Aldose reductase (AC)n gene polymorphism in Iranian patients with type 2 diabetic microangiopathy; a case–control study

Abstract

Aim

(AC)n promoter region of the aldose reductase (ALR) genes polymorphism has been associated with diabetic microvascular complications (MVCs). The aim of this study was to find the relationship between dinucleotide repeat (AC)n polymorphisms of the ALR gene and the occurrence of MVCs, such as diabetic retinopathy, neuropathy, and nephropathy in Iranian type 2 diabetic (T2D) patients.

Methods

This prospective case–control study was performed on T2D patients who were categorized into two groups based on the presence or absence of diabetic microangiopathy. All patients were provided informed consent. After extracting genomic DNA, the (AC)n of the ALR gene was determined using Polymerase chain reaction (PCR).

Results

Thirteen alleles of the (AC)n gene polymorphism were detected including Z + 16, Z + 14, Z + 8, Z + 6, Z + 4, Z + 2, Z, Z − 2, Z − 4, Z − 6, Z − 8, Z − 10, and Z − 12. The frequency of the Z − 4 allele was significantly higher in patients with retinopathy, nephropathy, and autonomic neuropathy compared with those with long-term uncomplicated diabetes (P < 0.001, P < 0.001, P = 0.031, respectively). After controlling for baseline risk factors, we found that the carrier of the Z − 4 allele of ALR (AC)n polymorphism had a higher risk of diabetic retinopathy and diabetic nephropathy (P < 0.001). The homozygosity for the Z − 4 allele was found to be associated with diabetic microangiopathy.

Conclusion

Our results showed that ALR (AC)n gene polymorphism in Iranian patients with type 2 diabetes independently, predispose retinal, renal and neural microvascular to diabetic complications.

Introduction

International federation of diabetes rated the Middle East and North Africa (MENA) with the second-highest worldwide prevalence of diabetes at 9.2% [1].

According to the fourth round of the Iranian periodic national survey of risk factors for non-communicable diseases project in 2011, 11.4% of the adult population suffers from diabetes and it is estimated that by the year 2030, this prevalence will grow up to 9.2 million individuals [2, 3].

Diabetic microangiopathy is specified as a microvascular disorder accounts for the serious microvascular complications (MVCs) of diabetes mellitus [4]. According to reports, around 21.9, 17.6, 28.0, and 6.2% of patients with diabetes suffer from diabetic microangiopathy including retinopathy, nephropathy, peripheral neuropathy, and diabetic foot, respectively [3].

Many studies on pedigree have demonstrated that genetic susceptibility affects the evolution of these complications [5–8]. Furthermore, many biochemical mechanisms have described the structural and functional abnormalities associated with the overexposure of the microvascular tissues to hyperglycemia including advanced glycation end products, an increase in aldose reductase activity, free radicals’ accumulation and protein kinase C activation. Although disease duration is the main predictive factor, an associated condition including glycemic control, blood pressure and genetic susceptibility especially in the polyol pathway may modify the development of diabetic MVCs [9, 10].

Aldose reductase (ALR) is the first and rate-limiting enzyme of the polyol pathway, and its gene coding is a candidate gene for diabetic MVCs [11]. DNA sequence differences in the ALR locus may cause increased expression of the gene and thereby, increase the risk of diabetic complications [12]. An (AC)n dinucleotide repeat polymorphism in the promoter region has been identified in the ALR gene [13].

The most common allele has been designated as Z, which consists of (AC) 24 repeats, whilst, the Z − 2 allele and conversely the Z + 2 allele contain (AC) 23 and (AC) 25 repeats, respectively. The Z − 2 allele of the (AC)n repeat marker has been linked as the most common risk allele for diabetic nephropathy, retinopathy, and neuropathy. However, some reports revealed negative results therefore, its association is still controversial [14–16]

Hence, this study was designed to provide information on (AC)n dinucleotide repeat polymorphism at the 5′-end of the aldose reductase gene in type 2 diabetic (T2D) patients with MVCs in Iran.

Materials and methods

This prospective case–control study was conducted with a focus on diabetic microangiopathy. Individuals with T2D whose ages ranged between 35 and 70 years old were enrolled from the eye clinic and the diabetes outpatient clinic of the Diabetes Research Centre in Sari, Iran between December 2014 and November 2018. Exclusion criteria include the duration of diabetes less than 5 years, a positive history of eye, kidney or neurological diseases unrelated to diabetes and patients’ unwillingness to participate in the study.

Cases were T2D patients who had any of the MVCs. With the goal of a thorough comparison, a secondary control group of 114 nondiabetic individuals was also obtained in the study as a secondary target. Informed consent was obtained from all participants and the research protocol was approved by Mazandaran University of Medical Sciences Ethical Committee (IR.MAZUMS.REC.1394.1514). Also, this study was designed according to the Declaration of Helsinki.

The diagnosis of T2D was based on the World Health Organization/American Diabetes Association description of diabetes [17]. Diabetic microangiopathy is specified as a small vessel disorder. The intended microangiopathies were categorized into diabetic retinopathy, nephropathy, and neuropathy [18].

The presence of diabetic retinopathy was determined by dilated ophthalmic examination and fundus fluorescein angiography, which was performed by an expert ophthalmologist. Diabetic retinopathy was classified as non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) [19, 20].

Diabetic nephropathy was determined by assessing two separate 24-h urine collection samples. Nephropathy was classified as an albumin excretion rate of 30–300 mg/l/24 h as microalbuminuria and > 300 mg/l/24 h as gross albuminuria in a timed urine collection after excluding urinary tract infection [21].

Clinical evaluation for peripheral neuropathy was carried out by neurophysiologic studies with nerve conduction velocity (NCV). The number of nerves involvement of the median, ulnar, peroneal, posterior tibial motor nerves and median, ulnar, sural sensory nerves determined the clinical staging of peripheral neuropathy as follows; Stage 0 as no neuropathy; Stage 1 as one or two nerves affected (mild); Stage 2 as three or four nerves affected (moderate); Stage 3 as five or more nerves affected (severe) [22].

Autonomic dysfunction was evaluated based on the heart rate and blood pressure responses to a variety of stimuli. The cardiac parasympathetic response was tested by immediate heart rate response to standing, (30th beat: 15th beat ratios) < 1.00 was abnormal. Postural fall in blood pressure to standing more than 30 mmHg indicates sympathetic cardiac damage [23].

Peripheral blood samples were collected from the antecubital vein from all subjects into Ethylenediaminetetraacetic Acid (EDTA) and processed for ALR genotyping. A fasting blood sample was collected with the purpose of determination of Blood Sugar (FBS) level, as well as the percentage of glycosylated hemoglobin (HbA1c) [24]. Genomic DNA was extracted using a Macrogen DNA purification kit (Macrogen Inc., South Korea). All kits and reagents were utilized according to their manufacturer’s instructions.

The structural and functional polymorphism of the ALR gene polymorphism was determined using conventional polymerase chain reaction (PCR) [25]. The fragment containing the (AC)n repeat sequence was amplified using the following sense primer (59-GAATCTTAACATGCTCTGAACC-39) and the antisense primer (59-GCCCAGCCCTATACCTAGT-39) bounding the 138-bp region was used in PCR amplification of the sequence containing the (AC)n repeat. After thirty cycles of PCR amplification consisted of denaturation and annealing-extension, the PCR product was combined with an aliquot of the internal lane. The blend was then resolved by electrophoresis on a denaturing gel (6% urea-PAGE) using ABI 373 DNA sequencing. The internal lane standard (Liz 500) was used to create a calibration curve of peak arrival time, which, in turn, was used to calculate the length of unknown PCR product automatically by peak scanner software. The amplified PCR products were purified using a Qiaquick spin column to remove the primer impurity. The cycle sequencing products were electrophoresed on 6% urea-PAGE denaturing gel to confirm the (AC)n repeat sequence of the PCR products.

Statistical analysis was performed using SPSS version 18.0. Descriptive statistics for each group were computed for the following variables: age, sex, blood pressure, Body Mass index (BMI), serum lipid, HbA1c, and ALR. Data were tested for normality. Normally distributed data were presented as the mean and SD. Data that were not normally distributed were log-transformed and tested again for normality. To test for differences between groups, we used ANOVA when the raw or transformed data were normally distributed. For data that were not normally distributed, we used the nonparametric Kruskal–Wallis procedure. Differences in the distributions of specific alleles among the four study groups were evaluated using Fisher’s exact test. P value < 0.05 was considered statistically significant.

Results

A dataset of two hundred-Six T2D patients and 114 diabetes-free participants was included in this study. Mean age, sex, BMI, duration of diabetes, serum HbA1c level, history of hypertension and hyperlipidemia of the examined subjects are summarized in Table 1.

Table 1.

Clinical characteristics

Variables	Nondiabetic [n = 114]	Uncomplicated [n = 72]	Microangiopathic [n = 134]	P value
Age (years)	59.16 ± 13.21	55.69 ± 8.49	56.97 ± 10.46	0.376
Sex (M/F)	45/69	21/51	42/92	0.46
BMI (kg/m2)	25.7 ± 2.5	25.5 ± 2.4	25.9 ± 2.7	0.642
Diabetes duration (years)	–	11.26 ± 5.32	15.20 ± 5.37	< 0.001
HbA1c (%)	–	7.18 ± 0.52	8.87 ± 1.09	< 0.001
BP (systolic)	138 ± 13	141 ± 13	139 ± 12	0.376
BP (diastolic)	82 ± 7	84 ± 7	83 ± 6	0.224
TC (mg/dl)	201 ± 93	196 ± 91	218 ± 69	0.116
TG (mg/dl)	215 ± 88	201 ± 87	222 ± 87	0.239
Treated with insulin n (%)	–	26 (36.1%)	84 (62.7%)	< 0.001
Treated with OAD n (%)	–	37 (51.4%)	45 (33.6%)	0.064
Treated with anti-hypertensive n (%)	64 (56.1%)	39 (54.2%)	75 (55.9%)	0.218

The data are expressed as mean ± standard deviation (SD) or % (n)

BMI body mass index, BP blood pressure, DM diabetes mellitus, HbA1c hemoglobin A1c, M male, F female, OAD oral antidiabetic drug, TC total cholesterol, TG triglyceride

According to Table 1, analysis of the distribution by age and sex proved no significant differences between the study groups, therefore homogeneity between groups was observed. After adjustment regarding age and sex, diabetic microangiopathy was seen more in those who had higher HbA1c and longer duration of diabetes in the case group. Fourteen (6.6%) diabetic subjects were treated with diet only, 82 (39.9%) with oral hypoglycemic drugs, 110 (53.3%) with insulin and 69 (33.5%) combination therapy by insulin plus oral hypoglycemic agents. The frequency of oral medication was as follows: Metformin (62.1%), Glibenclamide (58.7%), Alpha- glucosidase inhibitors (4.4%), Repaglinide (2.4%), dipeptidyl peptidase four inhibitors (1.4%), and thiazolidinediones (1.4%).

Data analyses revealed that the significance of diabetes duration in non-microangiopathy versus microangiopathy diabetic patients was less than 0.001 and that the magnitude of elevated HbA1c in non-microangiopathy versus microangiopathy diabetic patients was less than 0.001. A total of 109 (52.9%) patients with microangiopathy suffered from retinopathy, 57 (27.6%) from nephropathy and 110 (53.4%) from neuropathy. Among the patients with diabetic nephropathy, 64.9% were categorized into microalbuminuria and 35.1% into gross albuminuria. In patients with T2D, peripheral neuropathy was observed in 102 subjects (57.8% mild, 28.4% moderate, and 13.7% severe), whereas, autonomic dysfunction was observed in 60 subjects (16.7% sympathetic, 50% parasympathetic, and 33.3% both). Therefore, 65% of diabetic patients had at least one form of microangiopathy.

We detected 13 alleles ranging from Z − 12 to Z + 16. These alleles were identified as Z + 16, Z + 14, Z + 8, Z + 6, Z + 4, Z + 2, Z, Z − 2, Z − 4, Z − 6, Z − 8, Z − 10, and Z − 12 where Z is the most common allele and consists of 24 AC repeats. Additionally, the relation between different alleles of the (AC)n repeat and the age, sex, BMI, blood pressure and lipid profile of the study participants were not significant. The frequency of the 5′ ALR alleles is shown in Table 2. The patients with retinopathy had an increased frequency of the Z − 4 allele compared with the patients with uncomplicated diabetes (31.6 and 5.6%, respectively; χ² = 5.2, P < 0.001). The Z − 4 allele was also increased in the patients with nephropathy and autonomous neuropathy compared with the patients with uncomplicated diabetes (29.8 and 31.6%, vs. 5.6% respectively; χ² = 6.4, P < 0.001).

Table 2.

Frequency of 5′ ALR alleles found in the study population

5′ ALR allele	Nondiabetic [n = 114]	Uncomplicated [n = 72]	Microangiopathic [n = 134]
			Retinopathy [n = 109]	Nephropathy [n = 57]	Neuropathy [n = 110]
					Autonomic [n = 60]	Peripheral [n = 102]
Z − 12	0.0 [0]	0.0 [0]	0.4 [1]	0.8 [1]	0.8 [1]	0.4 [1]
Z − 10	0.9 [2]	0.0 [0]	0.8 [2]	2.6 [3]	1.6 [2]	0.4 [1]
Z − 8	1.3 [3]	0.0 [0]	0.8 [2]	0.8 [1]	0.8 [1]	0.9 [2]
Z − 6	1.7 [4]	1.4 [2]	0.8 [2]	0.8 [1]	0.0 [0]	1.4 [3]
Z − 4	16.7 [38]	5.6 [8]	31.6 [68]*	29.8 [53]†	31.6 [3]‡	26.4 [54]
Z − 2	9.6 [49]	22.9 [33]	12.4 [27]	10.5 [12]	18.3 [22]	17.1 [35]
Z	33.3 [76]	40.2 [58]	29.8 [65]	21.9 [25]	25.0 [30]	28.4 [58]
Z + 2	17.6 [40]	20.8 [30]	10.0 [22]	7.9 [9]	5.0 [6]	8.8 [18]
Z + 4	3.1 [7]	3.5 [5]	4.1 [9]	1.7 [2]	6.6 [8]	5.4 [11]
Z + 6	1.3 [3]	2.1 [3]	3.6 [8]	3.5 [4]	4.2 [5]	4.4 [9]
Z + 8	1.3 [3]	1.4 [2]	1.3 [3]	0.0 [0]	1.6 [2]	1.9 [4]
Z + 14	0.44 [1]	1.4 [2]	0.0 [6]	1.7 [2]	2.5 [3]	2.4 [5]
Z + 16	0.9 [2]	0.7 [1]	1.3 [3]	0.8 [1]	1.6 [2]	1.4 [3]

Figures in brackets are number of subjects

*Frequency compared with uncomplicated DM χ² = 5.2, P < 0.001

^†Frequency compared with uncomplicated DM χ² = 6.1, P < 0.001

^‡Frequency compared with uncomplicated DM χ² = 6.4, P = 0.031

The frequency of the 5′ ALR genotypes in diabetic patients and nondiabetic control subjects is shown in Table 3. The presence of the Z − 4 allele was increased in diabetics microangiopathy compared with that in uncomplicated diabetes (27.6 and 5.6%, respectively; χ² = 5.3, P = 0.005). Of the 109 patients with retinopathy, 33.9% had the Z − 4/Y 5′ ALR genotype (where Y is not Z − 2) compared with only 5.6% in the uncomplicated diabetes group (χ² = 7.4, P < 0.001). As well as, the Z − 4/Y genotype (where Y is not Z − 2) was found in 50.8% of the patients with nephropathy but 5.6% of the patients with uncomplicated diabetes (χ² = 6.9, P < 0.001).

Table 3.

Frequency of 5′ ALR genotypes found in the study population

5′ ALR genotype	Nondiabetic [n = 114]	Uncomplicated [n = 72]	Microangiopathic [n = 134]
			Retinopathy [n = 109]	Nephropathy [n = 57]	Neuropathy [n = 110]
					Autonomic [n = 60]	Peripheral [n = 102]
Z − 2/X	35.1 [40]	38.8 [28]	20.2 [22]	19.3 [11]	33.3 [20]	30.4 [31]
Z − 4/Y	22.8 [26]	5.6 [4]	33.9 [37]*	50.8 [29]†	31.6 [19]	28.4 [28]
Z − 2/Z − 4	7.8 [9]	4.2 [3]	4.5 [5]	3.5 [2]	5.0 [3]	4.9 [5]
X/Y	34.2 [9]	51.3 [37]	41.3 [45]	26.3 [15]	30.0 [18]	37.2 [38]

Figures in brackets are number of subjects

X is any allele other than Z − 4 and Y is any allele other than Z − 2

A total of 13 alleles were detected designated Z + 16 to Z − 12 where Z = 24 AC repeats

*Frequency compared with uncomplicated DM χ² = 7.4, P < 0.001

^†Frequency compared with uncomplicated DM χ² = 6.9, P < 0.001

Based on the results of our study in Table 4, among diabetics, 23.8% of the retinopathy group and 38.6% of the nephropathy group were homozygous for Z − 4. Moreover, 38.5 and 54.4% of the group’s retinopathy and nephropathy, respectively, had 1 copy or more of Z − 4. In contrast, among nondiabetics, 2.6% was homozygous for Z − 4, and 30.7% had one copy or more of Z − 4.

Table 4.

Z − 4 genotypes in the study population

Group	Z − 4
	Homo	Hetero	No
Nondiabetic [n = 114]	2.6 [3]	28.1 [32]	69.3 [79]
Uncomplicated [n = 72]	1.4 [1]	8.4 [6]	90.2 [65]
Microangiopathic [n = 134]
Retinopathy [n = 109]	23.8 [26]	14.7 [16]	61.5 [67]
Nephropathy [n = 57]	38.6 [22]	15.8 [9]	45.6 [26]
Neuropathy [n = 110]
Autonomic [n = 60]	26.7 [16]	10.0 [6]	63.3 [38]
Peripheral [n = 102]	20.6 [21]	11.7 [12]	67.7 [69]

Fisher’s exact test (two-tailed), P = 0.005

Discussion

There is rational evidence that indicates the role of the ALR gene as a biologically probable candidate gene for the development of diabetic microvascular diseases. Several studies have reported that the ALR gene is associated with diabetic retinopathy, nephropathy, and neuropathy, signifying a possible role of the polyol pathway in the pathogenesis of the small vessels’ complications in diabetes [25–28]. Moreover, as in animal studies revealed, the transgenic mice that overexpressed the ALR gene, the increased ALR levels actuated an accelerated rate of microangiopathies, and the application of the ALR inhibitors in animals displayed an alleviation of diabetes MVCs [29–31]. Also, the gene expression of the ALR (AC)n gene polymorphism in diabetes may mediate MVCs [32]. It is considered that the length of the microsatellite (AC)n repeat can impress the expression of the gene [33].

Previous reports about the (AC)n repeat marker of the ALR gene concerning MVCs have been conflicting. In other words, most studies have described the Z − 2 allele of the (AC)n repeat could be a risk element for diabetic retinopathy or nephropathy, but contradictory results have also been considered [34–38]. Besides, the Z + 2 allele of the (AC)n repeat was reported to be associated with early progression of nephropathy to microalbuminuria in a Japanese population of Type 1 diabetic patients [39]. The Z − 4 allele of the (AC)n repeat marker appears to be a risk allele for MVCs in Japanese T2D patients [28]. Demaine et al. found that the Z − 2 ALR allele is strongly associated with the susceptibility to diabetic nephropathy, neuropathy, and retinopathy [13]. In another study, the protective effect of the Z + 2 allele was reported in Caucasians with type 1 diabetes [40]. Sivenius et al. found that the T allele of the C-106 T polymorphism, but not the Z − 2 allele of the (AC)n dinucleotide repeat marker, was independently associated with an increased risk of the early development of microalbuminuria in Finnish T2D patients [12]. Petrovic et al. reported that the Z − 2 allele of the ALR gene polymorphism was a risk factor for the development of diabetic retinopathy in T2D Caucasian patients [41]. The Chinese and Japanese studies could not prove that the Z + 2 allele had a protective effect against diabetic retinopathy [27, 41]. Xu et al. demonstrated a correlation between the (AC)n dinucleotide repeat polymorphism and the occurrence of diabetic nephropathy in Caucasian type 1 diabetic subjects (Z − 2 as the risk allele and Z + 2 as the protective allele) [16]. Ikegishi et al. in a study showed that the Z − 4 allele of the (AC)n repeat marker appears to be a risk allele for MVCs in Japanese T2D patients. The frequency of the Z − 4 allele was significantly increased in patients with proliferative retinopathy, and the erythrocyte ALR protein content was significantly higher in patients carrying the Z − 4 allele compared with those exhibiting other alleles. Further, in transfection experiments, the Z − 4 allele was associated with a significantly higher transcription of the luciferase reporter gene compared with constructs without the (AC)n repeat or with Z − 2, Z or Z + 2 alleles. The results of the mentioned study suggested that the Z − 4 allele in the ALR gene may enhance gene transcription and may be a genetic risk factor, which explains the molecular mechanism for diabetic MVCs [28].

In this study, we found that individuals with retinopathy, nephropathy, and autonomic neuropathy have a significantly increased frequency of the Z − 4 allele compared with those with long-term uncomplicated diabetes. Moreover, ALR genotype containing Z − 4/other than Z − 2 was significantly increased in patients with retinopathy and nephropathy. Among diabetics, the homozygosity for the Z − 4 allele that was associated with diabetic microangiopathy is consistent with the hypothesis that specific homozygous polymorphisms may modulate the level of ALR gene expression and the risk for small vessel disease in patients with T2D.

The present study is not family or population-based to provide a more accurate evaluation of the genetic association which alongside a small sample size are the limitations of the study. Nevertheless, we were able to demonstrate an association between ALR gene polymorphisms and diabetic microangiopathy.

Conclusion

In conclusion, we have studied polymorphic regions in the promoter of the ALR gene and confirmed the role of the gene in the susceptibility to diabetic microangiopathy. This suggests a potential interaction between ALR polymorphisms’ genotype and hyperglycemia and diabetes. These findings are consistent with the role of ALR in the pathogenesis of diabetic MVCs and identify a possible mechanistic relationship among ALR genotype, hyperglycemia, and microangiopathy.

Author contributions

All authors whose names appear on the submission (1) made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; (2) drafted the work or revised it critically for important intellectual content; (3) approved the version to be published; and (4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Conceptualization: MBH-S, AAA; methodology: MBH-S; formal analysis and investigation: SO; writing—original draft preparation: AAA; writing—review and editing: AAA; funding acquisition: MRSR; validation: AAA; supervision: AAA.

Funding

Mazandaran University of Medical Sciences provided funding for the study.

Compliance with ethical standards

Conflict of interest

Mohammad Bagher Hashemi-Soteh, Ali Ahmadzadeh Amiri, Majid Reza Sheikh Rezaee, Amir Ahmadzadeh Amiri, Soleiman Olfat, and Ahmad Ahmadzadeh Amiri declare that they have no potential conflict of interest.

Ethical approval

This study was designed according to the Declaration of Helsinki and was approved by Mazandaran University of Medical Sciences Ethical Committee on 2014 (IR.MAZUMS.REC.1394.1514).