MOLECULAR GENETIC STUDIES ON METHYLMALONYL COA MUTASE

甲基丙二酰辅酶A催化 甲基丙二酰辅酶A到琥珀酰辅酶A，这是一个中间步骤， 分支链氨基酸，奇链脂肪酸， 和胆固醇 这种酶的遗传缺陷导致 甲基丙二酸血症，一种有机酸代谢障碍 与智力迟钝、发育迟缓、暴发性 酸中毒甚至新生儿死亡 约1：20- 50，00新生儿 患有这种致命的疾病。 甲基丙二酰辅酶A 维生素B12是人体内需要酶的两种钴胺素之一。 因此，恶性贫血中缺乏B12，或遗传性贫血， 缺乏B12代谢，也会导致 甲基丙二酰辅酶A酶活性。 我们最近获得了甲基丙二酰辅酶A的全长克隆 从人类肝脏中克隆，并证明该克隆可以 用于通过遗传学方法将MCM活性转移到培养的细胞中， 转移 这个克隆的可用性使得分子遗传学 甲基丙二酰的结构、功能和进化研究 CoA酶与遗传学、病理化学、诊断和治疗 甲基丙二酸血症 具体而言，我们建议 描述甲基丙二酰辅酶A β基因的结构 通过绘制和测序cDNA和基因组克隆。 我们将 使用这些克隆作为探针来鉴定患有 甲基丙二酸血症 我们将生产重组甲基丙二酰 CoA在体外、真核细胞培养物或原核生物中表达 为了研究其结构、加工、酶活性和 这种酶的代谢作用。 我们将探索基因手段， 抑制培养细胞中甲基丙二酰辅酶A的活性 为了建立甲基丙二酰辅酶A的细胞模型， 缺乏和研究继发代谢畸变， 导致甲基丙二酸血症的病理学改变。 我们将 开发携带甲基丙二酰辅酶A的重组病毒载体 能够在甲基丙二酰中重建酶活性的基因 辅酶A缺乏的细胞，并探索体细胞的潜力， 甲基丙二酸血症的基因替代疗法。

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.