Studies on clinical and molecular heterogeneity in beta-ketothiolase deficiency.

β-酮硫解酶缺乏症（3 KTD）是一种常染色体隐性遗传的有机酸代谢疾病，涉及异亮氨酸分解代谢中间体和酮体，由线粒体乙酰辅酶A硫解酶（MAAT）缺乏引起。本研究从3 KTD的分子基础、表型/基因型相关性以及分子生物学技术在该病诊断中的应用等方面进行了研究。结果表明：1）在3 KTD中发现了几个新的突变，这些突变为RNA的剪接机制提供了线索。B）两个突变，N158 D和A301 P，与正常MAAT肽相比，由其翻译的肽具有较慢的电泳迁移率; c）内含子7的3'剪接位点的一个新的68-bp缺失，导致外显子8跳跃。2）我们在14个3 KTD家系中发现了19种基因突变，结论：3例KTD患者的基因突变和临床表现具有高度异质性。3）建立了免疫印迹法检测杂合子的方法，并首次应用PCR/MDE凝胶电泳技术对该病进行了产前诊断，4）制备了抗人胞浆型乙酰乙酰辅酶A硫解酶（CT）的抗体，并对其进行了初步的分子生物学研究本研究从分子水平上阐明了3 KTD的病因和发病机制，并且将能够临床应用于诊断患有3 KTD和相关酮体病症的患者以及其杂合子。

Beta-ketothiolase deficiency (3KTD) is an autosomal recessive metabolic disease of organic acids involving isoleucine catabolic intermediates and ketone bodies, caused by a deficiency of mitochondrial aceroacetyl-CoA thiolase (MAAT) . In this research project, we studied on the molecular basis of 3KTD,phenotype/genotype correlation, and application of molecular techniques for the diagnosis of this disease. The following results were obtained :1) Several novel mutations that would give clues for splicing mechanisms of the RNA were identified in 3KTD : eg. a) an exonic mutation at-13 that is a nonsense mutation, causing simultaneously an exon skipping ; b) two mutations, N158D and A301P,from which translated peptides had slower electrophoretic mobilities compared with that of the normal MAAT peptide ; c) a novel 68-bp deletion involving 3' splice site of intron 7, causing exon 8 skipping.2) We identified 19 types of gene mutations in 14 families with 3KTD,and concluded that the gene mutations as well as clinical findings were highly heterogenous among 3KTD patients. Furthermore, it was seemed that there is no phenotype/genotype correlation.3) We developped a method for heterozygote detection using immunoblot analysis, and performed a prenatal diagnosis of this disease by PCR/MDE gel electrophoresis for the first time, as clinical applications of the molecular studies of this disease.4) We prepared an antibody against human cytosolic acetoacety-CoA thiolase (CT) and cloned human CT cDNA.These should certainly contribute the molecular study of CT deficiency, which is another ketone body metabolic disease in the related field of 3KTD.This study made clear the causes and mechanisms of 3KTD at the molecular level, and would enable clinical application for the diagnosis of patients with 3KTD and related ketone body disorders as well as their heterozygotes.