Analysis of tRNA Synthetase Variants in the Undiagnosed Diseases Program

描述(由申请人提供)：DNA测序技术的快速进步使患者样本能够全面分析与疾病相关的突变。一个主要的复杂因素是，突变经常在小家系或零星的疾病病例中发现，因此很难为致病性建立强有力的遗传论据。在这些情况下，功能分析对于评估稀有变异的致病性至关重要。此外，功能评估可以为疾病的分子机制提供洞察，也可以为开发治疗方法提供平台。未诊断疾病计划(UDP)正在编目在具有无数临床表型的患者中发现的有希望的罕见变异。这些变异体的一个子集存在于编码氨基酰-tRNA合成酶(ARS)的基因中，ARS是参与向tRNA充电的关键酶。UDP已经确定了六个ARS变体-甘氨酰-tRNA合成酶(GARS)、丙氨酰-tRNA合成酶(AARS)和天冬氨酰-tRNA合成酶(DARS)各两个。我们的实验室专门研究ARS变异体在体外和体内的致病性。事实上，我们在2003年发现了GARS基因中第一个与疾病相关的ARS突变，并从那时起一直在不断地研究这类基因。我们提出了两个具体的目标来评估六个ARS变体的致病性，并了解它们是如何导致人类遗传病的。首先，我们将在生化、酵母互补和细胞定位分析中测试每个ARS变体的功能丧失效应。其次，我们将确定每个ARS变异是否能够在斑马鱼中发挥主导的有毒表型，类似于其他与疾病相关的ARS突变。这些努力构成了对每个ARS变异的致病潜力的彻底评估，并将为开发针对受影响个人的治疗方法提供基础。

DESCRIPTION (provided by applicant): Rapid advances in DNA sequencing technologies have allowed comprehensive analysis of patient samples for disease-associated mutations. A major complicating factor is that mutations are often found in small pedigrees or sporadic cases of disease making it difficult to build a strong genetic argument for pathogenicity. In these cases functional analyses are critical for evaluating the pathogenicity of rare variants. Furthermore, functional evaluation can provide insight into the molecular mechanism of disease as well as platforms for developing therapeutics. The Undiagnosed Diseases Program (UDP) is cataloging promising rare variants identified in patients with myriad clinical phenotypes. A subset of these variants reside in genes encoding aminoacyl-tRNA synthetases (ARSs), which are critical enzymes involved in charging tRNA with cognate amino acids. The UDP has identified six ARS variants-two each in glycyl-tRNA synthetase (GARS), alanyl-tRNA synthetase (AARS), and aspartyl-tRNA synthetase (DARS). Our laboratory specializes in evaluating the pathogenicity of ARS variants in vitro and in vivo. Indeed, we identified the first disease-associated ARS mutations in the GARS gene in 2003 and have been continuously studying this class of genes ever since. We propose two specific aims to evaluate the pathogenicity of the six ARS variants and to understand how they cause human genetic disease. First, we will test each ARS variant for a loss-of-function effect in biochemical, yeast complementation, and cellular localization assays. Second, we will determine if each ARS variant is able to exert a dominant, toxic phenotype in zebrafish, similar to other disease-associated ARS mutations. These efforts compose a thorough evaluation of the pathogenic potential of each ARS variant and will provide the basis for developing therapies for affected individuals.