Splice-Site Mutations: A Novel Genetic Mechanism of Crigler-Najjar Syndrome Type 1

Abstract

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations 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 intron, between exon 1 and exon 2. The other patient (B) was heterozygous 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-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.

Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome

“…13). Many instances in which a single-base substitution at a splice junction resulted in abnormal splicing have been reported (3,14). Members of the TSU family (Fig.…”

Outcome of donor splice site mutations accounting for congenital afibrinogenemia reflects order of intron removal in the fibrinogen alpha gene (FGA)

“…Although it is not immediately possible to conclude from the experiments in COS-7 cells that exactly the same aberrant mRNAs are being produced in the patient's hepatocytes, since cell-specific mRNA splicing of numerous genes, including the fibrinogen ␥-chain (FGG) gene, 20 has been proven, Gantla et al previously demonstrated the utility of the COS-7 model in evaluating the effects of potential splice-site mutations, particularly for genes expressed only in inaccessible tissues. 21 Two different outcomes were found for the common IVS4ϩ1GϾT mutation (cryptic splice-site activation) and the IVS3ϩ1_ϩ4delGTAA mutation (exon 3 skipping). Outcome of splice-site mutations in the fibrillar collagen genes COL1A1, COL1A2, COL3A1, and COL5A1 has been proposed to differ according to the order of intron removal.…”

Homodimerization of Human Bilirubin-Uridine-diphosphoglucuronate Glucuronosyltransferase-1 (UGT1A1) and Its Functional Implications

“…The entire 2.2-kilobase pair mutagenized hUGT1A1 cDNA was excised by digestion with NotI and XhoI and subcloned into the pcDNA3.1/Zeo(ϩ) expression vector (Invitrogen). Introduction of the desired mutation was confirmed by nucleotide sequence determination by the dideoxy chain termination method (22)(23)(24).…”

“…A 646-bp segment (nucleotides 320 -1066) was amplified by reverse transcription-primed polymerase chain reaction using the following amplimers: sense, 5Ј-GCGTGTGAT-CAAAACATACAA-3Ј; and antisense, 5Ј-GCCACTTAACAAGTATCGT-GTTG-3Ј. The sequence of the amplimer was determined by the dideoxy chain termination method using the cycle sequencing procedure as described (22)(23)(24).…”