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

项目总结 我们推测编码线粒体的核ARS2基因家族中的致病变异 氨基酰-tRNA合成酶(mt-aars)主要通过破坏营养感官和细胞而致病。 发信号。每个mt-aars负责向其同源线粒体tRNA(mt-tRNA)充电其特定的 氨基酸，这是专门的线粒体蛋白质合成机制所需的。功能丧失变异体 每一种ARS2都与人类疾病有关，显示出中枢神经系统受累，肌病， 感觉神经性耳聋、卵巢发育不全和肺动脉高压。然而，这种关联性很差。 个体变异的酶活性与疾病表型的表现之间的关系。这表明 这种疾病不一定是通过失去tRNA电荷，而是通过mt-AARs的其他功能来介导的。vt.给出 胞质中对应的ARS1基因通常与非规范功能有关， ，我们将检验ARS2介导营养感知和细胞信号传递过程的假设。 被导致疾病发展的致病变异所破坏的。为了验证这一假设，作为一名飞行员 模型，我们将产生一种人类受试者来源的诱导多能干细胞(IPSC)系，其具有致病性 YARS2基因的变异，编码mt-TyrRS。YARS2的致病变异导致MLASA(肌病， 乳酸中毒和铁粒母细胞贫血)综合征。以前对致病YARS2变种的研究表明 对tRNA氨酰化无明显影响。在这里，我们重点研究一对新型化合物杂合子 我们在一名患有新生儿致命疾病的人类受试者身上发现的突变，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，其中营养感知和细胞信号的最大变化 预计将会发生。在目标1中，我们将从已建立的人类成纤维细胞系中创建一个IPSC系 主题。我们将使用CRISPR/CAS来产生纠正基因突变的等基因控制系。在AIM 2、我们将根据疾病相关的细胞类型来区分患者和对照IPSC系。我们将重点关注肌细胞， 由于骨骼肌病是YARS2变异临床病例中最显著的特征。我们还将重点关注 外周神经元，在大多数其他ARS2变种的临床病例中受到影响。我们将确定以下变化 被试品系的营养感应或细胞信号中的基因表达。结合在一起，这项工作将产生一个 将与研究社区共享的一对同基因的受试者和控制者IPSC系。这双iPSC LINES将作为理解营养传感和细胞信号转导的基础 YARS2变种，这将提供一个模板，可推广到其他ARS2相关疾病。

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.