Association of polymorphisms of interleukin-18 gene promoter region with polycystic ovary syndrome in chinese population

A total of 118 patients with PCOS were included in this prospective, case-controlled study. Mean age at diagnosis was 28.60 years (S.D. = 3.45). For this study, data from women with PCOS visiting the Department of Obstetrics and Gynecology, Division of Reproductive Center, Peking University Third Hospital, Beijing, from October 2006 to January 2007 were analyzed. All patients were Chinese and had not taken medications, including contraceptive pills, for the last 6 months. None of the patients had any clinical evidence (history or examination) of a recent or ongoing infection. Women having received any hormonal treatment or insulin-lowering agent during the last 3 months were excluded from the study. Institutional Review Board approval was obtained for this study and patients' consent was obtained from all women prior to inclusion in the study.

The diagnosis of PCOS was based on the 2003 Rotterdam ESHRE/ASRM criteria: (1) oligoovulation(cycle intervals > 35 days) and/or anovulation (absence of menstruation for 3 consecutive months); (2) clinical and/or biochemical signs of hyperandrogenism (patients presented with hirsute (Ferriman-Gallwey scale ≥ 6), acne or alopecia, and/or increased circulating levels of testosterone, T ≥ 2.8 nmol/L); (3) polycystic ovaries(12 or more follicles 2-9 mm in diameter and/or increased ovarian volume of more than 10 mL). All other aetiologies (congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome) were excluded [8].

The control group consisted of 79 subjects with a mean age of 29.82 years (S.D. = 3.86). Controls were recruited by word of mouth and hospital advertisements calling for "healthy women". All controls underwent a brief history, physical exam and chemical examination of serum hormones, ensuring that all control women were non-hirsute and had regular menstrual cycles, normal androgen levels and had birthed at least one child.

For further analysis the PCOS patients were divided into two sub-groups three times. Firstly, PCOS IR group and PCOS without IR group, insulin resistance was judged by using the homeostatic model IR index (HOMA-IR), and 2.69 was selected as a cutoff point [9, 10]. Secondly, the PCOS patients were divided into obese PCOS patients and lean PCOS patients, BMI < 25 kg/m² had been used as a cutoff point. Finally, PCOS with hyperandrogenism group and PCOS without hyperandrogenism: group, we assessed total testosterone, 2.8 nmol/L was used as a cutoff point.

All subjects underwent a brief physical examination, including: height, weight, body mass index [BMI was calculated as follows: weight (kilograms)/height² (meters)] and blood sampling for hormonal measurement. Blood samples were obtained between days 2 and 3 of the menstrual cycle. In patients with amenorrhea, bleeding was induced by progestogens, with blood samples taken thereafter. If no bleeding occurred, blood samples were taken after validating the lack of an established pregnancy by a commercially available pregnancy test. Blood was taken from the antecubital vein after 12-hours of overnight fasting. Samples were immediately centrifuged, with the serum separated and frozen at -20°C until assayed.

Fasting insulin (FIN), follicle- stimulating hormone (FSH), luteinizing hormone (LH) and total testosterone (T) levels were determined by chemiluminescence- immunoassay (CLIA) and fasting glucose (FBG) levels were detected using the glucose oxidase method.

DNA was extracted from EDTA- collected peripheral whole blood with standard techniques following the manufacturer's instructions and stored at -20°C.

Fasting glucose and insulin values were also obtained for each of the subjects. The homeostasis model assessment (HOMA) was used to calculate indices of insulin resistance and insulin secretion for each patient [11]. Homeostasis model assessment index for IR (HOMA-IR) was calculated as follows: fasting insulin (mIU/l) × fasting glucose (mmol/L)/22.5.

Polymorphisms were analyzed by using PCR-SSP, at positions -607 and -137 in the promoter of the IL-18 gene [7]. For the position -607C/A- specific PCR, a common reverse primer 5'-TAACCTCATTCAGGACTTCC-3' and two sequence-specific forward primers 5'-GTTGCAGAAAGTGTAAAAATTATTA C-3' and 5'-GTTGCAGAAAGTGTAAAAATTATTAA-3' were used. An amplification product of 196 bp was detected. A control forward primer 5'-CTTTGC TATCATTCCAGGAA-3' was used to amplify a 301 bp fragment covering the polymorphic site as an internal positive amplification control. PCR reaction was performed in a final volume of 25 μL consisting of 2.5 μL 10 × PCR buffer, 0.5 μL dNTP, 1.5 μL genomic DNA and 0.5 μL Taq polymerase. One sequence-specific primer (for the C allele or A allele), the common reverse primer, and the internal position control primer were included in every reaction mixture at a concentration of 0.25 μL, respectively. Therefore, two PCR reactions were performed for every individual DNA.

Reactions were carried out in a Gene Amp PCR System 9600 (Perkin Elmer, USA) thermal cycler. Denaturation was performed for 2 min at 94°C, followed by 30 cycles at 94°C for 30 s, 57°C for 30 s, 72°C for 30 s and 72°C for 5 min. PCR products were visualized by 3% agarose gel electrophoresis. The molecular weight of PCR products was estimated using a 100-bp DNA ladder (Roche, Germany). PCR products were visualized and photographed under UV with an Alpha Imager 1220 v 5.5 Camera software

For the -137 genotyping, a common reverse primer 5'-AGGAGGGCAAAATGC ACTGG-3' and two sequence-specific forward primers 5'-CCCCAACTTTTACGG AAGAAAAG-3' and 5'-CCCCAACTTTTACGGAAGAAAAC-3' were used. An amplification product of 261 bp was detected. A control forward primer 5'-CCAATA GGACTGATTATTCCGCA-3' was used to amplify a 446 bp fragment covering the polymorphic site to serve as an internal positive amplification control. PCR reaction was performed in a final volume of 25 μL consisting of 2.5 μL 10 × PCR buffer, 0.5 μL dNTP, 1.5 μL genomic DNA and 0.5 μL Taq polymerase. The quantity of the control primer, the reverse primer, and sequence-specific primers was 0.25 μL, respectively. The PCR was run as follows: denaturation for 2 min at 94°C, followed by 30 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 30 s and 72°C for 5 min.

The primary phenotype for genetic association analysis was the presence or absence of PCOS, given that such analysis utilizes all available subjects to maximize power. Secondary analyses included the insulin-related traits (fasting insulin, fasting glucose, HOMA-IR), adiposityrelated traits (BMI) and androgen-related traits (T), within PCOS.

The frequencies of genotypes and alleles in the promoter region of the IL-18 gene at positions -607 and -137 were calculated by counting. Data were analyzed with SPSS statistical software system (SPSS 11.5; SPSS Inc., Chicago, IL) and the Hardy-Weinberg equilibrium was determined by means of chi square testing. Statistical significance was assumed for P values less than 0.05.

Polymorphisms at positions -607 and -137 in the promoter of the IL-18 gene were analyzed by PCR-SSP. At every polymorphic site, a common reverse primer and two sequence-specific forward primers were used, resulting in two PCR reactions performed for every individual DNA. The specific PCR products from homozygous individuals showed one DNA segment, while heterozygous individuals exhibited two specific fragments, as expected. In total, 197 unrelated Chinese subjects were studied for IL-18 promoter polymorphisms. As shown in Figure 1, there were CC, CA and AA genotypes at position -607, and GG, GC and CC genotypes at position -137.

Genotype and allele frequencies for IL-18 polymorphisms are summarized in Table 1. The genotype frequencies were in agreement with the Hardy-Weinberg equilibrium (P > 0.1 for all analyses). For the -607 genotypes from the 118 PCOS patients, 39 had the CC type (33%), 61 the CA type (51.7%), and 18 the AA (15.3%). Of the 79 control subjects, 22 had the CC type (27.8%), 48 the CA type (60.8%), and 9 the AA type (11.4%). Regarding the -137 genotypes, 92 of the 118 PCOS patients had the GG type (78.0%), 25 the GC type (21.2%), and 1 the CC type (0.8%). Sixty-three of the 79 control subjects were type GG (79.7%) and 16 were GC (20.3%). No significant differences were observed in the genotype distribution or allele frequency between the PCOS and control groups.

Based on genotypes of the IL-18 promoter polymorphisms, haplotype frequencies were estimated by the expectation maximization method. Four haplotypes of the IL-18 promoter at positions -607 and -137 were present in both patients and controls (haplotypes I, II, III and IV in Table 2). The frequencies of haplotypes I, II, III and IV in the PCOS patients were 57.0%, 0.6%, 30.7% and 11.7%, respectively. The frequencies of haplotypes I, II, III and IV in the controls were 28.2%, 0%, 31.6% and 10.2%, respectively. No significant differences were observed in the haplotype frequencies between the PCOS and the controls.

For further analysis of the relationship between IL-18 gene promoter polymorphisms and PCOS IR. Insulin resistance was judged by using the homeostatic model IR index (HOMA-IR), and 2.69 was selected as a cutoff point [9, 10]. The PCOS patients were divided into two sub-groups: PCOS IR: HOMA-IR ≥ 2.69; PCOS without IR: HOMA-IR < 2.69. Genotype and allele frequencies for IL-18 polymorphisms are summarized in Table 3. There were no significant differences in the genotype frequencies between the two sub-groups. As for the -137 allele frequencies, G allele frequency was 93.5% and C allele frequency was 6.5% in the PCOS IR group. In the PCOS without IR group, the G allele frequency was 85.4% and C allele frequency was 14.6%. There was indeed a significant difference in the allele frequency between the two sub-groups (P = 0.048). Four haplotypes of the IL-18 promoter at positions -607 and -137 were present in the two sub-groups; they are summarized in Table 4. There were no significant differences in the haplotype frequencies between the PCOS IR and PCOS without IR groups.

For further analysis of the relationship between the IL-18 gene promoter polymorphisms and PCOS IR, the PCOS patients were again divided into two sub-groups: obese PCOS: BMI ≥ 25 kg/m²; lean PCOS: BMI < 25 kg/m². Genotype and allele frequencies for IL-18 polymorphisms are summarized in Table 5; four haplotypes of the IL-18 promoter at positions -607 and -137 were present in the two sub-groups, which are summarized in Table 6. There were no significant differences in the genotype distribution, allele frequencies, or haplotype frequencies between obese and lean PCOS women.

For further analysis of the relationship between the IL-18 gene promoter polymorphisms and PCOS IR, the PCOS patients were again divided into two sub-groups: PCOS with hyperandrogenism: T ≥ 2.8 nmol/L; PCOS without hyperandrogenism: T < 2.8 nmol/L. Genotype and allele frequencies for IL-18 polymorphisms are summarized in Table 7; four haplotypes of the IL-18 promoter at positions -607 and -137 were present in the two sub-groups, and are summarized in Table 8. There were no significant differences in the genotype distribution, allele frequencies, or haplotype frequencies between PCOS subjects with and without hyperandrogenism.