This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (
The study aimed to elucidate whether the polymorphisms of the aldose reductase regulatory gene were risk factors for Diabetic Retinopathy (DR) in type-2 diabetes mellitus (T2DM) patients in Bali.
This is a case-control study including 35 cases of T2DM patients with DR paired with 35 cases with non-DR as controls. PCR analysis and DNA-sequencing were carried out to detect the C(-106)T and C(-12)G polymorphisms at the regulatory region of Aldose Reductase (ALR2) gene. Genotype and allele distributions were analyzed by Chi-squared test and independent t-and Mann-Whitney U tests were used to analyze other data.
Among all subjects in both groups, the baseline characteristics were homogenous except for systolic blood pressure, fasting blood glucose and 2-hours post-prandial blood glucose. This study found two polymorphisms, C(-104)T and C(-9)G, in the regulatory region of ALR2 gene. The result showed that the C(-104)T polymorphism was a risk factor for DR (OR=36; 95% CI = 4.43-292.85;
It can be concluded that C(-104)T polymorphism in the regulatory region of Aldose Reductase (ALR2) gene was the risk factor for DR among T2DM patients in Bali, Indonesia. However, small sample size, systolic blood pressure, fasting blood glucose and 2-hours post-prandial blood glucose could affect our finding.
The incidence of diabetes mellitus, especially Type-2 Diabetes Mellitus (T2DM) increases not only in developed countries but also in developing countries. Indonesia as one of a developing country has been estimated to be the fourth country with the highest number of diabetic cases in 2030. Various efforts have been attempted to overcome this problem, including new drugs development as well as changes in dietary pattern and lifestyle, but the number of cases is still increasing [
Type-2 DM is a multifactorial disease with micro and macro-vascular complications. The commonest microvascular complication in DM is Diabetic Retinopathy (DR) and the leading cause of blindness among diabetic patient in the productive age. DR can occur in the early stage of the disease in diabetic patients with well-controlled blood sugar level, while other patients who have diabetes for many years, do not complicate with DR. This suggests that the occurrence of DR cannot be explained based on the hyperglycaemic state alone. Several evidences have shown that complications of DM are also affected by genetic factors [
Aldose Reductase (ALR2) is a rate-limiting enzyme in the polyol pathway. This enzyme catalyzes the reduction of carbonyl group into their alcohol product, converts D-glucose into sorbitol and galactose into galactycol [
Aldose Reductase (ALR2) gene polymorphism (C→T) at position -106 of the promoter region has been reported previously [
This study was conducted based on the polymorphic characteristics of ALR2 gene in the regulatory region. Currently, studies on ALR2 polymorphisms, expression, and haplotype identification associated with microvascular complications of T2DM in Indonesia are still limited.Therefore, the primary objective of this study was to determine whether polymorphisms at the regulatory region of ALR2 gene were risk factors for DR in T2DM patients in Bali, Indonesia.
A total of 70 subjects (35 T2DM patients with DR and 35 T2DM without DR) with Balinese ethnic were enrolled from Sanglah General Hospital, Public Health Care Center at the West and East Denpasar between February 2013 and February 2014. The sample size was calculated using the formula for case study control for hypothesis test against Odds Ratio [
Body Mass Index (BMI) was calculated as body weight in kilograms divided by the square of height in meters. Body weight was measured using a digital scale, while body height was assessed using stature meter. Waist Circumference (WC) was measured using flexible non-elastic tape precisely at the middle level of the abdomen. Systolic and diastolic blood pressures were measured in a sitting position for two times using a mercury sphygmomanometer.
Fasting and 2 hours after meal blood samples were collected and separated into two vacutainer tubes with EDTA. One tube was used to examine blood glucose level and HbA1C, while the other was stored at -20oC for DNA isolation and Polymerase Chain Reaction (PCR) analysis. The study protocol has been approved by the Ethics Commission of the Faculty of Medicine, Udayana University/Sanglah General Hospital (No. 759/UN.14.2/Litbang/2012), and all subjects were provided with written informed consent.
A case-control study was designed, to elucidate whether the ALR2 polymorphisms were risk factors for DR in T2DM patients in Bali, Indonesia.
PCR analysis followed by DNA sequencing was used to detect polymorphisms. Genotyping of C(-106)T polymorphism of the ALR2 gene was performed using primer pair 5’-TTC GCT TTC CCA CCA GAT-3’ between nucleotide -225 and -235 and 5’CGC CGT TGT TGA GCA CGA GAC-3’ between nucleotide 50 and 71 that amplify a 326 base pair fragment. The PCR conditions consisted of pre-denaturation at 95o C for 2 minutes, followed by 35 cycles of denaturation at 95oC for 60 seconds, annealing at 53oC for 60 seconds, and extension at 72oC for 60 seconds. The final extension was at 72oC for 7 minutes [
Genotyping of ALR2 C(-12)G polymorphism was performed using primer pair 5'-CGC TAA AGC TTT CGC TTT CCC ACC AGA TAC AGC-3' and 5'-ATG GCT GCA GCG CTC CCC AGA CCC CCG CCC AGT-3'. The PCR conditions were as follows: denaturation at 95o C for 2 minutes, followed by 35 cycles of denaturation at 95 o C for 60 seconds, annealing at 55oC for 60 seconds, and a final extension at 72oC for 60 seconds. The final extension was at 72oC for 1 minute [
All PCR products were then purified and sequenced. Polymorphisms were analyzed based on the electropherograms, confirmed by BLAST with the reference sequence in gene bank (sequence ID:
Kolmogorov–Smirnov Test was used to evaluate the normality of variables and normally distributed values were expressed as a mean ± standard deviation. Genotype and allele distributions were analyzed using Chi-square test, while other data were analyzed using independent t- and Mann-Whitney U tests with
Thirty-five subjects with DR (cases) and 35 subjects without DR (control) were chosen randomly in this study. As shown in Table
Parameter | DR |
Non-DR |
|
---|---|---|---|
Sex | – | – | – |
Male (n) | 16 | 21 | 0.231 |
Female (n) | 19 | 14 | – |
Family History of DM | – | – | – |
Yes (n) | 21 | 25 | 0.314 |
No (n) | 14 | 10 | – |
Age (years) | 50.60±7.33 | 52.86±8.47 | 0.237 |
Duration of DM (years) | 5.70±3.56 | 5.79±3.87 | 0.995 |
Systolic BP (mmHg) | 132.43±13.69 | 125.14±11.91 | |
Diastolic BP (mmHg) | 84.57±7.41 | 81.71±7.17 | 0.096 |
Waist Circumference (cm) | 89.83±10.32 | 89.12±10.06 | 0.772 |
BMI (kg/m2) | 24.50±3.82 | 24.56±3.99 | 0.986 |
FBG (mg/dL) | 183.17±69.65 | 146.23±48.81 | |
2hppBG (mg/dL) | 267.89±103.34 | 217.94±93.98 | |
HBA1c | 9.04± 2.33 | 8.45±2.62 | 0.147 |
The genotyping result of C(-106)T and C(-12)G polymorphisms at the regulatory region of ALR2 gene was determined by DNA sequencing as shown in Fig. (
SNPs | Genotype | DR | Non-DR |
---|---|---|---|
C(-104)T | CC | 17 (48.57) | 34 (97.14) |
– | CT | 18 (51.43) | 1 (2.86) |
C(-9)G | CC | 19 (54.29) | 22 (62.86) |
– | CG | 14 (40.00) | 12 (34.29) |
– | GG | 2 (5.71) | 1 (2.86) |
Based on genotypic distribution analysis, both C(-104)T and C(-9)G polymorphisms showed no significant departure from Hardy-Weinberg Equilibrium (HWE) with
Chi-square test was used to determine the role of C(-104)T and C(-9)G polymorphisms as risk factors for DR in T2DM patients. Table
Variable | Group | OR | 95% CI |
|
||
---|---|---|---|---|---|---|
DR
|
Non-DR
|
Lower | Upper | |||
C(-104)T | 18 (51.43) | 1 (2.86) | 36 | 4.43 | 292.85 | |
C(-9)G | 14 (40.00) | 12 (34.28) | 1.28 | 0.48 | 3.38 | 0.621 |
Haplotype | – | – | – | – | – | – |
CC/CC | 8 (22.85) | 21 (60.00) | 0.19 | 0.07 | 0.56 | |
CC/CG | 6 (17.14) | 12 (34.28) | 0.39 | 0.13 | 1.28 | 0.101 |
CC/GG | 2 (5.71) | 1 (2.86) | 2.06 | 0.18 | 23.83 | 0.555 |
CT/CC | 11 (31.40) | 1 (2.86) | 15.58 | 1.88 | 128.89 | |
CT/CG | 8 (22.85) | 0 | 2.29 | 1.73 | 3.05 |
There are five haplotypes found in this study: CC/CC, CC/CG, CC/GG, CT/CC and CT/CG. As shown in Table
In this study, we found two new polymorphisms, the C-(-104)T dan C(-9)G at the regulatory region of ALR2 gene that has not been reported previously. Previous studies conducted in Australia, Chinese, Japanese, and Indonesian population found a C(-106)T polymorphism, which was reported to be correlated with DR [
Another ALR2 gene polymorphism, the C(-12)G also located at the regulatory region, was reported to be associated with DR in 123 patients with DR and 145 without DR in Chinese population [
Glycemic control, blood pressure, and duration of DM are important risk factors for DR. Thomas
In our study, we observed that C(-104)T in the regulatory region of ALR2 gene is a risk factor for DR in T2DM patients in Bali. The promoter region of ALR2 gene contains TATA box and CCAAT promoter elements showed significant transcription activities, located between +31 and -609 bases (640 bp). Cloning of this 640 bp fragment showed that deletion of -609 until -186 bases did not affect transcriptional activities of ALR2 gene [
Our study identified five haplotypes, CC/CC, CC/CG, CC/GG, CT/CC and CT/CG, among which CC/CC haplotype was a protective factor (OR=0.198,
In the present study, we used a smaller number of samples compared to other studies. This number of samples was determined based on the low prevalence rate of polymorphism in our previous pilot study. We acknowledge that the limitation of our study was a small number of samples that may affect the result.
In this study, we found two polymorphisms C(-104)T and C(-9)G, among which C(-104)T was a risk factor for DR in T2DM patients in Bali, Indonesia while C(-9)G was not. Five haplotypes were found,
The study protocol has been approved by the Ethics Commission of the Faculty of Medicine, Udayana University/Sanglah General Hospital (No. 759/UN.14.2/Litbang/2012).
No Animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2008.
Written informed consent was obtained from all the participants.
The authors declare no conflict of interest, financial or otherwise.
The authors were grateful to all patients and participants that were involved in this study. This work was supported by grants from the High Professional Education Project (HPEQ), The Ministry of Education and Culture, Republic of Indonesia, contract number: 14/UN.14.2/PHK-PKPD/SPK/X/2012.