GENETICS & DIAGNOSIS OF HERIDITARY FRUCTOSE INTOLERANCE

这一提议是为了理解一种 人类代谢紊乱遗传性果糖不耐受(HFI)，其 病因、发病率、分布、生化和生理学 影响。这种疾病是由肝醛缩酶B基因突变引起的 受影响个体的基因。了解这些遗传问题将会 为医生和遗传咨询师提供更好的诊断和 为患有这种潜在灾难性疾病的家庭提供咨询。 此外，对突变蛋白质的生化分析将导致 对底物专一性和结构/功能有更深入的了解 这种重要的糖酵解酶之间的关系。的具体目标 建议的调查是：1)鉴定基因突变。 美国人群中肾综合征出血热感染者的DNA，2) 酶分析国家级标准实验室的建立 对这种和其他果糖代谢的正确诊断是必要的 肝活检引起的疾病；3)生化测定 以及这种疾病的生理根源通过酶的检查 由最常见的HFI等位基因产生。聚合酶链式反应 将采用聚合酶链式反应(PCR)从美国人的血液样本中扩增DNA 根据等位基因特异性识别已知等位基因的患者 寡核苷酸杂交(ASO)和用于鉴定任何新的 突变的等位基因。对于后者，所有的醛缩酶B的PCR扩增 在几个片段中的基因编码区，鉴定 因变性过程中的迁移而含有突变的片段 梯度凝胶电泳(DGGE)，然后直接测序 将使用第三种底物进行测定。一个必需品 对这些分子研究的补充将是确定 肝活检的醛缩酶活性。这是最好、最可靠的 肾综合征出血热的诊断方法。将对以下患者进行肝活检分析 临床医生提交的样本以确认或排除疑似HFI 个人。该设施还将确定可能的主题 分子研究。最后，将进行定点突变。 产生含有A149P突变的突变酶，这是 在55%的欧洲肾综合征出血热患者中存在。重组蛋白的表达和纯化 这种突变的酶，随后进行结构和功能分析，将 确定这种酶是否有任何残存活性，这可能解释 这些患者在缺乏糖异生的情况下表现出的正常的糖异生 果糖。

This proposal is directed toward understanding the molecular basis of a human metabolic disorder, hereditary fructose intolerance (HFI), its causes, incidence, distribution, and biochemical and physiological affects. This disease is caused by mutations in the liver aldolase B gene of affected individuals. Understanding these genetic issues will offer the physician and genetic counselor better tools for diagnosis and counseling of families with this potentially disastrous disease. Moreover, the biochemical analysis of mutant proteins will lead to a greater understanding of substrate specificity and structure/function relationships for this important glycolytic enzyme. The specific aims of the proposed investigations are: 1) the identification of mutations in the DNA from individuals with HFI in the American population, 2) the establishment of a national reference laboratory for the enzyme assays necessary for proper diagnosis of this and other fructose metabolic disorders from liver biopsy, and 3) the determination of the biochemical and physiological roots of this disorder by examination of the enzyme produced from the most common HFI allele. The polymerase chain reaction (PCR) will be employed to amplify DNA from blood samples of American patients for the identification of known alleles by allele specific oligonucleotide hybridization (ASO) and for the identification of any new mutant alleles. For the latter, PCR amplification of all the aldolase B encoded regions of the gene in several fragments, the identification of fragments which contain mutations by their migration in denaturing gradient gel electrophoresis (DGGE), followed by the direct sequence determination using a third primer, will be employed. A necessary complement to these molecular studies will be the determination of aldolase activity from liver biopsy. This is the best and most reliable method for diagnosis of HFI. Assays of liver biopsy will be performed on samples submitted by clinicians to confirm or rule out HFI in suspected individuals. This facility will also identify possible subjects for the molecular studies. Finally, site-directed mutagenesis will be performed to generate the mutant enzyme containing the A149P mutation, which is present in 55% of European HFI patients. Expression and purification of this mutant enzyme, followed by structural and functional analysis, will determine if this enzyme has any residual activity which may explain the normal gluconeogenesis which these patients exhibit in the absence of fructose.