Prenatal diagnosis of bilirubin‐UDP‐glucuronosyltransferase deficiency in rats by genomic DNA analysis

Hepatic bilirubin excretion requires UDP‐glucuronosyltransferase–mediated glucuronidation. Patients with type I Crigler‐Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP‐glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler‐Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP‐glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP‐glucuronyltransferase activity toward bilirubin and the 3‐methylcholanthrene‐inducible phenol–UDP‐glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3′ regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17‐day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern‐blot analysis with a double‐stranded DNA probe for the common region of UDP‐glucuronyltransferase activity toward bilirubin and the 3‐methylcholanthrene–inducible UDP‐glucuronyltransferase messenger RNAs. DNA samples from Gunn rats showed 600‐bp fragments, whereas normal rat DNA showed a 400‐bp and a 200‐bp band. In heterozygous Gunn rats, three bands at 600 bp, 400 bp and 200 bp were observed. Homozygosity and heterozygosity of the fetal rats was confirmed by analysis of bile pigments excreted in the meconium. This principle may be adapted for the intrauterine diagnosis of type 1 Crigler‐Najjar syndrome and identification of carriers who should receive genetic counseling. However, because individual patients with type 1 Crigler‐Najjar syndrome may have genetic lesions at different regions, more extensive genetic analysis will be required. (H