A cell model of YARS2-associated childhood-onset mitochondrial disease

YARS2 相关的儿童期发病线粒体疾病的细胞模型

• 批准号: 10575369
• 负责人: Ya-Ming Hou
• 金额: $ 8.99万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10575369
• 关键词: Address; Affect; Alleles; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Award; Biological Assay; Cell Line; Cell Signaling Process; Cell model; Central Nervous System; Charge; Childhood; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Communities; Complex; Cytosol; DNA Sequence Alteration; Data; Dedications; Dermal; Development; Disease; Disease Progression; Enzymes; Failure to Thrive; Family; Fibroblasts; Foundations; Functional disorder; Gene Expression; Genes; Genetic; Genotype; Health; Health Care Costs; Heterozygote; Homeostasis; Human; Human Cell Line; Impairment; Individual; Intellectual functioning disability; Joints; Lactic Acidosis; Link; Manuscripts; Mediating; Methods; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Modeling; Motor Neurons; Muscle Cells; Mutation; Myopathy; Neonatal; Neuromuscular Diseases; Neurons; Nuclear Family; Nutrient; Organ; Ovarian; Pathogenicity; Patients; Peripheral; Phenotype; Positioning Attribute; Protein Biosynthesis; Public Health; Publishing; Pulmonary Hypertension; Recurrence; Research; Role; Secure; Sensorineural Hearing Loss; Severities; Severity of illness; Shapes; Sideroblastic Anemia; Signal Transduction; Skeletal Muscle; Speed; Spinal; Stem Cell Research; Testing; Tissue Model; Tissues; Transfer RNA; Transfer RNA Aminoacylation; Translations; Tyrosine-Specific tRNA; Tyrosine-tRNA Ligase; United States National Institutes of Health; Variant; Vertebral column; Work; autosome; bench to bedside; cell type; clinical diagnosis; clinical phenotype; clinically relevant; detection of nutrient; disease phenotype; emerging adult; enzyme activity; established cell line; frontier; human disease; human subject; induced pluripotent stem cell; infancy; insight; loss of function; mutation correction; myopathy-lactic acidosis-sideroblastic anemia syndrome; neonate; novel; prognostication; skeletal; stem cell differentiation; stem cell model; transcriptome; transcriptome sequencing

PROJECT SUMMARY We hypothesize that pathogenic variants in the family of the nuclear ARS2 genes, encoding mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), cause disease primarily by disrupting nutrient sensing and cell signaling. Each mt-aaRS is responsible for charging its cognate mitochondrial tRNA (mt-tRNA) with its specific amino acid, as required for the dedicated mitochondrial protein synthesis machinery. Loss-of-function variants of each ARS2 have been linked to human diseases, showing central nervous system involvement, myopathy, sensorineural hearing loss, ovarian dysgenesis, and pulmonary hypertension. However, the correlation is poor between the enzymatic activity of individual variants and manifestation of the disease phenotypes. This suggests that disease is mediated not necessarily via loss of tRNA charging, but by other functions of mt-aaRSs. Given that the cytosolic counterpart ARS1 genes are often implicated in non-canonical functions, beyond aminoacylation, we will test the hypothesis that ARS2s mediate nutrient-sensing and cell-signaling processes that are disrupted by pathogenic variants resulting in disease development. To test this hypothesis, as a pilot model, we will generate a human subject-derived induced pluripotent stem cell (iPSC) line with pathogenic variants in the YARS2 gene, encoding mt-TyrRS. Pathogenic variants of YARS2 result in the MLASA (myopathy, lactic acidosis and sideroblastic anemia) syndrome. Previous studies of pathogenic YARS2 variants have shown no appreciable effect on tRNA aminoacylation. Here we focus on a pair of novel compound heterozygous mutations that we identified in a human subject with neonatal fatal disease, the most severe clinical case observed to date. This provides a unique model where the largest changes in nutrient sensing and cell signaling are expected to occur. In Aim 1, we will create an iPSC line from an established fibroblast line of the human subject. We will use CRISPR/Cas to generate an isogenic control line that corrects the genetic mutations. In Aim 2, we will differentiate the patient and control iPSC lines to disease-relevant cell types. We will focus on myocytes, as skeletal myopathy is the most notable feature of clinical cases of YARS2 variants. We will also focus on peripheral neurons, which are affected in most clinical cases of other ARS2 variants. We will identify changes of gene expression in nutrient sensing or cell signaling of the subject line. Combined, this work will produce an isogenic pair of subject and control iPSC lines that will be shared with the research community. This pair of iPSC lines will serve as the foundation for understanding the implications of nutrient sensing and cell signaling in YARS2 variants, which will provide a template that is generalizable to other ARS2-associated disease.

项目摘要 我们假设，在核ARS 2基因家族中的致病性变异，编码线粒体 氨酰-tRNA合成酶（mt-aaRS）主要通过扰乱营养传感和细胞而引起疾病 发信号。每个mt-aaRS负责用其特定的线粒体tRNA（mt-tRNA）来充电其同源线粒体tRNA（mt-tRNA）。 氨基酸，如专用线粒体蛋白质合成机器所需。功能丧失变体 每种ARS 2都与人类疾病有关，表现为中枢神经系统受累，肌病， 感音神经性听力损失、卵巢发育不全和肺动脉高压。然而，相关性较差 个体变体的酶活性与疾病表型的表现之间的关系。这表明 该疾病不一定通过tRNA充电的丧失介导，而是通过mt-aaRS的其他功能介导。给定 细胞溶质对应物ARS 1基因通常涉及非典型功能， 通过氨酰化，我们将检验ARS 2介导营养感应和细胞信号传导过程的假设 被致病性变异破坏导致疾病发展。为了验证这一假设，作为一名飞行员 模型中，我们将产生一个人受试者来源的诱导多能干细胞（iPSC）系， 编码mt-TyrRS的YARS 2基因的变体。YARS 2的致病性变体导致MLASA（肌病， 乳酸性酸中毒和铁粒幼细胞性贫血）综合征。先前对致病性YARS 2变体的研究表明， 对tRNA氨酰化无明显影响。在这里，我们专注于一对新的复合杂合 我们在患有新生儿致命疾病的人类受试者中发现了突变，这是最严重的临床病例， 观察至今。这提供了一个独特的模型，其中营养传感和细胞信号传导的最大变化 预计会发生。在目标1中，我们将从已建立的人成纤维细胞系产生iPSC系。 话题吧我们将使用CRISPR/Cas来产生纠正基因突变的同基因对照系。在Aim中 2，我们将使患者和对照iPSC系分化为疾病相关的细胞类型。我们将重点关注肌细胞， 因为骨骼肌病变是YARS 2变体临床病例的最显著特征。我们也将关注 外周神经元，其在其他ARS 2变体的大多数临床病例中受到影响。我们将确定 在主题品系的营养感测或细胞信号传导中的基因表达。结合起来，这项工作将产生一个 将与研究界共享的受试者和对照iPSC系的同基因对。这对iPSC 线将作为基础，了解营养传感和细胞信号转导的影响， YARS 2变体，这将提供一个模板，可推广到其他ARS 2相关疾病。