AARS2 Research Articles

Abstract 18698: PCBP1 Regulates Alternative Splicing of Aars2 in Congenital Cardiomyopathy

Rationale: Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its alternative splicing, are linked to congenital cardiomyopathy in humans. However, the underlying molecular mechanism of how Aars2 regulates heart development remains unknown. Hypothesis: The alternative splicing of Aars2 and its function is essential for cardiac development. Methods and Results: We found that alternative splicing of the Aars2 transcript is mediated by poly(rC) binding protein 1 (PCBP1), an RNA binding protein, through direct interaction. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Therefore, Pcbp1 is critical for the expression and function of Aars2. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these data provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Conclusion: Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.

Abstract P2059: Poly(rC)-binding Protein-1 (Pcbp1) Is Essential For Heart Development

Rationale: Proper development of the heart relies on a tightly regulated, yet diverse, pattern of gene expression that is governed by transcriptional, post-transcriptional, and translational processes. Congenital heart disease (CHD) is the most common birth defect and a leading cause of morbidity and mortality in children. While the genetic cause of some types of CHD has been identified, the molecular basis for the rest remains elusive. Poly(rC)-binding protein 1 (Pcbp1) is a RNA-binding protein that regulates RNA processing as well as post-transcriptional and translational processes in a variety of biological systems. Hypothesis: Pcbp1 play a critical role in regulating heart development by governing Notch and UPR pathways and mediating proper Aars2 gene splicing. Methods and Results: Germline deletion of Pcbp1 results in lethality before embryonic day (E) 8.5. We generated a cardiac-specific deletion of Pcbp1 by crossing Pcbp1-Flox with cTNT-Cre mice (Pcbp1-cKO cTNT ) and found that Pcbp1-cKO cTNT mice is 50% lethal perinatally. Embryonic hearts of Pcbp1-cKO cTNT mice displayed ventricular non-compaction and abnormal ventricular apex formation. Deep RNA sequencing of Pcbp1-cKO cTNT hearts revealed alteration of gene expression profiles reflective on ventricular maturation delay and dysregulation of Notch and UPR pathways. Interestingly, loss of Pcbp1 in cardiomyocytes disrupts alternative splicing of many important genes including Aars2, a gene associated with congenital mitochondrial cardiomyopathy. Pcbp1 deficiency resulted in creation of an Aars2 exon16-skipping variant, leading to its premature termination. eCLIP-seq showed that Pcbp1 primarily binds to CU-rich motifs at 3’UTR, distal intron and CDS regions of targets, and it interacts with regions of Aars2 transcript near exon 16. Using CRISPR/Cas9 technology, we knocked in loxP sites flanking the exon 16 of Aars2 (Aars2-Flox), and cross the floxed mice with cTNT-Cre mice to generate cardiac-specific exon16 deletion mutant of Aars2 (Aars2-cKO cTNT ). Intriguingly, abnormality in Aars2-cKO cTNT embryonic heart phenocopy aspects of ventricular non-compaction and malformation observed in Pcbp1-cKO cTNT heart. Accordingly, the transcriptome from hearts of Pcbp1-cKO cTNT and Aars2-cKO cTNT display high concordance and similarity and share strikingly common dysregulated pathways. Conclusions: We discover a novel function of Pcbp1 in heart development by regulating Notch and UPR pathways. Additionally, Pcbp1 is indispensable for Aars2 gene splicing, whose deficiency is associated with congenital cardiomyopathy. Our findings suggest modulating Pcbp1 in developing hearts may offer a novel therapeutic intervention for congenital heart failure.

Towards Bacterial Expression of Unstable Mutants of a Mitochondrial Enzyme

Mutations in the human AARS2 gene, which codes for the mitochondrial alanyl‐tRNA synthetase, are linked with severe disease, including infantile cardiomyopathy and leukodystrophy. Our laboratory is interested in understanding, on a molecular level, why different mutations result in different disease phenotypes. Attempts to produce some mutants of AARS2 using a bacterial expression system have resulted in poor expression of proteins contaminated with proteolytic fragments. To improve expression of these mutants, we have used solubility tags to facilitate the purification of full‐length mutant protein. Addition of an MBP solubility tag greatly improved expression and purification of the wildtype and mutant enzymes. We are currently investigating strategies for solubility tag removal and characterization of wildtype and mutant thermal stability.

Management of mitochondrial leukoencephalopathy-related ataxia and cognitive impairment in a primary health care setup

Abstract Rationale: Ataxia is defined as an impaired coordination of voluntary muscle movement. In cases of slowly progressing ataxia in young people, we should first consider a genetic origin. Herein, we describe the case of a 25-year-old patient with severe and advanced neurological disease which was diagnosed and treated by a family medicine physician, leading to the discovery of a new form of hereditary ataxia. Patient concerns: In 2016, a 25-year-old professional driver arrived at our family medicine office with a gait disorder that he had been struggling with for the past 5 years. During clinical examination, gait ataxia was evident. In 2018, the patient returned to our family medicine office, with difficulty in speaking and could no longer retain urine. During the examination, he was not oriented in time and space, no longer recognized the staff, and responded only in a few words. Clinically, we observed postural and action tremors, bradykinesia, and increased muscle tone. Diagnoses: The patient was sent to a neurologist for further management. There, a magnetic resonance imaging scan of the head was prescribed, leading to a diagnosis of adrenoleukodystrophy; however, genetic testing and biochemical analysis performed in 2016 did not confirm that. With additional testing in 2018, we confirmed a hereditary mitochondrial leukoencephalopathy (the identified version c.2884C>T in the AARS2 gene has not been previously reported as a pathogen in relation to human diseases). Interventions: Unfortunately, no specific treatment is available, therefore, the reluctance of neurologists to provide treatment is understandable. Family medicine doctors are long-term companions to patients and are sometimes the only support for them and their families when it comes to such an incurable disease. Outcomes: Currently, the patient only needs palliative care. The diagnostic path was completed but no specific therapy is in view. Lessons: Our case shows a 25-year-old patient with severe and advanced neurological disease diagnosed and treated by a family medicine physician. Family doctors play an important role due to their long-term relationship with the patient and the comprehensive care they provide, even in cases where the disease is severe, and the prognosis is unfavorable.

Mutations in aminoacyl-tRNA synthetase genes: an analysis of 10 cases

To study the clinical features of the diseases associated with aminoacyl-tRNA synthetases (ARS) deficiency. A retrospective analysis was performed of the clinical and gene mutation data of 10 children who were diagnosed with ARS gene mutations, based on next-generation sequencing from January 2016 to October 2019. The age of onset ranged from 0 to 9 years among the 10 children. Convulsion was the most common initial symptom (7 children). Clinical manifestations included ataxia and normal or mildly retarded intellectual development (with or without epilepsy; n=4) and onset of epilepsy in childhood with developmental regression later (n=2). Some children experienced disease onset in the neonatal period and had severe epileptic encephalopathy, with myoclonus, generalized tonic-clonic seizure, and convulsive seizure (n=4); 3 had severe delayed development, 2 had feeding difficulty, and 1 had hearing impairment. Mutations were found in five genes: 3 had novel mutations in the AARS2 gene (c.331G>C, c.2682+5G>A, c.2164C>T, and c.761G>A), 2 had known mutations in the DARS2 gene (c.228-16C>A and c.536G>A), 1 had novel mutations in the CARS2 gene (c.1036C>T and c.323T>G), 1 had novel mutations in the RARS2 gene (c.1210A>G and c.622C>T), and 3 had novel mutations in the AARS gene (c.1901T>A, c.229C>T, c.244C>T, c.961G>C, c.2248C>T, and Chr16:70298860-70316687del). A high heterogeneity is observed in the clinical phenotypes of the diseases associated with the ARS deficiency. A total of 14 novel mutations in 5 genes are reported in this study, which enriches the clinical phenotypes and genotypes of the diseases associated with ARS deficiency.

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.

An adolescence-onset male leukoencephalopathy with remarkable cerebellar atrophy and novel compound heterozygous AARS2 gene mutations: a case report

Mutations in the mitochondrial alanyl-transfer (t)RNA synthetase 2 (AARS2; OMIM 612035) have been linked to leukoencephalopathy recently. Until now, there have been only 13 cases reported in the literature. Hence, the clinical and genetic characteristics of this disease are not fully understood. Here, we reported an adolescence-onset male leukoencephalopathy patient characterized by progressive limb tremor at the age of 17 years. He had no signs of a cardiomyopathy. Magnetic resonance imaging scanning demonstrated severe cerebellar atrophy and white matter abnormalities involving descending tracts. Focused exome sequencing revealed he had novel compound heterozygous mutations in AARS2 gene (c.2265dupA; p.Arg756fs and c.650C>T; p.Pro217Leu). The patient was diagnosed with AARS2 mutation-related leukodystrophy (AARS2-L). We report a case with novel AARS2 gene mutations with developed striking cerebellar atrophy and leukoencephalopathy, which helps to further understand the clinical and genetic heterogeneity of AARS2-L.

Inborn errors of metabolism in a cohort of pregnancies with non-immune hydrops fetalis: a single center experience.

The purpose of this study was to determine the frequency of non-immune hydrops fetalis (NIHF) among all pregnancies referred for prenatal care at Sultan Qaboos University Hospital (SQUH) during the study period and to evaluate the underlying etiologies of NIH. All pregnancies referred to SQUH between February 2014 and December 2015 were identified, and all pregnancies meeting the diagnosis of NIHF were included in this study. All cases of NIHF referred to our center during this period underwent standard systematic diagnostic work-up that included biochemical and molecular studies in addition to the standard investigations for hydrops fetalis. Clinical characteristics and results of the diagnostic work-up were retrospectively reviewed. A total of 3234 pregnancies were referred for prenatal care at SQUH during the study period, and 12 pregnancies were affected by NIHF. An underlying diagnosis was established in nine cases, and the majority of cases (7/9) were caused by inborn errors of metabolism (IEM). These included a novel homozygous variant in the AARS2 gene (5/7) and two cases of galactosialidosis (2/7). IEM was a major cause of NIHF in this cohort. The AARS2 variant accounts for a significant number of cases with NIHF in this cohort of Omani patients.

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.