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The Pro12Ala polymorphism of PPARγ2 gene and susceptibility to type 2 diabetes mellitus in a Polish population
Diabetes Research and Clinical Practice 62 (2003) 105 /111 www.elsevier.com/locate/diabres
The Pro12Ala polymorphism of PPARg2 gene and susceptibility to type 2 diabetes mellitus in a Polish population Maciej T. Malecki a,*, Jakub Frey a, Tomasz Klupa a, Jan Skupien a, Malgorzata Walus a, Wojciech Mlynarski b, Jacek Sieradzki a a
Department of Metabolic Diseases, Medical College, Jagiellonian University, 15 Kopernika Street, 31-501 Krakow, Poland b Section on Genetics and Epidemiology, Joslin Diabetes Center, Boston, MA, USA Received 17 January 2003; received in revised form 16 June 2003; accepted 30 June 2003
Abstract Introduction: It has recently been shown that polymorphisms of some genes might influence the genetic susceptibility to complex, multifactorial forms of type 2 diabetes mellitus (T2DM). One of those genes is peroxisome proliferator activated receptor g (PPARg). The PPARg gene product is a nuclear hormone receptor that regulates adipogenesis and is a target for thiazolidinediones, medications enhancing sensitivity to insulin. The Pro12Ala amino acid variant of the PPARg2 isoform is associated with T2DM in several populations. Aims: (1) To determine the allele and genotype frequency of the Pro12Ala PPARg2 amino acid variant in a Polish population; (2) To search for the association of the Pro12Ala polymorphism with T2DM in the examined population. Methods: We included 644 individuals in this study: 366 T2DM patients with age of diagnosis greater than 35 years and 278 non-diabetic control subjects. The fragment of the PPARg2 gene which contains the examined amino acid variant was amplified by polymerase chain reaction (PCR). Alleles and genotypes were determined based on electrophoresis of the DNA digestion products by the specific restriction enzyme BshI. Differences in distribution between the groups were examined by x2 test. Results: The frequency of Pro/Ala alleles was similar in T2DM patients and in the control subjects (83.5%/16.5% vs. 84.5%/15.5%, respectively, P/0.607). Similarly, there was no difference between the groups when we analysed the genotype distribution. Stratification analyses based on age of diagnosis, body mass index (BMI), and family history of T2DM were performed. The Pro/Ala and Ala/Ala genotypes tended to be more frequent in T2DM cases with age of diagnosis /50 years than in controls (36.2% vs. 27.3%, P/0.046). This difference was not significant after Sheffe correction for multiple comparisons. The other stratification analyses did not show any difference between the groups. Conclusion: The frequency of the Pro12Ala PPARg2 polymorphism in the Polish population studied is similar to that in other Caucasian populations. In the case-control study, we were not able to confirm earlier reports that the Pro allele conferred an increased risk for development of T2DM. Moreover, the results of the stratified analysis suggest an opposite trend in late onset T2DM. # 2003 Published by Elsevier Ireland Ltd. Keywords: Type 2 diabetes mellitus; PPARg gene; Genetic susceptibility
* Corresponding author. Tel.: /48-12-424-8305; fax: /48-12-421-9786. E-mail address: [email protected] (M.T. Malecki). 0168-8227/03/$ - see front matter # 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0168-8227(03)00164-5
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1. Introduction Genetic and environmental factors together create the clinical picture of T2DM [1 /3]. Knowledge of the molecular background of T2DM is important from scientific, prognostic, and prophylactic points of view. In the future, this knowledge will also influence therapeutic decisions. So far, mutations in several genes are linked with monogenic forms of this disease [4,5]. Monogenic forms are relatively rare and overall they constitute less than 10% of all type 2 diabetes mellitus (T2DM) cases. They are consequences of severe mutations in genes that code proteins or tRNA. Those forms are characterised by very high phenotypic penetration and usually, except for insulin receptor mutations, cause severe impairment in insulin secretion [6,7]. Efforts aimed at identifying the genes responsible for susceptibility to more common, polygenic, multifactorial forms of T2DM that appear in middle/late age and occur with both impaired insulin secretion and insulin resistance have been less successful. It has recently been shown that polymorphisms of several genes might influence genetic predisposition to complex, multifactorial forms of type 2 diabetes T2DM [7,8]. One of those genes is peroxisome proliferator activated receptor g (PPARg) [9]. The protein coded by this gene belongs to the family of nuclear receptors that bind to specific promoter sequences and regulate expression of other genes (peroxisome / proliferator response elements */PPRE) [10]. Through this mechanism, for example, PPARg is responsible for differentiation of fibroblasts to adipocytes and regulation of their function [11]. Sequence differences in the PPARg gene are associated in humans with several metabolic diseases. For example, some rare mutations are responsible for syndromes of severe insulin resistance [12] and monogenic forms of obesity [13]. The Pro12Ala polymorphism in the PPARg2 isoform is suggested to be linked with the multifactorial form of T2DM [9]. In a large genetic meta-analysis supported by a family based study design and replication samples, Pro, the more frequent allele, was associated with a modest increase in the risk of T2DM [9]. This finding was also upheld by the results of studies on healthy
individuals showing that the presence of Pro at residue 12 results in decreased insulin sensitivity [14,15]. On the other hand, some reports were not able to replicate this finding and even showed an opposite trend [16 /19]. The aim of this study was: (1) To determine the allele and genotype frequency of the PPARg2 Pro12Ala amino acid variant in a Polish population; (2) To search for the association of this polymorphism with T2DM in the examined population.
2. Subjects and methods 2.1. Subjects We included 644 unrelated individuals in this study. All of them were Caucasians and residents of the Malopolska region of South-Eastern Poland, mainly from Krakow, its capital. During the ascertainment that was conducted as previously described [20,21], the current WHO definitions and criteria for diagnosing diabetes were used [22]. Briefly, the patients completed a standard questionnaire that contained questions regarding age at T2DM diagnosis, family history, the treatment method, and other medical issues. Only patients with presenting T2DM after the age of 35 years and with no insulin therapy for at least 2 years after diagnosis were recruited. The individuals in the control group had normal fasting glucose levels. This group consisted mainly of the spouses of the T2DM patients and volunteers from medical personnel. Inclusion preference in the control group was given to individuals with no family history of T2DM among first and second degree relatives. The study individuals underwent a basic physical examination that included the measurement of height, weight, and blood pressure. Fasting glucose level was determined by the enzymatic technique (Automated Analysis Boehringer Mannheim Glucose GOD/PAP Method). HbA1c was measured in the T2DM patient group by the HLPC method (Biorad). This study was performed according to the Helsinki Declaration with approval of the Ethical Committee of the Jagiellonian University Medical College. All of the
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individuals included in this study gave informed consent prior to their inclusion in the study.
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first and/or second degree relatives). This analysis was computed by SAS software, version 8.02 for Windows (SAS Institute, Cary, NC).
2.2. Genotyping Alleles and genotypes were determined by the restriction fragment length polymorphism (RLFP) method using previously published primers [23]. PCR was performed in 200 ml tubes on a Biometra Uno II Thermocycler (Biometra). The total reaction volume was 15 ml. For each reaction, 40 ng of genomic DNA, 0.5 units of Taq polymerase (Perkin Elmer) and 1.5 ml 10 / PCR buffer were used. Magnesium chloride concentration was 1.8 mM. The following PCR conditions were used: initial denaturation at 94 8C for 5 min, followed by 39 cycles of denaturation at 94 8C, annealing at 56 8C and elongation at 72 8C. Each of these steps lasted 45 s. This was followed by final extension at 72 8C for 10 min. The PCR product had the entire length of 270 bp (base pairs) and it was digested by the specific restriction BshI enzyme. When alanine was present at residue 12 of PPARg2, the 270 bp DNA fragment was split into 227 and 43 bp pieces. The digestion products were then separated by electrophoresis on 3.2% agarose gel. The results were documented by digital camera and stored as computer files in Biocapt software (Vilbert-Lourmant). 2.3. Statistics Differences in distribution of alleles and genotypes between the groups were assessed by the x2 test. Deviations from Hardy /Weinberg equilibrium were tested using the x2 goodness-of-fit test. Comparisons between study groups were made with Student’s t-test for quantitative traits, while qualitative traits were analysed using the x2 test. For these analyses STATISTICA for Windows was used (Statsoft, Tulsa, OK). The Sheffe’s correction for multiple comparisons was applied to stratified analyses based on the age of onset of T2DM ( 5/50 years and /50 years, 50 being the mean age in the T2DM group, body mass index (BMI) (5/31 and /31, 31 being the mean BMI in the T2DM group), and family history of T2DM (positive and negative family history of T2DM in
3. Results Overall, the amino acid variants were determined in 644 individuals: 366 T2DM patients and 278 non-diabetic controls. The details of the clinical characteristics of both groups are provided in Table 1. Genotypes were in Hardy /Weinberg equilibrium for both T2DM cases and controls when analyzed for each group separately and in the total cohort. The distribution of the alleles and genotypes, shown in Table 2, was not different between the groups. In addition to the results presented in this table, we performed some analyses to verify the hypothesis that the examined polymorphism may be associated with T2DM in a subgroup of patients. The T2DM group was stratified with respect to BMI, age of disease onset, and family history of T2DM. We observed that the carriers of the Pro/Ala and Ala/Ala genotypes tended to be more prevalent in the sub-group of 177 T2DM patients with age of onset /50 years (50 being the mean for the entire T2DM group) than in the controls (36.2% vs. 27.3%, P /0.046). This difference was not significant after Sheffe’s correction for multiple comparisons (P/0.128). The results of additional comparisons based on BMI and family history of T2DM did not produce any positive findings. Additionally, we looked at the clinical characteristics of the Ala allele diabetic carriers. Overall, there were 110 hetero- or homozygous carriers of this variant. On average, the patients with the Ala allele were 50.69/9.8 years of age at diagnosis and 60.69/9.3 years old at the examination, while the average ages for the Pro allele homozygotes in the T2DM group were 49.59/9.1 and 59.39/9.3, respectively. The BMI among hetero- and homozygous Ala carriers was 30.99/6.6 kg/m2 which was very similar to the non-carriers in T2DM group (30.99/5.4 kg/m2).
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Table 1 Characteristics of the study groups Characteristics
T2DM
Controls
P
Women/men Age at examination (years)a Age of diagnosis (years)a Duration of the disease (years)a BMI (kg/m2)a Fasting glucose (mmol/l)a HbA1c (%)a % on insulin treatment Positive/negative family history of T2DM in first or second degree relatives
196/170 59.79/9.3 49.99/9.3 9.89/7.6 30.99/5.8 8.19/3.1 7.79/1.6 53.4% 180/186
166/112 52.49/14.9 N/A N/A 28.59/6.3 4.79/0.8 N/A N/A 49/229
0.12 B/0.001
B/0.001 B/0.001
B/0.001
Note: The high proportion of insulin-treated (either alone or in combination with oral medications) T2DM patients is related to the relatively long mean duration of disease. Fasting glucose was measured in T2DM patients prior to administration of morning dose of oral hypoglycaemic agents or insulin. N/A/not applicable. a Data are as mean9/S.D.
4. Discussion Population based, case-control, association studies are considered the gold standard in the search for susceptibility genes of complex diseases. In recent years their popularity has substantially increased over the family based study design [24]. The major advantage of the population-based studies over the pedigree-based approach is greater power [24,25]. However, there are many concerns due to the lack of reproducibility of the results generated by this approach [9,24,26]. Particularly, some researchers expressed the opinion that false positive results might arise from population stratification if case and control subjects are drawn from several subpopulations [26]. Recent analyses and simulations, however, showed that the problem of population stratification was overestimated [24]. Other factors were also indicated as potentially important for the reproducibility issue:
overinterpretation of marginal findings, inappropriate corrections for multiple hypothesis testing, publication bias, differences in clinical definitions, sample sizes, and risk-alleles among different populations [24]. Efforts to identify the T2DM complex form susceptibility variants reflect the general problems with genetic studies. Many reports on the identification of the susceptibility allele could not be replicated [9]. One of a very few sequence differences that seemed to show a well supported association with the complex T2DM form was the Pro12Ala variant of PPARg2. The initial report indicated that the alanine allele reduced the risk of T2DM [14]. Four out of five subsequent studies failed to replicate this finding, but in all of them a consistent trend was observed [27 /31]. Finally, the meta-analysis of Altshuler et al. [9] strengthened by the family-based study and replication sample seemed to dispel all doubts around
Table 2 Allele and genotype counts (percentages) with results of the case-control analysis. Allele Pro12 T2DM Controls
Genotype Ala12
611 (0.835) 121 (0.165) 470 (0.845) 86 (0.155) x2 /0.264 (P /0.607)
* Comparison between Pro12/Pro12 vs. other genotypes.
Pro12/Pro12
Pro12/Ala12
256 (0.699) 99 (0.270) 202 (0.726) 66 (0.237) x2 /0.567 (P /0.451)*
Ala12/Ala12 11 (0.03) 10 (0.036)
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this association. Moreover, this finding was later supported by data from the euglycemic-hyperinsulinemic clamp that revealed a significantly lower insulin sensitivity index in the Pro non-diabetic carriers [32]. Two other studies on pre-diabetic phenotypes also sustained this notion [14,15]. However, the results of some recently published studies contradict the conclusion that the Pro allele was the susceptibility allele for T2DM. One of these is the report of Lindi et al. [33] which showed that, in a very innovative, prospective approach, the alanine at residue 12 might predispose to the development of T2DM in obese subjects with IGT. Two other case-control studies on Central European populations suggested that the Ala allele was associated with increased susceptibility to T2DM in German and Czech populations [17,34]. These three recent reports together with our finding and some earlier publications [16,18,19] open again the dispute of whether the Pro12Ala is indeed a variant that predisposes to T2DM and which amino acid allele carries an increased risk of T2DM. It is appealing to speculate that the alleles of the Pro12Ala polymorphism may influence glucose homeostasis in two different mechanisms: by affecting insulin sensitivity and insulin secretion. While the carriers of the Pro allele were characterised by increased resistance to insulin over the Ala carriers [14,15,32], evidence exists that the latter may have decreased insulin secretion capacity [33 /36]. Thus, the phenotypic differences between the T2DM populations that were examined for the association might, at least in part, be responsible for the discrepancy in reported results. Further studies are necessary to prove this hypothesis. Finally, the results of another study from the Polish population on the Pro12Ala variant warrant additional comment [24]. The primary goal of Ardlie et al. [24] was to study the problem of population stratification in case-control studies rather than to perform the association study. However, they used, as a tool, the ethnically mixed population of American-Caucasians and the Polish homogenous group in an effort to replicate the findings of Altshuler et al. on the Pro12Ala variant [9]. The Polish population that they used showed evidence that Pro was indeed the susceptibility
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allele for T2DM. The results of the study in the American sample were not significant. There was no evidence for population stratification in either sample, however the homogenous Polish population showed the advantage of greater power in the analysis. A few points should be raised in an attempt to explain the discrepancies between these results and our study. First, no clinical data (for example, age of T2DM onset, age at examination, BMI, proportion of the patients on insulin, or biochemical results) were provided for the patients studied by Ardlie and his colleagues. Second, older, no longer valid criteria were used for the diagnosis of T2DM. Third, the authors provided no data of the specific region of Poland from which the group was recruited. Lastly, random variation and lack of power of our study could also have the impact on the observed difference between the studies. It should be pointed out, however, that using even a smaller sample of T2DM cases and controls, we were able to describe a moderate effect of the calpain 10 gene in a Polish population [20]. In conclusion: (1) The frequency of the Pro12Ala PPARg2 polymorphism in the Polish population studied is similar to that in other Caucasian populations. (2) In this case-control study we were not able to confirm earlier reports that the Pro allele conferred an increased risk for development of T2DM. Moreover, the results of stratified analysis suggest an opposite trend in late onset T2DM.
Acknowledgements This research was supported by the NIH FIRCA 1 R03 TW01351-01 Grant and the Medical College, Jagiellonian University Grant 501/KL/ 439/L. We are grateful to Professor Andrzej Krolewski from the Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA for scientific discussion and to Julianne Kenton and Paul Greenlaw for their help in the preparation of this manuscript.
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The Pro12Ala polymorphism of PPARg2 gene and susceptibility to type 2 diabetes mellitus in a Polish population Maciej T. Malecki a,*, Jakub Frey a, Tomasz Klupa a, Jan Skupien a, Malgorzata Walus a, Wojciech Mlynarski b, Jacek Sieradzki a a
Department of Metabolic Diseases, Medical College, Jagiellonian University, 15 Kopernika Street, 31-501 Krakow, Poland b Section on Genetics and Epidemiology, Joslin Diabetes Center, Boston, MA, USA Received 17 January 2003; received in revised form 16 June 2003; accepted 30 June 2003
Abstract Introduction: It has recently been shown that polymorphisms of some genes might influence the genetic susceptibility to complex, multifactorial forms of type 2 diabetes mellitus (T2DM). One of those genes is peroxisome proliferator activated receptor g (PPARg). The PPARg gene product is a nuclear hormone receptor that regulates adipogenesis and is a target for thiazolidinediones, medications enhancing sensitivity to insulin. The Pro12Ala amino acid variant of the PPARg2 isoform is associated with T2DM in several populations. Aims: (1) To determine the allele and genotype frequency of the Pro12Ala PPARg2 amino acid variant in a Polish population; (2) To search for the association of the Pro12Ala polymorphism with T2DM in the examined population. Methods: We included 644 individuals in this study: 366 T2DM patients with age of diagnosis greater than 35 years and 278 non-diabetic control subjects. The fragment of the PPARg2 gene which contains the examined amino acid variant was amplified by polymerase chain reaction (PCR). Alleles and genotypes were determined based on electrophoresis of the DNA digestion products by the specific restriction enzyme BshI. Differences in distribution between the groups were examined by x2 test. Results: The frequency of Pro/Ala alleles was similar in T2DM patients and in the control subjects (83.5%/16.5% vs. 84.5%/15.5%, respectively, P/0.607). Similarly, there was no difference between the groups when we analysed the genotype distribution. Stratification analyses based on age of diagnosis, body mass index (BMI), and family history of T2DM were performed. The Pro/Ala and Ala/Ala genotypes tended to be more frequent in T2DM cases with age of diagnosis /50 years than in controls (36.2% vs. 27.3%, P/0.046). This difference was not significant after Sheffe correction for multiple comparisons. The other stratification analyses did not show any difference between the groups. Conclusion: The frequency of the Pro12Ala PPARg2 polymorphism in the Polish population studied is similar to that in other Caucasian populations. In the case-control study, we were not able to confirm earlier reports that the Pro allele conferred an increased risk for development of T2DM. Moreover, the results of the stratified analysis suggest an opposite trend in late onset T2DM. # 2003 Published by Elsevier Ireland Ltd. Keywords: Type 2 diabetes mellitus; PPARg gene; Genetic susceptibility
* Corresponding author. Tel.: /48-12-424-8305; fax: /48-12-421-9786. E-mail address: [email protected] (M.T. Malecki). 0168-8227/03/$ - see front matter # 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0168-8227(03)00164-5
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1. Introduction Genetic and environmental factors together create the clinical picture of T2DM [1 /3]. Knowledge of the molecular background of T2DM is important from scientific, prognostic, and prophylactic points of view. In the future, this knowledge will also influence therapeutic decisions. So far, mutations in several genes are linked with monogenic forms of this disease [4,5]. Monogenic forms are relatively rare and overall they constitute less than 10% of all type 2 diabetes mellitus (T2DM) cases. They are consequences of severe mutations in genes that code proteins or tRNA. Those forms are characterised by very high phenotypic penetration and usually, except for insulin receptor mutations, cause severe impairment in insulin secretion [6,7]. Efforts aimed at identifying the genes responsible for susceptibility to more common, polygenic, multifactorial forms of T2DM that appear in middle/late age and occur with both impaired insulin secretion and insulin resistance have been less successful. It has recently been shown that polymorphisms of several genes might influence genetic predisposition to complex, multifactorial forms of type 2 diabetes T2DM [7,8]. One of those genes is peroxisome proliferator activated receptor g (PPARg) [9]. The protein coded by this gene belongs to the family of nuclear receptors that bind to specific promoter sequences and regulate expression of other genes (peroxisome / proliferator response elements */PPRE) [10]. Through this mechanism, for example, PPARg is responsible for differentiation of fibroblasts to adipocytes and regulation of their function [11]. Sequence differences in the PPARg gene are associated in humans with several metabolic diseases. For example, some rare mutations are responsible for syndromes of severe insulin resistance [12] and monogenic forms of obesity [13]. The Pro12Ala polymorphism in the PPARg2 isoform is suggested to be linked with the multifactorial form of T2DM [9]. In a large genetic meta-analysis supported by a family based study design and replication samples, Pro, the more frequent allele, was associated with a modest increase in the risk of T2DM [9]. This finding was also upheld by the results of studies on healthy
individuals showing that the presence of Pro at residue 12 results in decreased insulin sensitivity [14,15]. On the other hand, some reports were not able to replicate this finding and even showed an opposite trend [16 /19]. The aim of this study was: (1) To determine the allele and genotype frequency of the PPARg2 Pro12Ala amino acid variant in a Polish population; (2) To search for the association of this polymorphism with T2DM in the examined population.
2. Subjects and methods 2.1. Subjects We included 644 unrelated individuals in this study. All of them were Caucasians and residents of the Malopolska region of South-Eastern Poland, mainly from Krakow, its capital. During the ascertainment that was conducted as previously described [20,21], the current WHO definitions and criteria for diagnosing diabetes were used [22]. Briefly, the patients completed a standard questionnaire that contained questions regarding age at T2DM diagnosis, family history, the treatment method, and other medical issues. Only patients with presenting T2DM after the age of 35 years and with no insulin therapy for at least 2 years after diagnosis were recruited. The individuals in the control group had normal fasting glucose levels. This group consisted mainly of the spouses of the T2DM patients and volunteers from medical personnel. Inclusion preference in the control group was given to individuals with no family history of T2DM among first and second degree relatives. The study individuals underwent a basic physical examination that included the measurement of height, weight, and blood pressure. Fasting glucose level was determined by the enzymatic technique (Automated Analysis Boehringer Mannheim Glucose GOD/PAP Method). HbA1c was measured in the T2DM patient group by the HLPC method (Biorad). This study was performed according to the Helsinki Declaration with approval of the Ethical Committee of the Jagiellonian University Medical College. All of the
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individuals included in this study gave informed consent prior to their inclusion in the study.
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first and/or second degree relatives). This analysis was computed by SAS software, version 8.02 for Windows (SAS Institute, Cary, NC).
2.2. Genotyping Alleles and genotypes were determined by the restriction fragment length polymorphism (RLFP) method using previously published primers [23]. PCR was performed in 200 ml tubes on a Biometra Uno II Thermocycler (Biometra). The total reaction volume was 15 ml. For each reaction, 40 ng of genomic DNA, 0.5 units of Taq polymerase (Perkin Elmer) and 1.5 ml 10 / PCR buffer were used. Magnesium chloride concentration was 1.8 mM. The following PCR conditions were used: initial denaturation at 94 8C for 5 min, followed by 39 cycles of denaturation at 94 8C, annealing at 56 8C and elongation at 72 8C. Each of these steps lasted 45 s. This was followed by final extension at 72 8C for 10 min. The PCR product had the entire length of 270 bp (base pairs) and it was digested by the specific restriction BshI enzyme. When alanine was present at residue 12 of PPARg2, the 270 bp DNA fragment was split into 227 and 43 bp pieces. The digestion products were then separated by electrophoresis on 3.2% agarose gel. The results were documented by digital camera and stored as computer files in Biocapt software (Vilbert-Lourmant). 2.3. Statistics Differences in distribution of alleles and genotypes between the groups were assessed by the x2 test. Deviations from Hardy /Weinberg equilibrium were tested using the x2 goodness-of-fit test. Comparisons between study groups were made with Student’s t-test for quantitative traits, while qualitative traits were analysed using the x2 test. For these analyses STATISTICA for Windows was used (Statsoft, Tulsa, OK). The Sheffe’s correction for multiple comparisons was applied to stratified analyses based on the age of onset of T2DM ( 5/50 years and /50 years, 50 being the mean age in the T2DM group, body mass index (BMI) (5/31 and /31, 31 being the mean BMI in the T2DM group), and family history of T2DM (positive and negative family history of T2DM in
3. Results Overall, the amino acid variants were determined in 644 individuals: 366 T2DM patients and 278 non-diabetic controls. The details of the clinical characteristics of both groups are provided in Table 1. Genotypes were in Hardy /Weinberg equilibrium for both T2DM cases and controls when analyzed for each group separately and in the total cohort. The distribution of the alleles and genotypes, shown in Table 2, was not different between the groups. In addition to the results presented in this table, we performed some analyses to verify the hypothesis that the examined polymorphism may be associated with T2DM in a subgroup of patients. The T2DM group was stratified with respect to BMI, age of disease onset, and family history of T2DM. We observed that the carriers of the Pro/Ala and Ala/Ala genotypes tended to be more prevalent in the sub-group of 177 T2DM patients with age of onset /50 years (50 being the mean for the entire T2DM group) than in the controls (36.2% vs. 27.3%, P /0.046). This difference was not significant after Sheffe’s correction for multiple comparisons (P/0.128). The results of additional comparisons based on BMI and family history of T2DM did not produce any positive findings. Additionally, we looked at the clinical characteristics of the Ala allele diabetic carriers. Overall, there were 110 hetero- or homozygous carriers of this variant. On average, the patients with the Ala allele were 50.69/9.8 years of age at diagnosis and 60.69/9.3 years old at the examination, while the average ages for the Pro allele homozygotes in the T2DM group were 49.59/9.1 and 59.39/9.3, respectively. The BMI among hetero- and homozygous Ala carriers was 30.99/6.6 kg/m2 which was very similar to the non-carriers in T2DM group (30.99/5.4 kg/m2).
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Table 1 Characteristics of the study groups Characteristics
T2DM
Controls
P
Women/men Age at examination (years)a Age of diagnosis (years)a Duration of the disease (years)a BMI (kg/m2)a Fasting glucose (mmol/l)a HbA1c (%)a % on insulin treatment Positive/negative family history of T2DM in first or second degree relatives
196/170 59.79/9.3 49.99/9.3 9.89/7.6 30.99/5.8 8.19/3.1 7.79/1.6 53.4% 180/186
166/112 52.49/14.9 N/A N/A 28.59/6.3 4.79/0.8 N/A N/A 49/229
0.12 B/0.001
B/0.001 B/0.001
B/0.001
Note: The high proportion of insulin-treated (either alone or in combination with oral medications) T2DM patients is related to the relatively long mean duration of disease. Fasting glucose was measured in T2DM patients prior to administration of morning dose of oral hypoglycaemic agents or insulin. N/A/not applicable. a Data are as mean9/S.D.
4. Discussion Population based, case-control, association studies are considered the gold standard in the search for susceptibility genes of complex diseases. In recent years their popularity has substantially increased over the family based study design [24]. The major advantage of the population-based studies over the pedigree-based approach is greater power [24,25]. However, there are many concerns due to the lack of reproducibility of the results generated by this approach [9,24,26]. Particularly, some researchers expressed the opinion that false positive results might arise from population stratification if case and control subjects are drawn from several subpopulations [26]. Recent analyses and simulations, however, showed that the problem of population stratification was overestimated [24]. Other factors were also indicated as potentially important for the reproducibility issue:
overinterpretation of marginal findings, inappropriate corrections for multiple hypothesis testing, publication bias, differences in clinical definitions, sample sizes, and risk-alleles among different populations [24]. Efforts to identify the T2DM complex form susceptibility variants reflect the general problems with genetic studies. Many reports on the identification of the susceptibility allele could not be replicated [9]. One of a very few sequence differences that seemed to show a well supported association with the complex T2DM form was the Pro12Ala variant of PPARg2. The initial report indicated that the alanine allele reduced the risk of T2DM [14]. Four out of five subsequent studies failed to replicate this finding, but in all of them a consistent trend was observed [27 /31]. Finally, the meta-analysis of Altshuler et al. [9] strengthened by the family-based study and replication sample seemed to dispel all doubts around
Table 2 Allele and genotype counts (percentages) with results of the case-control analysis. Allele Pro12 T2DM Controls
Genotype Ala12
611 (0.835) 121 (0.165) 470 (0.845) 86 (0.155) x2 /0.264 (P /0.607)
* Comparison between Pro12/Pro12 vs. other genotypes.
Pro12/Pro12
Pro12/Ala12
256 (0.699) 99 (0.270) 202 (0.726) 66 (0.237) x2 /0.567 (P /0.451)*
Ala12/Ala12 11 (0.03) 10 (0.036)
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this association. Moreover, this finding was later supported by data from the euglycemic-hyperinsulinemic clamp that revealed a significantly lower insulin sensitivity index in the Pro non-diabetic carriers [32]. Two other studies on pre-diabetic phenotypes also sustained this notion [14,15]. However, the results of some recently published studies contradict the conclusion that the Pro allele was the susceptibility allele for T2DM. One of these is the report of Lindi et al. [33] which showed that, in a very innovative, prospective approach, the alanine at residue 12 might predispose to the development of T2DM in obese subjects with IGT. Two other case-control studies on Central European populations suggested that the Ala allele was associated with increased susceptibility to T2DM in German and Czech populations [17,34]. These three recent reports together with our finding and some earlier publications [16,18,19] open again the dispute of whether the Pro12Ala is indeed a variant that predisposes to T2DM and which amino acid allele carries an increased risk of T2DM. It is appealing to speculate that the alleles of the Pro12Ala polymorphism may influence glucose homeostasis in two different mechanisms: by affecting insulin sensitivity and insulin secretion. While the carriers of the Pro allele were characterised by increased resistance to insulin over the Ala carriers [14,15,32], evidence exists that the latter may have decreased insulin secretion capacity [33 /36]. Thus, the phenotypic differences between the T2DM populations that were examined for the association might, at least in part, be responsible for the discrepancy in reported results. Further studies are necessary to prove this hypothesis. Finally, the results of another study from the Polish population on the Pro12Ala variant warrant additional comment [24]. The primary goal of Ardlie et al. [24] was to study the problem of population stratification in case-control studies rather than to perform the association study. However, they used, as a tool, the ethnically mixed population of American-Caucasians and the Polish homogenous group in an effort to replicate the findings of Altshuler et al. on the Pro12Ala variant [9]. The Polish population that they used showed evidence that Pro was indeed the susceptibility
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allele for T2DM. The results of the study in the American sample were not significant. There was no evidence for population stratification in either sample, however the homogenous Polish population showed the advantage of greater power in the analysis. A few points should be raised in an attempt to explain the discrepancies between these results and our study. First, no clinical data (for example, age of T2DM onset, age at examination, BMI, proportion of the patients on insulin, or biochemical results) were provided for the patients studied by Ardlie and his colleagues. Second, older, no longer valid criteria were used for the diagnosis of T2DM. Third, the authors provided no data of the specific region of Poland from which the group was recruited. Lastly, random variation and lack of power of our study could also have the impact on the observed difference between the studies. It should be pointed out, however, that using even a smaller sample of T2DM cases and controls, we were able to describe a moderate effect of the calpain 10 gene in a Polish population [20]. In conclusion: (1) The frequency of the Pro12Ala PPARg2 polymorphism in the Polish population studied is similar to that in other Caucasian populations. (2) In this case-control study we were not able to confirm earlier reports that the Pro allele conferred an increased risk for development of T2DM. Moreover, the results of stratified analysis suggest an opposite trend in late onset T2DM.
Acknowledgements This research was supported by the NIH FIRCA 1 R03 TW01351-01 Grant and the Medical College, Jagiellonian University Grant 501/KL/ 439/L. We are grateful to Professor Andrzej Krolewski from the Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA for scientific discussion and to Julianne Kenton and Paul Greenlaw for their help in the preparation of this manuscript.
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