tRNAs in those organisms where this process occurs. In plants, it has been shown that mutation of U(70) to C(70) in Arabidopsis thaliana tRNA(Ala)(UGC) blocks aminoacylation and also prevents import of the tRNA into mitochondria. This suggests that interaction of tRNA(Ala) with alanyl-tRNA synthetase (AlaRS) is necessary for import to occur. To test whether this interaction is sufficient to drive import, we co-expressed A. thaliana tRNA(Ala)(UGC) and the precursor to the A. thaliana mitochondrial AlaRS in Saccharomyces cerevisiae. The A. thaliana enzyme and its cognate tRNA were correctly expressed in yeast in vivo. However, although the plant AlaRS was efficiently imported into mitochondria in the transformed strains, we found no evidence for import of the A. thaliana tRNA(Ala) nor of the endogenous cytosolic tRNA(Ala) isoacceptors. We conclude that at least one other factor besides the mitochondrial AlaRS precursor must be involved in mitochondrial import of tRNA(Ala) in plants.