Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia
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Increased levels of retinol binding protein 4 (RBP4) in serum is associated with insulin resistance. To examine this further, the genomic region of RBP4 was genetically surveyed in Mongolian people, who as a group are suffering from a recent rapid increase in diabetes. The RBP4 gene was screened by DHPLC system, and the PCR fragments which showed heteroduplex peaks in multiple samples were followed by direct sequencing to identify common polymorphisms in 48 Mongolian diabetic samples. Identified single nucleotide polymorphisms (SNPs) were genotyped in 511 control and 281 type 2 diabetes samples. The functions of SNPs in the regulatory region were assessed by reporter gene assay and electrophoretic mobility shift assay. Possible association between functional SNPs and serum RBP4 levels or metabolic parameters was statistically assessed. Nine SNPs were identified in the RBP4 gene. A case-control study revealed that the rare alleles of four SNPs were associated with increased risk of diabetes, even after Bonferroni correction (−803, G > A, P = 0.0054; +5169, C > T, P = 0.0025; +6969, G > C, P = 0.0015; +7542, T > del, P = 0.0015). The −803 G > A SNP influenced the transcription efficiency in a hepatocarcinoma cell line as well as the binding efficiency of hepatocyte nuclear factor 1 alpha to the motif. In addition, the −803 A allele was associated with increased serum RBP4 levels in diabetic patients. We have identified a functional SNP in the RBP4 gene associated with type 2 diabetes in Mongolian people.
KeywordsHepG2 Cell Electrophoretic Mobility Shift Assay Reporter Gene Assay DHPLC System Electrophoretic Mobility Shift Assay Reaction
Retinol binding protein 4
Hepatocyte nuclear factor 1 alpha
Single nucleotide polymorphism
Electrophoretic mobility shift assay
The study was supported by grants in aid (No.17390205 and F-27 as a center of excellence) from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and a grant from the Heiwa Nakajima Foundation (Tokyo, Japan). We thank Oyamada T, Nagashima K, Oh-hashi Y, Ishikawa T, Awano A for valuable technical assistance. We thank Drs. Shinetuya D, Tsetsgee D, Munkhchimeg Ch and Batjargal Ch (Health Sciences University of Mongolia) for sample collection, and Drs. Ishibashi S and Nagasaka S (Department of Endocrinolgy, Jichi Medical School) for valuable discussion.
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