MOLECULAR CHARACTERIZATION OF BIOTINIDASE DEFICIENCY

生物素酶缺乏症的分子特征

• 批准号: 2148430
• 负责人: BARRY WOLF
• 金额: $ 23.76万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2148430
• 关键词: alleles; amidohydrolases; biopsy; blood chemistry; enzyme activity; enzyme deficiency; enzyme therapy; family genetics; gene expression; gene mutation; genetic library; genetic mapping; human genetic material tag; human subject; in situ hybridization; inborn metabolism disorder; introns; molecular cloning; northern blottings; nucleic acid probes; nucleic acid sequence; phenotype; southern blotting; structural genes

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.

生物素酶缺乏症是一种遗传性代谢紊乱， 特征为神经和皮肤异常， 无法回收维生素生物素。 幸运的是，这种疾病可以 用药理剂量的生物素成功治疗。我们有 以前描述的临床和许多生化特征 生物素酶和生物素酶缺乏症，包括几种新的 酶的功能。 我们现在建议阐明 生物素酶的特性及其缺陷，以了解 酶的生化和代谢功能及其在 生物素酶缺乏 我们克隆并测序了其全长cDNA 正常生物素酶的cDNA文库。 我们将 通过筛选人胎盘组织克隆生物素酶基因组DNA 用全长cDNA构建基因组DNA文库。 启动子区， 基因组DNA的外显子/内含子组织和内含子区域 将确定与外显子相邻的序列。 的原位杂交 将使用全长cDNA至中期染色体进行物理作图 将生物素酶基因导入特定染色体，形成体细胞杂交系 将用于将该映射细化到子带级别。分子 正常个体中生物素酶基因的特征将 有助于阐明导致生物素酶的突变 缺陷 将使用Southern印迹分析来分析总的 基因的变异 将使用北方印迹分析来鉴定 异常转录本和评估信息量。 异源 单链构象多态性分析将用于 识别小的遗传病变，包括错义突变。 细菌 表达系统将被用来评估推定的错义 酶活性的突变。 所鉴定的分子突变将是 与其临床和生化表型相关。 等 相关性将有助于确定是否所有被识别为 通过新生儿筛查患有生物素酶缺乏症的患者应接受以下治疗 生物素。 拟议的研究将提供一个更完整的了解 生物素酶和其他酶的生化和代谢作用 循环维生素和它们对正常营养的贡献， 维生素缺乏症