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Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subjects with type 2 diabetes mellitus
Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subjects with type 2 diabetes mellitus Kazunori Morita a, Junji Saruwatari a, Takahiro Tanaka a, Kentaro Oniki a, Ayami Kajiwara a, Hiroko Miyazaki a, Akira Yoshida b, Hideaki Jinnouchi b, Kazuko Nakagawa a, c,⁎ a b c
Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Jinnouchi Clinic, Diabetes Care Center, Kumamoto, Japan Center for Clinical Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
a r t i c l e
i n f o
Article history: Received 6 May 2015 Received in revised form 6 June 2016 Accepted 7 June 2016 Available online xxxx Keywords: Diabetic retinopathy Hemoglobin A1c Hepatocyte nuclear factor-1A Longitudinal study Normal-weight
a b s t r a c t Aim: This study investigated the associations between the common hepatocyte nuclear factor-1A (HNF1A) variants and the risk of diabetic retinopathy (DR) in relation to the glycemic control and weight status. Methods: A retrospective longitudinal analysis was conducted among 354 Japanese patients with type 2 diabetes mellitus (T2DM) (mean follow-up duration: 5.8 ± 2.5 years). The multivariable-adjusted hazard ratio (HR) for the cumulative incidence of DR was calculated using a Cox proportional hazard model. During the observation period, the longitudinal associations of the HNF1A diplotypes with the risk of DR and the clinical parameters were also analyzed using the generalized estimating equations approach. Results: The combination of risk variants, i.e., rs1169288-C, rs1183910-A and rs2464196-A, was defined as the H1 haplotype. The incidence of DR was higher in the H1/H1 diplotype cases than in the others (HR 2.75 vs. non-H1/non-H1; p = 0.02). Only in normal-weight subjects, the risks of DR and poor glycemic control were higher in the H1/H1 diplotype cases than in the others [odds ratio 4.08 vs. non-H1/non-H1, p = 0.02; odds ratio 3.03, p = 0.01; respectively]. Conclusions: This study demonstrated that the common HNF1A diplotype of three risk variants may be an independent risk factor for the development of DR resulting from poor glycemic control in normal-weight patients with T2DM. These results need to be replicated in larger and more varied study populations. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The hepatocyte nuclear factor-1A (HNF1A) gene encodes the transcription factor, HNF-1α, a homeodomain protein that contributes to the transcriptional regulation of genes involved in glucose and lipid metabolism in the liver and regulates several genes that maintain normal beta cell function in the pancreas (Allin & Nordestgaard, 2014; Fajans, Bell, & Polonsky, 2001; Odom et al., 2004; Owen, Skupien, Malecki, & Consortium, 2009; Uchizono et al., 2009). In accordance with the biological functions, the HNF1A locus is known to harbor mutations causing monogenic diabetes, maturity-onset diabetes of the young type 3 (MODY3) (Fajans et al., 2001; Owen et al., 2009). However, the
Conflict of interest: The authors declare that they have no conflicts of interest concerning this article. ⁎ Corresponding author at: Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1, Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan. Tel./fax: +81 96 371 4545. E-mail address: [email protected] (K. Nakagawa).
frequencies of genetic mutations causing MODY3 are rare (about 1% in the Japanese population) (Iwasaki et al., 1997; Nishigori et al., 1998; Yamada et al., 1997). The HNF1A locus is also known to harbor a risk variant (rs7957187) for late-onset T2DM identified by genome-wide association study (GWAS) meta-analyses (Parra et al., 2011; Voight et al., 2010). However, no variant at rs7957187 was found in Japanese individuals according to the International HapMap Project (http://www.ncbi.nlm.nih.gov/ projects/SNP/). Recently, the HNF1A rs1183910 minor A variant has been associated with an increased risk of T2DM (Allin & Nordestgaard, 2014), and it has been suggested that other HNF1A common variants, including I27L (rs1169288), A98V (rs1800574) and S487N (rs2464196), in this gene are associated with either impaired insulin secretion (IIS) or an increased risk of T2DM in normal-weight individuals who are less insulin-resistant than obese individuals (Chiu, Chuang, Chu, & Wang, 2003; Morita et al., 2015; Ramachandran, Ma, & Snehalatha, 2010; Yamakawa-Kobayashi et al., 2012). Patients with rare defective HNF1A variants, i.e., MODY3, showed a high prevalence of diabetic retinopathy (DR), which was strongly related to poor glycemic control due to a defect in insulin secretion (Isomaa et al., 1998; Skupien et al., 2008). Therefore, it is hypothesized
http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
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K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
that common genetic variants in the HNF1A gene, e.g., I27L, would affect not only poor glycemic control, but also the risk for DR among subjects with T2DM; however, there are presently no data available regarding the relationships between these common HNF1A variants and the risk of DR in late-onset T2DM. According to this information, the present exploratory study aimed to investigate the associations of the HNF1A variants and haplotypes with the prevalence or incidence of DR, while also paying careful attention to the weight status of the subjects, in Japanese patients with T2DM. The body mass index (BMI) cutoff levels for screening overweight or obese Asian Americans for prediabetes and T2DM were changed from 25 kg/m 2 to 23 kg/m 2 in the "Standards of Medical Care in Diabetes–2015", in order to reflect the growing evidence that this population is at an increased risk for diabetes at lower BMI levels than other ethic Americans (Hsu, Araneta, Kanaya, Chiang, & Fujimoto, 2015). Therefore, this study evaluated the associations separately in normal-weight (BMI b 23 kg/m 2) and overweight (BMI ≥ 23 kg/m2) individuals. 2. Methods In this clinic-based retrospective longitudinal study, 383 consecutive cases with T2DM who visited the Jinnouchi Clinic, Diabetes Care Center in Kumamoto, Japan, between February 2002 and January 2011 were reviewed. Among them, patients who were treated for less than six months and/or those with incomplete data for analysis were excluded; consequently 354 subjects (229 males and 125 females) were included for the present analysis. The study protocol was approved by the ethics committee of the Faculty of Life Sciences, Kumamoto University (Approval No. 169) according to the Ethical Guidelines for Human Genome/Gene Analysis Research of Ministries of Japan. All of the subjects provided their written informed consent to participate in the study. The clinical information was recorded at each follow-up visit. The laboratory tests were performed using the standard methods of the Japan Society of Clinical Chemistry. The postprandial plasma glucose level was measured using a glucose oxidase method with glucose analyzers (GA-1160 and GA-1170; Arkray, Inc., Kyoto, Japan) using 2 mL venous blood samples, which were collected in heparinized tubes 2 ± 0.5 h after a meal. The blood pressure (BP) was measured after the subjects rested in a sitting position. An overweight status was defined as a BMI of ≥23 kg/m 2 (Hsu et al., 2015). The information regarding smoking habits and alcohol intake was obtained via face-to-face interviews with health care providers. T2DM was defined as a fasting plasma glucose level of ≥7.0 mmol/L or a casual or 2-h glucose level of ≥11.1 mmol/L after a 75 g OGTT and a hemoglobin A1c (HbA1c) level of ≥6.5% (46.0 mmol/mol) or history of diabetes (Seino et al., 2010). Poor glycemic control was defined as a HbA1c level of ≥8.0% (64.0 mmol/mol) (Inzucchi et al., 2012). All patients were diagnosed with DR by a professional ophthalmologist using direct ophthalmoscopy, and 23 of the patients were also examined by fluorescein angiography to detect the progression of DR during the observation period. DR was staged as no retinopathy, non-proliferative DR (NPDR) or proliferative DR (PDR) according to the criteria determined at the third national ophthalmology conference held in 1985 (Klein et al., 1986). The occurrence of DR was defined as having no DR signs in both eyes at baseline and developing NPDR or PDR in either of the eyes during the observation period. The estimated glomerular filtration rate (eGFR) of each patient was calculated from the serum creatinine (SCr) level, age and gender using the following Japanese eGFR equation determined by the Japanese Society of Nephrology: eGFR (ml/min/1.73m2) = 194 × SCr (mg/dl)−1.094 × age−0.287(×0.739 if female) (Matsuo et al., 2009). Genomic DNA was isolated from EDTA-preserved blood samples using an automated DNA isolation system (NA-3000; Kurabo, Osaka, Japan). The four HNF1A variants, i.e., rs1169288 (79ANC, I27L),
rs1800574 (293CNT, A98V), rs1183910 (326+3910GNA) and rs2464196 (1460GNA, S487N), which are frequently found in Asian populations and/or may result in clinical consequences (Allin & Nordestgaard, 2014; Holmkvist et al., 2006; Holmkvist et al., 2008; Morita et al., 2015), were determined by real-time polymerase chain reaction (PCR) with 5′-nuclease allele discrimination assays (Step One Plus Real-Time PCR system version 2.1; Applied Biosystems, Tokyo, Japan). Genotyping was performed using commercially available assays (assay IDs: C_7474231_10, C_5478_10, C_7474214_10 and C_1263617_10 for rs1169288, rs1800574, rs1183910 and rs2464196, respectively), according to the manufacturer’s protocol. Pairwise linkage disequilibrium was evaluated using the SNPAlyze V6.0 Standard software program (Dynacom Co. Ltd, Chiba, Japan), and the haplotype frequencies were estimated by the expectation– maximization algorithm. Student’s t-test or a one-way analysis of variance and Fisher's exact test were used for comparisons of the continuous and categorical variables, respectively. Among the subjects who did not have DR at baseline, the DR-free survival according to the HNF1A variants or haplotypes was estimated using the Kaplan–Meier survival curves, and a comparison of the cumulative incidence among diplotypes was carried out using the generalized Wilcoxon test. The bi-variable and multivariable-adjusted hazard ratios (HRs) were calculated using a Cox proportional hazard model. Among all subjects enrolled in this study, the longitudinal associations of the HNF1A variants or haplotypes with the risks of DR and poor glycemic control, and with the values of clinical parameters, were also analyzed using the generalized estimating equations approach. The independent and autoregressive models were used for the analyses of the risk of DR and poor glycemic control, respectively, with calculations of the odds ratio (OR) and 95% confidence intervals (95% CIs). The autoregressive models were used with calculations of the adjusted partial regression coefficient (Β) and the standard error (SE) for the analysis of clinical parameters (i.e., HbA1c, eGFR, BP and lipid profiles) during the observation period. In all multiple regression models, the following variables were used as covariates: gender, weight status at the baseline, HbA1c, diabetes duration and systolic BP. A value of p b 0.05 was considered to be statistically significant. Multiple comparisons were corrected using Bonferroni’s method when comparing the demographic characteristics between the diplotypes and analyzed after stratifying the subjects by their weight status. In this method, values of p b 0.05/n were considered to be statistically significant after correcting for the number of comparisons made. Statistical power at a significance (alpha) level of 0.05 (two-tailed) based on the sample size of this study was calculated using IBM SPSS SamplePower software (version 2.0). All other statistical analyses were performed using the SPSS software package (version 17.0, IBM Japan Inc., Tokyo, Japan). 3. Results The allele frequencies of the HNF1A rs1169288-C, rs1800574-T, rs1183910-A and rs2464196-A variants were 49.6%, 0%, 44.6% and 53.4%, respectively. Because the rs1800574-T variant was not identified in this population, the association analysis of rs1800574 was not done. The observed genotype frequencies were consistent with the Hardy– Weinberg equilibrium (p N 0.05), and significant linkage disequilibrium was detected among rs1169288, rs1183910 and rs2464196 (p b 0.05). Therefore, the combination of the rs1169288-C, rs1183910-A and rs2464196-A variants (i.e., CAA haplotype) was defined as the H1 haplotype, because every variant comprising the H1 haplotype has been reported to be a risk factor for T2DM by our group and others (Allin & Nordestgaard, 2014; Holmkvist et al., 2006; Holmkvist et al., 2008; Morita et al., 2015). The H1 haplotype frequency was 41.8% (Supplemental Table 1), and the diplotypes were devided into three groups: H1/H1, non-H1/H1 and non-H1/non-H1, which had frequencies of 14.7%, 54.5% and 30.8%, respectively.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Table 1 Clinical characteristics of subjects stratified by the HNF1A diplotypes at baseline.
Female Age (years) Age of diagnosis (years) Diabetes duration (years) BMI (kg/m2) Overweight Fasting plasma glucose (mmol/l) Postprandial plasma glucose (mmol/l) HbA1c (%) HbA1c (mmol/mol) Systolic BP (mmHg) Diastolic BP (mmHg) Triglycerides (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) AST (IU/L) ALT (IU/L) GGT (IU/L) eGFR (ml/min/1.73m2) DR Hypertension Dyslipidemia Ever smoker Alcohol intake Therapy components Hypoglycemic agents Sulfonylureas Biguanides α-Glucosidase inhibitors Thiazolidines Other oral hypoglycemic agents Insulin Antihypertensive agents ACE inhibitors or ARBs Others Agents for hyperlipidemia Fibrates Statins Others
All subjects (n = 354)
H1/H1 (n = 52)
non-H1/H1 (n = 193)
non-H1/non-H1 (n = 109)
p value
125 (35.3) 58.8 ± 11.0 48.0 ± 11.9 10.8 ± 8.3 24.2 ± 3.7 214 (60.5) 9.4 ± 3.4 11.5 ± 4.7 8.7 ± 2.0 71.2 ± 21.9 139.8 ± 19.9 83.0 ± 12.2 1.8 ± 1.7 1.4 ± 0.4 3.2 ± 0.9 27.3 ± 16.0 30.5 ± 25.8 39.6 ± 42.7 84.3 ± 26.7 133 (37.6) 107 (30.2) 107 (30.2) 179 (50.6) 178 (50.3)
22 (42.3) 59.2 ± 9.8 48.4 ± 12.7 10.8 ± 7.1 24.1 ± 3.4 31 (59.6) 9.9 ± 3.7 11.8 ± 4.3 8.9 ± 2.3 73.8 ± 25.4 141.5 ± 20.3 83.8 ± 11.3 1.7 ± 0.8 1.4 ± 0.3 3.3 ± 0.9 27.0 ± 14.1 28.6 ± 21.5 37.1 ± 41.5 83.5 ± 26.1 21 (40.4) 11 (21.2) 15 (28.8) 21 (40.4) 25 (48.1)
63 (32.6) 59.0 ± 11.4 47.6 ± 11.9 11.4 ± 8.7 24.2 ± 3.8 114 (59.1) 9.6 ± 3.8 11.9 ± 4.8 8.9 ± 2.1⁎
73.3 ± 22.8⁎ 139.1 ± 20.1 82.3 ± 12.6 1.9 ± 2.0 1.5 ± 0.4 3.1 ± 0.9 26.2 ± 14.8 28.7 ± 19.9 40.2 ± 46.8 84.7 ± 27.5 79 (40.9) 63 (32.6) 58 (30.1) 100 (51.8) 98 (50.8)
40 (36.7) 58.2 ± 10.8 48.6 ± 11.7 9.7 ± 8.0 24.3 ± 3.5 69 (63.3) 9.0 ± 2.7 10.7 ± 4.8 8.2 ± 1.6 66.4 ± 17.2 140.2 ± 19.5 83.8 ± 12.0 1.7 ± 1.3 1.4 ± 0.4 3.2 ± 0.8 29.2 ± 18.7 34.6 ± 35.1 39.7 ± 35.7 84.0 ± 25.7 33 (30.3) 33 (30.3) 34 (31.2) 58 (53.2) 55 (50.5)
0.41 0.80 0.76 0.21 0.97 0.80 0.25 0.15 0.02 0.02 0.71 0.53 0.62 0.61 0.71 0.32 0.14 0.90 0.95 0.17 0.27 0.96 0.29 0.95
133 (37.6) 77 (21.8) 64 (18.1) 13 (3.7) 20 (5.7) 91 (25.7)
19 (36.5) 14 (26.9) 4 (7.7) 1 (1.9) 5 (9.6) 14 (26.9)
80 (41.5) 38 (19.8) 35 (18.2) 6 (3.1) 6 (3.1) 53 (27.5)
34 (31.2) 25 (22.9) 25 (22.9) 6 (5.5) 9 (8.3) 24 (22.0)
0.21 0.50 0.06 0.53 0.053 0.59
64 (18.1) 91 (25.0)
5 (9.6) 13 (25.0)
41 (21.4) 54 (28.1)
18 (16.5) 24 (22.0)
0.13 0.51
4 (1.1) 25 (7.1) 2 (0.6)
0 (0.0) 6 (11.5) 0 (0.0)
3 (1.6) 11 (5.7) 1 (0.5)
1 (0.9) 8 (7.3) 1 (0.9)
1.00 0.34 1.00
Bold typeface indicates data that are statistically significant (p b 0.05). Data are number (%) or mean ± standard deviation. HNF1A, hepatocyte nuclear factor 1A; BMI, body mass index; HbA1c, hemoglobin A1c; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, γ-glutamyltransferase; eGFR, estimated glomerular filtration rate; DR, diabetic retinopathy; ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker. ⁎ p b 0.025, compared with the non-H1/non-H1 diplotype.
The mean follow-up duration was 5.8 ± 2.5 years. In the demographic characteristics of the subjects at baseline, only the mean values of HbA1c were significantly different among the patients with the different HNF1A diplotypes (Table 1). Each rs1169288-C, rs1183910-A and rs2464196-A variant carrier group also had significantly higher HbA1c levels than the non-carriers (Supplemental Table 2). In addition, the rs1169288-C variant carriers exhibited a significantly longer duration of diabetes than the non-carriers (Supplemental Table 2). On the other hand, the demographic characteristics of the subjects stratified by the weight status at baseline are shown in Supplemental Table 3. The overweight subjects had significantly higher BMIs, postprandial plasma glucose, BP, triglycerides and transaminase levels, and lower age and HDL-C level than the normal-weight subjects. In addition, the overweight subjects had significantly higher frequencies of the use of biganides and antihypertensive agents than the normal-weight subjects. At baseline, 133 (37.6%) patients had DR (Table 1), and 121 and 12 of the patients were examined by direct ophthalmoscopy alone or in combination with fluorescein angiography, respectively. Among all 354 subjects, the H1 haplotype carriers tended to have a higher frequency of DR at baseline than the non-carriers [100 (40.8%) vs. 33 (30.3%), p = 0.07]. On the other hand, rs1183910-A and rs2464196-A
variant carriers had significantly higher frequencies of DR at baseline than the non-carriers (Supplemental Table 2). During the observation period, 49 (13.8%) patients were newly diagnosed with DR by direct ophthalmoscopy, and 11 (3.1%) patients were examined by fluorescein angiography. Among the 221 subjects who did not have DR at baseline, the subjects with the H1/H1 diplotype exhibited a higher incidence of DR than did those with the non-H1/non-H1 diplotype [10 (32.3%) vs. 12 (15.8%), p = 0.03] (Fig. 1a). In a bi-variable Cox proportional hazard model, the H1/H1 diplotype was significantly associated with the incidence of DR, in addition to HbA1c (Table 2). In a multivariable Cox proportional hazard model, HbA1c, but not the H1/H1 diplotype, was found to be an independent risk factor for incidence of DR (Table 2). When the multivariable analyses were performed separately in normal-weight and overweight subjects, the H1/H1 diplotype was significantly associated with incidence for DR in normal-weight, but not in overweight, subjects (Table 3, Fig. 1b and c). The rs1169288-C and rs2464196-A variants also tended to be risk factors for DR in normal-weight subjects, although the associations did not reach statistical significance (Supplemental Table 4). In a longitudinal association analysis, the H1/H1 diplotype was also an independent risk factor for DR only in normal-weight subjects (Table 4). Additionally, the risk of DR significantly increased with a
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
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K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx Table 2 Association between the risk of DR and the covariates during the observation period (year) in the Cox proportional hazard model. Bi-variable
HNF1A diplotype † Non-H1/H1 H1/H1 Female‡ HbA1c Diabetes duration Systolic BP Overweight§
Multivariable
HR
95% CI
p value
HR
95% CI
p value
1.78 2.75 1.48 1.20 1.00 1.01 1.01
0.90, 3.52 1.19, 6.37 0.82, 2.65 1.07, 1.34 0.96, 1.04 0.998, 1.03 0.57, 1.78
0.10 0.02 0.19 b0.01 0.83 0.10 0.97
1.55 2.32 1.55 1.22 1.02 1.01 0.92
0.77, 3.10 0.97, 5.53 0.84, 2.86 1.08, 1.38 0.97, 1.06 0.999, 1.03 0.50, 1.69
0.22 0.06 0.16 b0.01 0.45 0.06 0.78
Bold typeface indicates data that are statistically significant (p b 0.05). HNF1A, hepatocyte nuclear factor 1A; DR, diabetic retinopathy; HR, hazard ratio; CI, confidence interval; HbA1c, hemoglobin A1c; BP, blood pressure. † Compared with the non-H1/non-H1 diplotype. ‡ Compared with male. § Compared with normal-weight.
with the non-H1/non-H1 diplotype carriers in normal-weight individuals (Table 5). The H1 haplotype carriers were also associated with higher HbA1c values [B 0.70% (7.57 mmol/mol); SE 0.19% (2.02 mmol/mol); p b 0.01] and poor glycemic control (OR 2.52; 95% CI 1.39–4.57; p b 0.01) than the non-carriers, but only in normal-weight subjects. In the normal-weight subjects, each of the variants (rs1169288-C, rs1183910-A and rs2464196-A) also had a significant association with a higher HbA1c value and poor glycemic control (Supplemental Table 5). On the other hand, no association was observed between the HNF1A diplotypes and the longitudinal changes in the other clinical parameters, i.e., eGFR, BP and lipid profiles, irrespective of the weight status (Supplemental Table 6). The statistical power of the association analyses between the HNF1A haplotypes and the risk for DR ranged from 56% to 64%. The power of the associations in normal-weight and overweight subjects was 53%–78% and 15%–26%, respectively. All of the values were below the necessary limit of power (i.e., 80%) to predict the development of disease, indicating the necessity of a larger sample size to confirm the importance of these findings. 4. Discussion To the best of our knowledge, this is the first report to show that the common HNF1A H1/H1 diplotype is associated with the development of DR in normal-weight, but not in overweight, patients with T2DM. The H1 haplotype/diplotype or variants were also associated with poor glycemic control only in normal-weight patients, confirming our previous findings (Morita et al., 2015). Table 3 Association between the risk of DR and the covariates stratified by the weight status during the observation period (year) in the multivariable Cox proportional hazard model.
Fig. 1. Kaplan–Meier curves for the DR-free survival among all subjects (a), normal-weight subjects (b) and overweight subjects (c) according to the HNF1A diplotypes. And comparison of the cumulative incidence among diplotypes was carried out using the generalized Wilcoxon test. An overweight status was defined as a BMI ≥ 23 kg/m2.
longer duration of diabetes or an increase in the systolic BP, irrespective of the weight status (Table 4). During the observation period, the H1/H1 and non-H1/H1 diplotypes carriers had a higher HbA1c values and poor glycemic control compared
HNF1A diplotype † Non-H1/H1 H1/H1 Female‡ HbA1c Diabetes duration Systolic BP
Normal-weight (n = 94)
Overweight (n = 127)
HR
95% CI
p value
HR
95% CI
p value
1.72 6.63 3.10 1.36 0.99 1.01
0.51, 5.79 1.60, 27.53 1.14, 8.42 1.11, 1.67 0.93, 1.06 0.98, 1.03
0.38 b0.01 0.03 b0.01 0.72 0.61
1.28 1.39 1.11 1.17 1.05 1.02
0.53, 3.10 0.41, 4.71 0.48, 2.59 0.97, 1.42 0.98, 1.12 0.997, 1.04
0.58 0.60 0.81 0.09 0.19 0.10
Bold typeface indicates data that are statistically significant (p b 0.025). HNF1A, hepatocyte nuclear factor 1A; DR, diabetic retinopathy; HR, hazard ratio; CI, confidence interval; HbA1c, hemoglobin A1c; BP, blood pressure. † Compared with the non-H1/non-H1 diplotype. ‡ Compared with male.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
variants and the risk of DR could be attributed to poor glycemic control, although further investigation is needed to confirm this. Significant inter-ethnic differences in the HNF1A risk allele frequency have been reported, i.e., the frequencies of DR-related variants, rs1183910-A and rs2464196-A, in this study population (44.6% and 53.4%, respectively) were similar to those in Han Chinese (44.4% and 52.3%, respectively) and are higher than those in Europeans (29.2% and 29.6%, respectively), according to the International HapMap project (http://www.ncbi.nlm.nih.gov/projects/SNP/). Asians with T2DM are often lean compared to Caucasians, and predominantly exhibit IIS and onset at a relatively lower BMI (Chan et al., 2009; Cho, Lee, Park, & Nho, 2012; Morimoto et al., 2013; Ramachandran et al., 2010; Yamakawa-Kobayashi et al., 2012; Yu, Hu, & Jia, 2012). On the other hand, most of the susceptibility loci for T2DM identified through GWAS to date are considered to be likely to affect insulin secretion and to accumulate in normal-weight patients (Cho et al., 2012; Kahn, 2003; Murea et al., 2012; Parra et al., 2011; Ramachandran et al., 2010; Voight et al., 2010; Yamakawa-Kobayashi et al., 2012; Yu et al., 2012). In fact, an increased cumulative number of risk variants identified in a GWAS was associated with an increased risk of T2DM only in normal-weight individuals in Japanese (Yamakawa-Kobayashi et al., 2012). These facts suggest that the genetic architecture of T2DM may be different in obese/ overweight and normal-weight individuals and that the variants of genes involved in insulin secretion, e.g., HNF1A, would contribute to the development of T2DM and complications in normal-weight East Asians. In this study, the duration of diabetes and systolic BP were also found to be independent risk factors for DR in a longitudinal association analysis, irrespective of the weight status (Table 4). These results may support the previous findings that the duration of diabetes and hypertension are the primary risk factors for DR (Chan et al., 2010; Ola et al., 2012; Simo-Servat et al., 2013; Stitt et al., 2013; Yau et al., 2012). Additionally, overweight subjects had worse glycemic and metabolic profiles than normal-weight subjects in the present study (Supplemental Table 3). It is speculated that the effect of these factors on the risk for DR may be more pronounced in overweight subjects than in normal-weight subjects, and thus the HNF1A diplotype may affect the risk of DR only in normal-weight subjects. There are some limitations associated with the present study. First, this study was a retrospective investigation of a small number of patients, as shown by the sample power analysis. Because the power of the samples in the analyses regarding the effects of the HNF1A variants or haplotypes on the risk for DR was small in normal-weight and overweight subjects, large-scaled studies are necessary to confirm the current findings. Second, the number of the patients diagnosed using direct ophthalmoscopy with fluorescein angiography was also small (n = 23); therefore, further investigations are needed to confirm the effect of the HNF1A common variants on the severity of DR. Third, this study could not collect information regarding the plasma insulin and
Table 4 Association between the risk of DR and the covariates stratified by the weight status during the observation period (year) in the independent logistic regression models.
HNF1A diplotype† Non-H1/H1 H1/H1 Female‡ HbA1c Diabetes duration Systolic BP
Normal-weight (n = 140)
Overweight (n = 214)
OR
95% CI
p value
OR
95% CI
p value
2.27 4.08 1.95 1.23 1.07 1.02
0.94, 5.48 1.23, 13.55 0.96, 3.96 1.02, 1.47 1.02, 1.12 1.003, 1.03
0.07 0.02 0.07 0.03 b0.01 0.02
1.67 1.37 1.41 1.14 1.20 1.02
0.79, 3.54 0.55, 3.39 0.71, 2.78 0.96, 1.35 1.13, 1.27 1.01, 1.03
0.18 0.50 0.32 0.15 b0.01 b0.01
5
Bold typeface indicates data that are statistically significant (p b 0.025). HNF1A, hepatocyte nuclear factor 1A; DR, diabetic retinopathy; HR, hazard ratio; CI, confidence interval; HbA1c, hemoglobin A1c; BP, blood pressure. † Compared with the non-H1/non-H1 diplotype. ‡ Compared with male.
A number of susceptibility loci for DR have been reported with inconsistent results (Kuo, Wong, & Rotter, 2014; Murea, Ma, & Freedman, 2012; Simo-Servat, Hernandez, & Simo, 2013; Stitt, Lois, Medina, Adamson, & Curtis, 2013), and no variant related to hyperglycemia has been identified as a risk factor for DR to date. On the other hand, MODY3 is characterized by a defect in insulin secretion and a high prevalence of DR (Fajans et al., 2001; Isomaa et al., 1998; Skupien et al., 2008). Hyperglycemia was indicated to be the main predictor of DR in MODY3 patients (Isomaa et al., 1998). In a Polish study, 47.7% of MODY3 patients had DR and they also had significantly higher HbA1c levels than those without DR (Skupien et al., 2008). Furthermore, there is strong evidence that initial tight glycemic control soon after the diagnosis of diabetes delays the natural process of the diabetes, reducing the development and progression of DR (Chan, Kanwar, & Kowluru, 2010; Hammes, Feng, Pfister, & Brownlee, 2011; Inzucchi et al., 2012; Massin et al., 2011; Ola, Nawaz, Siddiquei, Al-Amro, & Abu El-Asrar, 2012; Sabanayagam et al., 2014; Stitt et al., 2013; Tsugawa et al., 2012; Xu & Weng, 2013). The HbA1c and fasting plasma glucose levels predicted the presence of DR 10 years after the diagnosis, while hypertension and the lipid profiles did not (Massin et al., 2011). A previous longitudinal Japanese study determined the threshold HbA1c level for the development of DR to be 6.5% (Tsugawa et al., 2012). In this study, the HNF1A H1/H1 diplotype was significantly associated with a higher prevalence of DR (Tables 2-4). In the H1 haplotype carriers, the HbA1c values were significantly higher at baseline (Table 1), and the higher HbA1c values were also observed throughout the observation period (Table 5), however, none of the other clinical parameters differed between the H1 haplotype carriers and non-carriers (Table 1 and Supplemental Table 6). These results indicate that our present findings regarding the associations between the common HNF1A diplotype or
Table 5 The combined effects of the HNF1A diplotypes and the weight status at baseline on the longitudinal change of glycemic control during the observation period (year) in the independent logistic regression models. Weight status
Normal-weight
Overweight
HNF1A diplotypes Non-H1/non-H1 (reference) Non-H1/H1 H1/H1 Non-H1/non-H1 (reference) Non-H1/H1 H1/H1
Poor glycemic control†
n
‡
HbA1c (%)
95% CI
p value
Β
‡
HbA1c (mmol/mol) SE
p value
Β‡
SE
p value
40
1
–
–
0
–
–
0
–
–
79 21 69
2.41 3.03 1
1.31, 4.44 1.28, 7.17 –
b0.01 0.01 –
0.67 0.80 0
0.20 0.28 –
b0.01 b0.01 –
7.29 8.61 0
2.19 3.07 –
b0.01 b0.01 –
114 31
1.08 1.75
0.71, 1.65 1.02, 3.02
0.73 0.04
0.16 0.55
0.17 0.23
0.34 0.02
1.72 5.96
1.80 2.47
0.34 0.02
OR
Bold typeface indicates data that are statistically significant (p b 0.025). HNF1A, hepatocyte nuclear factor 1A; HbA1c, hemoglobin A1c; OR, odds ratio; CI, confidence interval; B, partial regression coefficient; SE, standard error. † HbA1c ≥ 8.0% (64.0 mmol/mol). ‡ Adjusted by gender, duration of diabetes and systolic BP.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
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C-reactive protein levels. Lastly, studying only four variants may not cover the entire genetic variation in the HNF1A region. 5. Conclusion We herein demonstrated that the common HNF1A diplotype or variants are independent risk factors for poor glycemic control and for the subsequent development of DR in normal-weight Japanese patients with T2DM, but these results need to be replicated in larger and more varied study populations. T2DM in East Asians is characterized by a low BMI, onset at a relatively young age and a rapidly rising prevalence (Chan et al., 2009; Cho et al., 2012; Morimoto et al., 2013; Ramachandran et al., 2010; Yu et al., 2012). These distinctive features may be attributable to the ethnicity-specific genetic differences underlying the pathophysiology of T2DM (Tremblay & Hamet, 2015; Yu et al., 2012). Although the present study was exploratory in nature and further investigations in larger cohorts of other ethnic groups are needed to verify the results, the present study suggests that East Asians should be warned about the likely increase in DR in the near future, following the rapidly increasing prevalence of T2DM (Chan et al., 2009; Cho et al., 2012; Ramachandran et al., 2010; Yau et al., 2012). Acknowledgments The authors wish to thank all of the study participants. This study was supported by KAKENHI (No. 23510348). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jdiacomp.2016.06.007. References Allin, K. H., & Nordestgaard, B. G. (2014). Pleiotropic effects of HNF1A rs1183910 in a population-based study of 60,283 individuals. Diabetologia, 57(4), 729–737. Chan, P. S., Kanwar, M., & Kowluru, R. A. (2010). Resistance of retinal inflammatory mediators to suppress after reinstitution of good glycemic control: Novel mechanism for metabolic memory. Journal of Diabetes and its Complications, 24(1), 55–63. Chan, J. C., Malik, V., Jia, W., Kadowaki, T., Yajnik, C. S., Yoon, K. H., et al. (2009). Diabetes in Asia: Epidemiology, risk factors, and pathophysiology. JAMA, 301(20), 2129–2140. Chiu, K. C., Chuang, L. M., Chu, A., & Wang, M. (2003). Transcription factor 1 and beta-cell function in glucose-tolerant subjects. Diabetic Medicine, 20(3), 225–230. Cho, Y. S., Lee, J. Y., Park, K. S., & Nho, C. W. (2012). Genetics of type 2 diabetes in East Asian populations. Current Diabetes Reports, 12(6), 686–696. Fajans, S. S., Bell, G. I., & Polonsky, K. S. (2001). Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. The New England Journal of Medicine, 345(13), 971–980. Hammes, H. P., Feng, Y., Pfister, F., & Brownlee, M. (2011). Diabetic retinopathy: Targeting vasoregression. Diabetes, 60(1), 9–16. Holmkvist, J., Almgren, P., Lyssenko, V., Lindgren, C. M., Eriksson, K. F., Isomaa, B., et al. (2008). Common variants in maturity-onset diabetes of the young genes and future risk of type 2 diabetes. Diabetes, 57(6), 1738–1744. Holmkvist, J., Cervin, C., Lyssenko, V., Winckler, W., Anevski, D., Cilio, C., et al. (2006). Common variants in HNF-1 alpha and risk of type 2 diabetes. Diabetologia, 49(12), 2882–2891. Hsu, W. C., Araneta, M. R., Kanaya, A. M., Chiang, J. L., & Fujimoto, W. (2015). BMI cut points to identify at-risk Asian Americans for type 2 diabetes screening. Diabetes Care, 38(1), 150–158. Inzucchi, S. E., Bergenstal, R. M., Buse, J. B., Diamant, M., Ferrannini, E., Nauck, M., et al. (2012). Management of hyperglycaemia in type 2 diabetes: A patient-centered approach. Position statement of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetologia, 55(6), 1577–1596. Isomaa, B., Henricsson, M., Lehto, M., Forsblom, C., Karanko, S., Sarelin, L., et al. (1998). Chronic diabetic complications in patients with MODY3 diabetes. Diabetologia, 41(4), 467–473. Iwasaki, N., Oda, N., Ogata, M., Hara, M., Hinokio, Y., Oda, Y., et al. (1997). Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with earlyand late-onset NIDDM. Diabetes, 46(9), 1504–1508.
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Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subjects with type 2 diabetes mellitus Kazunori Morita a, Junji Saruwatari a, Takahiro Tanaka a, Kentaro Oniki a, Ayami Kajiwara a, Hiroko Miyazaki a, Akira Yoshida b, Hideaki Jinnouchi b, Kazuko Nakagawa a, c,⁎ a b c
Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Jinnouchi Clinic, Diabetes Care Center, Kumamoto, Japan Center for Clinical Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
a r t i c l e
i n f o
Article history: Received 6 May 2015 Received in revised form 6 June 2016 Accepted 7 June 2016 Available online xxxx Keywords: Diabetic retinopathy Hemoglobin A1c Hepatocyte nuclear factor-1A Longitudinal study Normal-weight
a b s t r a c t Aim: This study investigated the associations between the common hepatocyte nuclear factor-1A (HNF1A) variants and the risk of diabetic retinopathy (DR) in relation to the glycemic control and weight status. Methods: A retrospective longitudinal analysis was conducted among 354 Japanese patients with type 2 diabetes mellitus (T2DM) (mean follow-up duration: 5.8 ± 2.5 years). The multivariable-adjusted hazard ratio (HR) for the cumulative incidence of DR was calculated using a Cox proportional hazard model. During the observation period, the longitudinal associations of the HNF1A diplotypes with the risk of DR and the clinical parameters were also analyzed using the generalized estimating equations approach. Results: The combination of risk variants, i.e., rs1169288-C, rs1183910-A and rs2464196-A, was defined as the H1 haplotype. The incidence of DR was higher in the H1/H1 diplotype cases than in the others (HR 2.75 vs. non-H1/non-H1; p = 0.02). Only in normal-weight subjects, the risks of DR and poor glycemic control were higher in the H1/H1 diplotype cases than in the others [odds ratio 4.08 vs. non-H1/non-H1, p = 0.02; odds ratio 3.03, p = 0.01; respectively]. Conclusions: This study demonstrated that the common HNF1A diplotype of three risk variants may be an independent risk factor for the development of DR resulting from poor glycemic control in normal-weight patients with T2DM. These results need to be replicated in larger and more varied study populations. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The hepatocyte nuclear factor-1A (HNF1A) gene encodes the transcription factor, HNF-1α, a homeodomain protein that contributes to the transcriptional regulation of genes involved in glucose and lipid metabolism in the liver and regulates several genes that maintain normal beta cell function in the pancreas (Allin & Nordestgaard, 2014; Fajans, Bell, & Polonsky, 2001; Odom et al., 2004; Owen, Skupien, Malecki, & Consortium, 2009; Uchizono et al., 2009). In accordance with the biological functions, the HNF1A locus is known to harbor mutations causing monogenic diabetes, maturity-onset diabetes of the young type 3 (MODY3) (Fajans et al., 2001; Owen et al., 2009). However, the
Conflict of interest: The authors declare that they have no conflicts of interest concerning this article. ⁎ Corresponding author at: Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1, Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan. Tel./fax: +81 96 371 4545. E-mail address: [email protected] (K. Nakagawa).
frequencies of genetic mutations causing MODY3 are rare (about 1% in the Japanese population) (Iwasaki et al., 1997; Nishigori et al., 1998; Yamada et al., 1997). The HNF1A locus is also known to harbor a risk variant (rs7957187) for late-onset T2DM identified by genome-wide association study (GWAS) meta-analyses (Parra et al., 2011; Voight et al., 2010). However, no variant at rs7957187 was found in Japanese individuals according to the International HapMap Project (http://www.ncbi.nlm.nih.gov/ projects/SNP/). Recently, the HNF1A rs1183910 minor A variant has been associated with an increased risk of T2DM (Allin & Nordestgaard, 2014), and it has been suggested that other HNF1A common variants, including I27L (rs1169288), A98V (rs1800574) and S487N (rs2464196), in this gene are associated with either impaired insulin secretion (IIS) or an increased risk of T2DM in normal-weight individuals who are less insulin-resistant than obese individuals (Chiu, Chuang, Chu, & Wang, 2003; Morita et al., 2015; Ramachandran, Ma, & Snehalatha, 2010; Yamakawa-Kobayashi et al., 2012). Patients with rare defective HNF1A variants, i.e., MODY3, showed a high prevalence of diabetic retinopathy (DR), which was strongly related to poor glycemic control due to a defect in insulin secretion (Isomaa et al., 1998; Skupien et al., 2008). Therefore, it is hypothesized
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Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
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that common genetic variants in the HNF1A gene, e.g., I27L, would affect not only poor glycemic control, but also the risk for DR among subjects with T2DM; however, there are presently no data available regarding the relationships between these common HNF1A variants and the risk of DR in late-onset T2DM. According to this information, the present exploratory study aimed to investigate the associations of the HNF1A variants and haplotypes with the prevalence or incidence of DR, while also paying careful attention to the weight status of the subjects, in Japanese patients with T2DM. The body mass index (BMI) cutoff levels for screening overweight or obese Asian Americans for prediabetes and T2DM were changed from 25 kg/m 2 to 23 kg/m 2 in the "Standards of Medical Care in Diabetes–2015", in order to reflect the growing evidence that this population is at an increased risk for diabetes at lower BMI levels than other ethic Americans (Hsu, Araneta, Kanaya, Chiang, & Fujimoto, 2015). Therefore, this study evaluated the associations separately in normal-weight (BMI b 23 kg/m 2) and overweight (BMI ≥ 23 kg/m2) individuals. 2. Methods In this clinic-based retrospective longitudinal study, 383 consecutive cases with T2DM who visited the Jinnouchi Clinic, Diabetes Care Center in Kumamoto, Japan, between February 2002 and January 2011 were reviewed. Among them, patients who were treated for less than six months and/or those with incomplete data for analysis were excluded; consequently 354 subjects (229 males and 125 females) were included for the present analysis. The study protocol was approved by the ethics committee of the Faculty of Life Sciences, Kumamoto University (Approval No. 169) according to the Ethical Guidelines for Human Genome/Gene Analysis Research of Ministries of Japan. All of the subjects provided their written informed consent to participate in the study. The clinical information was recorded at each follow-up visit. The laboratory tests were performed using the standard methods of the Japan Society of Clinical Chemistry. The postprandial plasma glucose level was measured using a glucose oxidase method with glucose analyzers (GA-1160 and GA-1170; Arkray, Inc., Kyoto, Japan) using 2 mL venous blood samples, which were collected in heparinized tubes 2 ± 0.5 h after a meal. The blood pressure (BP) was measured after the subjects rested in a sitting position. An overweight status was defined as a BMI of ≥23 kg/m 2 (Hsu et al., 2015). The information regarding smoking habits and alcohol intake was obtained via face-to-face interviews with health care providers. T2DM was defined as a fasting plasma glucose level of ≥7.0 mmol/L or a casual or 2-h glucose level of ≥11.1 mmol/L after a 75 g OGTT and a hemoglobin A1c (HbA1c) level of ≥6.5% (46.0 mmol/mol) or history of diabetes (Seino et al., 2010). Poor glycemic control was defined as a HbA1c level of ≥8.0% (64.0 mmol/mol) (Inzucchi et al., 2012). All patients were diagnosed with DR by a professional ophthalmologist using direct ophthalmoscopy, and 23 of the patients were also examined by fluorescein angiography to detect the progression of DR during the observation period. DR was staged as no retinopathy, non-proliferative DR (NPDR) or proliferative DR (PDR) according to the criteria determined at the third national ophthalmology conference held in 1985 (Klein et al., 1986). The occurrence of DR was defined as having no DR signs in both eyes at baseline and developing NPDR or PDR in either of the eyes during the observation period. The estimated glomerular filtration rate (eGFR) of each patient was calculated from the serum creatinine (SCr) level, age and gender using the following Japanese eGFR equation determined by the Japanese Society of Nephrology: eGFR (ml/min/1.73m2) = 194 × SCr (mg/dl)−1.094 × age−0.287(×0.739 if female) (Matsuo et al., 2009). Genomic DNA was isolated from EDTA-preserved blood samples using an automated DNA isolation system (NA-3000; Kurabo, Osaka, Japan). The four HNF1A variants, i.e., rs1169288 (79ANC, I27L),
rs1800574 (293CNT, A98V), rs1183910 (326+3910GNA) and rs2464196 (1460GNA, S487N), which are frequently found in Asian populations and/or may result in clinical consequences (Allin & Nordestgaard, 2014; Holmkvist et al., 2006; Holmkvist et al., 2008; Morita et al., 2015), were determined by real-time polymerase chain reaction (PCR) with 5′-nuclease allele discrimination assays (Step One Plus Real-Time PCR system version 2.1; Applied Biosystems, Tokyo, Japan). Genotyping was performed using commercially available assays (assay IDs: C_7474231_10, C_5478_10, C_7474214_10 and C_1263617_10 for rs1169288, rs1800574, rs1183910 and rs2464196, respectively), according to the manufacturer’s protocol. Pairwise linkage disequilibrium was evaluated using the SNPAlyze V6.0 Standard software program (Dynacom Co. Ltd, Chiba, Japan), and the haplotype frequencies were estimated by the expectation– maximization algorithm. Student’s t-test or a one-way analysis of variance and Fisher's exact test were used for comparisons of the continuous and categorical variables, respectively. Among the subjects who did not have DR at baseline, the DR-free survival according to the HNF1A variants or haplotypes was estimated using the Kaplan–Meier survival curves, and a comparison of the cumulative incidence among diplotypes was carried out using the generalized Wilcoxon test. The bi-variable and multivariable-adjusted hazard ratios (HRs) were calculated using a Cox proportional hazard model. Among all subjects enrolled in this study, the longitudinal associations of the HNF1A variants or haplotypes with the risks of DR and poor glycemic control, and with the values of clinical parameters, were also analyzed using the generalized estimating equations approach. The independent and autoregressive models were used for the analyses of the risk of DR and poor glycemic control, respectively, with calculations of the odds ratio (OR) and 95% confidence intervals (95% CIs). The autoregressive models were used with calculations of the adjusted partial regression coefficient (Β) and the standard error (SE) for the analysis of clinical parameters (i.e., HbA1c, eGFR, BP and lipid profiles) during the observation period. In all multiple regression models, the following variables were used as covariates: gender, weight status at the baseline, HbA1c, diabetes duration and systolic BP. A value of p b 0.05 was considered to be statistically significant. Multiple comparisons were corrected using Bonferroni’s method when comparing the demographic characteristics between the diplotypes and analyzed after stratifying the subjects by their weight status. In this method, values of p b 0.05/n were considered to be statistically significant after correcting for the number of comparisons made. Statistical power at a significance (alpha) level of 0.05 (two-tailed) based on the sample size of this study was calculated using IBM SPSS SamplePower software (version 2.0). All other statistical analyses were performed using the SPSS software package (version 17.0, IBM Japan Inc., Tokyo, Japan). 3. Results The allele frequencies of the HNF1A rs1169288-C, rs1800574-T, rs1183910-A and rs2464196-A variants were 49.6%, 0%, 44.6% and 53.4%, respectively. Because the rs1800574-T variant was not identified in this population, the association analysis of rs1800574 was not done. The observed genotype frequencies were consistent with the Hardy– Weinberg equilibrium (p N 0.05), and significant linkage disequilibrium was detected among rs1169288, rs1183910 and rs2464196 (p b 0.05). Therefore, the combination of the rs1169288-C, rs1183910-A and rs2464196-A variants (i.e., CAA haplotype) was defined as the H1 haplotype, because every variant comprising the H1 haplotype has been reported to be a risk factor for T2DM by our group and others (Allin & Nordestgaard, 2014; Holmkvist et al., 2006; Holmkvist et al., 2008; Morita et al., 2015). The H1 haplotype frequency was 41.8% (Supplemental Table 1), and the diplotypes were devided into three groups: H1/H1, non-H1/H1 and non-H1/non-H1, which had frequencies of 14.7%, 54.5% and 30.8%, respectively.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Table 1 Clinical characteristics of subjects stratified by the HNF1A diplotypes at baseline.
Female Age (years) Age of diagnosis (years) Diabetes duration (years) BMI (kg/m2) Overweight Fasting plasma glucose (mmol/l) Postprandial plasma glucose (mmol/l) HbA1c (%) HbA1c (mmol/mol) Systolic BP (mmHg) Diastolic BP (mmHg) Triglycerides (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) AST (IU/L) ALT (IU/L) GGT (IU/L) eGFR (ml/min/1.73m2) DR Hypertension Dyslipidemia Ever smoker Alcohol intake Therapy components Hypoglycemic agents Sulfonylureas Biguanides α-Glucosidase inhibitors Thiazolidines Other oral hypoglycemic agents Insulin Antihypertensive agents ACE inhibitors or ARBs Others Agents for hyperlipidemia Fibrates Statins Others
All subjects (n = 354)
H1/H1 (n = 52)
non-H1/H1 (n = 193)
non-H1/non-H1 (n = 109)
p value
125 (35.3) 58.8 ± 11.0 48.0 ± 11.9 10.8 ± 8.3 24.2 ± 3.7 214 (60.5) 9.4 ± 3.4 11.5 ± 4.7 8.7 ± 2.0 71.2 ± 21.9 139.8 ± 19.9 83.0 ± 12.2 1.8 ± 1.7 1.4 ± 0.4 3.2 ± 0.9 27.3 ± 16.0 30.5 ± 25.8 39.6 ± 42.7 84.3 ± 26.7 133 (37.6) 107 (30.2) 107 (30.2) 179 (50.6) 178 (50.3)
22 (42.3) 59.2 ± 9.8 48.4 ± 12.7 10.8 ± 7.1 24.1 ± 3.4 31 (59.6) 9.9 ± 3.7 11.8 ± 4.3 8.9 ± 2.3 73.8 ± 25.4 141.5 ± 20.3 83.8 ± 11.3 1.7 ± 0.8 1.4 ± 0.3 3.3 ± 0.9 27.0 ± 14.1 28.6 ± 21.5 37.1 ± 41.5 83.5 ± 26.1 21 (40.4) 11 (21.2) 15 (28.8) 21 (40.4) 25 (48.1)
63 (32.6) 59.0 ± 11.4 47.6 ± 11.9 11.4 ± 8.7 24.2 ± 3.8 114 (59.1) 9.6 ± 3.8 11.9 ± 4.8 8.9 ± 2.1⁎
73.3 ± 22.8⁎ 139.1 ± 20.1 82.3 ± 12.6 1.9 ± 2.0 1.5 ± 0.4 3.1 ± 0.9 26.2 ± 14.8 28.7 ± 19.9 40.2 ± 46.8 84.7 ± 27.5 79 (40.9) 63 (32.6) 58 (30.1) 100 (51.8) 98 (50.8)
40 (36.7) 58.2 ± 10.8 48.6 ± 11.7 9.7 ± 8.0 24.3 ± 3.5 69 (63.3) 9.0 ± 2.7 10.7 ± 4.8 8.2 ± 1.6 66.4 ± 17.2 140.2 ± 19.5 83.8 ± 12.0 1.7 ± 1.3 1.4 ± 0.4 3.2 ± 0.8 29.2 ± 18.7 34.6 ± 35.1 39.7 ± 35.7 84.0 ± 25.7 33 (30.3) 33 (30.3) 34 (31.2) 58 (53.2) 55 (50.5)
0.41 0.80 0.76 0.21 0.97 0.80 0.25 0.15 0.02 0.02 0.71 0.53 0.62 0.61 0.71 0.32 0.14 0.90 0.95 0.17 0.27 0.96 0.29 0.95
133 (37.6) 77 (21.8) 64 (18.1) 13 (3.7) 20 (5.7) 91 (25.7)
19 (36.5) 14 (26.9) 4 (7.7) 1 (1.9) 5 (9.6) 14 (26.9)
80 (41.5) 38 (19.8) 35 (18.2) 6 (3.1) 6 (3.1) 53 (27.5)
34 (31.2) 25 (22.9) 25 (22.9) 6 (5.5) 9 (8.3) 24 (22.0)
0.21 0.50 0.06 0.53 0.053 0.59
64 (18.1) 91 (25.0)
5 (9.6) 13 (25.0)
41 (21.4) 54 (28.1)
18 (16.5) 24 (22.0)
0.13 0.51
4 (1.1) 25 (7.1) 2 (0.6)
0 (0.0) 6 (11.5) 0 (0.0)
3 (1.6) 11 (5.7) 1 (0.5)
1 (0.9) 8 (7.3) 1 (0.9)
1.00 0.34 1.00
Bold typeface indicates data that are statistically significant (p b 0.05). Data are number (%) or mean ± standard deviation. HNF1A, hepatocyte nuclear factor 1A; BMI, body mass index; HbA1c, hemoglobin A1c; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, γ-glutamyltransferase; eGFR, estimated glomerular filtration rate; DR, diabetic retinopathy; ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker. ⁎ p b 0.025, compared with the non-H1/non-H1 diplotype.
The mean follow-up duration was 5.8 ± 2.5 years. In the demographic characteristics of the subjects at baseline, only the mean values of HbA1c were significantly different among the patients with the different HNF1A diplotypes (Table 1). Each rs1169288-C, rs1183910-A and rs2464196-A variant carrier group also had significantly higher HbA1c levels than the non-carriers (Supplemental Table 2). In addition, the rs1169288-C variant carriers exhibited a significantly longer duration of diabetes than the non-carriers (Supplemental Table 2). On the other hand, the demographic characteristics of the subjects stratified by the weight status at baseline are shown in Supplemental Table 3. The overweight subjects had significantly higher BMIs, postprandial plasma glucose, BP, triglycerides and transaminase levels, and lower age and HDL-C level than the normal-weight subjects. In addition, the overweight subjects had significantly higher frequencies of the use of biganides and antihypertensive agents than the normal-weight subjects. At baseline, 133 (37.6%) patients had DR (Table 1), and 121 and 12 of the patients were examined by direct ophthalmoscopy alone or in combination with fluorescein angiography, respectively. Among all 354 subjects, the H1 haplotype carriers tended to have a higher frequency of DR at baseline than the non-carriers [100 (40.8%) vs. 33 (30.3%), p = 0.07]. On the other hand, rs1183910-A and rs2464196-A
variant carriers had significantly higher frequencies of DR at baseline than the non-carriers (Supplemental Table 2). During the observation period, 49 (13.8%) patients were newly diagnosed with DR by direct ophthalmoscopy, and 11 (3.1%) patients were examined by fluorescein angiography. Among the 221 subjects who did not have DR at baseline, the subjects with the H1/H1 diplotype exhibited a higher incidence of DR than did those with the non-H1/non-H1 diplotype [10 (32.3%) vs. 12 (15.8%), p = 0.03] (Fig. 1a). In a bi-variable Cox proportional hazard model, the H1/H1 diplotype was significantly associated with the incidence of DR, in addition to HbA1c (Table 2). In a multivariable Cox proportional hazard model, HbA1c, but not the H1/H1 diplotype, was found to be an independent risk factor for incidence of DR (Table 2). When the multivariable analyses were performed separately in normal-weight and overweight subjects, the H1/H1 diplotype was significantly associated with incidence for DR in normal-weight, but not in overweight, subjects (Table 3, Fig. 1b and c). The rs1169288-C and rs2464196-A variants also tended to be risk factors for DR in normal-weight subjects, although the associations did not reach statistical significance (Supplemental Table 4). In a longitudinal association analysis, the H1/H1 diplotype was also an independent risk factor for DR only in normal-weight subjects (Table 4). Additionally, the risk of DR significantly increased with a
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
4
K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx Table 2 Association between the risk of DR and the covariates during the observation period (year) in the Cox proportional hazard model. Bi-variable
HNF1A diplotype † Non-H1/H1 H1/H1 Female‡ HbA1c Diabetes duration Systolic BP Overweight§
Multivariable
HR
95% CI
p value
HR
95% CI
p value
1.78 2.75 1.48 1.20 1.00 1.01 1.01
0.90, 3.52 1.19, 6.37 0.82, 2.65 1.07, 1.34 0.96, 1.04 0.998, 1.03 0.57, 1.78
0.10 0.02 0.19 b0.01 0.83 0.10 0.97
1.55 2.32 1.55 1.22 1.02 1.01 0.92
0.77, 3.10 0.97, 5.53 0.84, 2.86 1.08, 1.38 0.97, 1.06 0.999, 1.03 0.50, 1.69
0.22 0.06 0.16 b0.01 0.45 0.06 0.78
Bold typeface indicates data that are statistically significant (p b 0.05). HNF1A, hepatocyte nuclear factor 1A; DR, diabetic retinopathy; HR, hazard ratio; CI, confidence interval; HbA1c, hemoglobin A1c; BP, blood pressure. † Compared with the non-H1/non-H1 diplotype. ‡ Compared with male. § Compared with normal-weight.
with the non-H1/non-H1 diplotype carriers in normal-weight individuals (Table 5). The H1 haplotype carriers were also associated with higher HbA1c values [B 0.70% (7.57 mmol/mol); SE 0.19% (2.02 mmol/mol); p b 0.01] and poor glycemic control (OR 2.52; 95% CI 1.39–4.57; p b 0.01) than the non-carriers, but only in normal-weight subjects. In the normal-weight subjects, each of the variants (rs1169288-C, rs1183910-A and rs2464196-A) also had a significant association with a higher HbA1c value and poor glycemic control (Supplemental Table 5). On the other hand, no association was observed between the HNF1A diplotypes and the longitudinal changes in the other clinical parameters, i.e., eGFR, BP and lipid profiles, irrespective of the weight status (Supplemental Table 6). The statistical power of the association analyses between the HNF1A haplotypes and the risk for DR ranged from 56% to 64%. The power of the associations in normal-weight and overweight subjects was 53%–78% and 15%–26%, respectively. All of the values were below the necessary limit of power (i.e., 80%) to predict the development of disease, indicating the necessity of a larger sample size to confirm the importance of these findings. 4. Discussion To the best of our knowledge, this is the first report to show that the common HNF1A H1/H1 diplotype is associated with the development of DR in normal-weight, but not in overweight, patients with T2DM. The H1 haplotype/diplotype or variants were also associated with poor glycemic control only in normal-weight patients, confirming our previous findings (Morita et al., 2015). Table 3 Association between the risk of DR and the covariates stratified by the weight status during the observation period (year) in the multivariable Cox proportional hazard model.
Fig. 1. Kaplan–Meier curves for the DR-free survival among all subjects (a), normal-weight subjects (b) and overweight subjects (c) according to the HNF1A diplotypes. And comparison of the cumulative incidence among diplotypes was carried out using the generalized Wilcoxon test. An overweight status was defined as a BMI ≥ 23 kg/m2.
longer duration of diabetes or an increase in the systolic BP, irrespective of the weight status (Table 4). During the observation period, the H1/H1 and non-H1/H1 diplotypes carriers had a higher HbA1c values and poor glycemic control compared
HNF1A diplotype † Non-H1/H1 H1/H1 Female‡ HbA1c Diabetes duration Systolic BP
Normal-weight (n = 94)
Overweight (n = 127)
HR
95% CI
p value
HR
95% CI
p value
1.72 6.63 3.10 1.36 0.99 1.01
0.51, 5.79 1.60, 27.53 1.14, 8.42 1.11, 1.67 0.93, 1.06 0.98, 1.03
0.38 b0.01 0.03 b0.01 0.72 0.61
1.28 1.39 1.11 1.17 1.05 1.02
0.53, 3.10 0.41, 4.71 0.48, 2.59 0.97, 1.42 0.98, 1.12 0.997, 1.04
0.58 0.60 0.81 0.09 0.19 0.10
Bold typeface indicates data that are statistically significant (p b 0.025). HNF1A, hepatocyte nuclear factor 1A; DR, diabetic retinopathy; HR, hazard ratio; CI, confidence interval; HbA1c, hemoglobin A1c; BP, blood pressure. † Compared with the non-H1/non-H1 diplotype. ‡ Compared with male.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
variants and the risk of DR could be attributed to poor glycemic control, although further investigation is needed to confirm this. Significant inter-ethnic differences in the HNF1A risk allele frequency have been reported, i.e., the frequencies of DR-related variants, rs1183910-A and rs2464196-A, in this study population (44.6% and 53.4%, respectively) were similar to those in Han Chinese (44.4% and 52.3%, respectively) and are higher than those in Europeans (29.2% and 29.6%, respectively), according to the International HapMap project (http://www.ncbi.nlm.nih.gov/projects/SNP/). Asians with T2DM are often lean compared to Caucasians, and predominantly exhibit IIS and onset at a relatively lower BMI (Chan et al., 2009; Cho, Lee, Park, & Nho, 2012; Morimoto et al., 2013; Ramachandran et al., 2010; Yamakawa-Kobayashi et al., 2012; Yu, Hu, & Jia, 2012). On the other hand, most of the susceptibility loci for T2DM identified through GWAS to date are considered to be likely to affect insulin secretion and to accumulate in normal-weight patients (Cho et al., 2012; Kahn, 2003; Murea et al., 2012; Parra et al., 2011; Ramachandran et al., 2010; Voight et al., 2010; Yamakawa-Kobayashi et al., 2012; Yu et al., 2012). In fact, an increased cumulative number of risk variants identified in a GWAS was associated with an increased risk of T2DM only in normal-weight individuals in Japanese (Yamakawa-Kobayashi et al., 2012). These facts suggest that the genetic architecture of T2DM may be different in obese/ overweight and normal-weight individuals and that the variants of genes involved in insulin secretion, e.g., HNF1A, would contribute to the development of T2DM and complications in normal-weight East Asians. In this study, the duration of diabetes and systolic BP were also found to be independent risk factors for DR in a longitudinal association analysis, irrespective of the weight status (Table 4). These results may support the previous findings that the duration of diabetes and hypertension are the primary risk factors for DR (Chan et al., 2010; Ola et al., 2012; Simo-Servat et al., 2013; Stitt et al., 2013; Yau et al., 2012). Additionally, overweight subjects had worse glycemic and metabolic profiles than normal-weight subjects in the present study (Supplemental Table 3). It is speculated that the effect of these factors on the risk for DR may be more pronounced in overweight subjects than in normal-weight subjects, and thus the HNF1A diplotype may affect the risk of DR only in normal-weight subjects. There are some limitations associated with the present study. First, this study was a retrospective investigation of a small number of patients, as shown by the sample power analysis. Because the power of the samples in the analyses regarding the effects of the HNF1A variants or haplotypes on the risk for DR was small in normal-weight and overweight subjects, large-scaled studies are necessary to confirm the current findings. Second, the number of the patients diagnosed using direct ophthalmoscopy with fluorescein angiography was also small (n = 23); therefore, further investigations are needed to confirm the effect of the HNF1A common variants on the severity of DR. Third, this study could not collect information regarding the plasma insulin and
Table 4 Association between the risk of DR and the covariates stratified by the weight status during the observation period (year) in the independent logistic regression models.
HNF1A diplotype† Non-H1/H1 H1/H1 Female‡ HbA1c Diabetes duration Systolic BP
Normal-weight (n = 140)
Overweight (n = 214)
OR
95% CI
p value
OR
95% CI
p value
2.27 4.08 1.95 1.23 1.07 1.02
0.94, 5.48 1.23, 13.55 0.96, 3.96 1.02, 1.47 1.02, 1.12 1.003, 1.03
0.07 0.02 0.07 0.03 b0.01 0.02
1.67 1.37 1.41 1.14 1.20 1.02
0.79, 3.54 0.55, 3.39 0.71, 2.78 0.96, 1.35 1.13, 1.27 1.01, 1.03
0.18 0.50 0.32 0.15 b0.01 b0.01
5
Bold typeface indicates data that are statistically significant (p b 0.025). HNF1A, hepatocyte nuclear factor 1A; DR, diabetic retinopathy; HR, hazard ratio; CI, confidence interval; HbA1c, hemoglobin A1c; BP, blood pressure. † Compared with the non-H1/non-H1 diplotype. ‡ Compared with male.
A number of susceptibility loci for DR have been reported with inconsistent results (Kuo, Wong, & Rotter, 2014; Murea, Ma, & Freedman, 2012; Simo-Servat, Hernandez, & Simo, 2013; Stitt, Lois, Medina, Adamson, & Curtis, 2013), and no variant related to hyperglycemia has been identified as a risk factor for DR to date. On the other hand, MODY3 is characterized by a defect in insulin secretion and a high prevalence of DR (Fajans et al., 2001; Isomaa et al., 1998; Skupien et al., 2008). Hyperglycemia was indicated to be the main predictor of DR in MODY3 patients (Isomaa et al., 1998). In a Polish study, 47.7% of MODY3 patients had DR and they also had significantly higher HbA1c levels than those without DR (Skupien et al., 2008). Furthermore, there is strong evidence that initial tight glycemic control soon after the diagnosis of diabetes delays the natural process of the diabetes, reducing the development and progression of DR (Chan, Kanwar, & Kowluru, 2010; Hammes, Feng, Pfister, & Brownlee, 2011; Inzucchi et al., 2012; Massin et al., 2011; Ola, Nawaz, Siddiquei, Al-Amro, & Abu El-Asrar, 2012; Sabanayagam et al., 2014; Stitt et al., 2013; Tsugawa et al., 2012; Xu & Weng, 2013). The HbA1c and fasting plasma glucose levels predicted the presence of DR 10 years after the diagnosis, while hypertension and the lipid profiles did not (Massin et al., 2011). A previous longitudinal Japanese study determined the threshold HbA1c level for the development of DR to be 6.5% (Tsugawa et al., 2012). In this study, the HNF1A H1/H1 diplotype was significantly associated with a higher prevalence of DR (Tables 2-4). In the H1 haplotype carriers, the HbA1c values were significantly higher at baseline (Table 1), and the higher HbA1c values were also observed throughout the observation period (Table 5), however, none of the other clinical parameters differed between the H1 haplotype carriers and non-carriers (Table 1 and Supplemental Table 6). These results indicate that our present findings regarding the associations between the common HNF1A diplotype or
Table 5 The combined effects of the HNF1A diplotypes and the weight status at baseline on the longitudinal change of glycemic control during the observation period (year) in the independent logistic regression models. Weight status
Normal-weight
Overweight
HNF1A diplotypes Non-H1/non-H1 (reference) Non-H1/H1 H1/H1 Non-H1/non-H1 (reference) Non-H1/H1 H1/H1
Poor glycemic control†
n
‡
HbA1c (%)
95% CI
p value
Β
‡
HbA1c (mmol/mol) SE
p value
Β‡
SE
p value
40
1
–
–
0
–
–
0
–
–
79 21 69
2.41 3.03 1
1.31, 4.44 1.28, 7.17 –
b0.01 0.01 –
0.67 0.80 0
0.20 0.28 –
b0.01 b0.01 –
7.29 8.61 0
2.19 3.07 –
b0.01 b0.01 –
114 31
1.08 1.75
0.71, 1.65 1.02, 3.02
0.73 0.04
0.16 0.55
0.17 0.23
0.34 0.02
1.72 5.96
1.80 2.47
0.34 0.02
OR
Bold typeface indicates data that are statistically significant (p b 0.025). HNF1A, hepatocyte nuclear factor 1A; HbA1c, hemoglobin A1c; OR, odds ratio; CI, confidence interval; B, partial regression coefficient; SE, standard error. † HbA1c ≥ 8.0% (64.0 mmol/mol). ‡ Adjusted by gender, duration of diabetes and systolic BP.
Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007
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K. Morita et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
C-reactive protein levels. Lastly, studying only four variants may not cover the entire genetic variation in the HNF1A region. 5. Conclusion We herein demonstrated that the common HNF1A diplotype or variants are independent risk factors for poor glycemic control and for the subsequent development of DR in normal-weight Japanese patients with T2DM, but these results need to be replicated in larger and more varied study populations. T2DM in East Asians is characterized by a low BMI, onset at a relatively young age and a rapidly rising prevalence (Chan et al., 2009; Cho et al., 2012; Morimoto et al., 2013; Ramachandran et al., 2010; Yu et al., 2012). These distinctive features may be attributable to the ethnicity-specific genetic differences underlying the pathophysiology of T2DM (Tremblay & Hamet, 2015; Yu et al., 2012). Although the present study was exploratory in nature and further investigations in larger cohorts of other ethnic groups are needed to verify the results, the present study suggests that East Asians should be warned about the likely increase in DR in the near future, following the rapidly increasing prevalence of T2DM (Chan et al., 2009; Cho et al., 2012; Ramachandran et al., 2010; Yau et al., 2012). Acknowledgments The authors wish to thank all of the study participants. This study was supported by KAKENHI (No. 23510348). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jdiacomp.2016.06.007. References Allin, K. H., & Nordestgaard, B. G. (2014). Pleiotropic effects of HNF1A rs1183910 in a population-based study of 60,283 individuals. Diabetologia, 57(4), 729–737. Chan, P. S., Kanwar, M., & Kowluru, R. A. (2010). Resistance of retinal inflammatory mediators to suppress after reinstitution of good glycemic control: Novel mechanism for metabolic memory. Journal of Diabetes and its Complications, 24(1), 55–63. Chan, J. C., Malik, V., Jia, W., Kadowaki, T., Yajnik, C. S., Yoon, K. H., et al. (2009). Diabetes in Asia: Epidemiology, risk factors, and pathophysiology. JAMA, 301(20), 2129–2140. Chiu, K. C., Chuang, L. M., Chu, A., & Wang, M. (2003). Transcription factor 1 and beta-cell function in glucose-tolerant subjects. Diabetic Medicine, 20(3), 225–230. Cho, Y. S., Lee, J. Y., Park, K. S., & Nho, C. W. (2012). Genetics of type 2 diabetes in East Asian populations. Current Diabetes Reports, 12(6), 686–696. Fajans, S. S., Bell, G. I., & Polonsky, K. S. (2001). Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. The New England Journal of Medicine, 345(13), 971–980. Hammes, H. P., Feng, Y., Pfister, F., & Brownlee, M. (2011). Diabetic retinopathy: Targeting vasoregression. Diabetes, 60(1), 9–16. Holmkvist, J., Almgren, P., Lyssenko, V., Lindgren, C. M., Eriksson, K. F., Isomaa, B., et al. (2008). Common variants in maturity-onset diabetes of the young genes and future risk of type 2 diabetes. Diabetes, 57(6), 1738–1744. Holmkvist, J., Cervin, C., Lyssenko, V., Winckler, W., Anevski, D., Cilio, C., et al. (2006). Common variants in HNF-1 alpha and risk of type 2 diabetes. Diabetologia, 49(12), 2882–2891. Hsu, W. C., Araneta, M. R., Kanaya, A. M., Chiang, J. L., & Fujimoto, W. (2015). BMI cut points to identify at-risk Asian Americans for type 2 diabetes screening. Diabetes Care, 38(1), 150–158. Inzucchi, S. E., Bergenstal, R. M., Buse, J. B., Diamant, M., Ferrannini, E., Nauck, M., et al. (2012). Management of hyperglycaemia in type 2 diabetes: A patient-centered approach. Position statement of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetologia, 55(6), 1577–1596. Isomaa, B., Henricsson, M., Lehto, M., Forsblom, C., Karanko, S., Sarelin, L., et al. (1998). Chronic diabetic complications in patients with MODY3 diabetes. Diabetologia, 41(4), 467–473. Iwasaki, N., Oda, N., Ogata, M., Hara, M., Hinokio, Y., Oda, Y., et al. (1997). Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with earlyand late-onset NIDDM. Diabetes, 46(9), 1504–1508.
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Please cite this article as: Morita, K., et al., Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subject..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.06.007