title: Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase.
abstract: Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.
authors.affiliation: Department of Gastrointestinal and Liver Diseases, Academic Medical Centre, Amsterdam, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Seppen
authors.forename: P J
authors.initials: PJ
authors.lastname: Bosma
authors.forename: B G
authors.initials: BG
authors.lastname: Goldhoorn
authors.forename: C T
authors.initials: CT
authors.lastname: Bakker
authors.forename: J R
authors.initials: JR
authors.lastname: Chowdhury
authors.forename: N R
authors.initials: NR
authors.lastname: Chowdhury
authors.forename: P L
authors.initials: PL
authors.lastname: Jansen
authors.forename: R P
authors.initials: RP
authors.lastname: Oude Elferink
mesh_terms: D000293:Adolescent; D000818:Animals; D001663:Bilirubin / Q000031:analogs & derivatives / Q000096:biosynthesis; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003414:Crigler-Najjar Syndrome / Q000145:classification* / Q000201:enzymology / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007223:Infant; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; C019787:bilirubin glucuronoside glucuronosyltransferase; C016266:bilirubin glucuronate; D014453:Glucuronosyltransferase; D001663:Bilirubin