IDENTIFICATION OF 2 SINGLE-BASE SUBSTITUTIONS IN THE UGT1 GENE LOCUS WHICH ABOLISH BILIRUBIN URIDINE-DIPHOSPHATE GLUCURONOSYLTRANSFERASE ACTIVITY IN-VITRO

Accumulating evidence indicates that mutations in the human UGT1 gene locus abolish hepatic bilirubin UDP-glucuronosyltransferase activity a nd cause the subsequent accumulation of bilirubin to toxic levels in p atients with Crigler-Najjar type 1 (CN-I). Genetic and biochemical cri teria are required to link CN-I with mutations in UGT1. Here we presen t analysis of mutations at the UGT1 locus in three individuals that we re clinically diagnosed with CN-I (two related and one unrelated). Eac h patient carries a single base substitution that alters conserved res idues in the transferase enzyme molecule, serine to phenylalanine at c odon 376 and glycine to glutamic acid at codon 309. Each was homozygou s for the defect as demonstrated by sequencing and RFLPs. Mutant cDNAs , constructed by site-directed mutagenesis, inserted into expression v ectors, and transfected into COS-1 cells, supported the synthesis of t he bilirubin transferase protein but only cells transfected with the w ild-type cDNA expressed bilirubin UDP-glucuronosyltransferase activity . The data provide conclusive evidence that alterations at Gly 309 and Ser 376 are the genetic basis for CN-I in these families. These resul ts suggest that the two codons, located in conserved regions of the mo lecule, are part of the active site of the bilirubin enzyme.