MOLECULAR GENETIC STUDIES ON METHYLMALONYL COA MUTASE

甲基丙二酰辅酶A变位酶催化异构化 甲基丙二酰辅酶A到琥珀酰辅酶A，这是 支链氨基酸、单链脂肪酸、 和胆固醇。这种酶的遗传缺陷导致 甲基丙二酸血症--一种有机酸代谢紊乱 与智力低下、发育迟缓、暴发性 酸中毒，甚至新生儿死亡。大约1：20-50，00新生儿 患有这种往往致命的疾病。甲基丙二酰辅酶A变位酶 S是体内两种需要酶的钴胺(维生素B12)之一。 因此，B12缺乏在恶性贫血，或遗传性 B12新陈代谢的缺陷也会导致 甲基丙二酰辅酶A变位酶活力。 我们最近获得了甲基丙二酰辅酶A的全长克隆 从人肝脏中提取突变酶，并证明该克隆可以 用于通过基因转导MCM活性到培养细胞中 调职。这种克隆的出现使分子遗传 甲基丙二酸基的结构、功能及进化研究 辅酶A突变酶与遗传学、病理化学、诊断和治疗 甲基丙二酸血症。具体来说，我们建议 甲基丙二酰辅酶A变位基因的结构特征 通过对cdna和基因组克隆进行作图和测序。我们会 用这些克隆作为探针识别慢性粒细胞白血病患者的突变 甲基丙二酸血症。我们将生产重组甲基丙二酸甲酯 CoA变位酶在体外、真核细胞培养或原核生物中的应用 为了研究其结构、加工工艺、酶活性和 这种酶的代谢作用。我们将探索基因手段来 抑制培养细胞中甲基丙二酰辅酶A变位酶的活性 为了建立甲基丙二酰辅酶A变位酶的细胞模型 缺乏和研究次生代谢异常 是甲基丙二酸血症的致病因素之一。我们会 携带甲基丙二酰辅酶A变位酶重组病毒载体的研制 甲基丙二酰基酶活性重建的基因 辅酶A变位酶缺陷细胞及其在体细胞移植中的应用 甲基丙二酸血症的基因替代治疗。

Methymalonyl CoA mutase catalyzes the isomerization of methylmalonyl CoA to succinyl CoA which is an intermediate step in the degradation of branch chain amino acids, odd chain fatty acids, and cholesterol. Genetic deficiency of this enzyme causes methylmalonic acidemia, a disorder of organic acid metabolism associated with mental retardation, developmental delay, fulminant acidosis, or even neonatal death. Approximately 1:20-50,00 newborn suffer from this often fatal disorder. Methylmalonyl CoA mutase s one of two cobalamin (vitamin B12) requiring enzymes in the body. Thus, deficiency of B12 in pernicious anemia, or genetic deficiencies in B12 metabolism, also cause deficits in methylmalonyl CoA mutase activity. We have recently obtained a full length clone for methylmalonyl CoA mutase from human liver and demonstrated that this clone can be used to transduce MCM activity into cultured cells by genetic transfer. The availability of this clone enables molecular genetic studies of the structure, function, and evolution of methylmalonyl CoA mutase and the genetics, pathochemistry, diagnosis, and therapy of methylmalonic acidemia. Specifically, we propose to characterize the structure, of the methylmalonyl CoA mutase gene by mapping and sequencing both cDNA and genomic clones. We will use these clones as probes to identify mutations in patients with methylmalonic acidemia. We will produce recombinant methylmalonyl CoA mutase in vitro, in eukaryotic cell culture, or in prokaryotes in order to study the structure, processing, enzymatic activity and metabolic role of this enzyme. We will explore genetic means for suppression of methylmalonyl CoA mutase activity in cultured cells in order to create cellular models of methylmalonyl CoA mutase deficiency and study the secondary metabolic aberrations which contribute to the pathology of methylmalonic acidemia. We will develop recombinant viral vectors carrying methylmalonyl CoA mutase genes capable of reconstitution of enzyme activity in methylmalonyl CoA mutase deficient cells and explore the potential for somatic gene replacement therapy of methylmalonic acidemia.