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

β-酮硫解酶缺乏症(3KTD)是由线粒体乙酰乙酰-辅酶A硫解酶(MAAT)缺陷引起的涉及异亮氨酸分解中间体和酮体的有机酸常染色体隐性代谢性疾病。在本研究项目中，我们对3KTD的分子基础、表型/基因相关性以及分子技术在该病诊断中的应用进行了研究。获得了以下结果：1)在3KTD中发现了几个新的突变，这些突变可能为RNA的剪接机制提供线索：例如。A)位于-13位的外显子突变是一个无义突变，同时导致外显子跳跃；b)两个突变N158D和A301P，其翻译的多肽与正常MAAT多肽相比具有较慢的电泳迁移率；c)涉及内含子7的3‘剪接点的新的68bp缺失，导致外显子8跳跃。2)我们在14个3KTD家系中鉴定了19种类型的基因突变，得出结论：3KTD患者的基因突变和临床表现具有高度的异质性。3)建立了免疫印迹杂合子检测方法，并首次应用PCR/MDE凝胶电泳法对本病进行了产前诊断，为本病的分子研究奠定了基础。4)制备了抗人胞浆乙酰乙酸辅酶A硫解酶(CT)抗体，克隆了人CT cDNA，为3KTD相关领域的又一酮体代谢性疾病CT缺乏症的分子研究奠定了基础。本研究从分子水平上阐明了3KTD的病因和发病机制。为3KTD及相关酮体疾病患者及其杂合子的诊断提供了临床应用。

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.