Identification of KCNJ15 as a susceptibility gene in Asian patients with type 2 diabetes mellitus.

Recent advances in genome research have enabled the identification of new genomic variations that are associated with type 2 diabetes mellitus (T2DM). Via fine mapping of SNPs in a candidate region of chromosome 21q, the current study identifies potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15) as a new T2DM susceptibility gene. KCNJ15 is expressed in the beta cell of the pancreas, and a synonymous SNP, rs3746876, in exon 4 (C566T) of this gene, with T allele frequency among control subjects of 3.1%, showed a significant association with T2DM affecting lean individuals in three independent Japanese sample sets (p = 2.5 x 10(-7), odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.76-3.67) and with unstratified T2DM (p = 6.7 x 10(-6), OR = 1.76, 95% CI = 1.37-2.25). The diabetes risk allele frequency was, however, very low among Europeans in whom no association between this variant and T2DM could be shown. Functional analysis in human embryonic kidney 293 cells demonstrated that the risk allele of the synonymous SNP in exon 4 increased KCNJ15 expression via increased mRNA stability, which resulted in the higher expression of protein as compared to that of the nonrisk allele. We also showed that KCNJ15 is expressed in human pancreatic beta cells. In conclusion, we demonstrated a significant association between a synonymous variant in KCNJ15 and T2DM in lean Japanese patients with T2DM, suggesting that KCNJ15 is a previously unreported susceptibility gene for T2DM among Asians.

Genetic polymorphisms in organic cation transporter 1 (OCT1) in Chinese and Japanese populations exhibit altered function.

Organic cation transporter 1 (OCT1; SLC22A1) seems to play a role in the efficacy and disposition of the widely used antidiabetic drug metformin. Genetic variants in OCT1 have been identified largely in European populations. Metformin is increasingly being used in Asian populations where the incidence of type 2 diabetes (T2D) is on the rise. The goal of this study is to identify genetic variants of OCT1 in Chinese and Japanese populations, which may potentially modulate response to metformin. We used recent data from the 1000 Genomes Project (Chinese and Japanese) and direct sequencing of selected amplicons of OCT1 in 66 DNA samples from Japanese patients with T2D. A total of six nonsynonymous variants were identified. Three of them (Q97K, P117L, and R206C) had not been functionally characterized previously and had allele frequencies of 0.017, 0.023 and 0.008, respectively. The uptake of metformin in cells expressing Q97K, P117L, and R206C was significantly reduced relative to the OCT1 reference (62 ± 4.3, 55 ± 6.8, and 22 ± 1.5% for Q97K, P117L, and R206C, respectively). Kinetic studies indicated that P117L and R206C exhibited a reduced V(max), whereas Q97K showed an increased K(m). The green fluorescent protein (GFP)-tagged Q97K and P117L variants localized to the plasma membrane, whereas the GFP-tagged R206C was retained mainly in the endoplasmic reticulum. Replacement of the highly conserved R206 with different amino acids modulated the subcellular localization and function of the transporter. This study suggests that nonsynonymous variants of OCT1 in Chinese and Japanese populations may affect the differential response to metformin.

Impact of variants in the VEGF gene on progression of proliferative diabetic retinopathy.

BACKGROUND: The development of diabetic retinopathy is associated with the duration of diabetes and HbA1c levels. However, the familial aggregation of diabetic retinopathy is consistent with genetic susceptibility. Recently, a -634C/G polymorphism in the vascular endothelial growth factor (VEGF) gene was shown to be associated with diabetic retinopathy. To clarify the contribution of the VEGF gene in the development of diabetic retinopathy we analyzed variants in this gene among 469 Japanese patients with type 2 diabetes. METHODS: DNA from each patient was typed for -634C/G and -2578C/A polymorphisms using conventional polymerase chain reaction techniques. The vitreous fluid samples were obtained from 40 patients with PDR for measurement of VEGF levels. RESULTS: We found a significantly higher frequency of the A allele in the group with proliferative diabetic retinopathy (PDR) than in the control group at -2578C/A polymorphism (p = 0.036). Moreover, if the subjects were grouped according to the duration of diabetes and status of diabetic retinopathy (a first group consisting of subjects with longer duration (>20 y) of diabetes without any retinopathy (n = 102), and a second group of those with shorter diabetes (<15 y) but having retinopathy (n = 35), the genotype distribution at -2578 C/A polymorphism was again significantly higher in the second group (p = 0.005) and differed significantly (p = 0.002) in a recessive model. The risk of the AA for PDR was 7.7 (95%, CI: 1.8-30.9). CONCLUSIONS: The AA genotype at -2578C/A polymorphism in the VEGF gene is associated with proliferative diabetic retinopathy. No significant association with -634 C/G polymorphism was confirmed.

Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor are related to diabetic macular edema.

PURPOSE: To investigate whether vitreous levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) are related to diabetic macular edema (DME). DESIGN: Retrospective case-control study. PARTICIPANTS: Thirty-six patients with DME, 6 diabetic patients without retinopathy, and 13 patients with nondiabetic ocular disease. METHODS: After vitreous fluid samples were obtained at vitreoretinal surgery, VEGF and PEDF levels in the vitreous fluid were measured by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Correlations between vascular permeability and the vitreous fluid levels of VEGF and PEDF. RESULTS: The vitreous level of VEGF was significantly higher in patients with DME than in nondiabetic patients and diabetic patients without retinopathy (P<0.0001 and P<0.0001, respectively). Conversely, the vitreous level of PEDF was significantly lower in patients with DME than in nondiabetic patients and diabetic patients without retinopathy (P<0.0001 and P<0.0001, respectively). The vitreous level of PEDF did not correlate significantly with that of VEGF (P = 0.1806). The vitreous level of VEGF was significantly higher in patients with hyperfluorescent DME than in those with minimally fluorescent DME (P = 0.0022). Conversely, the vitreous PEDF level was significantly lower in patients with hyperfluorescent DME than in those with minimally fluorescent DME (P = 0.0172). Vitreous levels of VEGF and PEDF were related to the retinal thickness at the central fovea (P<0.0001 and P = 0.0469, respectively). CONCLUSIONS: Our retrospective study suggests that VEGF and PEDF have an independent association with vascular permeability in the eye and on the DME, and we recommend that prospective validation of our findings be undertaken to confirm these observations.

Questionnaire survey on periodic ocular examination in Japanese diabetic patients.

PURPOSE: To identify the reasons why diabetic patients do not undergo periodic examination. DESIGN: Cohort study. METHODS: A questionnaire survey of 1,333 Japanese type 2 diabetic patients was conducted at Tokyo Women's Medical University. Performance of ocular examinations was investigated over 5 years and the reasons for not undergoing examination were analyzed. RESULTS: Periodic ocular examination was performed in 69.5% of patients, but not in 30.5%. The questionnaire survey showed that physicians usually explained the risk of ocular complications and recommended periodic examination. More than 98% of the patients were aware of diabetic eye disease. Multivariate logistic regression analysis showed the main reason for skipping examination as a reason was that patients believed they had no diabetic retinopathy. CONCLUSIONS: Patients should be informed about the necessity of receiving periodic ocular examination even if they do not yet have retinopathy.

Quantitative measurement of retinal thickness in patients with diabetic macular edema is useful for evaluation of therapeutic agents.

The effect of lisinopril (an angiotensin-converting enzyme inhibitor) on diabetic macular edema (DME) was investigated by quantitative measurement of macular thickness. In a nonrandomized clinical trial, 19 normotensive type 2 diabetic patients with DME prospectively received oral lisinopril therapy for 2 months. Another 10 normotensive type 2 diabetic patients with similar DME were prospectively followed for two months without treatment. Central macular thickness was measured with a retinal thickness analyzer (RTA). In the lisinopril group, visual acuity improved by two lines or more in two out of 19 eyes (11%), was unchanged in 15 eyes (78%), and deteriorated by two lines or more in two eyes (11%). The mean central macular thickness was significantly reduced after 2 months of treatment (381.3 +/- 121.1 microm) compared with that before administration (475.2 +/- 171.0 microm, P = 0.0093). In the control group, central macular thickness was not significantly decreased after 2 months (458.5 +/- 113.7 microm, P = 0.2178) compared with the baseline value (464.7 +/- 152.2). Fluorescein angiography showed that macular leakage was decreased in 10 patients from the lisinopril group (53%) and was unchanged in nine patients (47%). There was a significant difference of central macular thickness between the patients with and without improvement of macular leakage (P = 0.0040). Lisinopril therapy may reduce macular thickness in patients with DME, as shown by this quantitative study. In addition, quantitative measurement of retinal thickness is useful when evaluating therapeutic agents for DME.

[Attitude survey of diabetic patients visiting the department of internal medicine as outpatients].

PURPOSE: To investigate the awareness and understanding of ophthalmologic complications by diabetic patients and assess the problems. SUBJECTS AND METHODS: In August 1999, a questionnaire survey was done of 3,613 diabetic outpatients attending the Department of Internal Medicine at Tokyo Women's Medical University Diabetes Center. RESULTS: Patients who understood diabetic retinopathy as ophthalmological complications were 54.4% and those to whom physicians explained the complications were 33.8%. Patients who were recommended to receive an eye examination by physicians were 66.4% and those who continued to attend the eye clinic were 61.8%. Patients who had ophthalmological complications explained by physicians were 74.2%. CONCLUSION: Few patients understood diabetic retinopathy as a diabetic ophthalmological complication. To encourage the awareness and understanding concerning diabetic retinopathy, it is important to form a tight relationship between physicians and ophthalmologists and to improve the system for educating patients.

Aqueous humor levels of cytokines are related to vitreous levels and progression of diabetic retinopathy in diabetic patients.

BACKGROUND: Cytokine levels are elevated in the ocular fluid of diabetic patients. It is unclear whether aqueous humor levels of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) are related to the vitreous fluid levels of these substances and to the progression of diabetic retinopathy. METHODS: Aqueous humor and vitreous fluid samples were obtained during cataract and vitreous surgery from ten eyes of ten patients with diabetic macular edema and 26 eyes of 26 patients with proliferative diabetic retinopathy (PDR). The VEGF and IL-6 levels in aqueous humor, vitreous fluid, and plasma were measured by enzyme-linked immunosorbent assay. RESULTS: VEGF and IL-6 levels in aqueous humor were significantly correlated with those in vitreous fluid (rho=0.793 and rho=0.737, respectively). VEGF levels in aqueous humor and vitreous fluid were significantly correlated with the corresponding IL-6 levels (rho=0.631 and rho=0.687, respectively). The aqueous and vitreous levels of VEGF were significantly correlated with the severity of diabetic retinopathy (rho=0.659 and rho=0.771, respectively). Aqueous and vitreous levels of IL-6 were also significantly correlated with the severity of diabetic retinopathy (rho=0.742 and rho=0.746, respectively). Aqueous and vitreous levels of both VEGF and IL-6 were significantly higher in the patients with active PDR than those in quiescent PDR. CONCLUSIONS: Our results suggest that there is a significant relationship between VEGF and IL-6 levels in aqueous humor and in vitreous fluid. Measurement of the aqueous levels of VEGF and IL-6 may be useful to analyze the pathogenesis of diabetic retinopathy and to predict disease activity.