Identification of two 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 and cause the subsequent accumulation of bilirubin to toxic levels in patients with Crigler-Najjar type 1 (CN-I). Genetic and biochemical criteria are required to link CN-I with mutations in UGT1. Here we present analysis of mutations at the UGT1 locus in three individuals that were clinically diagnosed with CN-I (two related and one unrelated). Each patient carries a single base substitution that alters conserved residues in the transferase enzyme molecule, serine to phenylalanine at codon 376 and glycine to glutamic acid at codon 309. Each was homozygous for the defect as demonstrated by sequencing and RFLPs. Mutant cDNAs, constructed by site-directed mutagenesis, inserted into expression vectors, and transfected into COS-1 cells, supported the synthesis of the bilirubin transferase protein but only cells transfected with the wild-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 results suggest that the two codons, located in conserved regions of the molecule, are part of the active site of the bilirubin enzyme.