GENETICS/DIAGNOSIS OF HEREDITARY FRUCTOSE INTOLERANCE

描述：（改编自申请人摘要）本提案是 旨在了解人类代谢的分子基础， 碳水化合物代谢障碍，遗传性果糖不耐受（HFI）， 以及它的成因、分布、生化和生理 方面的影响. 这些研究将直接检验可能解释 HFI患者在缺乏果糖的情况下表现出的正常的新生血管生成， 因此，可能会导致更好地了解葡萄糖稳态， 人类 拟议调查的具体目的是：1） 鉴定来自HFI个体的DNA突变， 美国人口，和2）的生化根源的测定， 通过检查正常的酶，醛缩酶B， 由含有错义突变的HFI等位基因产生的酶。 一是 聚合酶链反应（PCR）将用于扩增血液中的DNA 用于鉴定已知突变的美国HFI患者样本 通过等位基因特异性寡核苷酸杂交（ASO），直接PCR分析， 或扩增难治性突变（ARM）分析。 第二，对于HFI 不携带21种已知突变中的任何一种的受试者， 扩增编码整个醛缩酶B的几个基因片段 序列将用于鉴定新的突变。 这些突变 将通过使用第三引物的直接序列测定来鉴定， PCR辅助循环测序。 任何新突变的分离分析 将提供基因证据证明这些突变导致HFI。 第三、 进行定点诱变以产生突变酶 含有HFI错义突变。 这些HFI酶将被表达， 纯化为谷胱甘肽-S-转移酶（GST）的融合蛋白。 第四、 结构和功能分析将确定HFI酶是否具有 任何残余活动。 第五，正常醛缩酶B的酶学将是 通过测量几种共价反应的水平进行检查 中间体（席夫碱、烯胺等）通过化学捕集， 它们的形成和相互转化率（席夫碱，碳-碳 键断裂，质子提取）通过在单次转换期间快速淬灭 实验 最后，稳定的HFI酶将在类似的环境中进行检查。 实验来评估导致HFI的生化缺陷的性质。

DESCRIPTION: (Adapted from the applicant's abstract) This proposal is directed toward understanding the molecular basis of a human metabolic disorder of carbohydrate metabolism, hereditary fructose intolerance (HFI), as well as its causes, distribution, and biochemical and physiological effects. These studies will directly test hypotheses that may explain the normal gluconeogenesis that HFI patients exhibit in the absence of fructose, and therefore, may lead to a better understanding of glucose homeostasis in humans. The specific aims of the proposed investigations are: 1) the identification of mutations in the DNA from individuals with HFI in the American population, and 2) the determination of the biochemical roots of this disorder by examination of both the normal enzyme, aldolase B, and the enzymes produced from HFI alleles containing missense mutations. First, the polymerase chain reaction (PCR) will be employed to amplify DNA from blood samples of American HFI patients for the identification of known mutations by allele specific oligonucleotide hybridization (ASO), direct PCR analysis, or amplification refractory mutation (ARM) analysis. Second, for those HFI subjects who do not carry any of the 21 known mutations, similar PCR amplification of several gene fragments encoding the entire aldolase B sequence will be used for identification of new mutations. These mutations will be identified by direct sequence determination using a third primer in PCR assisted cycle sequencing. Segregation analysis of any new mutations will give genetic evidence that these mutations cause HFI. Third, site-directed mutagenesis will be performed to generate mutant enzymes containing HFI missense mutations. These HFI enzymes will be expressed and purified as fusion proteins to glutathione-S-transferase (GST). Fourth, structural and functional analyses will determine if the HFI enzymes have any residual activity. Fifth, the enzymology of normal aldolase B will be examined by measurement of the levels of several covalent reaction intermediates (Schiff base, enamine, etc.) by chemical trapping and the rates of their formation and interconversion (Schiff base, carbon-carbon bond cleavage, proton abstraction) by rapid quenching during single turnover experiments. Lastly, stable HFI enzymes will be examined in similar experiments to assess the nature of the biochemical defects that causes HFI.