Carbonic Anhydrase 5A Dysfunction in Complex V Deficiency

复合物 V 缺乏时碳酸酐酶 5A 功能障碍

• 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
• 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

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.

摘要 线粒体复合物V（CV）亚单位基因突变导致多种严重的代谢疾病， 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 临床批准的CV功能测定，因此，作出诊断是具有挑战性的。的发现 在CV患者中，碳酸酐酶5A（CA 5A）下游的代谢产物复发性异常 缺乏对开发针对这种深刻能量的有针对性的诊断测试具有深远的影响 缺陷我们假设（a）CV和碳酸酐酶5A（CA 5A）物理关联，因此， CV中的突变导致CA 5 A缺乏，和（B）对CA 5 A下游代谢物的仔细检查将提供 CV缺陷的新生物标志物。这项工作的具体目的是[目的1]评价CV的发生率 新生儿筛查与CA 5A缺乏症一致的患者中的缺乏症; [目的2]确定 CV疾病的人成纤维细胞、胞质杂种和肝细胞系模型中CA 5A损伤的存在和程度; 和[目的3]确定CA 5A和CV之间是否存在物理关联。方法将测序CV 与CA 5A功能障碍一致的异常新生儿筛查受试者的亚基基因（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CA 5A功能 靶向和非靶向代谢物的细胞评估（Orbitrap液相色谱/质谱 在来自健康个体、基于遗传的CV疾病和 药理学抑制（寡霉素）。靶向代谢组学将重点关注CA 5A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/质谱）。CA 5A测定 使用稳定同位素研究（13 C标记的乙酸盐孵育，同位素比质谱法） 来自健康个体的跨线粒体胞质杂交体、肝细胞和成纤维细胞系、基于遗传的CV疾病 和药理学CV抑制（寡霉素）。我们还将研究CA 5A的体外物理相互作用， 和CV（免疫共沉淀，蓝色天然凝胶）在正常和基于遗传的CV疾病肝细胞中- 线这些研究将严格调查CA 5A功能和CV之间的相互作用。 缺乏和新的潜在生物标志物诊断CV疾病。我们的长期目标是扩大 可用于人类CV缺陷的诊断和治疗选择，目前尚无治疗方法。 这些研究将为开发未来的临床诊断检测方法奠定基础， 精确诊断CV疾病，并将导致对CV在协调 线粒体生化途径，为未来的治疗提供了一个新的目标。