Carbonic Anhydrase 5A Dysfunction in Complex V Deficiency

复合物 V 缺乏时碳酸酐酶 5A 功能障碍

基本信息

批准号：
10215509
负责人：
Rebecca Ganetzky
金额：
$ 13.2万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10215509
关键词：
Acetates Acids Adenosine Triphosphate Affect Algorithms Amino Acids Ataxia Binding Biochemical Biochemical Pathway Bioenergetics Biological Assay Blindness Blood Platelets Brain Cardiomyopathies Carnitine Cell Line Cells Chemicals Child Development Child Health Chloroplasts Citrulline Clinical Clinical Research Co-Immunoprecipitations Complex Data Defect Development Diagnosis Diagnostic Diagnostic tests Disease Electron Transport Energy-Generating Resources Enzymes Etiology Evaluation Eye Fibroblasts Foundations Functional disorder Future Gel Gene Mutation Genes Genetic Genetic Diseases Genome Genotype Goals Haplogroup Heart HepG2 Hepatic Hepatocyte Human Hydroxyl Radical Impairment In Vitro Incidence Individual Inherited Isotopes Label Lead Link Liquid substance Liver Mass Fragmentography Mass Spectrum Analysis Measurement Measures Membrane Potentials Metabolic Metabolic Diseases Methods Mitochondria Mitochondrial DNA Mitochondrial Respiratory Chain Deficiencies Modeling Molecular Mutation Neonatal Screening Neuropathy New Jersey Newborn Infant Nuclear Oligomycins Pathway interactions Patients Pennsylvania Performance Pharmacology Polyacrylamide Gel Electrophoresis Prevalence Propionic Acids Prospective cohort Proteomics Pump Recurrence Retrospective cohort Role Screening Result Source Stable Isotope Labeling Stroke Structure Suggestion Testing Work base body system carbonate dehydratase clinical diagnostics clinical phenotype diagnostic assay diagnostic biomarker experimental study follow-up indexing insight liquid chromatography mass spectrometry metabolomics mutant novel novel diagnostics novel marker oligomycin sensitivity-conferring protein organic acid potential biomarker stable isotope therapeutically effective

项目摘要

ABSTRACT Mitochondrial complex V (CV) subunit gene mutations cause a variety of severe metabolic diseases that impair child health and development, with strokes, neuropathy, ataxia, vision loss, and cardiomyopathy. There is no clinically-approved assay of CV function, therefore, making the diagnosis is challenging. The discovery that metabolites downstream of Carbonic Anhydrase 5A (CA5A) are recurrently abnormal in patients with CV deficiency has profound implications on developing targeted diagnostic testing for this profound energy deficiency. We Hypothesize that (a) CV and Carbonic Anhydrase 5A (CA5A) physically associate, therefore, mutations in CV cause CA5A deficiency and (b) scrutiny of the metabolites downstream of CA5A will provide novel biomarkers for CV deficiency. Specific Aims of this work are to [Aim 1] Evaluate incidence of CV deficiency among patients with a newborn screen consistent with CA5A deficiency; [Aim 2] Determine the presence and degree of CA5A impairment in human fibroblast, cybrid and liver cell line models of CV disease; and [Aim 3] Determine if there is a physical association between CA5A and CV. Methods will sequencing CV subunit genes in subjects with abnormal newborn screens consistent with CA5A dysfunction (prospective and retrospective cohort), direct measurement of CA5A function using stable isotope-labeled acetate and in vitro cellular assessment of targeted and untargeted metabolites (Orbitrap liquid chromatography/mass spectrometry) in fibroblasts and hepatic cell lines from healthy individuals, genetic-based CV diseases and pharmacologic inhibition (oligomycin). Targeted metabolomics will focus on metabolites downstream of CA5A: amino acids, particularly citrulline (ultra-high-performance liquid chromatrography) and the organic acids propionic acid and hydroxyisovaleric acid (gas chromatography/mass spectrometry). determination of CA5A function using stable isotope studies (13C-labeled acetate incubation, isotope-ratio mass spectrometry) in transmitochondrial cybrid, hepatic and fibroblast cell lines from healthy individuals, genetic-based CV diseases and pharmacologic CV inhibition (oligomycin). We will also investigate the in vitro physical interaction of CA5A and CV (co-immunoprecipitation, blue native gel) in normal and genetic-based CV disease hepatocyte cell- lines. These studies will rigorously investigate the interaction between CA5A function and CV deficiency and new potential biomarkers for diagnosing CV disease. Our long-term goal is to expand the diagnostic and treatment options available for human CV deficiency, for which no treatment currently exists. These studies will establish the foundation of which to develop future clinical diagnostic assays to rapidly and precisely diagnosis CV disease and will lead to a new understanding of the role of CV in coordinating mitochondrial biochemical pathways, providing a novel target for future therapies.

抽象的线粒体复合物V（CV）亚基基因突变会导致多种严重的代谢疾病损害儿童健康和发育，中风，神经病，共济失调，视力丧失和心肌病。没有因此，临床上批准的简历功能测定法是具有挑战性的。发现 CV患者的碳酸酐酶5a（CA5A）下游的代谢产物反复异常缺乏对这一深刻能量的有针对性的诊断测试具有深远的影响不足。我们假设（a）CV和碳酸酐酶5A（CA5A）物理相关， CV中的突变会导致CA5A缺乏症和（b）CA5A下游代谢物的审查将提供简历缺乏的新型生物标志物。这项工作的具体目的是[AIM 1]评估简历的发生率新生儿筛查患者缺乏与CA5A缺乏一致的患者； [AIM 2]确定人类成纤维细胞，cybrid和肝细胞系模型的CA5A损伤的存在和程度； [AIM 3]确定CA5A和CV之间是否存在物理关联。方法将测序CV 与CA5A功能障碍一致的新生儿筛查异常筛查受试者的亚基基因（前瞻性和回顾性队列），使用稳定的同位素标记的乙酸盐和体外直接测量Ca5a功能靶向和非靶向代谢物的细胞评估（Orbitrap液相色谱/质量来自健康个体的成纤维细胞和肝细胞系中的光谱法），基于遗传的CV疾病和药理学抑制（寡霉素）。靶向代谢组学将专注于CA5A下游的代谢物：氨基酸，尤其是瓜氨酸（超高液体色谱）和有机酸丙酸和羟基异甲酸酸（气相色谱/质谱法）。 CA5A的确定使用稳定的同位素研究（13C标记的乙酸盐孵育，同位素比率质谱法）的功能来自健康个体的转叶片脊髓软骨，肝和成纤维细胞系，基于遗传的CV疾病和药理学简历抑制（寡霉素）。我们还将研究CA5A的体外物理相互作用和基于遗传的CV疾病肝细胞中的CV（共免疫沉淀，蓝色天然凝胶）线。这些研究将严格研究CA5A功能与CV之间的相互作用缺乏症和新的潜在生物标志物来诊断简历疾病。我们的长期目标是扩展可用于人类简历缺乏症的诊断和治疗方案，目前尚无治疗。这些研究将建立一个基础，以迅速开发未来的临床诊断测定法精确诊断CV疾病，将对CV在协调中的作用有了新的了解线粒体生化途径为未来疗法提供了新的靶标。