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Association Between Two Common Polymorphisms of PPARγ Gene and Metabolic Syndrome Families in a Chinese Population
Archives of Medical Research 40 (2009) 89e96
ORIGINAL ARTICLE
Association Between Two Common Polymorphisms of PPARg Gene and Metabolic Syndrome Families in a Chinese Population Li Lan Yang, Qi Hua, Rong Kun Liu, and Zheng Yang Department of Cardiology, Xuanwu Hospital of the Capital University of Medical Science, Beijing, China Received for publication August 11, 2008; accepted November 3, 2008 (ARCMED-D-08-00359). Previously published online January 22, 2009.
Background and Aims. We investigated the association between the two common polymorphisms, C1431T and Pro12Ala of PPARg gene, and metabolic syndrome (MS) in a Chinese population. Methods. We included 423 subjects with MS and families without MS. Subjects were divided into three groups: MS probands and first- and second-degree relatives of probands, spouses and controls. Each group was then divided into two subgroups according to genotype (Pro/Pro and Pro/Ala for Pro12Ala, CC and CT þ TT for 1431C/T). Anthropometric indices, fasting plasma glucose, lipid profile, Sv1 þ Rv5 of electrocardiogram and single nucleotide polymorphisms were detected. Results. Frequencies of C1431T genotypes, but not Pro12Ala, were different among the three groups. MS patients with Pro/Ala genotype had higher fasting blood sugar (FBS) levels and Sv1 þ Rv5. Controls with Ala allele had lower total cholesterol levels. In relatives, Ala carriers had higher high-density lipoprotein cholesterol (HDL-c) levels. BMI of the different groups were not significant. MS patients with T allele had higher FBS and Sv1 þ Rv5. In relatives of MS subjects, T-allele carriers had lower blood uric acid, creatinine and higher HDL-c levels and Sv1 þ Rv5. Conclusions. C1431T, but not Pro12Ala polymorphisms, are associated with MS in a Chinese population. In MS patients, Ala allele and T allele are both associated with higher fasting blood sugar and higher left ventricular voltage. In controls, Ala carriers have lower total cholesterol. In MS relatives, Ala carriers had higher HDL-c levels and T-allele carriers had lower uric acid, creatinine and higher HDL-c levels and left ventricular voltage. Ó 2009 IMSS. Published by Elsevier Inc. Key Words: PPARg, Polymorphism, Metabolic syndrome, Genotype.
Introduction Metabolic syndrome (MS) is a complex disorder that encompasses several metabolic diseases such as abdominal obesity, insulin resistance, elevated plasma triglyceride (TG) levels and low high-density lipoprotein cholesterol (HDL-c) levels, high blood pressure, and altered glucose homeostasis (1). Genetic and environmental factors contribute to the susceptibility to the MS. Many genes are candidates for MS. Peroxisome proliferator-activated receptor-g (PPARg) is one of the nuclear hormone receptor
Address reprint requests to: Qi Hua, Department of Cardiology, Xuanwu Hospital, Capital University of Medical Science, Changchun Street No.45, Beijing, China, 100053; E-mail: [email protected]
superfamily and heterodimerizes with retinoid X receptor (RXR). It is suggested that it regulates target genes in adipocyte differentiation (2,3) and insulin sensitization (4,5). PPARg has four isoforms of which PPARg-1 is expressed in most tissues. PPARg-2 is specific to adipose tissue and regulates adipocyte differentiation (6). PPARg3 seems to be mainly confined to macrophages, adipose tissue, and colon (7). Tissue distribution of PPARg4 has not yet been explored (8). The PPARg gene is located on chromosome 3 (9), and the specific isoforms are a result of alternative mRNA splicing. The prevalent human PPARg polymorphism is the Pro12Ala polymorphism (rs1801282) of the exon B in PPARg (10). This polymorphism has been associated with increased protection against the development of type 2 diabetes and insulin resistance (11e14). However,
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.11.005
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Yang et al./ Archives of Medical Research 40 (2009) 89e96
there are variations in Ala allele frequencies in different populations. There are also different relationships with MS (11e14). Another polymorphism, the C1431T silent substitution (rs3856806) in the 6th exon of PPARg, has been shown to modulate the effect of Pro12Ala on susceptibility to type 2 diabetes (15). However, there are some contradictory results of the association between these two polymorphisms and diabetes and MS (11e15). There are also few reports about these two polymorphisms of MS in Chinese families. We investigated the two polymorphisms in PPARg gene in Chinese families with MS probands.
Materials and Methods Subjects Since 2003, data on all patients attending the Cardiac Clinic of Xuanwu Hospital of Capital Medical University (Beijing, China) have been documented for detailed family history. Families including two patients with MS (probands) were recruited into the study. Families without MS patients were also recruited as the control group. The present study included 101 families with a total of 442 subjects. Most families were nuclear families. Subjects who were !30 years old (nine subjects) were excluded and subjects whose DNA could not be amplified were also excluded (ten subjects). We included 423 subjects investigated from January 2003 to September 2007. Subjects were divided into three groups: probands (n 5 146), first- and second-degree relatives (without MS) of probands (n 5 96) and spouses and control families without MS (n 5 181). Each group was then divided into two subgroups according to the genotype (Pro/Pro and Pro/ Ala for Pro12Ala, CC and CT þ TT for 1431C/T). Probands included 86 (58.9%) subjects with three characteristics of MS, 42 (28.8%) subjects with four characteristics of MS and 18 (12.3%) subjects with five characteristics of MS. All subjects signed informed consent. The protocol was approved by the ethics committee of our institution. MS was defined using criteria established in the third report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment in Adults (Adult Treatment Panel III) (16) as the presence of three or more of the following risk factors: waist circumference (WC) $88 cm in women and $102 cm in men, TG $150 mg/dL, HDL-c #50 mg/dL in women and #40 mg/dL in men, blood pressure (BP) $130/85 mm Hg, fasting blood sugar (FBS) $110 mg/dL or a diagnosis of diabetes. Type 2 diabetes diagnosis was defined on the basis of the OGTT using the plasma glucose criteria of the American Diabetes Association (2-h glucose $11.1 mmol/L or FBS $7.0 mmol/L). Measurements Weight, height, WC and hip circumference (HC) were measured by trained personnel, and body mass index (BMI) was
calculated according to the Quetelet equation. BP was measured on the right arm with the subject in a sitting position after a minimum 5-min rest using a standard mercury sphygmomanometer. The mean value of two consecutive BP readings was used. Venous blood samples were collected in the morning after subjects had fasted for 12 h. FBS was assayed by the glucose oxidase method. Serum TG levels were enzymatically measured. Total cholesterol (TC), TG, and HDL-c levels were measured after sodium phosphotungstate/magnesium chloride precipitation. All biochemical indices were measured with an automatic biochemistry machine (Hitachi 7170, Tokyo, Japan). LDL-c levels were calculated by the Friedewald equation. We also used the electrocardiogram (ECG) of subjects and the sum of Sv1 and Rv5 for investigation of left ventricular hypertrophy. All subjects were investigated with regard to their family medical history. Genotyping Genetic analyses were performed on genomic DNA extracted from human leukocytes. Single nucleotide polymorphism of Pro12Ala and 1431C/T were detected using restriction fragment length polymorphism and polymerase chain reaction (RFLP-PCR). PCR was performed with thermal cycler (Bio-Rad, Hercules, CA). Total volume of PCR reaction was 25 ml. For each reaction, 0.5 ml of genomic DNA, 0.725 units of Taq polymerase (MBI, Fermentas, Canada) and 2.5 ml 10X PCR buffer were used. Magnesium chloride concentration was 1.5 mM. Each primer concentration was 0.2 mM. Genotyping for Pro12Ala Primers used were 50 -CAAGCCCAGTCCTTTCTGTG-30 and 50 -AGTGAAGGAATCGCTTTCCG-30 . The base C (underlined) was mismatched for the site of the restriction enzyme HpaII (MBI). The following PCR conditions were used: initial denaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C, annealing at 52 C and elongation at 72 C. Each of these steps lasted 30 sec. This was followed by a final extension at 72 C for 10 min. PCR products spanned the entire length of 237 bp (base pairs). PCR products of 10 ml were digested with 10 U of HpaII at 37 C for 5 h or overnight. When alanine was present at residue 12 of PPARg2, the 237-bp DNA fragment was split into 217- and 20-bp pieces. Digestion products were then separated by electrophoresis on 3% agarose gel, and photos were taken under ultraviolet light. Genotyping for C1431T Primers used were 50 -GCTCCAGAAAATGACAGACC T-30 and 50 -TGGAAGAAGGGAAATGTTGG-3. Annealing temperature was 55 C and cycle times were 40 sec. Other conditions were the same with the genotyping for
Polymorphisms of PPARg Gene and Metabolic Syndrome
Pro12Ala. PCR product spanned the entire length of 170 bp and was digested by the specific restriction Eco72I enzyme. When allele C was present at C1431T of PPARg2, the 170bp DNA fragment was split into 127- and 43-bp pieces. Concordance rates between the PCR-RFLP assay used for genotyping and sequencing or repeat genotyping was 100%. Statistical Analysis We conducted analysis on normally distributed variables. Variables of quantitative traits were normally distributed. Differences between quantitative traits were compared with one-way ANOVA and post hoc multiple comparison (using LSD method). For genotypes and gender we used Pearson c2 to compare differences. The influence of covariates such as gender, age, family and other possible covariates in the comparison of means was adjusted by the general linear model. In order to abscise the effect of relationship of the same family, the family number was used as one covariant. Comparison of creatinine and uric acid, diabetes and FBS were also used as covariates. Comparison of the sum of Sv1 and Rv5, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were also taken as covariates. Two-sided p values !0.05 were considered statistically significant. SPSS (Chicago, IL) software v.13.0 was used. Hardy-Weinberg equilibrium tests were performed for genotypes using c2 test. The tests of linkage of disequilibrium were performed using the software of THESIAS (17).
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Results Subjects’ Characteristics Quantitative traits are presented as mean SD (standard deviation). Basic anthropometric and biochemical characteristics of the subjects are presented in Table 1. There were statistical differences among the three groups in terms of gender, age, height, weight, WC and HC, waist and hip circumference ratio (W/H ratio), BMI, abdominal circumference (AC), SBP, DBP, FBS levels, blood creatinine levels, TC, and TG levels. There were no statistical differences in HDL-c or LDL-c levels, blood uric acid levels and Sv1 þ Rv5 of ECG among the three groups. Subjects’ Genotypes Genotypes of subjects are presented in Table 2. Of the 423 subjects, Pro12Pro genotype was present in 91.02% (385 subjects) of the population and Pro12Ala genotype was present in 8.98% (38 subjects). There were no Ala12Ala genotypes. Allele frequencies were 0.955 for Pro allele and 0.045 for Ala allele. Genotypic frequencies of the PPAR exon 6 C1431T substitution were 56.97% (241 subjects) for CC, 36.41% (154 subjects) for CT, and 6.62% (28 subjects) for TT genotype. Allele frequency for the C allele was 0.751 and 0.249 for T allele. Allele frequencies of the two genotypes satisfied Hardy-Weinberg equilibrium. There were no differences in terms of Pro12Ala genotype frequencies among the three groups. There were statistical differences in terms of C1431T genotype frequencies
Table 1. Basic anthropometric and biochemical characteristics of different groups (probands, first- or second-degree relatives, controls) Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv2 þ Rv5 (mV)
Probands (n 5 146)
First- or second-degree relatives (n 5 96)
Controls (n 5 181)
p value
62/84 56.8 15.07 165.58 8.83 76.24 16.09 94.13 10.20 106.71 8.99 0.88 0.07 27.67 4.65 102.98 10.70 144.52 21.38 89.24 12.31 118.73 42.38 6.12 1.88 86.16 27.93 208.35 41.88 257.45 164.88 47.80 59.38 109.07 75.41 2.54 0.89
54/42 49.44 13.45a 167.04 7.92 70.83 16.27a 85.51 9.23a 100.53 8.58a 0.85 0.07a 25.27 5.07a 96.14 10.87a 133.84 22.53a 86.12 14.51 96.20 19.77a 6.14 5.64 80.73 18.82 192.27 37.01a 137.37 99.74a 56.42 38.39 106.94 51.28 2.54 0.86
76/105 47.32 15.9a 164.75 7.13b 68.15 14.63 86.32 13.17a 99.26 10.09a 0.87 0.12b 24.99 4.38a 94.10 11.99a 132.51 22.05a 84.36 13.91a 95.57 23.52a 5.37 1.39 77.92 19.83a 190.78 61.61a 122.69 89.27a 52.04 10.66 114.20 60.80 2.61 0.71
0.039 !0.0001 0.015 !0.0001 !0.0001 !0.0001 0.001 !0.0001 !0.0001 !0.0001 !0.0001 !0.0001 0.124 0.009 !0.0001 !0.0001 0.417 0.630 0.218
Data are mean SD. p value in the fifth column is for the comparison among three groups. BMI, body mass index; AC, abdominal circumference; W/H ratio, waist/hip ratio; WC, waist circumference; HC, hip circumference; FBS, fasting blood sugar; TC, total cholesterol; TG, triglyceride; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol. a Significant difference compared with the probands ( p !0.05). b Significant difference compared with the first- or second-degree relatives ( p !0.05).
Yang et al./ Archives of Medical Research 40 (2009) 89e96
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Table 2. Genotypes of subjects of three groups of metabolic syndrome Groups Pro12Ala Pro/Pro Pro/Ala C161T CC CT TT Pro12Ala þ C161T Pro/Pro þ CC Pro/Pro þCT or TT Pro/Ala þ CC Pro/Ala þCT or TT
Probands n 5 146
First- or second-degree relatives n 5 96
Control n 5 181
p value
137 (93.84%) 9 (6.16%)
83 (86.46%) 13 (13.54%)
165 (91.16%) 16 (8.84%)
0.281
91 (62.33%) 50 (34.25%) 5 (3.42%)
61 (63.54%) 34 (35.42%) 1 (1.04%)
89 (49.17%) 70 (38.67%) 22 (12.16%)
87 50 4 5
56 27 5 8
86 79 3 13
(59.59%) (34.25%) (2.74%) (3.42%)
among the three groups. The prevalence of CC genotype in controls was lower than that in MS probands and first- or second-degree relatives of MS (49.17 vs. 62.33%, 63.54%, p 5 0.002). Because the association between two polymorphisms was significant ( p 5 0.015), we compared the four genotypes of two polymorphisms in different groups of MS. The presence of Pro/Pro þ CC in controls was lower than that in probands and relatives of patients with MS. The presence of Pro/Ala þ CT or TT of controls was higher than that of probands with MS. We also tested the linkage disequilibrium between the two polymorphisms. The two polymorphisms were in linkage disequilibrium (D0 5 0.516, r2 5 0.031). To test the association between two polymorphisms and individual components of MS, we compared biochemical and anthropometric indices among different genotype groups of two polymorphisms. Data are presented in Tables 3e6. To test the differences between relatives and controls, we included these two groups in the same tables.
Pro12Ala Genotypes and Individual Components of MS As presented in Table 3, MS patients with Pro/Ala genotype had lower weight and height, but had higher FBS levels and Sv1 þ Rv5 than those without Ala allele. The differences of blood sugar levels (when corrected by age, gender and family, p 5 0.001) and Sv1 þ Rv5 (when corrected by age, gender, family and BP, p 5 0.018) were significant. There were no differences in terms of WC, HC, W/H ratio, BMI, AC, BP, creatinine, uric acid, TC, TG, LDL-c and HDL-c levels between the two groups. In relatives and controls (presented in Table 4), there were no differences in gender, age, weight, height, WC, BMI, AC, BP, FBS, uric acid, creatinine, TC and Sv1 þ Rv5 among the four groups. HDL-c levels of MC relatives with Pro/Ala genotype were higher than those of the other three groups. Controls with Pro/ Ala genotype had lower TC levels. Controls and relatives with Pro/Ala had lower LDL-c levels, but this was not statistically significant ( p 5 0.095).
(58.33%) (28.13%) (5.21%) (8.33%)
0.002
(47.51%) (43.65%) (1.66%) (7.18%)
0.048
C1431T Genotypes and Individual Components of MS As presented in Table 5, there were significant differences in terms of gender, FBS, LDL-c levels, TG and Sv1 þ Rv5 in ECG between CC genotype and CT þ TT genotype groups in patients with MS. There were no differences in terms of age, height, weight, WC, HC, W/H ratio, BMI, AC, BP, creatinine, uric acid, TC and HDL-c levels between the two groups. FBS, Sv1 þ Rv5 and TG ( p 5 0.008, after adjusted p 5 0.147) of CT þ TT genotype group were higher than those of CC genotype in MS Table 3. Biochemical characteristics of different genotype of Pro12Ala in MS probands Probands n 5 146 Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
p value
Pro/Pro Pro/Ala 61/76 54.90 14.6 166.30 8.58 77.27 16.44 94.18 10.26 106.78 9.22 0.88 0.07 27.80 4.72 102.94 11.07 144.56 20.81 89.42 12.43 113.96 37.10 6.07 1.81 87.47 28.77 208.77 252.54 48.88 109.37 2.47
43.94 151.06 63.26 79.58 0.78
70.11 160.56 69.06 93.78 106.22 0.88 26.79 103.22 144.22 88.00 151.92 6.51
1/8 10.50 9.17 11.37 10.04 7.35 0.06 4.04 7.80 15.71 11.72 60.24 2.29
0.048 !0.0001 0.004 0.036 0.888 0.814 0.974 0.390 0.923 0.951 0.677 0.001 0.604
77.03 19.45
0.069
0.79 0.209 0.388 0.882 0.018
205.44 291.51 40.17 106.98 2.99
23.90 243.37 13.69 36.10 1.36
p values were corrected by age, gender and family. p values of creatinine and uric acid were also adjusted by diabetes and fasting blood sugar. p value of the sum of Sv1 and Rv5 was also adjusted by systolic and diastolic blood pressures.
Polymorphisms of PPARg Gene and Metabolic Syndrome
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Table 4. Biochemical characteristics of different genotypes of Pro12Ala in first- or second-degree relatives and controls First- or second-degree relatives n 5 96
Controls n 5 181
Pro/Pro Pro/Ala
Pro/Pro Pro/Ala
Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
47/36 48.22 13.89 166.67 7.29 70.31 16.26 85.71 9.16 99.69 9.01 0.86 0.07 25.26 5.79 96.19 11.24 133.29 23.15 86.06 15.09 96.58 22.10 6.12 6.03 82.37 19.78 190.04 37.51 128.36 65.19 50.37 11.57 112.15 37.54 2.49 0.83
53.82 168.36 72.68 84.70 103.80 0.81 25.31 95.90 135.82 88.91 94.82 6.22 74.84 200.26 169.75 77.58 88.73 2.70
7/6 13.89 9.95 16.55 9.69 5.63 0.08 3.22 10.06 20.54 12.26 6.65 2.24 13.70 34.77 173.74 76.09a 82.00 0.96
69/96 47.41 15.73 164.70 7.16 68.41 15.17 86.62 13.63 99.46 10.08 0.87 0.13 25.09 4.49 94.20 11.81 132.97 22.43 85.01 13.80 96.76 24.96 5.37 1.47 78.09 19.83 195.09 64.05 123.99 94.49 52.21 10.82 118.08 63.55 2.58 0.71
7/9 46.85 165.08 66.73 84.69 98.15 0.86 25.47 93.54 130.00 80.69 89.07 5.42 77.02 167.25 115.53 51.15 92.99 2.81
p value
17.25 7.12 11.36 12.98 10.28 0.06 3.73 13.16 20.00 14.20 11.41 0.95 20.21 39.21 53.07 9.88 36.89 0.70
0.277 0.301 0.077 0.456 0.805 0.218 0.158 0.892 0.666 0.841 0.216 0.424 0.456 0.268 0.033 0.163 !0.0001 0.095 0.368
a
Difference from the first group. Difference from the second group. c Difference from the third group. p values in the sixth column are for the comparison among four groups. p value !0.05 was considered significant. p values of creatinine and uric acid were also adjusted by diabetes and FBS. p value of the sum of Sv1 and Rv5 was also adjusted by SBP and DBP. b
Table 5. Biochemical characteristics of different genotypes of C1431T in MS probands Probands (n 5 146) CC
CT þ TT
p value
39/52 55.96 13.49 165.76 8.80 76.53 17.53 93.36 11.23 105.99 9.23 0.88 0.08 27.71 5.18 102.39 11.54 142.65 18.02 88.92 11.88 112.94 32.69 6.01 1.82
23/32 59.22 17.01 165.15 9.10 75.13 13.67 95.28 8.77 107.81 8.43 0.88 0.05 27.40 3.45 103.72 9.02 147.17 25.31 89.26 13.09 127.20 54.85 6.20 2.00
0.920 0.215 0.663 0.610 0.339 0.261 0.852 0.710 0.505 0.239 0.886 0.006 0.746
84.86 23.74
88.19 34.47
0.406
0.225 0.147 0.169 0.093 0.048
Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
213.0 231.17 44.35 122.61 2.49
48.19 119.40 9.04 45.14 0.78
202.37 287.20 54.32 90.61 2.74
31.49 203.56 96.09 106.02 1.05
p values were corrected by age, gender and family. p values of creatinine and uric acid were also adjusted by diabetes and fasting blood sugar. p value of the sum of Sv1 and Rv5 was also adjusted by SBP and DBP.
patients. LDL-c levels ( p 5 0.005, after adjusted p 5 0.093) of CT þ TT genotype group were lower than those of CC genotype group. After the effects of age, gender, family and blood pressure were corrected, only FBS and Sv1 þ Rv5 were significant ( p !0.05). In relatives and controls (Table 6), there were no differences in terms of age, weight, height, WC, HC, W/H ratio, BMI, AC, DBP, FBS, TC, TG, and LDL-c levels. Relatives with CT þ TT genotypes had lower uric acid and blood creatinine (corrected by age, gender, family, diabetes and FBS) than those with CC genotypes. Relatives with CT þ TT genotypes had higher HDL-c levels and Sv1 þ Rv5 (corrected) than those with CC genotypes. Differences in controls were not significant.
Discussion This is the investigation of the association of PPARg polymorphisms and MS families in mainland China. We tested the polymorphisms of Pro12Ala of exon B and C1431T of exon 6 of PPARg. There was no Ala12Ala homozygote in the population. Allele frequency was 0.955 for Pro allele and 0.045 for Ala allele, similar to the rare frequency of Ala allele in the Asian population in previous studies (18e22) and lower than the frequency of Ala allele in a Hispanic population (11) and in Caucasians (12). The allele
Yang et al./ Archives of Medical Research 40 (2009) 89e96
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Table 6. Biochemical characteristics of different genotype of C1431T in first- and second-degree relatives and controls First- or second-degree relatives (n 5 96) Groups Gender (M/F) Age (years) Height (cm) Weight (kg) Waist circumference (cm) Hip circumference (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) Triglyceride (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
Control (n 5 181)
CC
CT þ TT
CC
CT þ TT
p value
38/23 49.08 13.35 167.46 7.72 70.71 10.72 85.15 15.15 101.10 9.38 0.84 0.08 25.18 3.14 96.07 12.59 131.17 22.82 85.27 15.32 95.52 20.38 6.85 7.10 85.22 20.20 194.31 32.97 129.07 58.29 50.22 10.80 115.95 30.54 2.39 0.86
16/19 49.87 12.83 166.10 8.17 71.18 22.15 85.86 7.83 99.51 7.28 0.86 0.07 25.58 7.07 96.07 7.77 137.74 22.18 88.82 13.38 97.21 19.52 5.11 1.94a 73.57 14.61a 190.32 43.24 152.61 142.81 65.26 58.73a 94.54 71.26 2.79 0.83a
31/58 45.75 15.81 164.83 7.55 68.17 14.35 87.42 15.74 99.04 11.34 0.89 0.16 24.97 4.27 95.39 11.14 128.76 20.46 83.82 15.70 94.41 15.01 5.47 1.36 77.41 20.04 199.45 81.65 117.04 62.07 53.61 11.29 122.43 80.75 2.52 0.71
45/47 48.83 16.45 164.88 7.24 68.51 15.66 85.62 11.41 99.84 8.72 0.86 0.08 25.08 4.70 93.00 13.21 135.04 23.69 84.64 13.04 96.74 29.74 5.36 1.48 80.61 19.92 184.99 42.84 126.85 110.64 50.74 10.32 108.88 41.90 2.67 0.73
0.011 0.435 0.156 0.645 0.453 0.661 0.141 0.939 0.359 0.147 0.358 0.903 !0.0001 !0.0001 0.43 0.314 0.005 0.105 !0.0001
a
Difference from the first group. Difference from the second group. p values in the sixth column are for the comparison among four groups. p value !0.05 was considered significant. p values were corrected by age, gender and family. p values of creatinine and uric acid were also adjusted by diabetes and FBS. p value of the sum of Sv1 and Rv5 was also adjusted by SBP and DBP. b
frequency for the C allele was 0.751 and 0.249 for T allele, demonstrating higher T-allele frequency than in previous studies (23e25) and similar frequency to that in another study (26). We calculated the allele frequency by including all family subjects. Allele frequencies were similar to the Asian population (nonrelated population). This demonstrated that there were no obvious aggregations of these two polymorphisms in MS familial population. Two polymorphisms are in linkage disequilibrium (D0 5 0.516, p 5 0.003). Several studies reported an association between the Ala allele of the codon 12 polymorphism of exon B and reduced risk of diabetes and decreased insulin resistance (25,27e32). However, other studies were not able to replicate these associations (18,33,34). We did not find differences of Ala frequencies among MS probands, relatives and controls ( p 5 0.281), which meant that there was no association between Pro/Ala polymorphism and MS, similar to the results of other studies (35,36). In a large French population-based study, Meirhaeghe et al. found no association between PPARg Pro12Ala polymorphism and MS (35). In the Chinese population, Donxia et al. also found no association between them (21). Our study is similar to this result. However, in the present study, MS patients with Ala allele had higher FBS levels similar to the previous study (22,33) and higher Sv1 þ Rv5 in ECG than those without Ala allele. When corrected, these differences were
significant. However, Sv1 þ Rv5 increase did not make the diagnosis of left ventricular hypertrophy. This demonstrates that Ala allele may cause the increase of left ventricular voltage in MS patients. Duan et al. developed a cardiocyte-specific PPAR-g knockout mouse model and showed that cardiocyte PPAR-g inactivation in vivo caused cardiac hypertrophy, likely through NF-kB activation (37). Further studies are needed to clarify the association between PPARg gene and left ventricular hypertrophy in MS. Whereas the effect of the Ala12 substitution on the structure of PPARg2 remains unknown, it has been shown that Ala substitution results in a 2-fold lower affinity of PPARg2 for binding to the PPRE (PPAR response element) and reduces transcriptional activity in the presence and absence of PPARg agonists (12). In patients with MS, relatives and controls, BMI was not different. We suggest that Ala allele has no effect on obesity in Chinese subjects. Dongxia et al. found similar results, but the presence of Pro12Ala and T allele simultaneously showed a potential effect on obesity (21). A study in the Spanish population suggested that Pro/Ala polymorphism was related to obesity in men and not in women (38). These contradictory results may be due to population differences, which need further studies to confirm. In controls, Ala allele carriers had lower TC levels. In relatives, Ala allele carriers had higher HDL-c levels. In an Asian study (22), Ala allele also had higher HDL-c
Polymorphisms of PPARg Gene and Metabolic Syndrome
levels. In relatives and controls, Ala carriers had lower LDL-c levels, but these were not significant. Other studies did not find the association between Ala allele and LDL-c level (21,36). This may be because our study discriminated among patients with MS, relatives and controls and others investigated from the total population. To some extent, Ala allele may have the potential protective effect from atherosclerosis for some non-MS subjects. This needs to be clarified in further studies. It was reported that polymorphism C1431T of exon 6 of PPARg was associated with diabetes risks (22,25) and leptin level in obese humans (23). However, not all studies had similar results (32,36). We found differences of C1431T frequencies among the three groups, which meant that there was association between C1431T polymorphism and MS. Dongxia et al. found no association between C1431T and MS in a Chinese population (21). In Meirhaeghe’s French population study (35), polymorphisms of C1431T were not individually associated with MS. However connected with the other three polymorphisms, 681COG, P2-689COT, Pro12Ala, haplotypes are associated with MS. We found the association between C1431T polymorphism and MS in Chinese people. It may be that our subjects were a family population and this contributed to the association. Other studies only had the patients and controls or total population. Patients with MS with T allele had higher FBS and Sv1 þ Rv5 levels than those without T allele and the differences were also significant when adjusted by gender, age and family. T-allele carriers had higher FBS, different from previous studies (22,25). The mechanism by which this silent polymorphism in PPARg affects its activity remains unclear, but one possibility may be that it is in linkage disequilibrium with the Pro12Ala polymorphism. In relatives, T-allele carriers had lower blood uric acid, creatinine, and higher HDL-c levels and Sv1 þ Rv5. Some studies showed the association between uric acid and MS (39). MS was found correlated with chronic kidney disease (40). However, there are few studies focused on the association among uric acid, creatinine and PPARg gene polymorphism. PPARg may play a role in uric acid and creatinine metabolism individually or by modulating MS. Further studies are needed to confirm this relationship. T-allele carriers in relatives had higher HDL-c levels. However, other studies did not show this effect. This effect may only be in MS relatives. T-allele carriers also had higher Sv1 þ Rv5 in relatives and patients with MS. In controls, it also had this effect but was not significant. Further studies are needed to verify the association between C1431T polymorphism and left ventricular hypertrophy in MS. Our investigation seems to be able to explain some contradictory results of PPARg polymorphisms and MS. These two polymorphisms are correlated with some components of MS. Some are protective and others are harmful. The limitation of our study was that the population was not large enough, so some groups had fewer subjects. This
95
may lead to some bias in results. Larger population studies should be done to confirm the results presented here. In conclusion, Pro12Ala polymorphism is not associated directly with MS. However, MS patients with Ala allele have higher FBS and higher left ventricular voltage. In controls, Ala carriers have lower TC. In relatives, Ala carriers have higher HDL-c levels. We find the association between C1431T polymorphism and MS in a Chinese population. MS patients with T allele have higher FBS and higher left ventricular voltage. In MS relatives, T-allele carriers have lower uric acid, creatinine and higher HDL-c levels and left ventricular voltage. Acknowledgments This study was also supported by a group of professors of the Capital Medical University, Beijing, China. We thank the volunteers for their enthusiastic participation. The research was funded by the Beijing Nature Foundation.
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27. Altshuler D, Hirschhorn JN, Klannemark M, et al. The common PPARg Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Genet 2000;26:76e80. 28. Hara K, Okada T, Tobe K, et al. The Pro12Ala polymorphism in PPAR-g2 may confer resistance to type 2 diabetes. Biochem Biophys Res Commun 2000;271:212e216. 29. Mori H, Ikegami H, Kawaguchi Y, et al. The pro12ala substitution in PPAR-g is associated with resistance to development of diabetes in the general population: possible involvement in impairment of insulin secretion in individuals with type 2 diabetes. Diabetes 2001;50:891e894. 30. Douglas JA, Erdos MR, Watanabe RM, et al. The peroxisome proliferator-activated receptorg2 Pro12Ala variant: association with type 2 diabetes and trait differences. Diabetes 2001;50:886e890. 31. Ek J, Andersen G, Urhammer SA, et al. Studies of the Pro12Ala polymorphism of the perioxisome proliferatoractivated receptor-g2 (PPAR-g2) gene in relation to insulin sensitivity among glucose tolerant Caucasians. Diabetologia 2001;44:1170e1176. 32. Poulsen P, Andersen G, Fenger M, et al. Impact of two common polymorphisms in the PPARg gene on glucose tolerance and plasma insulin profiles in monozygotic and dizygotic twins: thrifty genotype, thrifty phenotype, or both? Diabetes 2003;52:194e198. 33. Lindi VI, Uusitupa MIJ, Lindstrom MJ, et al. Association of the Pro12Ala polymorphism in the PPAR-g2 gene with 3-year incidence of type 2 diabetes and body weight change in the Finnish diabetes prevention study. Diabetes 2002;51:2581e2586. 34. Stefanski A, Majkowska L, Ciechanowicz A, et al. Lack of association between the Pro12Ala polymorphism in PPAR-g2 gene and body weight changes, insulin resistance and chronic diabetic complications in obese patients with type 2 diabetes. Arch Med Res 2006;37:736e743. 35. Meirhaeghe A, Cotte D, Amouye P, Dallongeville J. Association between peroxisome proliferator-activated receptor g haplotypes and the metabolic syndrome in French men and women. Diabetes 2005; 54:3043e3048. 36. Rhee EJ, Oh KW, Lee WY, et al. Effects of two common polymorphisms of peroxisome proliferator-activated receptor-g gene on metabolic syndrome. Arch Med Res 2006;37:86e94. 37. Duan SZ, Ivashchenko CY, Russell MW, Milstone DS, Mortensen RM. Cardiomyocyte-specific knockout and agonist of peroxisome proliferator-activated receptor-g both induce cardiac hypertrophy in mice. Circ Res 2005;97:372e379. 38. Gonzalez-Sanchez JL, Serrano-Rios M, Fernandez Perez C, Laakso M, Martinez Larrad MT. Effect of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptorg2 gene on adiposity, insulin sensitivity and lipid profile in the Spanish population. Eur J Endocrinol 2002;147:495e501. 39. Ford ES, Li C, Cook S, Choi HK. Serum concentrations of uric acid and the metabolic syndrome among US children and adolescents. Circulation 2007;115:2526e2532. 40. Chen J, Muntner P, Hamm LL, et al. The metabolic syndrome and chronic kidney disease in U.S. adults. Ann Intern Med 2004;140:167e174.
ORIGINAL ARTICLE
Association Between Two Common Polymorphisms of PPARg Gene and Metabolic Syndrome Families in a Chinese Population Li Lan Yang, Qi Hua, Rong Kun Liu, and Zheng Yang Department of Cardiology, Xuanwu Hospital of the Capital University of Medical Science, Beijing, China Received for publication August 11, 2008; accepted November 3, 2008 (ARCMED-D-08-00359). Previously published online January 22, 2009.
Background and Aims. We investigated the association between the two common polymorphisms, C1431T and Pro12Ala of PPARg gene, and metabolic syndrome (MS) in a Chinese population. Methods. We included 423 subjects with MS and families without MS. Subjects were divided into three groups: MS probands and first- and second-degree relatives of probands, spouses and controls. Each group was then divided into two subgroups according to genotype (Pro/Pro and Pro/Ala for Pro12Ala, CC and CT þ TT for 1431C/T). Anthropometric indices, fasting plasma glucose, lipid profile, Sv1 þ Rv5 of electrocardiogram and single nucleotide polymorphisms were detected. Results. Frequencies of C1431T genotypes, but not Pro12Ala, were different among the three groups. MS patients with Pro/Ala genotype had higher fasting blood sugar (FBS) levels and Sv1 þ Rv5. Controls with Ala allele had lower total cholesterol levels. In relatives, Ala carriers had higher high-density lipoprotein cholesterol (HDL-c) levels. BMI of the different groups were not significant. MS patients with T allele had higher FBS and Sv1 þ Rv5. In relatives of MS subjects, T-allele carriers had lower blood uric acid, creatinine and higher HDL-c levels and Sv1 þ Rv5. Conclusions. C1431T, but not Pro12Ala polymorphisms, are associated with MS in a Chinese population. In MS patients, Ala allele and T allele are both associated with higher fasting blood sugar and higher left ventricular voltage. In controls, Ala carriers have lower total cholesterol. In MS relatives, Ala carriers had higher HDL-c levels and T-allele carriers had lower uric acid, creatinine and higher HDL-c levels and left ventricular voltage. Ó 2009 IMSS. Published by Elsevier Inc. Key Words: PPARg, Polymorphism, Metabolic syndrome, Genotype.
Introduction Metabolic syndrome (MS) is a complex disorder that encompasses several metabolic diseases such as abdominal obesity, insulin resistance, elevated plasma triglyceride (TG) levels and low high-density lipoprotein cholesterol (HDL-c) levels, high blood pressure, and altered glucose homeostasis (1). Genetic and environmental factors contribute to the susceptibility to the MS. Many genes are candidates for MS. Peroxisome proliferator-activated receptor-g (PPARg) is one of the nuclear hormone receptor
Address reprint requests to: Qi Hua, Department of Cardiology, Xuanwu Hospital, Capital University of Medical Science, Changchun Street No.45, Beijing, China, 100053; E-mail: [email protected]
superfamily and heterodimerizes with retinoid X receptor (RXR). It is suggested that it regulates target genes in adipocyte differentiation (2,3) and insulin sensitization (4,5). PPARg has four isoforms of which PPARg-1 is expressed in most tissues. PPARg-2 is specific to adipose tissue and regulates adipocyte differentiation (6). PPARg3 seems to be mainly confined to macrophages, adipose tissue, and colon (7). Tissue distribution of PPARg4 has not yet been explored (8). The PPARg gene is located on chromosome 3 (9), and the specific isoforms are a result of alternative mRNA splicing. The prevalent human PPARg polymorphism is the Pro12Ala polymorphism (rs1801282) of the exon B in PPARg (10). This polymorphism has been associated with increased protection against the development of type 2 diabetes and insulin resistance (11e14). However,
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.11.005
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there are variations in Ala allele frequencies in different populations. There are also different relationships with MS (11e14). Another polymorphism, the C1431T silent substitution (rs3856806) in the 6th exon of PPARg, has been shown to modulate the effect of Pro12Ala on susceptibility to type 2 diabetes (15). However, there are some contradictory results of the association between these two polymorphisms and diabetes and MS (11e15). There are also few reports about these two polymorphisms of MS in Chinese families. We investigated the two polymorphisms in PPARg gene in Chinese families with MS probands.
Materials and Methods Subjects Since 2003, data on all patients attending the Cardiac Clinic of Xuanwu Hospital of Capital Medical University (Beijing, China) have been documented for detailed family history. Families including two patients with MS (probands) were recruited into the study. Families without MS patients were also recruited as the control group. The present study included 101 families with a total of 442 subjects. Most families were nuclear families. Subjects who were !30 years old (nine subjects) were excluded and subjects whose DNA could not be amplified were also excluded (ten subjects). We included 423 subjects investigated from January 2003 to September 2007. Subjects were divided into three groups: probands (n 5 146), first- and second-degree relatives (without MS) of probands (n 5 96) and spouses and control families without MS (n 5 181). Each group was then divided into two subgroups according to the genotype (Pro/Pro and Pro/ Ala for Pro12Ala, CC and CT þ TT for 1431C/T). Probands included 86 (58.9%) subjects with three characteristics of MS, 42 (28.8%) subjects with four characteristics of MS and 18 (12.3%) subjects with five characteristics of MS. All subjects signed informed consent. The protocol was approved by the ethics committee of our institution. MS was defined using criteria established in the third report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment in Adults (Adult Treatment Panel III) (16) as the presence of three or more of the following risk factors: waist circumference (WC) $88 cm in women and $102 cm in men, TG $150 mg/dL, HDL-c #50 mg/dL in women and #40 mg/dL in men, blood pressure (BP) $130/85 mm Hg, fasting blood sugar (FBS) $110 mg/dL or a diagnosis of diabetes. Type 2 diabetes diagnosis was defined on the basis of the OGTT using the plasma glucose criteria of the American Diabetes Association (2-h glucose $11.1 mmol/L or FBS $7.0 mmol/L). Measurements Weight, height, WC and hip circumference (HC) were measured by trained personnel, and body mass index (BMI) was
calculated according to the Quetelet equation. BP was measured on the right arm with the subject in a sitting position after a minimum 5-min rest using a standard mercury sphygmomanometer. The mean value of two consecutive BP readings was used. Venous blood samples were collected in the morning after subjects had fasted for 12 h. FBS was assayed by the glucose oxidase method. Serum TG levels were enzymatically measured. Total cholesterol (TC), TG, and HDL-c levels were measured after sodium phosphotungstate/magnesium chloride precipitation. All biochemical indices were measured with an automatic biochemistry machine (Hitachi 7170, Tokyo, Japan). LDL-c levels were calculated by the Friedewald equation. We also used the electrocardiogram (ECG) of subjects and the sum of Sv1 and Rv5 for investigation of left ventricular hypertrophy. All subjects were investigated with regard to their family medical history. Genotyping Genetic analyses were performed on genomic DNA extracted from human leukocytes. Single nucleotide polymorphism of Pro12Ala and 1431C/T were detected using restriction fragment length polymorphism and polymerase chain reaction (RFLP-PCR). PCR was performed with thermal cycler (Bio-Rad, Hercules, CA). Total volume of PCR reaction was 25 ml. For each reaction, 0.5 ml of genomic DNA, 0.725 units of Taq polymerase (MBI, Fermentas, Canada) and 2.5 ml 10X PCR buffer were used. Magnesium chloride concentration was 1.5 mM. Each primer concentration was 0.2 mM. Genotyping for Pro12Ala Primers used were 50 -CAAGCCCAGTCCTTTCTGTG-30 and 50 -AGTGAAGGAATCGCTTTCCG-30 . The base C (underlined) was mismatched for the site of the restriction enzyme HpaII (MBI). The following PCR conditions were used: initial denaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C, annealing at 52 C and elongation at 72 C. Each of these steps lasted 30 sec. This was followed by a final extension at 72 C for 10 min. PCR products spanned the entire length of 237 bp (base pairs). PCR products of 10 ml were digested with 10 U of HpaII at 37 C for 5 h or overnight. When alanine was present at residue 12 of PPARg2, the 237-bp DNA fragment was split into 217- and 20-bp pieces. Digestion products were then separated by electrophoresis on 3% agarose gel, and photos were taken under ultraviolet light. Genotyping for C1431T Primers used were 50 -GCTCCAGAAAATGACAGACC T-30 and 50 -TGGAAGAAGGGAAATGTTGG-3. Annealing temperature was 55 C and cycle times were 40 sec. Other conditions were the same with the genotyping for
Polymorphisms of PPARg Gene and Metabolic Syndrome
Pro12Ala. PCR product spanned the entire length of 170 bp and was digested by the specific restriction Eco72I enzyme. When allele C was present at C1431T of PPARg2, the 170bp DNA fragment was split into 127- and 43-bp pieces. Concordance rates between the PCR-RFLP assay used for genotyping and sequencing or repeat genotyping was 100%. Statistical Analysis We conducted analysis on normally distributed variables. Variables of quantitative traits were normally distributed. Differences between quantitative traits were compared with one-way ANOVA and post hoc multiple comparison (using LSD method). For genotypes and gender we used Pearson c2 to compare differences. The influence of covariates such as gender, age, family and other possible covariates in the comparison of means was adjusted by the general linear model. In order to abscise the effect of relationship of the same family, the family number was used as one covariant. Comparison of creatinine and uric acid, diabetes and FBS were also used as covariates. Comparison of the sum of Sv1 and Rv5, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were also taken as covariates. Two-sided p values !0.05 were considered statistically significant. SPSS (Chicago, IL) software v.13.0 was used. Hardy-Weinberg equilibrium tests were performed for genotypes using c2 test. The tests of linkage of disequilibrium were performed using the software of THESIAS (17).
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Results Subjects’ Characteristics Quantitative traits are presented as mean SD (standard deviation). Basic anthropometric and biochemical characteristics of the subjects are presented in Table 1. There were statistical differences among the three groups in terms of gender, age, height, weight, WC and HC, waist and hip circumference ratio (W/H ratio), BMI, abdominal circumference (AC), SBP, DBP, FBS levels, blood creatinine levels, TC, and TG levels. There were no statistical differences in HDL-c or LDL-c levels, blood uric acid levels and Sv1 þ Rv5 of ECG among the three groups. Subjects’ Genotypes Genotypes of subjects are presented in Table 2. Of the 423 subjects, Pro12Pro genotype was present in 91.02% (385 subjects) of the population and Pro12Ala genotype was present in 8.98% (38 subjects). There were no Ala12Ala genotypes. Allele frequencies were 0.955 for Pro allele and 0.045 for Ala allele. Genotypic frequencies of the PPAR exon 6 C1431T substitution were 56.97% (241 subjects) for CC, 36.41% (154 subjects) for CT, and 6.62% (28 subjects) for TT genotype. Allele frequency for the C allele was 0.751 and 0.249 for T allele. Allele frequencies of the two genotypes satisfied Hardy-Weinberg equilibrium. There were no differences in terms of Pro12Ala genotype frequencies among the three groups. There were statistical differences in terms of C1431T genotype frequencies
Table 1. Basic anthropometric and biochemical characteristics of different groups (probands, first- or second-degree relatives, controls) Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv2 þ Rv5 (mV)
Probands (n 5 146)
First- or second-degree relatives (n 5 96)
Controls (n 5 181)
p value
62/84 56.8 15.07 165.58 8.83 76.24 16.09 94.13 10.20 106.71 8.99 0.88 0.07 27.67 4.65 102.98 10.70 144.52 21.38 89.24 12.31 118.73 42.38 6.12 1.88 86.16 27.93 208.35 41.88 257.45 164.88 47.80 59.38 109.07 75.41 2.54 0.89
54/42 49.44 13.45a 167.04 7.92 70.83 16.27a 85.51 9.23a 100.53 8.58a 0.85 0.07a 25.27 5.07a 96.14 10.87a 133.84 22.53a 86.12 14.51 96.20 19.77a 6.14 5.64 80.73 18.82 192.27 37.01a 137.37 99.74a 56.42 38.39 106.94 51.28 2.54 0.86
76/105 47.32 15.9a 164.75 7.13b 68.15 14.63 86.32 13.17a 99.26 10.09a 0.87 0.12b 24.99 4.38a 94.10 11.99a 132.51 22.05a 84.36 13.91a 95.57 23.52a 5.37 1.39 77.92 19.83a 190.78 61.61a 122.69 89.27a 52.04 10.66 114.20 60.80 2.61 0.71
0.039 !0.0001 0.015 !0.0001 !0.0001 !0.0001 0.001 !0.0001 !0.0001 !0.0001 !0.0001 !0.0001 0.124 0.009 !0.0001 !0.0001 0.417 0.630 0.218
Data are mean SD. p value in the fifth column is for the comparison among three groups. BMI, body mass index; AC, abdominal circumference; W/H ratio, waist/hip ratio; WC, waist circumference; HC, hip circumference; FBS, fasting blood sugar; TC, total cholesterol; TG, triglyceride; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol. a Significant difference compared with the probands ( p !0.05). b Significant difference compared with the first- or second-degree relatives ( p !0.05).
Yang et al./ Archives of Medical Research 40 (2009) 89e96
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Table 2. Genotypes of subjects of three groups of metabolic syndrome Groups Pro12Ala Pro/Pro Pro/Ala C161T CC CT TT Pro12Ala þ C161T Pro/Pro þ CC Pro/Pro þCT or TT Pro/Ala þ CC Pro/Ala þCT or TT
Probands n 5 146
First- or second-degree relatives n 5 96
Control n 5 181
p value
137 (93.84%) 9 (6.16%)
83 (86.46%) 13 (13.54%)
165 (91.16%) 16 (8.84%)
0.281
91 (62.33%) 50 (34.25%) 5 (3.42%)
61 (63.54%) 34 (35.42%) 1 (1.04%)
89 (49.17%) 70 (38.67%) 22 (12.16%)
87 50 4 5
56 27 5 8
86 79 3 13
(59.59%) (34.25%) (2.74%) (3.42%)
among the three groups. The prevalence of CC genotype in controls was lower than that in MS probands and first- or second-degree relatives of MS (49.17 vs. 62.33%, 63.54%, p 5 0.002). Because the association between two polymorphisms was significant ( p 5 0.015), we compared the four genotypes of two polymorphisms in different groups of MS. The presence of Pro/Pro þ CC in controls was lower than that in probands and relatives of patients with MS. The presence of Pro/Ala þ CT or TT of controls was higher than that of probands with MS. We also tested the linkage disequilibrium between the two polymorphisms. The two polymorphisms were in linkage disequilibrium (D0 5 0.516, r2 5 0.031). To test the association between two polymorphisms and individual components of MS, we compared biochemical and anthropometric indices among different genotype groups of two polymorphisms. Data are presented in Tables 3e6. To test the differences between relatives and controls, we included these two groups in the same tables.
Pro12Ala Genotypes and Individual Components of MS As presented in Table 3, MS patients with Pro/Ala genotype had lower weight and height, but had higher FBS levels and Sv1 þ Rv5 than those without Ala allele. The differences of blood sugar levels (when corrected by age, gender and family, p 5 0.001) and Sv1 þ Rv5 (when corrected by age, gender, family and BP, p 5 0.018) were significant. There were no differences in terms of WC, HC, W/H ratio, BMI, AC, BP, creatinine, uric acid, TC, TG, LDL-c and HDL-c levels between the two groups. In relatives and controls (presented in Table 4), there were no differences in gender, age, weight, height, WC, BMI, AC, BP, FBS, uric acid, creatinine, TC and Sv1 þ Rv5 among the four groups. HDL-c levels of MC relatives with Pro/Ala genotype were higher than those of the other three groups. Controls with Pro/ Ala genotype had lower TC levels. Controls and relatives with Pro/Ala had lower LDL-c levels, but this was not statistically significant ( p 5 0.095).
(58.33%) (28.13%) (5.21%) (8.33%)
0.002
(47.51%) (43.65%) (1.66%) (7.18%)
0.048
C1431T Genotypes and Individual Components of MS As presented in Table 5, there were significant differences in terms of gender, FBS, LDL-c levels, TG and Sv1 þ Rv5 in ECG between CC genotype and CT þ TT genotype groups in patients with MS. There were no differences in terms of age, height, weight, WC, HC, W/H ratio, BMI, AC, BP, creatinine, uric acid, TC and HDL-c levels between the two groups. FBS, Sv1 þ Rv5 and TG ( p 5 0.008, after adjusted p 5 0.147) of CT þ TT genotype group were higher than those of CC genotype in MS Table 3. Biochemical characteristics of different genotype of Pro12Ala in MS probands Probands n 5 146 Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
p value
Pro/Pro Pro/Ala 61/76 54.90 14.6 166.30 8.58 77.27 16.44 94.18 10.26 106.78 9.22 0.88 0.07 27.80 4.72 102.94 11.07 144.56 20.81 89.42 12.43 113.96 37.10 6.07 1.81 87.47 28.77 208.77 252.54 48.88 109.37 2.47
43.94 151.06 63.26 79.58 0.78
70.11 160.56 69.06 93.78 106.22 0.88 26.79 103.22 144.22 88.00 151.92 6.51
1/8 10.50 9.17 11.37 10.04 7.35 0.06 4.04 7.80 15.71 11.72 60.24 2.29
0.048 !0.0001 0.004 0.036 0.888 0.814 0.974 0.390 0.923 0.951 0.677 0.001 0.604
77.03 19.45
0.069
0.79 0.209 0.388 0.882 0.018
205.44 291.51 40.17 106.98 2.99
23.90 243.37 13.69 36.10 1.36
p values were corrected by age, gender and family. p values of creatinine and uric acid were also adjusted by diabetes and fasting blood sugar. p value of the sum of Sv1 and Rv5 was also adjusted by systolic and diastolic blood pressures.
Polymorphisms of PPARg Gene and Metabolic Syndrome
93
Table 4. Biochemical characteristics of different genotypes of Pro12Ala in first- or second-degree relatives and controls First- or second-degree relatives n 5 96
Controls n 5 181
Pro/Pro Pro/Ala
Pro/Pro Pro/Ala
Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
47/36 48.22 13.89 166.67 7.29 70.31 16.26 85.71 9.16 99.69 9.01 0.86 0.07 25.26 5.79 96.19 11.24 133.29 23.15 86.06 15.09 96.58 22.10 6.12 6.03 82.37 19.78 190.04 37.51 128.36 65.19 50.37 11.57 112.15 37.54 2.49 0.83
53.82 168.36 72.68 84.70 103.80 0.81 25.31 95.90 135.82 88.91 94.82 6.22 74.84 200.26 169.75 77.58 88.73 2.70
7/6 13.89 9.95 16.55 9.69 5.63 0.08 3.22 10.06 20.54 12.26 6.65 2.24 13.70 34.77 173.74 76.09a 82.00 0.96
69/96 47.41 15.73 164.70 7.16 68.41 15.17 86.62 13.63 99.46 10.08 0.87 0.13 25.09 4.49 94.20 11.81 132.97 22.43 85.01 13.80 96.76 24.96 5.37 1.47 78.09 19.83 195.09 64.05 123.99 94.49 52.21 10.82 118.08 63.55 2.58 0.71
7/9 46.85 165.08 66.73 84.69 98.15 0.86 25.47 93.54 130.00 80.69 89.07 5.42 77.02 167.25 115.53 51.15 92.99 2.81
p value
17.25 7.12 11.36 12.98 10.28 0.06 3.73 13.16 20.00 14.20 11.41 0.95 20.21 39.21 53.07 9.88 36.89 0.70
0.277 0.301 0.077 0.456 0.805 0.218 0.158 0.892 0.666 0.841 0.216 0.424 0.456 0.268 0.033 0.163 !0.0001 0.095 0.368
a
Difference from the first group. Difference from the second group. c Difference from the third group. p values in the sixth column are for the comparison among four groups. p value !0.05 was considered significant. p values of creatinine and uric acid were also adjusted by diabetes and FBS. p value of the sum of Sv1 and Rv5 was also adjusted by SBP and DBP. b
Table 5. Biochemical characteristics of different genotypes of C1431T in MS probands Probands (n 5 146) CC
CT þ TT
p value
39/52 55.96 13.49 165.76 8.80 76.53 17.53 93.36 11.23 105.99 9.23 0.88 0.08 27.71 5.18 102.39 11.54 142.65 18.02 88.92 11.88 112.94 32.69 6.01 1.82
23/32 59.22 17.01 165.15 9.10 75.13 13.67 95.28 8.77 107.81 8.43 0.88 0.05 27.40 3.45 103.72 9.02 147.17 25.31 89.26 13.09 127.20 54.85 6.20 2.00
0.920 0.215 0.663 0.610 0.339 0.261 0.852 0.710 0.505 0.239 0.886 0.006 0.746
84.86 23.74
88.19 34.47
0.406
0.225 0.147 0.169 0.093 0.048
Groups Gender (M/F) Age (years) Height (cm) Weight (kg) WC (cm) HC (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmmol/L) TC (mg/dL) TG (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
213.0 231.17 44.35 122.61 2.49
48.19 119.40 9.04 45.14 0.78
202.37 287.20 54.32 90.61 2.74
31.49 203.56 96.09 106.02 1.05
p values were corrected by age, gender and family. p values of creatinine and uric acid were also adjusted by diabetes and fasting blood sugar. p value of the sum of Sv1 and Rv5 was also adjusted by SBP and DBP.
patients. LDL-c levels ( p 5 0.005, after adjusted p 5 0.093) of CT þ TT genotype group were lower than those of CC genotype group. After the effects of age, gender, family and blood pressure were corrected, only FBS and Sv1 þ Rv5 were significant ( p !0.05). In relatives and controls (Table 6), there were no differences in terms of age, weight, height, WC, HC, W/H ratio, BMI, AC, DBP, FBS, TC, TG, and LDL-c levels. Relatives with CT þ TT genotypes had lower uric acid and blood creatinine (corrected by age, gender, family, diabetes and FBS) than those with CC genotypes. Relatives with CT þ TT genotypes had higher HDL-c levels and Sv1 þ Rv5 (corrected) than those with CC genotypes. Differences in controls were not significant.
Discussion This is the investigation of the association of PPARg polymorphisms and MS families in mainland China. We tested the polymorphisms of Pro12Ala of exon B and C1431T of exon 6 of PPARg. There was no Ala12Ala homozygote in the population. Allele frequency was 0.955 for Pro allele and 0.045 for Ala allele, similar to the rare frequency of Ala allele in the Asian population in previous studies (18e22) and lower than the frequency of Ala allele in a Hispanic population (11) and in Caucasians (12). The allele
Yang et al./ Archives of Medical Research 40 (2009) 89e96
94
Table 6. Biochemical characteristics of different genotype of C1431T in first- and second-degree relatives and controls First- or second-degree relatives (n 5 96) Groups Gender (M/F) Age (years) Height (cm) Weight (kg) Waist circumference (cm) Hip circumference (cm) W/H ratio BMI (kg/m2) AC (cm) SBP (mm Hg) DBP (mm Hg) FBS (mg/dL) Blood uric acid (mg/dL) Blood creatinine (mmol/L) TC (mg/dL) Triglyceride (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) Sv1 þ Rv5 (mV)
Control (n 5 181)
CC
CT þ TT
CC
CT þ TT
p value
38/23 49.08 13.35 167.46 7.72 70.71 10.72 85.15 15.15 101.10 9.38 0.84 0.08 25.18 3.14 96.07 12.59 131.17 22.82 85.27 15.32 95.52 20.38 6.85 7.10 85.22 20.20 194.31 32.97 129.07 58.29 50.22 10.80 115.95 30.54 2.39 0.86
16/19 49.87 12.83 166.10 8.17 71.18 22.15 85.86 7.83 99.51 7.28 0.86 0.07 25.58 7.07 96.07 7.77 137.74 22.18 88.82 13.38 97.21 19.52 5.11 1.94a 73.57 14.61a 190.32 43.24 152.61 142.81 65.26 58.73a 94.54 71.26 2.79 0.83a
31/58 45.75 15.81 164.83 7.55 68.17 14.35 87.42 15.74 99.04 11.34 0.89 0.16 24.97 4.27 95.39 11.14 128.76 20.46 83.82 15.70 94.41 15.01 5.47 1.36 77.41 20.04 199.45 81.65 117.04 62.07 53.61 11.29 122.43 80.75 2.52 0.71
45/47 48.83 16.45 164.88 7.24 68.51 15.66 85.62 11.41 99.84 8.72 0.86 0.08 25.08 4.70 93.00 13.21 135.04 23.69 84.64 13.04 96.74 29.74 5.36 1.48 80.61 19.92 184.99 42.84 126.85 110.64 50.74 10.32 108.88 41.90 2.67 0.73
0.011 0.435 0.156 0.645 0.453 0.661 0.141 0.939 0.359 0.147 0.358 0.903 !0.0001 !0.0001 0.43 0.314 0.005 0.105 !0.0001
a
Difference from the first group. Difference from the second group. p values in the sixth column are for the comparison among four groups. p value !0.05 was considered significant. p values were corrected by age, gender and family. p values of creatinine and uric acid were also adjusted by diabetes and FBS. p value of the sum of Sv1 and Rv5 was also adjusted by SBP and DBP. b
frequency for the C allele was 0.751 and 0.249 for T allele, demonstrating higher T-allele frequency than in previous studies (23e25) and similar frequency to that in another study (26). We calculated the allele frequency by including all family subjects. Allele frequencies were similar to the Asian population (nonrelated population). This demonstrated that there were no obvious aggregations of these two polymorphisms in MS familial population. Two polymorphisms are in linkage disequilibrium (D0 5 0.516, p 5 0.003). Several studies reported an association between the Ala allele of the codon 12 polymorphism of exon B and reduced risk of diabetes and decreased insulin resistance (25,27e32). However, other studies were not able to replicate these associations (18,33,34). We did not find differences of Ala frequencies among MS probands, relatives and controls ( p 5 0.281), which meant that there was no association between Pro/Ala polymorphism and MS, similar to the results of other studies (35,36). In a large French population-based study, Meirhaeghe et al. found no association between PPARg Pro12Ala polymorphism and MS (35). In the Chinese population, Donxia et al. also found no association between them (21). Our study is similar to this result. However, in the present study, MS patients with Ala allele had higher FBS levels similar to the previous study (22,33) and higher Sv1 þ Rv5 in ECG than those without Ala allele. When corrected, these differences were
significant. However, Sv1 þ Rv5 increase did not make the diagnosis of left ventricular hypertrophy. This demonstrates that Ala allele may cause the increase of left ventricular voltage in MS patients. Duan et al. developed a cardiocyte-specific PPAR-g knockout mouse model and showed that cardiocyte PPAR-g inactivation in vivo caused cardiac hypertrophy, likely through NF-kB activation (37). Further studies are needed to clarify the association between PPARg gene and left ventricular hypertrophy in MS. Whereas the effect of the Ala12 substitution on the structure of PPARg2 remains unknown, it has been shown that Ala substitution results in a 2-fold lower affinity of PPARg2 for binding to the PPRE (PPAR response element) and reduces transcriptional activity in the presence and absence of PPARg agonists (12). In patients with MS, relatives and controls, BMI was not different. We suggest that Ala allele has no effect on obesity in Chinese subjects. Dongxia et al. found similar results, but the presence of Pro12Ala and T allele simultaneously showed a potential effect on obesity (21). A study in the Spanish population suggested that Pro/Ala polymorphism was related to obesity in men and not in women (38). These contradictory results may be due to population differences, which need further studies to confirm. In controls, Ala allele carriers had lower TC levels. In relatives, Ala allele carriers had higher HDL-c levels. In an Asian study (22), Ala allele also had higher HDL-c
Polymorphisms of PPARg Gene and Metabolic Syndrome
levels. In relatives and controls, Ala carriers had lower LDL-c levels, but these were not significant. Other studies did not find the association between Ala allele and LDL-c level (21,36). This may be because our study discriminated among patients with MS, relatives and controls and others investigated from the total population. To some extent, Ala allele may have the potential protective effect from atherosclerosis for some non-MS subjects. This needs to be clarified in further studies. It was reported that polymorphism C1431T of exon 6 of PPARg was associated with diabetes risks (22,25) and leptin level in obese humans (23). However, not all studies had similar results (32,36). We found differences of C1431T frequencies among the three groups, which meant that there was association between C1431T polymorphism and MS. Dongxia et al. found no association between C1431T and MS in a Chinese population (21). In Meirhaeghe’s French population study (35), polymorphisms of C1431T were not individually associated with MS. However connected with the other three polymorphisms, 681COG, P2-689COT, Pro12Ala, haplotypes are associated with MS. We found the association between C1431T polymorphism and MS in Chinese people. It may be that our subjects were a family population and this contributed to the association. Other studies only had the patients and controls or total population. Patients with MS with T allele had higher FBS and Sv1 þ Rv5 levels than those without T allele and the differences were also significant when adjusted by gender, age and family. T-allele carriers had higher FBS, different from previous studies (22,25). The mechanism by which this silent polymorphism in PPARg affects its activity remains unclear, but one possibility may be that it is in linkage disequilibrium with the Pro12Ala polymorphism. In relatives, T-allele carriers had lower blood uric acid, creatinine, and higher HDL-c levels and Sv1 þ Rv5. Some studies showed the association between uric acid and MS (39). MS was found correlated with chronic kidney disease (40). However, there are few studies focused on the association among uric acid, creatinine and PPARg gene polymorphism. PPARg may play a role in uric acid and creatinine metabolism individually or by modulating MS. Further studies are needed to confirm this relationship. T-allele carriers in relatives had higher HDL-c levels. However, other studies did not show this effect. This effect may only be in MS relatives. T-allele carriers also had higher Sv1 þ Rv5 in relatives and patients with MS. In controls, it also had this effect but was not significant. Further studies are needed to verify the association between C1431T polymorphism and left ventricular hypertrophy in MS. Our investigation seems to be able to explain some contradictory results of PPARg polymorphisms and MS. These two polymorphisms are correlated with some components of MS. Some are protective and others are harmful. The limitation of our study was that the population was not large enough, so some groups had fewer subjects. This
95
may lead to some bias in results. Larger population studies should be done to confirm the results presented here. In conclusion, Pro12Ala polymorphism is not associated directly with MS. However, MS patients with Ala allele have higher FBS and higher left ventricular voltage. In controls, Ala carriers have lower TC. In relatives, Ala carriers have higher HDL-c levels. We find the association between C1431T polymorphism and MS in a Chinese population. MS patients with T allele have higher FBS and higher left ventricular voltage. In MS relatives, T-allele carriers have lower uric acid, creatinine and higher HDL-c levels and left ventricular voltage. Acknowledgments This study was also supported by a group of professors of the Capital Medical University, Beijing, China. We thank the volunteers for their enthusiastic participation. The research was funded by the Beijing Nature Foundation.
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