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Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. 1994

J Seppen, and P J Bosma, and B G Goldhoorn, and C T Bakker, and J R Chowdhury, and N R Chowdhury, and P L Jansen, and R P Oude Elferink
Department of Gastrointestinal and Liver Diseases, Academic Medical Centre, Amsterdam, The Netherlands.

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.

UI MeSH Term Description Entries
D007223 Infant A child between 1 and 23 months of age. Infants
D008297 Male Males
D008862 Microsomes, Liver Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough. Liver Microsomes,Liver Microsome,Microsome, Liver
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D002648 Child A person 6 to 12 years of age. An individual 2 to 5 years old is CHILD, PRESCHOOL. Children
D002675 Child, Preschool A child between the ages of 2 and 5. Children, Preschool,Preschool Child,Preschool Children
D003414 Crigler-Najjar Syndrome A familial form of congenital hyperbilirubinemia transmitted as an autosomal recessive trait. It is characterized by icterus and brain damage caused by a glucuronyl transferase deficiency in the liver and faulty bilirubin conjugation. Crigler Najjar Syndrome,Crigler Najjar Syndrome, Type 1,Crigler-Najar Syndrome,Crigler-Najjar Syndrome, Type I,Familial Nonhemolytic Unconjugated Hyperbilirubinemia,Hereditary Unconjugated Hyperbilirubinemia,Crigler Najar Syndrome,Crigler Najjar Syndrome, Type I,Crigler Najjar Syndromes,Hereditary Unconjugated Hyperbilirubinemias,Hyperbilirubinemia, Hereditary Unconjugated,Najjar Syndrome, Crigler,Syndrome, Crigler Najjar,Unconjugated Hyperbilirubinemia, Hereditary
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked

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