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

用于诊断微创组织的线粒体功能测定：优化和临床实用性

基本信息

批准号：
10700003
负责人：
JOHAN L VAN HOVE
金额：
$ 17.82万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10700003
关键词：
Affect American Amino Acids Biochemical Biological Assay Biopsy Blood Blood Cells Blood Platelets Blood specimen Candidate Disease Gene Carbon Categories Cells Charge Clinical Complex Coupling Cytochrome-c Oxidase Deficiency DNA Maintenance DNA sequencing Data Defect Development Diagnosis Diagnostic Disease Electron Transport Complex III Environmental Risk Factor Enzymes Esters Exhibits Fibroblasts Folic Acid Formates Frustration Functional disorder Galactose Gel Genes Genetic Genetic Heterogeneity Genetic Transcription Goals Individual Iron Leucine-Specific tRNA Lysine-Specific tRNA Maintenance Measurement Metabolism Methionine Methods Mitochondria Mitochondrial DNA Mitochondrial Diseases Mitochondrial Proteins Mitochondrial Respiratory Chain Deficiencies Modification Molecular Mononuclear Muscle Mutation Nature Nutritional Oxidative Phosphorylation Pathogenicity Patient Participation Patients Performance Population Procedures Process Protein Biosynthesis Protein Import Research Resolution Respiratory Chain Sensitivity and Specificity Series Serine Skin Spirometry Stains Stress Sulfur Taurine Testing Therapeutic Tissues Transfer RNA Transfer RNA Aminoacylation Translations Uridine Validation base biobank biological adaptation to stress candidate identification clinical heterogeneity enzyme activity experience genetic disorder diagnosis improved lipoate liver biopsy minimally invasive mitochondrial dysfunction next generation next generation sequencing novel novel strategies oligomycin sensitivity-conferring protein performance tests posttranscriptional pyruvate dehydrogenase tissue culture treatment trial variant of unknown significance

项目摘要

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.

项目概要原发性线粒体疾病的特点是广泛的生化、临床和遗传异质性，使得他们的分析变得复杂，诊断过程非常具有挑战性、漫长且令人沮丧。尽管使用下一代测序在识别遗传原因方面取得了快速进展，但功能性研究表明，许多患者仍需要线粒体呼吸链功能障碍完成诊断。当前的诊断实践涉及呼吸链酶的测量肌肉或肝脏活检中的活性，但这些测定并不能提供线粒体的完整分析由于活检的侵入性，通常会避免进行活检。血液采样和皮肤活检成纤维细胞培养是微创手术。近年来，新的线粒体功能检测方法被提出。在研究环境中开发的适用于成纤维细胞甚至血细胞的技术开辟了新的机会，但是一系列线粒体遗传缺陷的表现和临床实用性仍有待探索。目标本研究的目的是开发功能测试方法并确定其在诊断和治疗中的临床实用性确认微创组织中的原发性线粒体疾病，重点关注皮肤成纤维细胞。我们假设对于疑似原发性线粒体疾病的患者，适当的一组测试可以在成纤维细胞（或血细胞）中开发和优化，具有临床稳健性、良好的灵敏度和特异性，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患有已知原发性线粒体疾病的患者的成纤维细胞，按功能类别进行组织，并确定敏感性和特异性。此外，我们还将研究组织培养条件的差异对功能测试的影响成纤维细胞的表现。培养条件反映的环境因素，例如氨基酸丰度影响这些功能并具有诊断和治疗意义。功能化的多重因素线粒体 tRNA 与氨基酸或一碳叶酸酯等营养因子相互作用。在遗传方面线粒体 tRNA 氨酰基转移酶 (ARS2) 的缺陷，与同源氨基酸相互作用，增加氨基酸浓度可能会改善功能性，而降低其浓度则会加剧线粒体功能障碍。我们将研究改变同源氨基酸的影响功能测定中 tRNA 氨酰化缺陷的浓度。一碳充电线粒体内叶酸影响 tRNA 过程，例如通过 MTFMT 甲硫氨酸甲酰化，以及 tRNALys 中摆动碱基转录后修饰为甲基尿苷或牛磺酸甲基尿苷和 tRNALeu(UUR)，它受到常见致病突变的影响，具有致病的功能影响。我们将评估牛磺酸和丝氨酸或甲酸盐调节叶酸充电的影响。