Introduction

Peroxisome proliferators-activated receptor gamma (PPARγ) is a nuclear hormone receptor of ligand-dependent transcription factor involved in adipogenesis and a molecular target of the insulin sensitizers, thiazolidinediones. PPARγ has been suggested to play important roles in obesity and in the development of type 2 diabetes (T2D) related with insulin sensitivity and lipid metabolism [1–3]. In an animal model, heterozygous deficiency of PPAR γ was shown to lead to the protection from high-fat diet-induced insulin resistance [4]. Barroso et al. [3], in their study of human subjects, reported that the first germ line loss-of-function mutations in the ligand binding domain of PPAR γ gene caused severe insulin resistance, early onset of T2D, and hypertension.

A more recent study has suggested that people with the Ala12 allele of the PPAR-γ2 gene could be more sensitive to insulin than those with the Pro12 allele among Brazilian Caucasians [5]. The Pro12Ala polymorphism in the PPAR γ gene has been shown to influence the risk for T2D and obesity in various ethnic populations worldwide [5–15]. A recent study in Qatar [8] revealed that obesity, consanguinity, blood pressure, total cholesterol, HDL-cholesterol, and triglyceride were more prevalent in the Qatari diabetic patients. The characterization of these factors will contribute to defining more effective and specific strategies to screen for and control diabetes and cardiovascular disease in a new developing country. The prevalence of the Pro12Ala mutation, however, varies according to the ethnic background [9–20].

In this study, we focused on the Pro12Ala polymorphism in Qatar. During this past decade, diabetes prevalence in Qatar has been dramatically increasing [8], which may be due to change of life styles such as increasing total calorie intake including fat, less exercise, and increasing level of stress under the unstable social–economic circumstances. There may, however, be genetic factors that contribute to the dramatic increase in diabetic patients under the interaction between genetic and environmental factors. Since the Pro12Ala polymorphism of the PPAR-γ2 gene has not yet been investigated in Qatar, we studied its prevalence among native Qataris and examined whether this polymorphism is associated with diabetes in Qatar.

Subjects and methods

Data collection

Data including age, sex, onset of diseases, family history of diabetes, and diabetic complications were obtained from the patients. All subjects were native Qataris, identified by themselves as being native Qataris, by at least two pedigrees (parents and grand parents). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using standard methods. The survey was based on standardized interviews performed by trained health professionals and nurses. The study was approved by the Hamad General Hospital, Hamad Medical Corporation. All human studies have been approved by the Research Ethics Committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All the persons who agreed to participate in this study gave their informed consent prior to their inclusion in the study.

Selection of diabetic subjects

Persons were classified as diabetic if they were currently taking diabetic medication. We studied Qatari diabetic subjects diagnosed for T2D in accordance with the established diagnostic criteria [21]. A total number of 400 diabetic Qatari patients aged 35–60 years selected by a simple random process from the Primary Health Care (PHC) Centers. Their health status was assessed by recording previous medical conditions, family history, and blood pressure.

Selection of control subjects

Control subjects were self-reported, aged 35–60 years with random glucose value of <6.1 mmol/l, and if had never taken any diabetic medication. Since T2D is a late onset disorder, the control subjects selected were above the age of 35. This group consisted of a random sample of 450 healthy Qatari subjects who visited the PHC Centers for any reason other than acute or chronic disease. Their health status was further assessed by recording previous medical conditions, family history, and blood pressure.

Height and weight were measured using standardized methods, and all the participants wore light clothes and no shoes for this part of the examination. Body mass index (BMI) was calculated as weight (kg)/height (m2). Subjects were classified into three categories: acceptable weight, BMI < 25; overweight, BMI 25–30; and obese, BMI > 30 [8].

Blood pressure measurement was carried out by trained practical nurses according to World Health Organization (WHO) standardized criteria [22]. The mean value obtained from three readings was used in the analysis. Hypertension was defined according to WHO criteria as SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg and/or the use of antihypertensive medication. Mean blood pressure was defined as 1/3 systolic pressure + 2/3 diastolic pressure. The pulse pressure was calculated as the difference between systolic and diastolic pressures.

Procedures

Blood samples were collected by venipuncture in vacutainer EDTA tubes and used for manual DNA purification using the QiaAmp mini DNA kits. Random glucose values were determined by using the One Touch Sure Step Kit.

The single nucleotide polymorphism (SNP) detection protocol was based on a modified procedure of Ghoussaini et al. [9] and consisted of an amplification step by polymerase chain reaction (PCR) followed by mutation analysis of the PCR product by real time PCR. The PCR primer set consisted of: PGE × 1F 5′-AAT AGG ACA GTG CCA GCC-3′ and PGE × 1R 5′-TAC ATA AAT GCC CCC ACG-3′. The reaction was performed in a final volume of 25 μl and contained: 1 × GeneAmp® PCR Gold buffer; 2.5 mM MgCl2, 1% DMSO, 0.25 mM each dNTP, two primers (PGE × 1F: 4.3 nM and PGE × 1R 3.58 nM), 0.75 U AmpliTaq Gold® DNA Polymerase, and 100–500 ng template DNA. The PCR cycles consisted of an initial denaturation at 95°C for 12 min followed by 35 cycles, which consisted of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, elongation at 72°C for 30 s.

The Pro12Ala in the PPARγ2 gene was detected on the LightCycler using two specific probes:(Sensor [G] 5′-CTC CTA TTG ACG CAG AAA GCG-FL and PPAR Anchor 5′ LC Red 640-TCC TTC ACT GAT ACA CTG TCT GCA AAC ATA TC-PH). The melting temperature profiles were interpreted as follows; wild type (CC): 54°C, mutant (GG): 60°C and heterozygous (CG): 54 and 60°C.

Sample size was calculated with 80% power at the 5% level of significance and 95% confidence interval which was found to be 410 subjects in each group. We have recruited 400 subjects and 450 controls for the current study.

Statistical analysis

Student’s t test was used to ascertain the significance of differences between mean values of two continuous variables and confirmed by a nonparametric Mann–Whitney test. Chi-square analysis was performed to test for differences in proportions of categorical variables between two or more groups. In 2 × 2 tables, the Fisher’s exact test (two-tailed) replaced the chi-square test if the assumptions underlying Chi-square violated, namely in case of small sample size and where the expected frequency is less than 5 in any of the cells. Odds Ratio (OR) and their 95% confidence intervals (CI) were calculated by using the Mantel–Haenszel test. The level P ≤ 0.05 was considered as the cut-off value for significance.

Results

Phenotypic characteristics of the studied populations

The characteristics of the type 2 diabetic and normal control subjects are summarized in Table 1. The average age of normal subjects was (50.0 ± 12.8 years) and that of diabetic subjects was (50.3 ± 9.8 years). Nearly half of the diabetic type 2 patients (48.5%) were obese (BMI > 30) compared with nondiabetic subjects (29.8%) (P < 0.001). The prevalence of T2D was significantly higher among the consanguineous marriages group (P < 0.001). Furthermore, among first and second degrees consanguinity, the chances of diabetes were even higher. Systolic and diastolic blood pressure levels were significantly higher in diabetes patients than nondiabetics healthy subjects (P < 0.001).

Table 1 Characteristics of the type 2 Diabetic and normal control subjects
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Association with T2D

Table 2 shows the distribution of the Pro12Ala polymorphism in 400 diabetic and 450 nondiabetic subjects. The distribution of the genotypes both in the diabetic and nondiabetic groups was in agreement with the Hardy–Weinberg equilibrium. The table also shows the calculated frequency of the Ala12 frequency of the PPAR γ gene in both groups. There was however no significant difference in the allelic frequency of the Ala12 variant between the type 2 diabetic subjects and the controls (0.055 vs 0.059, P = 0.669), indicating the absence of association between the Pro12Ala polymorphism and the T2D in the Qatar.

Table 2 Allele frequencies of the Pro12Ala polymorphism of PPAR γ 2 in type 2 diabetic patients and control subjects
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Comparison of the clinical characteristics

Tables 3 and 4 show a comparison of the clinical profiles between the Pro/Ala heterozygotes and the homozygous normal genotype (Pro/Pro) with and without diabetes concerning the Ala12 variant of the PPAR γ gene in relation to age, BMI, and blood pressure.

Table 3 Comparison of clinical characteristics between diabetic subjects with and without the Pro12Ala variant of PPAR γ
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Table 4 Comparison of clinical characteristics between nondiabetic subjects with and without the Pro12Ala variant of PPAR γ
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Discussion

T2D is a chronic condition in Qatar with an estimated prevalence rate of 11–15% affected and a further 11% showing impaired glucose tolerance [8].

Among the candidate genes known for T2D, the Pro12Ala polymorphism of the PPARγ gene has been widely implicated in the T2D in various ethnic populations. PPARγ gene codes for a transcription factor that is activated by certain fatty acids and the insulin-sensitizing antidiabetic thiazolidiones. The activated form of the factor binds specific DNA sites and facilitates transcription of a wide range of target genes [23]. The PPARγ-2 isoform is expressed predominantly in adipose tissues and is critical in adipogenesis and insulin action. The rs number for the studied polymorphism is 1805192.

The Pro12Ala allele frequencies in the PPAR γ2 gene in diabetic subjects or nondiabetic subjects vary among ethnic communities. Frequencies among Caucasians are generally higher than those of the other ethnic groups. Several studies of the Caucasians in different countries have indicated that their Pro12Ala polymorphism was greater than 10% [11, 15, 19]. In contrast, Asian populations have less prevalence of this polymorphism. A Japanese multicenter study involving a large number of samples demonstrated that the Ala12 allele frequencies of diabetic and nondiabetic subjects were 2.39 and 4.13%, respectively [12]. In both Chinese and Korean studies, their allele frequencies were less than 5% [16, 17]. A recent publication from the 1998 Singapore National Health Survey reported that the Ala12 allele frequency for Malaysians was also less than 5% [16]. Our study with native Qataris have also indicated rather low incidence of this polymorphism (5.5% for diabetic and 5.9% for nondiabetics) as compared to the previous reports in various Caucasians suggesting a common tendency of the Qataris with Asian populations.

Several studies have demonstrated that the Pro12Ala substitution in the PPAR γ gene is associated with protection against diabetes in both Caucasians and Asians [12, 13, 24–30]. The most extensive study was a meta-analysis involving 3000 individuals that found 1.25-fold (P = 0.002) increase in the diabetes risk associated with the Pro12 allele (85% frequency) [13]. The reduced frequency of the Ala12 allele among Qataris, relative to many Caucasian populations, potentially indicates that a larger number of the Qatari population is at risk of T2D. Furthermore, the controls in our study were self-reported and were only checked for diabetes by random glucose measurement. A better selection procedure for diabetes would require oral glucose tolerance test. Future studies should also include detailed patient and control characterization based on laboratory data such as triglycerides, total cholesterol, HDL, LDL, and glycated haemoglobin (HbA1c) measurements.

Our result is consistent with several previous studies, which reported an absence of association between the Pro12Ala polymorphism of the PPARγ gene and the T2D. However, a meta-analysis from 30 independent studies with a total number of 19,136 subjects demonstrated that the Pro12Ala carriers had significantly higher BMI than the noncarriers, particularly in those with the higher BMI (BMI > 27) [14].

There are some possible explanations for the effects of this polymorphism on BMI. Lindi et al. [25] reported an interesting finding in their 10-year follow-up study that the Pro12Ala carriers have a tendency to gain weight over time. They speculated that higher insulin sensitivity could explain why the subjects with the Pro12Ala allele gained more weight during follow-up. In our study, the association of Pro12Ala allele with higher BMI in nondiabetic controls was more prominent than in diabetic subjects. Accordingly to this, the controls with Pro12Ala may have higher insulin sensitivity and gain weight without developing diabetes.

The study by Luan et al. [27] suggested that when the ratio of dietary polyunsaturated fat and saturated fat is low, the BMI in Pro12Ala carriers is greater than that in Pro12 homozygotes, but when the ratio is high, the opposite is seen. In general, Qatari population consumes a high-carbohydrate and high-fat diet from many sources. Furthermore, in the most recent study, the impact of high consanguinity on the prevalence of common adult diseases in the Qatari population was investigated [31, 32], the rate of consanguinity in the present generation was 51% (95% CI = 47.7–54.4) with a coefficient of inbreeding 0.023724.

Similar to the observations from several groups [24, 26], among other parameters studied, blood pressures, especially the systolic blood pressure in Pro12Ala carriers, were significantly higher than Pro/Pro homozygotes in diabetic subjects. Although the small number of Pro/Ala carriers might restrict our interpretation, this observed phenomenon might be related to the notion that once diabetes has developed, the protective effect of Ala12 allele may be lost [2].

In summary, this is the first study on Pro12Ala polymorphism performed in Qatar, and in this population, we failed to find an association between this polymorphism and T2D, but found no association with obesity.