INHERITED DISORDERS OF HEPATIC BILIRUBIN GLUCURONIDATION

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UDP-glucuronosyltransferases (UGTs) are a family of enzymes that catalyze the glucuronidation of endogenous substrates, e.g. bilirubin and hormones and exogenous substances, e.g. toxins, carcinogens and drugs. Glucuronidation is critical for detoxication and elimination of bilirubin; inherited deficiencies of bilirubin-UGT (B-UGT) activity results in three grades of hyperbilirubinemia: (i) Crigler-Najjar syndrome, Type I (CN-I) (complete deficiency of B-UGT activity, potentially lethal); (ii) Crigler-Najjar syndrome, Type II (CN-II) (incomplete deficiency of B-UGT activity, usually responsive to phenobarbital) and (iii) Gilbert syndrome, a common mild disorder with partial deficiency of B-UGT activity. Immaturity of B-UGT activity is an important cause of neonatal jaundice. The mRNAs for two forms of B-UGT and that for another form with activity toward phenolic substrates (P-UGT) are expressed from one locus consisting of a series of exons encoding the unique NH2 terminal regions of these UGTs and four exons encoding their identical COOH-terminal region. We will determine the DNA structural abnormality that results in CN-I. In neonatal jaundice and in CN-II and Gilbert syndrome, the defect may be in the regulation of expression of UGTs. Although expression products of a single locus, B-UGT and P-UGT are differentially expressed during development, in various tissues and after administration of isoform-specific inducing agents. To determine the mechanism of the differential expression of the B-UGTs and P-UGT, we will identify and characterize regulatory elements present upstream of each unique region and also possibly at intragenic sites, by DNase I hypersensitivity and methylation analysis. Binding sites for regulatory elements will be localized by DNase foot-printing, methylation interference and mobility shift assays. In patients with partial deficiency of B-UGT activity (CN-II and Gilbert syndrome) the abnormality may lie in the regulatory elements. Therefore, sequence of these regions of CN-II and Gilbert syndrome will be determined. Function of the regulatory elements will be evaluated using reporter genes by in vitro transcription analysis and in vivo following receptor-mediated targeting to hepatocytes in rats. Possible role of transcription regulatory proteins on the enhancing or repressing the function of regulatory elements during development and enzyme induction will be determined by in vitro transcription analysis. Delineation of the expression of the multiple UGT isoforms from this uniquely complex gene should elucidate important and novel aspects of mammalian gene expression. Study of the hyperbilirubinemic patients and their family members will provide simple non-invasive methods for genotypic diagnosis, including prenatal diagnosis, and will clarify the molecular basis of inherited disorders of bilirubin glucuronidation.