Measurement of biochemical analyses and genotyping

The level of fasting plasma glucose (FPG) was measured using the hexokinase method. The levels of lipids were measured using the enzymatic methods for triglycerides (TGs) and total cholesterol (TC) measurements, and the clearance methods for high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) measurements. The measurements were performed using a kit assay (SEKISUI Medical Technology, Tokyo, Japan) and an automatic biochemical analyser (Hitachi 7060). The levels of adipocytokines (leptin, adiponectin and resistin) were measured by ELISA techniques.¹⁹

Genomic DNA was isolated from peripheral white blood cells using the salt fractionation method. Genotyping of rs3748024 was conducted using the TaqMan Allelic Discrimination Assay with the GeneAmp 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The genotyping call rate for the SNP was 98.3%. We repeated 70 samples randomly for the SNP to validate the accuracy of the genotyping and observed 100% concordance between the results of the two tests. We also sent 30 samples to direct sequencing and observed 100% concordance between the two genotyping methods.