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.
