MOLECULAR CHARACTERIZATION OF BIOTINIDASE DEFICIENCY

生物素酶缺乏症是一种遗传性代谢紊乱， 以神经和皮肤异常为特征的 不能回收维生素生物素。幸运的是，这种紊乱可以是 成功地用药理剂量的生物素治疗。我们有 以前的特点是临床和许多生化特征 生物素酶和生物素酶缺乏症，包括几个新的 酶的功能。我们现在建议阐明分子 了解生物素酶的特性及其缺陷 酶的生化和代谢功能及其在体内的作用 生物素酶缺乏症。我们已经克隆并测序了全长的cdna。 通过筛选人肝脏cDNA库，获得了正常生物素酶的表达。我们会 筛选人胎盘组织克隆生物素酶基因组DNA 含全长c DNA的基因组DNA文库。启动子区域， 基因组DNA的外显子/内含子组织以及内含子的区域 外显子的相邻位置将会被确定。白粉菌的原位杂交研究 全长cdna到中期染色体将被用于物理作图。 将生物素酶基因转入特定染色体、体细胞杂交系 将用于将此映射细化到子带级别。分子 正常人Will生物素酶基因的特征 促进阐明导致生物素酶的突变 缺乏症。Southern印迹分析将用于分析总 基因的变异。将使用Northern印迹分析来鉴定 并评估信息的数量。异质双工 单链构象多态分析将用于 识别微小的基因损伤，包括错义突变。一种细菌 表达系统将用于评估推定错义的影响 酶活性的突变。已确认的分子突变将是 与他们的临床和生化表型相关。是这样的 相关性将有助于确定是否所有被确认为 新生儿筛查发现生物素酶缺乏症应给予治疗 生物素。拟议的研究将提供一个更完整的理解 生物素酶和其他酶的生化和代谢作用 循环利用维生素及其对正常营养的贡献 维生素缺乏症。

Biotinidase deficiency is an inherited metabolic disorder that is characterized by neurologic and cutaneous abnormalities resulting from the inability to recycle the vitamin biotin. Fortunately, the disorder can be successfully treated with pharmacologic doses of biotin. We have previously characterized the clinical and many of the biochemical features of biotinidase and biotinidase deficiency, including several novel functions of the enzyme. We now propose to elucidate the molecular characteristics of biotinidase and its defects to understand the biochemical and metabolic functions of the enzyme and their roles in biotinidase deficiency. We have cloned and sequenced the full-length cDNA of normal biotinidase by screening a human hepatic cDNA library. We will clone the genomic DNA of biotinidase by screening a human placental genomic DNA library with the full-length cDNA. The promoter region, exon/intron organization of genomic DNA, and regions of the introns adjacent to the exons will be determined. In situ hybridization of the full-length cDNA to metaphase chromosomes will be used to physically map the biotinidase gene to a specific chromosome, Somatic cell hybrid lines will be used to refine this mapping to the subband level. Molecular characterization of the biotinidase gene in normal individuals will facilitate the elucidation of mutations that result in biotinidase deficiency. Southern blot analysis will be used to analyze gross aberrations of the gene. Northern blot analysis will be used to identify aberrant transcripts and to assess the quantity of message. Heteroduplex and single-stranded conformational polymorphism analyses will be used to identify small genetic lesions, including missense mutations. A bacterial expression system will be used to assess the effect of putative missense mutations on enzyme activity. The identified molecular mutations will be correlated with their clinical and biochemical phenotypes. Such correlations will be useful in determining if all infants identified as having biotinidase deficiency by newborn screening should be treated with biotin. The proposed research will provide a more complete understanding of the biochemical and metabolic role of biotinidase and other enzymes that recycle vitamins and their contributions to normal nutrition and vitamin deficiency states.