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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要我们假设编码线粒体的核 ARS2 基因家族中的致病性变异氨酰基-tRNA 合成酶 (mt-aaRS) 主要通过破坏营养感应和细胞引起疾病发信号。每个 mt-aaRS 负责为其同源线粒体 tRNA (mt-tRNA) 充电，并为其特定的氨基酸，这是专用线粒体蛋白质合成机制所需的。功能丧失变体每个 ARS2 都与人类疾病有关，显示中枢神经系统受累、肌病、感音神经性听力损失、卵巢发育不全和肺动脉高压。但相关性较差个体变体的酶活性与疾病表型的表现之间。这表明该疾病不一定是通过 tRNA 电荷损失介导的，而是通过 mt-aaRS 的其他功能介导的。给定细胞质对应 ARS1 基因通常涉及非规范功能，超出氨酰化，我们将测试 ARS2 介导营养感应和细胞信号传导过程的假设被致病变异破坏，导致疾病发展。为了检验这个假设，作为一名飞行员模型中，我们将生成具有致病性的人类受试者衍生的诱导多能干细胞 (iPSC) 系 YARS2 基因的变体，编码 mt-TyrRS。 YARS2 的致病变异导致 MLASA（肌病、乳酸性酸中毒和铁粒幼细胞贫血）综合征。先前对致病性 YARS2 变异的研究表明对 tRNA 氨酰化没有明显影响。这里我们重点关注一对新型复合杂合体我们在患有新生儿致命疾病的人类受试者中发现的突变，这是最严重的临床病例迄今为止观察到的。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类成纤维细胞系中创建 iPSC 系主题。我们将使用 CRISPR/Cas 生成纠正基因突变的等基因对照系。瞄准 2、我们将患者和对照 iPSC 系区分为疾病相关细胞类型。我们将重点关注肌细胞，因为骨骼肌病是 YARS2 变异临床病例最显着的特征。我们还将重点关注周围神经元，在大多数其他 ARS2 变体的临床病例中都会受到影响。我们将确定以下变化主题系的营养感应或细胞信号传导中的基因表达。结合起来，这项工作将产生受试者和对照 iPSC 系的同基因对，将与研究界共享。这对 iPSC 线将作为理解营养传感和细胞信号传导的影响的基础 YARS2 变体，它将提供一个可推广到其他 ARS2 相关疾病的模板。