OBJECTIVE: Peroxisome-proliferator-activated receptors γ (PPAR γ), is a key regulator of adipocyte differentiation and energy balance. Two naturally occurring mutations in the PPAR γ gene, Pro115Gln and Pro12Ala, have recently been shown to impair the function of the PPARγ2 isoform of the receptor and to be associated with obesity or diabetes- related phenotypes in different populations.
SUBJECTS: We studied the occurrence and possible associations of the Pro115Gln and Pro12Ala in the PPAR γ2 gene with several clinical and metabolic phenotypes in three independent large populations of non-obese non-diabetic, type 2 diabetic, and morbidly obese French Caucasians.
RESULTS: The Pro115Gln mutation was not found in any of the 1069 subjects screened including 626 obese patients. The frequency of the Pro12Ala mutation was similar in all groups (0.08, 0.11,0.09) and was not associated with BMI or any of the clinical parameters tested.
CONCLUSIONS: We conclude that the Pro115Gln mutation is not a frequent cause of morbid obesity in Caucasians and that the Pro12Ala mutation is not associated with clinically significant changes in these populations.
The peroxisome-proliferator-activated receptor γ (PPAR γ), is a member of the nuclear hormone receptor superfamily.1 This regulator of transcription has been identified as a functional receptor for the Thiazolinedione class of insulin sensitizing drugs.2,3 PPAR γ2, the predominantly adipose isoform of PPAR γ, is expressed selectively and at higher level in adipose tissue,4 where it modulates the expression of target genes implicated in adipocyte differentiation and glucose homeostasis.1,5,6 The PPAR γ2 gene is therefore a major candidate gene for type 2 diabetes or obesity, both complex phenotypes determined by the combination of multiple genetic and environmental factors.7,8
Two naturally occurring mutations in this gene have recently been shown to impair the function of the PPARγ2 isoform of the receptor and to be associated with obesity and/or diabetes and insulin sensitivity related phenotypes in different populations.9,10
The Pro115Gln10 mutation affects the serine 114 phosphorylation of PPARγ2 leading to a constitutively more active protein as assessed by the rate of adipocyte differentiation and fat accumulation in tissue-culture model of adipogenesis. This mutation was found in 1% (4/358) of subjects of a German population recruited for a study on the genetic of type 2 diabetes and was only present in a subgroup of obese patients accounting for 3% (4/121) of those cases in this population. All four Pro115Gln carriers described were severely obese (body mass index, BMI, range 37.9–47.3kg/m2). This result suggests that this mutation could account for a significant number of severe obesity cases in Caucasians. To date, no study has investigated the prevalence of this mutation in independent populations.
The Pro12Ala mutant was found to lower the transactivation capacity of PPARγ2.9 This mutant occurs with extremely variable frequencies in populations of different ethnic origins.11 It has been associated with a modest reduction in BMI and slightly increased insulin sensitivity in two large Finnish populations and with a reduced prevalence of diabetes in Japanese Americans.9 In contrast one study did not find an association with markers of adiposity and insulin resistance in a smaller population of Japanese men,12 two studies did not find an association of this variant with type 2 diabetes in Caucasians13,14 and one study found a positive relationship with BMI.15
Given the important role of PPARγ as a therapeutic target in metabolic ddiseases and given the physiologically meaningful data supporting a functional role for the PPARγ2 Pro115Gln and Pro12Ala mutations, more independent replications are clearly needed to evaluate the potential impact of those mutations in clinically relevant populations.
In this study, 1069 individuals from three independent populations of obese, diabetic and control Caucasians of comparable geographical origin were screened for the Pro12Ala and Pro115Gln PPARγ2 mutations. Associations between those mutations and obesity related phenotypes were evaluated.
Non-obese, non-diabetic controls (n=295) were randomly chosen among 3000 individuals participating in a prospective study evaluating the efficacy of daily supplementation with antioxidant vitamins and minerals in reducing the major health problems and the causes of premature death in a large population of healthy volunteer (SU-VI-MAX study).16 Subject characteristics were as follows: gender (f/m) 195/100; age (y) 53.2±6.7 (range 40–66), BMI (kg/m2) 22±1.8 (range 15–25); max BMI reached (kg/m2) 23.5±2.2 (range 16.5–29.7); waist/hip ratio 0.78±0.08 (range 0.6–1.04); none of those subjects had diabetes.
Morbidly obese patients (n=372) were randomly chosen from a panel of 800 French morbidly obese patients of an already described population.17 Main relevant clinical and metabolic parameters were as follows: gender (f/m) 295/77, age (y) 43.5±11.8 (range 15–74); BMI (kg/m2) 47.3±7.5 (range 40–82); maximum reached BMI (kg/m2) 49.5±8.5 (range 40–99), waist/hip ratio 0.93±0.11 (range 0.7–1.4). Their glucose tolerance status was evaluated by a 75 g oral glucose tolerance test. After the 0GTT, 165 were strictly normo-glycemic subjects (NG) and 207 had abnormalities of glucose tolerance; either an impaired fasting glucose (n=31), an impaired glucose tolerance (n=72) or type 2 diabetes (n=104).
Type 2 diabetic patients (n=402) were consecutively enrolled in an ongoing study on the genetics of diabetes complications. Those patients had all been referred to the same hospital located in the Paris area. Patient characteristics were: gender (f/m) 160/242; age (y) 57.5±10.7 (range 30–86), age of diabetes onset 44.74±11.1; HbAlc (%) 8.46±1.44 (range 4.5–14.7); BMI (kg/m2) 29.6±5.5 (range 17–58); max BMI reached (kg/m2) 32.74±6.5 (range 17–68); waist/hip ratio 0.96 ± 0.1 (range 0.67–1.5); hypertension (SBP>140 or DBP>85 or antihypertensive treatment) 72%.
DNA extracted from peripheral lymphocyte was used to genotype for the two variants by PCR-RFLP assays as described.10,15 To test for the presence of the rare Pro115Gln mutation, a plasmid containing the mutated PPAR γ2 cDNA was kindly provided by M. Ristow as a positive control.10
The Pro115Gln mutation in the PPAR γ2 gene was not found in any of the 1069 individuals tested. This population included a total of 626 subjects with a phenotype comparing well to that of the carriers of this mutation in the German population of Caucasian origin (372 morbidly obese patients, and 254 obese patients from the type 2 diabetes cohort). While our result does not argue against a causal effect of the Pro115Gln mutation in the previously described severely obese carriers, these data indicate that the Pro115Gln mutation does not account for a clinically significant number of morbidly obese cases in Caucasians with or without type 2 diabetes.
The Pro12Ala genotyping results, as well as some of the clinical and biological characteristics of the patient subgroups are indicated in Table 1. The observed genotype frequency of this mutant was in agreement with Hardy-Weinberg expectations in all three populations studied. Allelic frequencies of the Pro12Ala missense mutation in the PPAR γ2 was not different between controls and morbidly obese or type 2 diabetic patient respectively. In the analysis of phenotypical data according to PPAR γ2 genotype within the three groups, we found no significant difference in BMI or any obesity related traits in any of the three groups (Table 2). In addition, no significant difference in glucose homeostasis related phenotypes was found in obese subjects analyzed together or separated by their diabetes status or when men and women were analyzed separately.
In summary, our results show that the Pro12Ala and the Pro115Gln mutations in the PPAR γ2 gene are unlikely to serve as clinically useful predictor of type 2 diabetes and/or obesity in Caucasians. Lack of association of the two most frequent missense variants in the PPARγ gene with type 2 diabetes and/or obesity extends the results of previous linkage studies18 indicating that the PPARγ loci is not a major contributor to obesity or type 2 diabetes in French Caucasians.
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Informed consents were obtained for all subjects and the protocols were approved by the Ethics Committee of Paris (CCPPRB Hôtel-Dieu and Cochin). This work was supported by the Direction de la Recherche Clinique/Assistance Publique-Hopitaux de Paris, Programme Hospitalier de Recherche Clinique (PHRC no. AOM 96088) and the Danone Institute (Danone/Suvimax DNA Bank). We thank Genevieve Bonhomme and Annie Legal for their excellent technical help. We also thank M. Ristow who kindly provided a mutated PPARγ2 cDNA plasmid
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