DISCRIMINATION BETWEEN CRIGLER-NAJJAR TYPE-I AND TYPE-II BY EXPRESSION OF MUTANT BILIRUBIN URIDINE DIPHOSPHATE-GLUCURONOSYLTRANSFERASE

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-glucuronosyltransferase (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 mu M) 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 K-m of 5.1+/-0.9 mu M and 7.9+/-5.3 mu M, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K-m = 56+/-23 mu M. 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.