GENETICS & DIAGNOSIS OF HERIDITARY FRUCTOSE INTOLERANCE

这项建议是针对理解的分子基础， 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 影响。 这种疾病是由肝脏醛缩酶B突变引起的 受影响个体的基因。 了解这些基因问题将 为医生和遗传咨询师提供更好的诊断工具， 为患有这种潜在灾难性疾病的家庭提供咨询。 此外，突变蛋白的生化分析将导致一个 更好地理解底物特异性和结构/功能 这种重要的糖酵解酶的关系。 的具体目标 建议的研究是：1）鉴定突变， 来自美国人群中HFI个体的DNA，2） 建立一个国家参考实验室，进行酶测定 必要的正确诊断这种和其他果糖代谢 肝活检的疾病，和3）生化测定 和生理根源，这种疾病的检查酶 由最常见的HFI等位基因产生。 聚合酶链反应 (PCR)将用于扩增美国人血液样本的DNA， 通过等位基因特异性鉴定已知等位基因的患者 寡核苷酸杂交（阿索）和用于鉴定任何新的 突变等位基因 对于后者，PCR扩增所有的醛缩酶B 在几个片段的基因编码区，鉴定 通过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 使用第三引物进行测定。 一个必要 这些分子研究的补充将是确定 肝脏活检的醛缩酶活性。 这是最好最可靠的 HFI的诊断方法。 肝活检检测将在 临床医生提交的样本，以确认或排除疑似HFI 个体 该设施还将确定可能的主题， 分子研究 最后，将进行定点诱变 以产生含有A149 P突变的突变酶， 55%的欧洲HFI患者存在。 的表达和纯化 这种突变酶，随后进行结构和功能分析， 确定这种酶是否有任何残余活性，这可以解释 这些患者在缺乏 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.