SPLICE-SITE MUTATIONS - A NOVEL GENETIC MECHANISM OF CRIGLER-NAJJAR SYNDROME TYPE-1

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, pote ntially lethal disorder characterized by severe unconjugated hyperbili rubinemia resulting from deficiency of the hepatic enzyme bilirubin-UD P-glucuronosyltransferase. In all CN-1 patients studied, structural mu tations in one of the five exons of the gene (UGT1A1) encoding the uri dinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirub in-UGT(1), were implicated in the absence or inactivation of the enzym e. We report two patients in whom CN-1 is caused, instead, by mutation s in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the i ntron, between exon 1 and exon 2. The other patient (B) was heterozygo us for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT(1), mRNA is difficult to obtain, since i t is expressed in the liver only. To determine the effects of these sp lice-junction mutations, we amplified genomic DNA of the relevant spli ce junctions. The amplicons were expressed in COS-7 cells, and the exp ressed mRNAs were analyzed. In both cases, splice-site mutations led t o the use of cryptic splice sites, with consequent deletions in the pr ocessed mRNA. This is the first report of intronic mutations causing C N-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.