The Pharmacogenomics Journal (2014) 14, 488–492 & 2014 Macmillan Publishers Limited All rights reserved 1470-269X/14 www.nature.com/tpj
ORIGINAL ARTICLE
Association of PAX4 genetic variants with oral antidiabetic drugs efficacy in Chinese type 2 diabetes patients M Chen1, C Hu1, R Zhang, F Jiang, J Wang, D Peng, S Tang, X Sun, J Yan, Y Luo, Y Bao and W Jia The aim of this study was to investigate the association of PAX4 variants with therapeutic effect of oral antidiabetic drugs in Chinese type 2 diabtes mellitus (T2DM) patients. A total of 209 newly diagnosed T2DM patients were randomly assigned to treatment with repaglinide or rosiglitazone for 48 weeks, and the therapeutic effects were compared. In the rosiglitazone cohort, rs6467136 GA þ AA carriers showed greater decrease in 2-h glucose levels (P ¼ 0.0063) and higher cumulative attainment rates of target 2-h glucose levels (Plog rank ¼ 0.0093) than GG homozygotes. In the subgroup with defective b-cell function, rs6467136 GA þ AA carriers exhibited greater decrements of 2-h glucose level and improvement of homeostasis model assessment of insulin resistance (P ¼ 0.0143). Moreover, GA þ AA carriers were more likely to attain the target fasting and 2-h glucose level (Plog rank ¼ 0.0091 and 0.007, respectively). However, these single-nucleotide polymorphisms showed no effect on repaglinide efficacy. In conclusion, PAX4 variant rs6467136 was associated with the therapeutic effect of rosiglitazone in Chinese T2DM patients. The Pharmacogenomics Journal (2014) 14, 488–492; doi:10.1038/tpj.2014.18; published online 22 April 2014
INTRODUCTION Oral antidiabetic drugs, including metformin, sulfonylureas, glinides, a-glucosidase inhibitors and thiazolidinediones, are commonly used in the treatment of type 2 diabetes mellitus (T2DM). However, the clinical response to these oral agents varies among individuals. These individual variability is partly due to the factors such as age, sex, liver and renal function, severity of the disease and so on. In addition to the above, genetic factors involved in drug absorption, distribution, metabolism and target also have an important role in it. Paired box (PAX) proteins are a family of transcription factors that have an important role in tissue development and cellular differentiation. PAX protein family includes nine proteins divided into four groups based on structural domains, which are expressed in different organs and tissues.1 Herein, PAX4 is one of transcription factors that are mandatory for the differentiation of islet b-cells.2 PAX4 is expressed in the early pancreas, but is later restricted to b-cells.3 It has been proved that mutations and polymorphisms of PAX4 are associated with various types of diabetes, such as type 1 diabetes mellitus (T1DM), T2DM, maturityonset diabetes of the young (MODY) and ketosis-prone diabetes in different population.4–9 Recently, genome-wide association study identified two novel diabetes-associated loci near PAX4 (rs6467136 and rs10229583) in Chinese and other East Asian populations.10,11 However, whether these single-nucleotide polymorphisms (SNPs) influence the therapeutic effects of oral antihyperglycemic drugs has not been reported. Thus, we chose newly diagnosed patients treated with repaglinide or rosiglitazone to investigate the association of these SNPs with therapeutic effects.
MATERIALS AND METHODS Patients and study design A total of 209 newly diagnosed type 2 diabetes patients were recruited from the outpatient clinics in Shanghai, China. Detailed information on this study population has been described previously.12–14 After a 2-week run-in period (diet and exercise therapy only), 104 patients were treated with repaglinide (NovoNorm; Novo Nordisk, Copenhagen, Denmark), whereas the other 105 patients were given rosiglitazone treatment (Avandia; GlaxoSmithKline, Munich, Germany) for 48 weeks. Patients were visited at weeks 0, 2, 4, 12, 24, 32 and 48, and designed clinical assessments were taken. Repaglinide was administered initially in a mealtime dosage of 0.5 mg, whereas rosiglitazone treatment was started with a dosage of 4 mg. The dosage was increased stepwise to 1, 1.5 and 2 mg for repaglinide and to 8 mg for rosiglitazone when the patients failed to achieve glycemic targets of fasting plasma glucose (FPG) o7 mmol l 1 (126 mg dl 1) and/or 2 h plasma glucose o11 mmol l 1 (200 mg dl 1). Patients were excluded from the study when they showed any one of the following test results at two consecutive times (a maximal interval of 6 days): FPG 413 mmol l 1 (234 mg dl 1) or 2 h plasma glucose 418 mmol l 1 (324 mg dl 1) or glycated hemoglobin (HbA1c) X8%. In the analysis of SNPs and treatment attainment, the therapeutic targets of fasting glucose, 2-h glucose and HbA1c were, respectively, set as FPG o6.1 mmol l 1, 2-h glucose o7.8 mmol l 1, HbA1c o6.5% or over 20% decrease compared with the baseline after treatment. The study was approved by the institutional review board of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China. Written informed consent was obtained from each patient.
Anthropometric and clinical laboratory measurements Height (in m) and weight (in kg) were measured for all the patients, and body mass index (BMI; in kg m 2) was calculated. At the weeks 0, 2, 4, 12, 24, 32 and 48, fasting blood samples and 2-h blood samples were collected at each visit, following a 75 g oral glucose tolerance test. Plasma glucose
Department of Endocrinology and Metabolism, Shanghai Clinical Center for Diabetes, Shanghai Key Clinical Center for Metabolic Disease, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Diabetes Institute, Shanghai, China. Correspondence: Professor W Jia, Department of Endocrinology and Metabolism, Shanghai Clinical Center for Diabetes, Shanghai Key Clinical Center for Metabolic Disease, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Diabetes Institute, 600 Yishan Road, Shanghai 200233, China. E-mail: [email protected] 1 These authors contributed equally to this work. Received 2 January 2014; revised 14 February 2014; accepted 26 February 2014; published online 22 April 2014
Association of PAX4 with Chinese T2DM M Chen et al
489 concentrations were measured by means of the glucose oxidaseperoxidase method, using commercial kits (Shanghai Biological Products Institution, Shanghai, China). HbA1c values were determined by highperformance liquid chromatography, using a Bio-Rad Variant II hemoglobin testing system (Bio-Rad Laboratories, Hercules, CA, USA). Arginine stimulation tests were performed on all the patients at the beginning of the run-in period ( 2 weeks) and 48 weeks after treatment in overnight-fasted states (12 h fast). Blood samples were collected at 0 min, and then arginine hydrochloride (10% arginine hydrochloride of 50 ml, 5 g) was injected intravenously within 30–60 s. After injection, blood samples were taken at 2, 4 and 6 min. The acute insulin response (AIR) to arginine (AIR-Arg, in mU l 1) was calculated as the mean insulin value of 2, 4 and 6 min samples minus fasting insulin concentration to evaluate the potential function of b-cells. Defective b-cell function was defined as reported before.15 Briefly, the QL of AIR (24.95 mU l 1) in this study was regarded as cutting points of defective b-cell function. AIR o24.95 mU l 1 was regarded as defective b-cell function, whereas AIR X24.95 mU l 1 was normal b-cell function. Homeostasis model assessment was used to estimate the insulin resistance (HOMA-IR) and b-cell function (HOMA-B).16 HOMA-IR is calculated using the formula: fasting insulin concentration (in mU l 1) FPG concentration (in mmol l 1)/22.5, and HOMA-B is calculated using the formula: 20 fasting insulin concentration/(FPG concentration 3.5).
Genotype distribution of the SNPs (rs6467136 and rs10229583) was in agreement with the Hardy–Weinberg equilibrium.
Rosiglitazone cohort The association between SNPs and the clinic parameters in rosiglitazone cohort was shown in Table 1 and Supplementary Table 1. No significant differences were observed between two genotype groups of rs6467136 and rs10229583 in all the clinic parameters at baseline. After rosiglitazone therapy for 48 weeks, rs6467136 GA þ AA carriers show greater decrease in 2-h glucose levels than GG homozygotes ( 6.13±0.61 vs 3.60±0.51 mmol l 1, P ¼ 0.0063). Meanwhile, BMI in GA þ AA carriers decreased after 48 weeks treatment, whereas those with GG homozygotes increased (P ¼ 0.0192). There is no significant
Table 1. Association between PAX4 rs6467136 and clinical features in the rosiglitazone cohort GG
GA þ AA
P-value
Age (years) Gender (male/female) Dosage (mg per day)
51.62±1.11 44/20 5.55±0.25
53.25±1.86 20/8 5.38±0.38
0.4349 0.7972 0.7230
BMI(kg m 2) Baseline 48 Weeks DValue
25.01±0.39 25.23±0.44 0.12±0.18
24.80±0.59 23.97±0.69 0.52±2.22
0.7669 0.1260 0.0192
Fasting glucose(mmol l 1) Baseline 8.93±0.24 48 Weeks 6.67±0.18 DValue 2.33±0.28
9.17±0.28 6.35±0.15 2.74±0.30
0.2773 0.4176 0.3020
2-h Glucose(mmol l 1) Baseline 48 weeks DValue
12.91±0.39 9.24±0.33 3.60±0.51
14.35±0.57 8.09±0.33 6.13±0.61
0.0505 0.0517 0.0063
Glycated hemoglobin (%) Baseline 8.33±0.20 48 Weeks 6.42±0.11 DValue 1.75±0.23
8.30±0.25 6.23±0.14 2.10±0.26
0.6194 0.2563 0.1432
14.99±0.79 17.22±1.47 1.90±1.36
13.65±1.71 14.22±0.87 0.57±1.88
0.1033 0.594 0.6381
HOMA-IR Baseline 48 Weeks DValue
6.00±0.37 5.10±0.51 1.11±0.49
5.33±0.56 4.07±0.32 1.52±0.62
0.2831 0.5318 0.9565
HOMA-B Baseline 48 Weeks DValue
60.10±4.07 117.88±8.67 56.32±8.06
55.92±9.82 104.59±7.71 44.53±13.22
0.1042 0.804 0.5817
25.96±4.93 21.32±2.73 4.65±5.77
0.1135 0.0337 0.5513
Parameter
Genotyping Genomic DNA was extracted from peripheral blood leucocytes in whole blood samples. Genotyping was performed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, using a MassARRAY platform (MassARRAY Compact Analyzer; Sequenom, San Diego, CA, USA). The results were confirmed by DNA sequencing using 3130 Genetic Analyzer (Applied Biosystem, Foster City, CA, USA).
Statistical analysis The allele frequencies were determined by gene calculation and the Hardy– Weinberg equilibrium tests were performed. Data are shown as mean values±s.e.m. DValues were calculated by subtracting the initial value from the final value at week 48. Differences of quantitative traits between two genotypes groups were analyzed using multiple linear regression or Kruskal–Wallis test when appropriate. Kaplan–Meier method was used to estimate the attainment rates of clinical target levels. FPG o6.1 mmol l 1, 2-h glucose o7.8 mmol l 1 and HbA1c o6.5% or these parameters decreased more than 20% compared with the baseline after treatment was defined as attainment, respectively. Attainment rates between two genotype groups was compared by means of the log-rank test and Cox regression model analysis with adjustment for confounding factors, namely, age, gender and BMI at baseline. A two-tailed P-value of o0.05 was considered statistically significant. All the statistical analyses were performed using SAS for Windows (version 8.0; SAS Institute, Cary, NC, USA).
RESULTS Follow-up information of cohorts In the repaglinide cohort, of the total 104 patients recruited, 91 patients completed the 48-week study. Among the 13 patients withdrawn, 4 patients were with HbA1c X8% at two consecutive time points, and 9 patients were lost to follow-up. The GG, GA and AA genotypes distribution of rs6467136 and rs10229583 among the patients was 61, 23, 7 and 67, 23, 1, respectively. Of the total 105 patients recruited for rosiglitazone treatment, 93 patients completed the whole study. Twelve patients were withdrawn, among whom 1 patient had elevated hepatic enzymes and 5 patients attributed to inadequate control of blood glucose, and 6 patients were lost to follow-up. One patient was failed to be genotyped, and 92 patients was eventually included to statistics. The GG, GA and AA genotypes distribution of rs6467136 and rs10229583 among the patients was 64, 26, 2 and 65, 26, 1, respectively. Considering few subjects of rare allele homozygotes (AA), the genotype–phenotype associations were analyzed between common allele homozygotes and rare allele carriers. & 2014 Macmillan Publishers Limited
Fasting insulin (pmol l 1) Baseline 48 Weeks DValue
Acute insulin response (pmol l 1) Baseline 34.14±3.12 48 Weeks 35.02±3.93 DValue 0.62±3.96
Abbreviations: BMI, body mass index; HOMA-B, homeostasis model assessment of b-cell function; HOMA-IR, homeostasis model assessment of insulin resistance. Note: Data are shown as means±s.e.m. P-values o0.05 are shown in bold. Dvalue ¼ T48 weeks value baseline (T0) value. P-values were adjusted for age, gender and BMI at baseline when linear regression model was adopted.
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Figure 1. Association of rs6467136 and with the attainment rates of target values of fasting glucose, 2-h glucose and glycated hemoglobin among GA þ AA and GG in rosiglitazone cohort. (a) Plog rank ¼ 0.8359, PCox regression ¼ 0.9720; (b) Plog rank ¼ 0.0093, PCox regression ¼ 0.0169; and (c) Plog rank ¼ 0.6032, PCox regression ¼ 0.7202. PCox regression values were adjusted for age, gender and body mass index at baseline.
difference between two genotype groups of rs10229583 in all the clinic parameters after treatment. Then, we explored the associations between SNPs and the rate of attainment of target levels of FPG, 2-h glucose and HbA1c. Survival analyses showed that patients with rs6467136 GA þ AA genotype were significantly more likely to attain the standard 2-h glucose level than GG homozygotes at all assessment points (Plog rank ¼ 0.0093). Even after adjusting for age, gender and BMI at baseline, this difference was still significant (P ¼ 0.0169) (Figure 1b). We found no significant association between cumulative attainment and rs10229583 genotype (Supplementary Figure 1). As it has been proved that PAX4 was associated with cellular differentiation and b-cell function, we further divided the patients into two subgroups according to b-cell function assessed by arginine stimulation tests. In the group with defective b-cell function, rs6467136 GA þ AA genotype carriers showed greater decrease of BMI than GG homozygotes after 48 weeks rosiglitazone treatment (P ¼ 0.0033) (Table 2). Besides, there was a trend toward greater decrements of 2-h glucose level in individuals with rs6467136 GA þ AA genotype than GG carriers (P ¼ 0.0721). At the baseline, rs6467136 GA þ AA carriers showed worse insulin sensitivity assessed by HOMA-IR than GG homozygotes (P ¼ 0.0088), whereas the improvement of HOMA-IR was greater in GA þ AA carriers (P ¼ 0.0143). Survival analyses showed that patients with rs6467136 GA þ AA genotype were significantly more likely to attain the standard FPG and 2-h glucose level than GG homozygotes at all assessment points (Plog rank ¼ 0.0091 and 0.007, respectively) (Figures 2a and b). Even after adjusting for age, gender and BMI at baseline, there was still a trend toward higher rate of reaching the standard in GA þ AA carriers (P ¼ 0.0627 and 0.0543, respectively). In addition, we compared the clinical features between subgroups of rosiglitazone cohort stratified by b-cell function (Supplementary Table 2). Although the patients with defective b-cell function showed lower BMI and fasting insulin level, and better insulin sensitivity at baseline, the drug response was similar between the two subgroups after rosiglitazone treatment. The effect of PAX4 rs6467136 on rosiglitazone response was not observed in the subgroup with normal b-cell function (Supplementary Table 3). Repaglinide cohort Similarly, we analyzed the rs6467136 and rs10229583 genotype and therapeutic effects in repaglinide cohort. However, neither rs6467136 nor rs10229583 was found to affect efficacy of repaglinide. All the parameters have no difference between two The Pharmacogenomics Journal (2014), 488 – 492
Table 2. Association between PAX4 rs6467136 and clinical features in subgroup with defective b-cell function in the rosiglitazone cohort GG
GA þ AA
P-value
Age (year) Gender (male/female) Dosage (mg per day)
52.70±1.80 21/7 5.85±0.40
55.62±2.70 11/2 4.67±0.45
0.3692 0.4888 0.0873
BMI (kg m 2) Baseline 48 Weeks DValue
23.77±0.53 23.99±0.59 0.24±0.19
23.71±0.67 22.78±0.76 1.0±0.28
0.9416 0.0033 0.0033
Fasting glucose(mmol l 1) Baseline 8.93±0.40 48 weeks 6.28±0.19 DValue 2.77±0.43
9.29±0.46 6.16±0.19 3.0±0.43
0.4493 0.5713 0.7895
Parameter
2-h glucose(mmol l 1) Baseline 48 Weeks DValue
12.52±0.65 8.76±0.40 3.74±0.84
15.12±0.84 7.99±0.46 6.91±0.86
0.0433 0.4265 0.0721
Glycated hemoglobin (%) Baseline 48 Weeks DValue
8.34±0.31 6.36±0.11 1.71±0.29
8.35±0.48 6.07±0.17 2.28±0.44
0.9664 0.0707 0.2265
Fasting insulin (pmol l 1) Baseline 48 Weeks DValue
11.14±0.92 3.54±1.35 2.13±1.43
16.46±3.04 12.69±1.23 3.78±3.16
0.0849 0.9762 0.0923
HOMA-IR Baseline 48 Weeks DValue
4.53±0.54 3.85±0.51 0.81±0.70
6.38±0.88 3.41±0.38 3.09±0.93
0.0088 0.8203 0.0143
HOMA-B Baseline 48 Weeks DValue
45.89±4.93 108.28±11.21 63.93±10.36
Acute insulin response (pmol l 1) Baseline 13.97±1.32 48 Weeks 20.28±3.91 DValue 6.25±4.40
67.81±18.74 96.91±11.40 25.83±21.34
0.2807 0.5164 0.1013
9.60±3.78 18.48±4.03 8.88±5.69
0.441 0.8698 0.8698
Abbreviations: BMI, body mass index; HOMA-B, homeostasis model assessment of b-cell function; HOMA-IR, homeostasis model assessment of insulin resistance. Note: Data are shown as means±s.e.m. P-values o0.05 are shown in bold. Dvalue ¼ T48 weeks value baseline (T0) value. P-values were adjusted for age, gender and BMI at baseline when linear regression model was adopted.
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Figure 2. Association of rs6467136 and with the attainment rates of target values of fasting glucose, 2-h glucose and glycated hemoglobin among GA þ AA and GG in the subgroup with defective b-cell function in rosiglitazone cohort. (a) Plog rank ¼ 0.0091, PCox regression ¼ 0.0627; (b) Plog rank ¼ 0.0070, PCox regression ¼ 0.0543; and (c) Plog rank ¼ 0.9977, PCox regression ¼ 0.8762. PCox regression values were adjusted for age, gender and body mass index at baseline.
genotypes after 48 weeks repaglinide treatment (Supplementary Tables 4 and 5), and survival analyses showed no difference between two genotypes either (Supplementary Figures 2 and 3).
DISCUSSION TZDs are insulin-sensitizing agents that act as agonists of the nuclear factor peroxisome proliferator-activated receptor-g, thus leading to the improvements of the insulin sensitivity, especially in the peripheral tissues.17,18 As commonly used TZDs, rosiglitazone was effective in reducing glycemia in patients with type 2 diabetes for many years. Clinical trials showed that rosiglitazone could significantly improve insulin sensitivity and decrease FPG, 2 h plasma glucose and HbA1c, thereby improving glycemic control.19–21 PAX4 has been proved mandatory for the differentiation of islet b-cells. PAX4 gene has been shown to be implicated in pancreatic b-cell development, and polymorphism of PAX4 could lead to a reduction of adult b-cell mass and proliferation.2 The SNPs we investigated in the study, PAX4 rs6467136 and rs10229583, were identified as novel loci for type 2 diabetes in Chinese and other East Asian populations in genome-wide association study.10,11 Carriers of the risk allele G of rs6467136 and rs10229583 are more likely to develop type 2 diabetes. In our study, rs6467136 GG homozygotes from the subgroup with defective b-cell function showed better insulin sensitivity than the other genotype patients at baseline. This is perhaps attributed to the fact that carriers of G allele may affect b-cell function and insulin secretion, thus leading to compensatory better insulin sensitivity. In contrast, patients with rs6467136 GA þ AA genotype showed worse insulin sensitivity and attain better improvement of HOMA-IR after rosiglitazone treatment. Better fasting and 2-h glucose control, as well as BMI increment in individuals with rs6467136 GA þ AA genotype may due to greater improvement of insulin sensitivity. In our study, we identified association between PAX4 variants and therapeutic effect of rosiglitazone, which was an agonist of peroxisome proliferator-activated receptor-g. A functional study showed that peroxisome proliferatoractivated receptor-g regulates the expression of PDX-1, NKX6.1 and BETA2/NeuroD,22–24 which are all mandatory transcription factors for the differentiation of b-cells. PAX4 is also one of the protein in the pathway of b-cell differentiation, which was downstream of PDX-1, NKX6.1 and BETA2/NeuroD. Therefore, we suggested that the expression of PAX4 might also be affected by agonists of peroxisome proliferator-activated receptor-g, such as rosiglitazone. However, as PAX4 is a novel locus for T2DM, the mechanism under its effects on disease pathogenesis and drug & 2014 Macmillan Publishers Limited
efficacy has not been so clear, and further functional study was needed to reveal it. There are several limitations of this study that should be noted. First, the sample size was relatively small, thus we did not have enough statistical power to detect the effect of genetic variants on some of the parameters. Second, as most variables of our study are in skew distribution, the Kruskal–Wallis test was the main method used for their statistical analyses, thus the confounding factors could not be adjusted. Third, no adjustment of multiple testing was performed on the analyses of genotype and clinical parameters. Given the modest P-values, most of the tests would not be significant with a Bonferroni correction. However, as the variables tested in our study were related, Bonferroni correction was not proper and highly conservative. Fourth, we set the beginning of the run-in period as the baseline, rather than the initiation of drug treatment, which might lead to deviations in evaluating drug efficacy. However, given that all the participants were undergoing the same treatment (diet and exercise therapy) during this short run-in period, the impact of this limitation may be minimal. In conclusion, the present study demonstrates that the PAX4 variant rs6467136 was associated with the therapeutic effect of rosiglitazone treatment in Chinese patients with type 2 diabetes. GA þ AA genotype exhibited lower 2-h glucose levels as well as significantly higher cumulative attainment rates of target 2-h glucose levels than GG homozygotes. However, as our study is at an early stage of pharmacogenomics investigation on rosiglitazone, further investigations are needed to confirm our findings and reveal the underlying mechanisms. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGMENTS We thank all the patients who participated in this study and are appreciative of the doctors and nurses at the Shanghai Clinical Center for Diabetes. This project was funded by 973 Program (2011CB504001), grants from NSFC (81322010, 81170735 and 81200582), the Drug Innovation Program of the National Science and Technology Project (2011ZX09307-001-02), 863 Program (2012AA02A509), Excellent Young Medical Expert of Shanghai (XYQ2011041), Shanghai Talent Development Grant (2012041), Shanghai Raising Star Program (12QH1401700) and National Young Top Talent Supporting Program.
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492 2 Sosa-Pineda B, Chowdhury K, Torres M, Oliver G, Gruss P. The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. Nature 1997; 386: 399–402. 3 Habener JF, Kemp DM, Thomas MK. Minireview: transcriptional regulation in pancreatic development. Endocrinology 2005; 146: 1025–1034. 4 Kooptiwut S, Plengvidhya N, Chukijrungroat T, Sujjitjoon J, Semprasert N, Furuta H et al. Defective PAX4 R192H transcriptional repressor activities associated with maturity onset diabetes of the young and early onset-age of type 2 diabetes. J Diabetes Compl 2012; 26: 343–347. 5 Tokuyama Y, Matsui K, Ishizuka T, Egashira T, Kanatsuka A. The Arg121Trp variant in PAX4 gene is associated with beta-cell dysfunction in Japanese subjects with type 2 diabetes mellitus. Meta Clin Exp 2006; 55: 213–216. 6 Shimajiri Y, Sanke T, Furuta H, Hanabusa T, Nakagawa T, Fujitani Y et al. A missense mutation of Pax4 gene (R121W) is associated with type 2 diabetes in Japanese. Diabetes 2001; 50: 2864–2869. 7 Plengvidhya N, Kooptiwut S, Songtawee N, Doi A, Furuta H, Nishi M et al. PAX4 mutations in Thais with maturity onset diabetes of the young. J Clin Endocrinol Metab 2007; 92: 2821–2826. 8 Biason-Lauber A, Boehm B, Lang-Muritano M, Gauthier BR, Brun T, Wollheim CB et al. Association of childhood type 1 diabetes mellitus with a variant of PAX4: possible link to beta cell regenerative capacity. Diabetologia 2005; 48: 900–905. 9 Mauvais-Jarvis F, Smith SB, Le May C, Leal SM, Gautier JF, Molokhia M et al. PAX4 gene variations predispose to ketosis-prone diabetes. Hum Mol Genet 2004; 13: 3151–3159. 10 Cho YS, Chen CH, Hu C, Long J, Ong RT, Sim X et al. Meta-analysis of genome-wide association studies identifies eight new loci for type 2 diabetes in east Asians. Nat Genet 2012; 44: 67–72. 11 Ma RC, Hu C, Tam CH, Zhang R, Kwan P, Leung TF et al. Genome-wide association study in a Chinese population identifies a susceptibility locus for type 2 diabetes at 7q32 near PAX4. Diabetologia 2013; 56: 1291–1305. 12 Wang J, Bao YQ, Hu C, Zhang R, Wang CR, Lu JX et al. Effects of ABCA1 variants on rosiglitazone monotherapy in newly diagnosed type 2 diabetes patients. Acta Pharmacol Sin 2008; 29: 252–258.
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Supplementary Information accompanies the paper on the The Pharmacogenomics Journal website (http://www.nature.com/tpj)
The Pharmacogenomics Journal (2014), 488 – 492
& 2014 Macmillan Publishers Limited
ORIGINAL ARTICLE
Association of PAX4 genetic variants with oral antidiabetic drugs efficacy in Chinese type 2 diabetes patients M Chen1, C Hu1, R Zhang, F Jiang, J Wang, D Peng, S Tang, X Sun, J Yan, Y Luo, Y Bao and W Jia The aim of this study was to investigate the association of PAX4 variants with therapeutic effect of oral antidiabetic drugs in Chinese type 2 diabtes mellitus (T2DM) patients. A total of 209 newly diagnosed T2DM patients were randomly assigned to treatment with repaglinide or rosiglitazone for 48 weeks, and the therapeutic effects were compared. In the rosiglitazone cohort, rs6467136 GA þ AA carriers showed greater decrease in 2-h glucose levels (P ¼ 0.0063) and higher cumulative attainment rates of target 2-h glucose levels (Plog rank ¼ 0.0093) than GG homozygotes. In the subgroup with defective b-cell function, rs6467136 GA þ AA carriers exhibited greater decrements of 2-h glucose level and improvement of homeostasis model assessment of insulin resistance (P ¼ 0.0143). Moreover, GA þ AA carriers were more likely to attain the target fasting and 2-h glucose level (Plog rank ¼ 0.0091 and 0.007, respectively). However, these single-nucleotide polymorphisms showed no effect on repaglinide efficacy. In conclusion, PAX4 variant rs6467136 was associated with the therapeutic effect of rosiglitazone in Chinese T2DM patients. The Pharmacogenomics Journal (2014) 14, 488–492; doi:10.1038/tpj.2014.18; published online 22 April 2014
INTRODUCTION Oral antidiabetic drugs, including metformin, sulfonylureas, glinides, a-glucosidase inhibitors and thiazolidinediones, are commonly used in the treatment of type 2 diabetes mellitus (T2DM). However, the clinical response to these oral agents varies among individuals. These individual variability is partly due to the factors such as age, sex, liver and renal function, severity of the disease and so on. In addition to the above, genetic factors involved in drug absorption, distribution, metabolism and target also have an important role in it. Paired box (PAX) proteins are a family of transcription factors that have an important role in tissue development and cellular differentiation. PAX protein family includes nine proteins divided into four groups based on structural domains, which are expressed in different organs and tissues.1 Herein, PAX4 is one of transcription factors that are mandatory for the differentiation of islet b-cells.2 PAX4 is expressed in the early pancreas, but is later restricted to b-cells.3 It has been proved that mutations and polymorphisms of PAX4 are associated with various types of diabetes, such as type 1 diabetes mellitus (T1DM), T2DM, maturityonset diabetes of the young (MODY) and ketosis-prone diabetes in different population.4–9 Recently, genome-wide association study identified two novel diabetes-associated loci near PAX4 (rs6467136 and rs10229583) in Chinese and other East Asian populations.10,11 However, whether these single-nucleotide polymorphisms (SNPs) influence the therapeutic effects of oral antihyperglycemic drugs has not been reported. Thus, we chose newly diagnosed patients treated with repaglinide or rosiglitazone to investigate the association of these SNPs with therapeutic effects.
MATERIALS AND METHODS Patients and study design A total of 209 newly diagnosed type 2 diabetes patients were recruited from the outpatient clinics in Shanghai, China. Detailed information on this study population has been described previously.12–14 After a 2-week run-in period (diet and exercise therapy only), 104 patients were treated with repaglinide (NovoNorm; Novo Nordisk, Copenhagen, Denmark), whereas the other 105 patients were given rosiglitazone treatment (Avandia; GlaxoSmithKline, Munich, Germany) for 48 weeks. Patients were visited at weeks 0, 2, 4, 12, 24, 32 and 48, and designed clinical assessments were taken. Repaglinide was administered initially in a mealtime dosage of 0.5 mg, whereas rosiglitazone treatment was started with a dosage of 4 mg. The dosage was increased stepwise to 1, 1.5 and 2 mg for repaglinide and to 8 mg for rosiglitazone when the patients failed to achieve glycemic targets of fasting plasma glucose (FPG) o7 mmol l 1 (126 mg dl 1) and/or 2 h plasma glucose o11 mmol l 1 (200 mg dl 1). Patients were excluded from the study when they showed any one of the following test results at two consecutive times (a maximal interval of 6 days): FPG 413 mmol l 1 (234 mg dl 1) or 2 h plasma glucose 418 mmol l 1 (324 mg dl 1) or glycated hemoglobin (HbA1c) X8%. In the analysis of SNPs and treatment attainment, the therapeutic targets of fasting glucose, 2-h glucose and HbA1c were, respectively, set as FPG o6.1 mmol l 1, 2-h glucose o7.8 mmol l 1, HbA1c o6.5% or over 20% decrease compared with the baseline after treatment. The study was approved by the institutional review board of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China. Written informed consent was obtained from each patient.
Anthropometric and clinical laboratory measurements Height (in m) and weight (in kg) were measured for all the patients, and body mass index (BMI; in kg m 2) was calculated. At the weeks 0, 2, 4, 12, 24, 32 and 48, fasting blood samples and 2-h blood samples were collected at each visit, following a 75 g oral glucose tolerance test. Plasma glucose
Department of Endocrinology and Metabolism, Shanghai Clinical Center for Diabetes, Shanghai Key Clinical Center for Metabolic Disease, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Diabetes Institute, Shanghai, China. Correspondence: Professor W Jia, Department of Endocrinology and Metabolism, Shanghai Clinical Center for Diabetes, Shanghai Key Clinical Center for Metabolic Disease, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Diabetes Institute, 600 Yishan Road, Shanghai 200233, China. E-mail: [email protected] 1 These authors contributed equally to this work. Received 2 January 2014; revised 14 February 2014; accepted 26 February 2014; published online 22 April 2014
Association of PAX4 with Chinese T2DM M Chen et al
489 concentrations were measured by means of the glucose oxidaseperoxidase method, using commercial kits (Shanghai Biological Products Institution, Shanghai, China). HbA1c values were determined by highperformance liquid chromatography, using a Bio-Rad Variant II hemoglobin testing system (Bio-Rad Laboratories, Hercules, CA, USA). Arginine stimulation tests were performed on all the patients at the beginning of the run-in period ( 2 weeks) and 48 weeks after treatment in overnight-fasted states (12 h fast). Blood samples were collected at 0 min, and then arginine hydrochloride (10% arginine hydrochloride of 50 ml, 5 g) was injected intravenously within 30–60 s. After injection, blood samples were taken at 2, 4 and 6 min. The acute insulin response (AIR) to arginine (AIR-Arg, in mU l 1) was calculated as the mean insulin value of 2, 4 and 6 min samples minus fasting insulin concentration to evaluate the potential function of b-cells. Defective b-cell function was defined as reported before.15 Briefly, the QL of AIR (24.95 mU l 1) in this study was regarded as cutting points of defective b-cell function. AIR o24.95 mU l 1 was regarded as defective b-cell function, whereas AIR X24.95 mU l 1 was normal b-cell function. Homeostasis model assessment was used to estimate the insulin resistance (HOMA-IR) and b-cell function (HOMA-B).16 HOMA-IR is calculated using the formula: fasting insulin concentration (in mU l 1) FPG concentration (in mmol l 1)/22.5, and HOMA-B is calculated using the formula: 20 fasting insulin concentration/(FPG concentration 3.5).
Genotype distribution of the SNPs (rs6467136 and rs10229583) was in agreement with the Hardy–Weinberg equilibrium.
Rosiglitazone cohort The association between SNPs and the clinic parameters in rosiglitazone cohort was shown in Table 1 and Supplementary Table 1. No significant differences were observed between two genotype groups of rs6467136 and rs10229583 in all the clinic parameters at baseline. After rosiglitazone therapy for 48 weeks, rs6467136 GA þ AA carriers show greater decrease in 2-h glucose levels than GG homozygotes ( 6.13±0.61 vs 3.60±0.51 mmol l 1, P ¼ 0.0063). Meanwhile, BMI in GA þ AA carriers decreased after 48 weeks treatment, whereas those with GG homozygotes increased (P ¼ 0.0192). There is no significant
Table 1. Association between PAX4 rs6467136 and clinical features in the rosiglitazone cohort GG
GA þ AA
P-value
Age (years) Gender (male/female) Dosage (mg per day)
51.62±1.11 44/20 5.55±0.25
53.25±1.86 20/8 5.38±0.38
0.4349 0.7972 0.7230
BMI(kg m 2) Baseline 48 Weeks DValue
25.01±0.39 25.23±0.44 0.12±0.18
24.80±0.59 23.97±0.69 0.52±2.22
0.7669 0.1260 0.0192
Fasting glucose(mmol l 1) Baseline 8.93±0.24 48 Weeks 6.67±0.18 DValue 2.33±0.28
9.17±0.28 6.35±0.15 2.74±0.30
0.2773 0.4176 0.3020
2-h Glucose(mmol l 1) Baseline 48 weeks DValue
12.91±0.39 9.24±0.33 3.60±0.51
14.35±0.57 8.09±0.33 6.13±0.61
0.0505 0.0517 0.0063
Glycated hemoglobin (%) Baseline 8.33±0.20 48 Weeks 6.42±0.11 DValue 1.75±0.23
8.30±0.25 6.23±0.14 2.10±0.26
0.6194 0.2563 0.1432
14.99±0.79 17.22±1.47 1.90±1.36
13.65±1.71 14.22±0.87 0.57±1.88
0.1033 0.594 0.6381
HOMA-IR Baseline 48 Weeks DValue
6.00±0.37 5.10±0.51 1.11±0.49
5.33±0.56 4.07±0.32 1.52±0.62
0.2831 0.5318 0.9565
HOMA-B Baseline 48 Weeks DValue
60.10±4.07 117.88±8.67 56.32±8.06
55.92±9.82 104.59±7.71 44.53±13.22
0.1042 0.804 0.5817
25.96±4.93 21.32±2.73 4.65±5.77
0.1135 0.0337 0.5513
Parameter
Genotyping Genomic DNA was extracted from peripheral blood leucocytes in whole blood samples. Genotyping was performed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, using a MassARRAY platform (MassARRAY Compact Analyzer; Sequenom, San Diego, CA, USA). The results were confirmed by DNA sequencing using 3130 Genetic Analyzer (Applied Biosystem, Foster City, CA, USA).
Statistical analysis The allele frequencies were determined by gene calculation and the Hardy– Weinberg equilibrium tests were performed. Data are shown as mean values±s.e.m. DValues were calculated by subtracting the initial value from the final value at week 48. Differences of quantitative traits between two genotypes groups were analyzed using multiple linear regression or Kruskal–Wallis test when appropriate. Kaplan–Meier method was used to estimate the attainment rates of clinical target levels. FPG o6.1 mmol l 1, 2-h glucose o7.8 mmol l 1 and HbA1c o6.5% or these parameters decreased more than 20% compared with the baseline after treatment was defined as attainment, respectively. Attainment rates between two genotype groups was compared by means of the log-rank test and Cox regression model analysis with adjustment for confounding factors, namely, age, gender and BMI at baseline. A two-tailed P-value of o0.05 was considered statistically significant. All the statistical analyses were performed using SAS for Windows (version 8.0; SAS Institute, Cary, NC, USA).
RESULTS Follow-up information of cohorts In the repaglinide cohort, of the total 104 patients recruited, 91 patients completed the 48-week study. Among the 13 patients withdrawn, 4 patients were with HbA1c X8% at two consecutive time points, and 9 patients were lost to follow-up. The GG, GA and AA genotypes distribution of rs6467136 and rs10229583 among the patients was 61, 23, 7 and 67, 23, 1, respectively. Of the total 105 patients recruited for rosiglitazone treatment, 93 patients completed the whole study. Twelve patients were withdrawn, among whom 1 patient had elevated hepatic enzymes and 5 patients attributed to inadequate control of blood glucose, and 6 patients were lost to follow-up. One patient was failed to be genotyped, and 92 patients was eventually included to statistics. The GG, GA and AA genotypes distribution of rs6467136 and rs10229583 among the patients was 64, 26, 2 and 65, 26, 1, respectively. Considering few subjects of rare allele homozygotes (AA), the genotype–phenotype associations were analyzed between common allele homozygotes and rare allele carriers. & 2014 Macmillan Publishers Limited
Fasting insulin (pmol l 1) Baseline 48 Weeks DValue
Acute insulin response (pmol l 1) Baseline 34.14±3.12 48 Weeks 35.02±3.93 DValue 0.62±3.96
Abbreviations: BMI, body mass index; HOMA-B, homeostasis model assessment of b-cell function; HOMA-IR, homeostasis model assessment of insulin resistance. Note: Data are shown as means±s.e.m. P-values o0.05 are shown in bold. Dvalue ¼ T48 weeks value baseline (T0) value. P-values were adjusted for age, gender and BMI at baseline when linear regression model was adopted.
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Figure 1. Association of rs6467136 and with the attainment rates of target values of fasting glucose, 2-h glucose and glycated hemoglobin among GA þ AA and GG in rosiglitazone cohort. (a) Plog rank ¼ 0.8359, PCox regression ¼ 0.9720; (b) Plog rank ¼ 0.0093, PCox regression ¼ 0.0169; and (c) Plog rank ¼ 0.6032, PCox regression ¼ 0.7202. PCox regression values were adjusted for age, gender and body mass index at baseline.
difference between two genotype groups of rs10229583 in all the clinic parameters after treatment. Then, we explored the associations between SNPs and the rate of attainment of target levels of FPG, 2-h glucose and HbA1c. Survival analyses showed that patients with rs6467136 GA þ AA genotype were significantly more likely to attain the standard 2-h glucose level than GG homozygotes at all assessment points (Plog rank ¼ 0.0093). Even after adjusting for age, gender and BMI at baseline, this difference was still significant (P ¼ 0.0169) (Figure 1b). We found no significant association between cumulative attainment and rs10229583 genotype (Supplementary Figure 1). As it has been proved that PAX4 was associated with cellular differentiation and b-cell function, we further divided the patients into two subgroups according to b-cell function assessed by arginine stimulation tests. In the group with defective b-cell function, rs6467136 GA þ AA genotype carriers showed greater decrease of BMI than GG homozygotes after 48 weeks rosiglitazone treatment (P ¼ 0.0033) (Table 2). Besides, there was a trend toward greater decrements of 2-h glucose level in individuals with rs6467136 GA þ AA genotype than GG carriers (P ¼ 0.0721). At the baseline, rs6467136 GA þ AA carriers showed worse insulin sensitivity assessed by HOMA-IR than GG homozygotes (P ¼ 0.0088), whereas the improvement of HOMA-IR was greater in GA þ AA carriers (P ¼ 0.0143). Survival analyses showed that patients with rs6467136 GA þ AA genotype were significantly more likely to attain the standard FPG and 2-h glucose level than GG homozygotes at all assessment points (Plog rank ¼ 0.0091 and 0.007, respectively) (Figures 2a and b). Even after adjusting for age, gender and BMI at baseline, there was still a trend toward higher rate of reaching the standard in GA þ AA carriers (P ¼ 0.0627 and 0.0543, respectively). In addition, we compared the clinical features between subgroups of rosiglitazone cohort stratified by b-cell function (Supplementary Table 2). Although the patients with defective b-cell function showed lower BMI and fasting insulin level, and better insulin sensitivity at baseline, the drug response was similar between the two subgroups after rosiglitazone treatment. The effect of PAX4 rs6467136 on rosiglitazone response was not observed in the subgroup with normal b-cell function (Supplementary Table 3). Repaglinide cohort Similarly, we analyzed the rs6467136 and rs10229583 genotype and therapeutic effects in repaglinide cohort. However, neither rs6467136 nor rs10229583 was found to affect efficacy of repaglinide. All the parameters have no difference between two The Pharmacogenomics Journal (2014), 488 – 492
Table 2. Association between PAX4 rs6467136 and clinical features in subgroup with defective b-cell function in the rosiglitazone cohort GG
GA þ AA
P-value
Age (year) Gender (male/female) Dosage (mg per day)
52.70±1.80 21/7 5.85±0.40
55.62±2.70 11/2 4.67±0.45
0.3692 0.4888 0.0873
BMI (kg m 2) Baseline 48 Weeks DValue
23.77±0.53 23.99±0.59 0.24±0.19
23.71±0.67 22.78±0.76 1.0±0.28
0.9416 0.0033 0.0033
Fasting glucose(mmol l 1) Baseline 8.93±0.40 48 weeks 6.28±0.19 DValue 2.77±0.43
9.29±0.46 6.16±0.19 3.0±0.43
0.4493 0.5713 0.7895
Parameter
2-h glucose(mmol l 1) Baseline 48 Weeks DValue
12.52±0.65 8.76±0.40 3.74±0.84
15.12±0.84 7.99±0.46 6.91±0.86
0.0433 0.4265 0.0721
Glycated hemoglobin (%) Baseline 48 Weeks DValue
8.34±0.31 6.36±0.11 1.71±0.29
8.35±0.48 6.07±0.17 2.28±0.44
0.9664 0.0707 0.2265
Fasting insulin (pmol l 1) Baseline 48 Weeks DValue
11.14±0.92 3.54±1.35 2.13±1.43
16.46±3.04 12.69±1.23 3.78±3.16
0.0849 0.9762 0.0923
HOMA-IR Baseline 48 Weeks DValue
4.53±0.54 3.85±0.51 0.81±0.70
6.38±0.88 3.41±0.38 3.09±0.93
0.0088 0.8203 0.0143
HOMA-B Baseline 48 Weeks DValue
45.89±4.93 108.28±11.21 63.93±10.36
Acute insulin response (pmol l 1) Baseline 13.97±1.32 48 Weeks 20.28±3.91 DValue 6.25±4.40
67.81±18.74 96.91±11.40 25.83±21.34
0.2807 0.5164 0.1013
9.60±3.78 18.48±4.03 8.88±5.69
0.441 0.8698 0.8698
Abbreviations: BMI, body mass index; HOMA-B, homeostasis model assessment of b-cell function; HOMA-IR, homeostasis model assessment of insulin resistance. Note: Data are shown as means±s.e.m. P-values o0.05 are shown in bold. Dvalue ¼ T48 weeks value baseline (T0) value. P-values were adjusted for age, gender and BMI at baseline when linear regression model was adopted.
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Figure 2. Association of rs6467136 and with the attainment rates of target values of fasting glucose, 2-h glucose and glycated hemoglobin among GA þ AA and GG in the subgroup with defective b-cell function in rosiglitazone cohort. (a) Plog rank ¼ 0.0091, PCox regression ¼ 0.0627; (b) Plog rank ¼ 0.0070, PCox regression ¼ 0.0543; and (c) Plog rank ¼ 0.9977, PCox regression ¼ 0.8762. PCox regression values were adjusted for age, gender and body mass index at baseline.
genotypes after 48 weeks repaglinide treatment (Supplementary Tables 4 and 5), and survival analyses showed no difference between two genotypes either (Supplementary Figures 2 and 3).
DISCUSSION TZDs are insulin-sensitizing agents that act as agonists of the nuclear factor peroxisome proliferator-activated receptor-g, thus leading to the improvements of the insulin sensitivity, especially in the peripheral tissues.17,18 As commonly used TZDs, rosiglitazone was effective in reducing glycemia in patients with type 2 diabetes for many years. Clinical trials showed that rosiglitazone could significantly improve insulin sensitivity and decrease FPG, 2 h plasma glucose and HbA1c, thereby improving glycemic control.19–21 PAX4 has been proved mandatory for the differentiation of islet b-cells. PAX4 gene has been shown to be implicated in pancreatic b-cell development, and polymorphism of PAX4 could lead to a reduction of adult b-cell mass and proliferation.2 The SNPs we investigated in the study, PAX4 rs6467136 and rs10229583, were identified as novel loci for type 2 diabetes in Chinese and other East Asian populations in genome-wide association study.10,11 Carriers of the risk allele G of rs6467136 and rs10229583 are more likely to develop type 2 diabetes. In our study, rs6467136 GG homozygotes from the subgroup with defective b-cell function showed better insulin sensitivity than the other genotype patients at baseline. This is perhaps attributed to the fact that carriers of G allele may affect b-cell function and insulin secretion, thus leading to compensatory better insulin sensitivity. In contrast, patients with rs6467136 GA þ AA genotype showed worse insulin sensitivity and attain better improvement of HOMA-IR after rosiglitazone treatment. Better fasting and 2-h glucose control, as well as BMI increment in individuals with rs6467136 GA þ AA genotype may due to greater improvement of insulin sensitivity. In our study, we identified association between PAX4 variants and therapeutic effect of rosiglitazone, which was an agonist of peroxisome proliferator-activated receptor-g. A functional study showed that peroxisome proliferatoractivated receptor-g regulates the expression of PDX-1, NKX6.1 and BETA2/NeuroD,22–24 which are all mandatory transcription factors for the differentiation of b-cells. PAX4 is also one of the protein in the pathway of b-cell differentiation, which was downstream of PDX-1, NKX6.1 and BETA2/NeuroD. Therefore, we suggested that the expression of PAX4 might also be affected by agonists of peroxisome proliferator-activated receptor-g, such as rosiglitazone. However, as PAX4 is a novel locus for T2DM, the mechanism under its effects on disease pathogenesis and drug & 2014 Macmillan Publishers Limited
efficacy has not been so clear, and further functional study was needed to reveal it. There are several limitations of this study that should be noted. First, the sample size was relatively small, thus we did not have enough statistical power to detect the effect of genetic variants on some of the parameters. Second, as most variables of our study are in skew distribution, the Kruskal–Wallis test was the main method used for their statistical analyses, thus the confounding factors could not be adjusted. Third, no adjustment of multiple testing was performed on the analyses of genotype and clinical parameters. Given the modest P-values, most of the tests would not be significant with a Bonferroni correction. However, as the variables tested in our study were related, Bonferroni correction was not proper and highly conservative. Fourth, we set the beginning of the run-in period as the baseline, rather than the initiation of drug treatment, which might lead to deviations in evaluating drug efficacy. However, given that all the participants were undergoing the same treatment (diet and exercise therapy) during this short run-in period, the impact of this limitation may be minimal. In conclusion, the present study demonstrates that the PAX4 variant rs6467136 was associated with the therapeutic effect of rosiglitazone treatment in Chinese patients with type 2 diabetes. GA þ AA genotype exhibited lower 2-h glucose levels as well as significantly higher cumulative attainment rates of target 2-h glucose levels than GG homozygotes. However, as our study is at an early stage of pharmacogenomics investigation on rosiglitazone, further investigations are needed to confirm our findings and reveal the underlying mechanisms. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGMENTS We thank all the patients who participated in this study and are appreciative of the doctors and nurses at the Shanghai Clinical Center for Diabetes. This project was funded by 973 Program (2011CB504001), grants from NSFC (81322010, 81170735 and 81200582), the Drug Innovation Program of the National Science and Technology Project (2011ZX09307-001-02), 863 Program (2012AA02A509), Excellent Young Medical Expert of Shanghai (XYQ2011041), Shanghai Talent Development Grant (2012041), Shanghai Raising Star Program (12QH1401700) and National Young Top Talent Supporting Program.
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Supplementary Information accompanies the paper on the The Pharmacogenomics Journal website (http://www.nature.com/tpj)
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