Identification and functional characterization of a novel mutation of hepatocyte nuclear factor-1alpha gene in a Korean family with MODY3. Diabetologia 2003 May;46(5):721-7
Date
04/25/2003Pubmed ID
12712243DOI
10.1007/s00125-003-1079-7Scopus ID
2-s2.0-0038353162 (requires institutional sign-in at Scopus site) 23 CitationsAbstract
AIMS/HYPOTHESIS: After screening 16 Korean families with early onset Type 2 diabetes in search for hepatocyte nuclear factor (HNF) -1alpha gene mutation, we identified a novel missense mutation (R263L) associated with MODY phenotype. We studied the biological characteristics of the mutation and the potential functional consequences based on the crystallographic structure of HNF-1alpha in complex with DNA.
METHODS: DNA from subjects with a familial form of early onset diabetes was isolated and HNF-1alpha was sequenced. The R263L substitution was generated by PCR-based sited-directed mutagenesis. Functional and biochemical studies were conducted by reporter assay using glucose-transporter type 2 (GLUT2) or insulin promoters and electrophoretic mobility shift assay, respectively.
RESULTS: Transfection of wild-type HNF-1alpha increased the reporter activities of GLUT2 and insulin promoters in NIH3T3 and SK-Hep1 cells, while R263L mutant was defective in transactivation of those promoters. Both wild-type HNF-1alpha and R263L mutant could not transactivate GLUT2 and insulin promoters in MIN6N8 insulinoma cells. R263L mutant had a defective cooperation with its heterodimeric partner HNF-1beta or coactivator p300. R263L mutant protein displayed greatly reduced DNA binding ability, despite its comparable protein stability to the wild-type HNF-1alpha.
CONCLUSION/INTERPRETATION: These results suggest that the mutation of HNF-1alpha at codon 263 from arginine to leucine leads to the development of MODY3 through decreased insulin production and defective glucose sensing. These findings are in good agreement with the crystal structure in which R263 makes hydrogen bonds with phosphorus atoms of DNA backbone to mediate the stable binding of HNF-1alpha homeodomain to the promoter.
Author List
Kim KA, Kang K, Chi YI, Chang I, Lee MK, Kim KW, Shoelson SE, Lee MSAuthor
Young-In Chi PhD Assistant Professor in the Surgery department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SubstitutionArginine
Binding Sites
DNA-Binding Proteins
Diabetes Mellitus, Type 2
Female
Glucose Transporter Type 2
Hepatocyte Nuclear Factor 1
Hepatocyte Nuclear Factor 1-alpha
Hepatocyte Nuclear Factor 1-beta
Humans
Insulin
Korea
Leucine
Male
Models, Molecular
Monosaccharide Transport Proteins
Mutation, Missense
Nuclear Proteins
Pedigree
Promoter Regions, Genetic
Protein Conformation
Transcription Factors
