Clarification of the cause of disease for a patient suffering from Crigler-Najjar syndrome tupe II with a heterologous mutation on the molecular basis

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10671026
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Embryonic/Neonatal medicine
• Research Institution: Science University of Tokyo
• Principal Investigator: KOIWAI Osamu Science University of Tokyo, Applied biological science, Assistant Professor, 理工学部・応用生物科学, 助教授 (50132923)
• Project Period (FY): 1998 – 1999
• Keywords: Bilirubin / Chrigler-Najjar / hereditary disease / jaundice

Research Abstract

It had been believed that Crigler-Najjar syndrome type II (CN-II) is inherited as a recessive trait. I found a patient with CN-II, who had a heterozygous nonsense mutation on the gene for bilirubin UDP-glucuronosyltransferase (Br-UDPGT), indicating that CN-II is inherited both as a dominant and as a recessive trait. I speculated that the Br-UDPGT forms a tetramer on a lumen of endoplasmic reticulum. Because Peters et al. had reported that Br-UDPGT was composed of four subunits by radiation inactivation experiment. To elucidate the cause of the CN-II with a heterozygous nonsense mutation on a molecular level, I performed an SDS-PAGE, cross-linking experiment, protein-protein interaction experiment, and gel filtration column chromatography. Among thses experiments, I could clearly demonstrate the cause of the disease by protein-protein interaction experiment. At first, I constructed the plasmids of His-UGTCN-II and GST-UGTCN-II and overexpressed the His-UGTCN-II and GST-UGTCN-II fusion proteins. Then I performed Ni-affinity column chromatography. As expected, both fusion proteins were simultaniouly eluted with imidazol buffer, showing that the Br-UDPGT formed a complex. I could verify the CN-II is caused by a dominant negative mutation.