A total of 320 MetS patients [mean age ± standard deviation (SD), 50.94±8.434] and 320 healthy control subjects (mean age ± SD, 49.86±11.76 years) were included in this study. We used the National Cholesterol Education Program Adult Treatment Panel III definition for MetS (15) with slight modifications, including waist circumference (WC) (≥90 cm in men, ≥85 cm in women); increased triglycerides level (≥150 mg/dl); high-density lipoprotein (HDL)-cholesterol (<40 mg/dl in men, <50 mg/dl in women); increased blood pressure (≥130/85 mmHg or antihypertensive medication) and increased fasting blood glucose (≥100 mg/dl or type II DM). Individuals with ≥3 traits of the risk factors described above were defined as MetS patients. The control group comprised healthy individuals without a history of hypertension, DM, cardiovascular or other diseases to exclude those with MetS.

The biospecimen and data used in this study werew provided by the Jeju National University Hospital Biobank, a member of the National Biobank of South Korea, which is supported by the Ministry of Health and Welfare. All samples obtained were from Korean individuals and were collected together with written informed consent according to protocol approved by the Institutional Review Board of the Jeju National University Hospital in June 2002.

Total genomic DNA was extracted according to the manufacturer’s instructions (Qiagen, Inc., Valencia, CA, USA). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques were used to detect CYP11B2 polymorphisms with slight modifications of the method used by Gu et al (12). The PCR for the -344T/C polymorphism was performed using sense (5′-GTG TCA GGG CAG GGG GTA-3′) and antisense (5′-AGG CGT GGG GTC TGG ACT-3′) primers. DNA was amplified for 35 cycles with denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. The amplified PCR products were 228 bp and digested with a restriction endonuclease, HaeIII (New England Biolabs, Beverly, MA, USA) at 37°C for 17 h. The primer sequences used to detect the K173R polymorphism were 5′-GAA AAG GCT GCA GCT CGA ACA CAA-3′ (sense) and 5′-GCA TGG CCC ACA CCT TCT A-3′ (antisense). DNA was amplified for 35 cycles with denaturation at 94°C for 30 sec, annealing at 65°C for 30 sec and extension at 72°C for 1 min. Bands of 371 bp were amplified by PCR and digested with a restriction endonuclease Bsu36I (New England Biolabs). The IC polymorphism was analyzed using two separate PCRs, one to amplify the wild-type gene (W) and another to amplify the conversion gene (C). The forward primer sequence for the wild-type gene was 5′-TGG AGA AAA GCC CTA CCC TGT-3′, whereas 5′-CAG AAA ATC CCT CCC CCC TA-3′ was used for the conversion gene. The same reverse primer: 5′-AGG AAC CTC TGC ACG GCC-3′ was used for the two PCRs. PCR conditions were run for 35 cycles with denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. The size of PCR products in each reaction was ~418 bp and was directly detected by 2% agarose gel electrophoresis.

For measurements of biochemical traits, venous blood of the study subjects was collected into EDTA tubes after overnight fasting. The levels of plasma glucose, triglycerides and HDL-cholesterol were measured enzymatically on an automatic analyzer (TBA 200FR NEO; Toshiba Medical Systems, Tokyo, Japan).

Anthropometric traits containing body mass index (BMI) and WC were measured. BMI was calculated as weight (kg) divided by height (m²). Using a flexible tape, WC was measured at the smallest horizontal circumference between the costal margin and iliac crest. Measurements of blood pressures were obtained on the right arm at a seated position after 10 min of rest.

Data were expressed as means ± SD. Clinical characteristics were compared by the Student’s unpaired t-test. The χ² test was used to compare genotype and allele frequencies of CYP11B2 polymorphisms between the cases and controls and to test for Hardy-Weinberg equilibrium. Associations of the studied polymorphisms with MetS prevalence were calculated using adjusted odds ratios (AORs) and 95% confidence intervals (95% CIs) from multivariate logistic regression adjusted for age and gender. StatsDirect statistical software, version 2.4.4 (StatsDirect, Ltd., Altrincham, UK) was used to calculate the AOR and 95% CIs. P<0.05 was considered to indicate statistically significant differences. Haplotypes for multiple loci were estimated using the expectation-maximization algorithm with SNPAlyze (version 5.1; Dynacom Co., Ltd., Yokohama, Japan). The linkage disequilibrium (LD) between loci was measured using the absolute value of Lewontin’s D (16).

MetS patients had significantly higher values for MetS risk parameters such as BMI, glucose, blood pressure, triglycerides and WC (P<0.0001), but lower levels of HDL-cholesterol (P<0.0001) than the control subjects (Table I).

A comparison was made of the genotype and allele frequencies of the CYP11B2 -344T>C, K173R and IC polymorphisms between the MetS and control groups (Table II). The genotype frequencies for all of the polymorphisms were in accordance with the Hardy-Weinberg equilibrium in the two groups. The genotype and allele frequencies of the -344T>C, K173R and IC polymorphisms were not significantly different between the MetS and control groups. However, when the data were stratified by gender, the recessive (TT+TC vs. CC) distribution of the -344T>C polymorphism showed a significant association (OR=0.438, 95% CI: 0.208–0.922, P=0.030) with increased MetS risk only in the male group (Table III).

To expand the available association data, we evaluated the effectiveness of allelic combinations using haplotypes of CYP11B2 three polymorphic loci (-344T>C, K173R and IC) (Table IV). Haplotype analysis revealed that the C-R-W haplotype was significantly more frequent in the MetS patients than in the control subjects, suggesting an association with increased MetS susceptibility (OR=1.412, 95% CI: 1.075–1.856, P=0.016). In addition, the T-R-W, C-K-W and C-R-C haplotype showed a significantly lower association with the risk of MetS.

When the haplotype data were stratified by gender, the patterns of association with MetS were found to be gender-specific (Table V). The C-R-W haplotype was significantly associated with MetS susceptibility (OR=1.429, 95% CI: 1.006–2.031, P=0.047) in men only as compared to the total number of subjects. By contrast, the C-K-W haplotype in men and the T-K-C haplotype in women was significantly associated with a lower risk of MetS.

D prime (D′) was calculated to assess the level of LD between the K173R and -344T>C polymorphisms in the controls plus patients. Polymorphisms CYP11B2, K173R and -344T>C were in moderate level of LD (D′=0.67).