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：
10042614
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/10042614
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 Treatment Efficacy 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

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代谢疾病，损害人体健康儿童健康和发育，包括中风、神经病、共济失调、视力丧失和心肌病。没有临床上批准的 CV 功能测定，因此，做出诊断具有挑战性。发现 CV 患者碳酸酐酶 5A (CA5A) 下游代谢物经常出现异常缺陷对于开发这种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) 物理关联，因此， CV 突变导致 CA5A 缺乏，(b) 对 CA5A 下游代谢物的仔细检查将提供 CV 缺陷的新型生物标志物。这项工作的具体目标是 [目标 1] 评估 CV 的发生率新生儿筛查与 CA5A 缺陷一致的患者中的缺陷； [目标 2] 确定 CV疾病的人成纤维细胞、细胞杂种和肝细胞系模型中CA5A损伤的存在和程度； [目标 3] 确定 CA5A 和 CV 之间是否存在物理关联。方法对 CV 进行测序新生儿筛查异常的受试者中的亚基基因与 CA5A 功能障碍一致（前瞻性和回顾性队列），使用稳定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/质谱光谱法）在健康个体的成纤维细胞和肝细胞系、基于遗传的心血管疾病和药理学抑制（寡霉素）。靶向代谢组学将重点关注 CA5A 下游的代谢物：氨基酸，特别是瓜氨酸（超高效液相色谱法）和有机酸丙酸和羟基异戊酸（气相色谱法/质谱法）。 CA5A的测定使用稳定同位素研究（13C 标记的乙酸盐孵育、同位素比质谱法）健康个体的传输软骨细胞系、肝细胞系和成纤维细胞系、遗传性心血管疾病和药理学CV抑制（寡霉素）。我们还将研究 CA5A 的体外物理相互作用正常和遗传性 CV 疾病肝细胞中的 CV（免疫共沉淀，蓝色天然凝胶）- 线。这些研究将严格研究 CA5A 功能与 CV 之间的相互作用缺陷和诊断心血管疾病的新的潜在生物标志物。我们的长期目标是扩大针对人类心血管缺陷的诊断和治疗方案，目前尚无治疗方法。这些研究将为开发未来的临床诊断测定法奠定基础，以快速、准确地实现这一目标。精确诊断 CV 疾病，并将导致人们对 CV 在协调中的作用有新的认识线粒体生化途径，为未来的治疗提供了新的靶点。