Analysis of tRNA Synthetase Variants in the Undiagnosed Diseases Program

描述（由申请人提供）：DNA测序技术的快速发展已经允许对患者样本进行疾病相关突变的全面分析。一个主要的复杂因素是，突变往往是在小家系或零星病例中发现的，这使得很难建立一个强有力的致病性遗传论据。在这些情况下，功能分析对于评估罕见变异的致病性至关重要。此外，功能评估可以提供深入了解疾病的分子机制以及开发治疗方法的平台。未诊断疾病计划（UDP）正在对具有无数临床表型的患者中发现的有希望的罕见变异进行编目。这些变体的一个子集存在于编码氨酰-tRNA合成酶（ARS）的基因中，所述氨酰-tRNA合成酶是参与使tRNA与同源氨基酸结合的关键酶。UDP已鉴定出六种ARS变体--甘氨酰-tRNA合成酶（加尔斯）、丙氨酰-tRNA合成酶（阿尔斯）和氨酰-tRNA合成酶（DARS）各两种。我们的实验室专门评估ARS变异体的体外和体内致病性。事实上，我们在2003年发现了第一个与疾病相关的加尔斯基因ARS突变，并从那时起一直在研究这类基因。我们提出了两个具体的目标，以评估六种ARS变异的致病性，并了解它们如何导致人类遗传疾病。首先，我们将在生化、酵母互补和细胞定位测定中测试每种ARS变体的功能丧失效应。第二，我们将确定是否每个ARS变异体能够在斑马鱼中发挥显性毒性表型，类似于其他疾病相关的ARS突变。这些努力构成了对每种ARS变体致病潜力的全面评估，并将为受影响个体的治疗提供基础。

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.