Carbonic Anhydrase 5A Dysfunction in Complex V Deficiency

摘要 线粒体复合体V(CV)亚单位基因突变可导致多种严重的代谢性疾病 儿童健康和发育，有中风、神经病变、共济失调、视力丧失和心肌病。没有 临床批准的心血管功能测定，因此，作出诊断是具有挑战性的。这一发现 心血管疾病患者碳酸酐酶5A(CA5A)下游代谢产物的反复异常 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 缺乏症。我们假设(A)CV和碳水解酶5A(CA5A)在物理上相关，因此， CV的突变导致CA5A缺乏症和(B)对CA5A下游代谢物的检查将提供 CV缺乏症的新生物标志物。这项工作的具体目标是[目标1]评估心血管疾病的发生率 新生儿筛查与CA5A缺乏症一致的患者中的缺乏症；[目标2]确定 人血管病变成纤维细胞、囊虫和肝细胞系模型中CA5A损伤的存在和程度； 和[目标3]确定CA5A和CV之间是否存在物理关联。方法将排序简历 与CA5A功能障碍一致的新生儿筛查异常受试者的亚单位基因(前瞻性和 回溯队列)，使用稳定同位素标记的醋酸酯和体外直接测量CA5A功能 靶向和非靶向代谢物的细胞评估(Orbitrap液相色谱/质谱仪 光谱分析)来自健康人、遗传性心血管疾病和 药物抑制(寡霉素)。靶向代谢组学将专注于CA5A下游的代谢物： 氨基酸，特别是瓜氨酸(超高效液相色谱)和有机酸 丙酸和羟基异戊酸(气相色谱/质谱仪)。CA5A的测定 利用稳定同位素研究(13C标记醋酸盐孵育、同位素比质谱学)的功能 遗传性心血管疾病--来自健康人的线粒体胞质、肝和成纤维细胞系 和药物心血管抑制(奥奇霉素)。我们还将研究CA5A的体外物理相互作用 和CV(共免疫沉淀，蓝色天然凝胶)在正常和遗传性CV病肝细胞中的表达。 台词。这些研究将严格地研究CA5A功能和CV之间的相互作用 缺乏和新的潜在的诊断心血管疾病的生物标志物。我们的长期目标是扩大 诊断和治疗方案可用于人类CV缺乏症，但目前尚无治疗方法。 这些研究将为开发未来的临床诊断分析奠定基础，以快速和 准确诊断心血管疾病，将使人们对心血管疾病的协调作用有新的认识 线粒体生化途径，为未来的治疗提供了一个新的靶点。

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.