Mitochondrial functional assays for diagnosis in minimally invasive tissues:optimization and clinical utility

项目摘要 原发线粒体疾病的特点是广泛的生化、临床和遗传。 异构性，使得他们的分析复杂，诊断过程非常具有挑战性、冗长和 令人沮丧。尽管在使用下一代测序确定遗传原因方面取得了快速进展，但功能性 显示线粒体呼吸链功能障碍的研究在许多患者中仍然需要 完成诊断。目前的诊断实践包括呼吸链酶的测量 肌肉或肝脏活检中的活性，但这些分析不能提供对线粒体的完整分析。 由于活组织检查的侵袭性，通常会避免这种情况。抽血和皮肤活检 成纤维细胞培养是一种微创方法。近年来，新的线粒体功能分析方法 在适用于成纤维细胞甚至血细胞打开新机会的研究环境中开发，但 这种基因在一系列线粒体遗传缺陷中的表现和临床用途仍未被探索。目标是 这项研究的目的是开发功能测试方法，并建立其临床实用价值，以诊断和 确认以皮肤成纤维细胞为重点的微创组织中的原发线粒体疾病。我们 假设在怀疑患有原发线粒体疾病的患者中，适当的一组测试可以 在成纤维细胞(或血细胞)中进行开发和优化，具有临床稳健性、良好的敏感性和 特异性，以允许有效的线粒体功能测试，策略依赖于功能 类别。我们将检查一系列线粒体功能测试的诊断性能 已知的原发线粒体疾病患者的成纤维细胞，按功能类别组织，并确定 敏感性和特异性。 此外，我们还将检查组织培养条件的差异对功能测试的影响 成纤维细胞的性能。反映在培养条件中的环境因素，如氨基酸丰度 对这些功能产生诊断和治疗方面的影响。功能实现中的多重因素 线粒体tRNAs与氨基酸或单碳叶酸酯等营养因子相互作用。在遗传方面 与同源氨基酸相互作用的线粒体tRNA氨基转移酶(ARS2)的缺陷， 增加氨基酸浓度可能会改善功能，而降低其浓度将 加重线粒体功能障碍。我们将研究不同同源氨基酸的影响。 功能检测中tRNA氨酰化缺陷的浓度。一碳充电 线粒体内的叶酸影响tRNA过程，如MTFMT对启动子蛋氨酸的甲酰化， 在tRNALys中将摆动碱基转录后修饰为甲基尿苷或牛磺酸甲基尿苷 以及tRNALeu(UUR)，它受到常见的致病突变的影响，具有致病功能影响。 我们将评估牛磺酸的影响以及丝氨酸或甲酸盐对叶酸充电的调节作用。

Project Summary Primary mitochondrial diseases are characterized by extensive biochemical, clinical, and genetic heterogeneity, making their analysis complex and the diagnostic process very challenging, lengthy, and frustrating. Despite rapid progress in identifying a genetic cause using next-generation sequencing, functional studies showing dysfunction in the mitochondrial respiratory chain are still required in many patients to complete the diagnosis. Current diagnostic practice involves measurement of respiratory chain enzyme activities in muscle or liver biopsies, but these assays do not provide a complete analysis of mitochondrial function and are often avoided due to the invasive nature of the biopsies. Blood sampling and skin biopsy for fibroblast culture are minimally invasive procedures. In recent years, new mitochondrial functional assays were developed in the research setting applicable to fibroblasts or even blood cells opening new opportunities, but the performance in a range of mitochondrial genetic defects and the clinical utility remain unexplored. The goal of this study is to develop functional testing methods and to establish their clinical utility for the diagnosis and confirmation of primary mitochondrial disease in minimally invasive tissue with a focus on skin fibroblasts. We hypothesize that in patients with suspected primary mitochondrial diseases an appropriate panel of tests can be developed and optimized in fibroblasts (or blood cells) with clinical robustness, good sensitivity and specificity, to allow for effective mitochondrial function testing with the strategy dependent on functional category. We will examine a series of mitochondrial functional tests for their diagnostic performance in fibroblasts of patients with known primary mitochondrial diseases, organized by functional class, and determine sensitivity and specificity. Further, we will also examine the effect of differences in tissue culture conditions on the functional test performance in fibroblasts. Environmental factors reflected in culture conditions such as amino acid abundance impact these functions with diagnostic and therapeutic implications. Multiple factors in the functionalization of mitochondrial tRNAs interact with nutritional factors such as amino acids or one-carbon folate esters. In genetic defects of mitochondrial tRNA aminoacyltransferases (ARS2), which interact with the cognate amino acid, increasing the amino acid concentration may improve functionality, whereas decreasing its concentration will exacerbate mitochondrial dysfunction. We will examine the impact of varying the cognate amino acid concentration for tRNA aminoacylation defects on functional assays. The one-carbon charging of intramitochondrial folates affects tRNA processes such as formylation of the initiator methionine by MTFMT, and post transcriptional modification of the wobble base to methyl-uridine or taurinomethyl-uridine in tRNALys and tRNALeu(UUR), which is affected by common pathogenic mutations with disease-causing functional impact. We will evaluate the impact of taurine and of modulation of folate charging by serine or formate.