The specific aims of this project were (A) to perform a genotype-phenotype correlation in patients with Crigler-Najjar syndrome types I and II, and (B) to define the molecular basis of Gilbert syndrome. In addition to making progress on these aims, we have made additional advances in a closely related area, preclinical development of gene therapy methods for Crigler-Najjar syndrome type I. Genotype-phenotype correlation: We have continued to analyze the sequence of the coding region for the UGT1A1 gene. These studies have confirmed that mutations in any of the five exons encoding UGT1A1 (bilirubin-UGT) can markedly reduce the enzyme activity, causing Crigler-Najjar syndrome types I and II. The following mutations were of specific interest. (i) A novel mutation found in exon 5, downstream to sequences encoding the UDP-glucuronic acid-binding and the putative membrane-anchoring regions. This shows that deletion of a few aminoacid residues in the domains for which no specific function have been assigned can also be critical for enzyme activity. (ii) A mutation upstream to the putative region of the bilirubin-binding site was associated with severe hyperbilirubinemia, consistent with Crigler-Najjar syndrome type 1. However, in this case, the bile contained a small amount of bilirubin glucuronides. This case highlights the overlap between phenotypes of Crigler-Najjar syndrome types I and II. Our hypothesis is that mutations in the vicinity of the bilirubin-binding domain will increase the Km of the enzyme for bilirubin, thereby increasing serum bilirubin levels. However, in these cases, the Vmax may be relatively unaffected and, therefore, the bile may contain bilirubin glucuronides. This hypothesis is being tested by site-directed mutagenesis of the expression constructs. The significance of genotypic diagnosis of Crigler-Najjar syndrome has increased as a result of our recent work on site-directed gene repair in vivo. This strategy requires the generation and delivery of a synthetic RNA-DNA chimera that is capable of aligning at the site of mutation and triggering the cell's mismatch repair system. In contrast to other methods of gene therapy, this method requires identification of the specific genetic lesion. Molecular basis of Gilbert syndrome: We have continued to perform genetic analysis in a large number of patients with mild unconjugated hyperbilirubinemia. These studies have revealed the following interesting findings: (i) There is an extremely high correlation between the clinical diagnosis of Gilbert syndrome and a longer than normal TATAA element upstream to exon 1 of UGT1A1. In fact, among Caucasians, blacks and (Asian) Indians, we have found no exceptions to this rule. In some cases of Gilbert syndrome, the TATAA element is elongated by four nucleotides (TATA), rather than two (TA). However, TATAA elements that were shorter than normal by two nucleotides (TA) were not associated with Gilbert syndrome. (ii) A structural mutation in exon 1 of UGT1A1 has been reported to be associated with Gilbert syndrome in many Japanese patients. Our extensive studies have not revealed this mutation in Caucasians, blacks and (Asian) Indians. We will determine the significance of the mutation reported from Japan by site-directed mutagenesis followed by expression in COS cells.
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