The Study on the Relationship Between IRS-1 Gly972Arg and IRS-2 Gly1057Asp Polymorphisms and Type 2 Diabetes in the Kurdish Ethnic Group in West Iran

Abstract

An association between the IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms and type 2 diabetes mellitus (T2DM) in different ethnic groups is controversial. We aimed to identify the association of these polymorphisms with T2DM in the Kurdish ethnic group of Iran. Study groups included 336 T2DM and 341 normoglycemic subjects. Genotyping was determined by polymerase chain reaction-restriction fragment length polymorphism. Genotypic and allelic frequencies were then evaluated. GR and RR genotypes of IRS-1 Gly972Arg variant gave a higher risk for T2DM (odds ratios [OR]=1.76 and OR=3.86, respectively). IRS-1 Gly972Arg polymorphism was found to be significantly associated with T2DM (OR=1.63) for the dominant model (GG vs. GR+RR). GD genotypes of the IRS-2 Gly1057Asp variant gave a higher risk for T2DM (OR=1.63). The dominant model analysis of the IRS-2 Gly1057Asp genotypes (GG vs. GD+DD) also showed an enhanced association with T2DM (OR=1.69). Among several combinations, GR/GD gave the highest risk for T2DM (OR=3.1). Other combinations were also significantly associated with T2DM, including, GR/GG (OR=1.86), RR/GG (OR=1.76), GG/GD (OR=1.83), and GG/DD (OR=2.35). HbA1c, serum triglyceride, and systolic blood pressure were higher in the control subjects with GR+RR genotypes compared with the GG genotype. Among the T2DM subjects, fasting plasma glucose was significantly lower in subjects with the GG genotype in relation to those with the GR+RR genotypes. Normoglycemic subjects carrying GD+DD genotypes of IRS-2 Gly1057Asp variation had a significantly higher fasting plasma glucose and total cholesterol, as compared with those with the GG genotype. Our findings revealed that IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms are associated with T2DM in the Kurdish ethnic group.

Introduction

Type 2 diabetes mellitus (T2DM), the most common metabolic disorder, is characterized by a combination of insulin resistance and a relative deficiency in the pancreatic β-cell function. It represents 90% to 95% of all diabetes types (Goldstein, 2002).

T2DM is a classic example of multifactorial disease that drives from the coexistence of genetic and environmental factors (Hamman, 1992; Toye and Gauguier, 2003). The molecular mechanisms underlying T2DM are poorly understood, but it appears that genetic defects affecting insulin sensitivity may be involved as a common basis of susceptibility to T2DM (McCarthy and Hattersley, 2001). Identification of T2DM susceptibility genes has a great importance in the area of diabetes research because elucidation of the diabetes gene(s) will influence all efforts toward a mechanistic understanding of the disease, its complications, treatment, cure, and prevention (Toye and Gauguier, 2003; Bakhtiyari et al., 2010). However, the identification of putative gene(s) is difficult. A number of genes have been introduced to be associated with T2DM and related metabolic disorders, and it has been shown that variations of some genes may influence genetic predisposition to insulin resistance and T2DM (McCarthy and Hattersley, 2001; Bakhtiyari et al., 2010).

Insulin receptor substrate (IRS)-1 and IRS-2 genes have been considered candidate genes for T2DM (Almind et al., 1996; Hager et al., 1993; Kalidas et al., 1998; D'Alfonso et al., 2003). IRS-1 and IRS-2 proteins are critical to insulin signaling in insulin target tissues (Sesti et al., 2001) and also mediate most insulin-signaling effects, especially those associated with somatic growth and carbohydrate metabolism (White, 2002). Several single-nucleotide polymorphisms (SNPs) have been reported for IRS-1 gene, including Pro512Ala, Asn1137Asp, Arg158Pro, and Gly972Arg. Among those, the Gly972Arg polymorphism has been found to be related to the imbalance of insulin action and T2DM risk (Porzio et al., 1999; Hribal et al., 2000; Burguete-Garcia et al., 2010). Jellema et al. (2003) showed that carriers of the Gly972Arg polymorphism of the IRS-1 gene were at a 25% increased risk of having T2DM with respect to noncarriers. However, despite the body of evidence supporting the functionality of Gly972Arg polymorphism, the negative associations between this SNP and T2DM have also been reported in some populations (Florez et al., 2004; Zeggini et al., 2004).

In humans, several SNPs have been identified in the IRS-2 gene. Among those, the Gly1057Asp polymorphism has been found to be associated with increased risk for the T2DM in some populations (Mammarella et al., 2000; Stefan et al., 2003; Okazawa et al., 2005; Bodhini et al., 2007), but not in others (Kalidas et al., 1998; Bektas et al., 1999; D'Alfonso et al., 2003; Ouederni et al., 2009). Jellema et al. (Withers et al., 1998) showed that the disruption of IRS-2 leads to T2DM in mice. A study conducted in an Italian population showed this polymorphism to be associated with an increased risk of type 2 diabetes, particularly among obese individuals (Mammarella et al., 2000). In the context of the aforementioned studies, these findings suggest that IRS-2 Gly1057Asp polymorphism may play a major role in the etiology of T2DM.

To our knowledge, because these relationships have not been addressed in Iranian subjects and the paucity of data on the IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms in other populations, we decided to study the relationship between these polymorphisms and T2DM in the Kurdish ethnic group of Iran. In view of these considerations, we aimed to determine the allele and genotype frequency of the IRS-1 Gly972Arg and IRS-2 Gly1057Asp variants and to investigate the association of these polymorphisms with insulin resistance and T2DM risk factors in an Iranian case-control population.

Material and Methods

Patients

The studied population included 336 (males/females: 166/170; age: 53.40±8.65 years) Iranian unrelated patients with type 2 diabetes and 341 (males/females: 173/168; age: 54.10±9.22 years) Iranian unrelated healthy controls. These subjects were selected in an age- and sex-matched manner. All subjects originated from the Ilam and Kermanshah provinces situated in the west of Iran, and belonged to the Kurdish ethnic group. Diabetic and healthy controls were distinguished based on their glycemic status, which was defined according to WHO 1997 criteria (American Diabetes Association, 1997). The control group only contained individuals with a normal fasting glucose level and negative family history of type 2 diabetes among first-degree relatives. The written informed consent was obtained from all participants before enrollment in the study.

Screening included standardized questionnaires on personal data and clinical measurements, such as age, sex, obesity, drug consumption during the past month, and medical or family history of diabetes (Saberi et al., 2011). All those who were not taking diabetes medication underwent a 2-h oral glucose tolerance test (OGTT) after an overnight fast. Criteria for control selection were fasting glucose <6.1 mM and 2-h plasma glucose <7.8 mM after OGTT. Diabetes was defined as fasting glucose ≥7.0 mM, 2-h glucose ≥11.1 mM after OGTT, or use of hypoglycemic medication. Systolic and diastolic blood pressure was measured twice in the right arm of the subjects who had been resting for at least 10 min in a comfortable position. Signed informed consent was obtained from all subjects, and the study was approved by the Institutional Ethics Committee. All participants were interviewed to obtain clinical data and T2DM family history. Body mass index (BMI) was estimated through dividing weight in kilograms by the height in meters squared. Insulin resistance was assessed from glucose and insulin concentrations using homeostasis model assessment of insulin resistance (HOMA-IR) equation (Matthews et al., 1985).

Sample preparation

Blood samples were collected from patients and controls in the morning, after an overnight (12-14 h) fasting. Five micro liters of venous blood was withdrawn from the antecubital vein into Vacutainer tubes containing ethylenediaminetetraacetic acid as the anticoagulant. To separate plasma, blood samples were centrifuged at 3000 rpm. A supernatant was used for biochemical experiments, and the Buffy coat and the red blood cell pellet were used for DNA extraction.

Biochemical assays

Serum blood glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured by the standard enzymatic method (Pars Azmon kit, Iran), using the auto analyzer system BT3000 (Biotechnica, Italy). Serum concentrations of insulin were assessed using a RIA kit specific for human insulin (LINCO Research, Inc., St. Louis, MO).

Genotyping

Leukocyte genomic DNA was extracted from the blood specimen using the standard procedure involving proteinase K and phenol/chloroform. The IRS-1 Gly972Arg polymorphic site was amplified by the polymerase chain reaction (PCR) method using the primer pair 5′-CTTCTGTCAGGTGTCCATCC-3′ (forward) and 5′-CGATGCACCTGTGGAGCGGT-3′ (reverse). PCR was performed in a final volume of 25 μL containing 100 ng genomic DNA, 1.5 mM MgCl2, 0.5 mM of each dNTPs, and 0.5 pmol of each primer. After an initial denaturation of 2 min at 94°C, the samples were subjected to 30 cycles at 94°C for 1 min, 59°C for 40 s, and 72°C for 40 s, with a final extension of 10 min at 72°C. The 263-bp product was digested with BstN1 for 2 h at 37°C followed by polyacrylamide gel electrophoresis. The digested products were stained with ethidium bromide. The resulting wild-type samples contained three bands sized 23, 81, and 159 bp and the mutant samples contained four bands sized 23, 81, 108, and 51 bp. We noted five bands sized 23, 81, 159, 108, and 51 bp for heterozygotes and four bands sized 23, 81, 108, and 51 bp for Gly972Arg homozygotes.

The IRS-2 Gly1057Asp polymorphic site was amplified using the primer pair 5′-TCCTTGGAC GGCCTCCTGT-3′ (forward) and 5′-AAGGCCTCGACTCCCGACA-3′ (reverse). PCR was carried out under conditions described above. The PCR products were digested overnight with the restriction enzyme HhaI at 37°C followed by polyacrylamide gel electrophoresis. The resulting wild-type samples contained one band sized 188 bp, and the mutant samples contained three bands sized 188 bp, 153 bp, and 35 bp for heterozygote and two bands sized 153 bp and 35 bp for Gly1057Asp homozygotes. We regenotyped approximately 5% of random samples for quality control. The rate of genotyping success for the polymorphisms was 99%.

Statistical analysis

All statistical analyses were carried out using the statistical program SPSS (version 18; SPSS, Chicago, IL); Differences in genotype/allele frequencies were compared by the Chi-square test or the Fisher exact test, where appropriate. One-way analysis of variance (ANOVA) was utilized to compare the clinical features among groups with different genotypes. Odds ratios (ORs) and 95% confidence interval (CI) were calculated by logistic regression analysis using diabetes as a dependant variable and the genotypes as independent variables.

We used the χ² test to evaluate deviation from Hardy-Weinberg equilibrium. Baseline quantitative results are expressed as mean±standard deviation (SD) and compared with the Student's t test; the continuous variables that failed the normality test were logarithmically transformed before analysis. The variables transformed were triglyceride, insulin, and HOMA-IR. Statistical differences are based on analyses of log-transformed data, but means of untransformed data are presented in tables.

Results

The clinical, anthropometrical, and biochemical characteristics of the participants are shown in Table 1. The mean of age and gender distribution were almost same in T2DM patients and their controls (p=0.56 and p=0.31, respectively). Other clinical and metabolic characteristics differed significantly between T2DM and normoglycemic control. T2DM subjects had significantly higher values for diastolic and systolic blood pressure, BMI, waist to hip ratio, glucose, HbA1c, cholesterol, triglyceride, LDL-C, insulin, and HOMA-IR, and lower levels of HDL-C than normoglycemic control subjects (All p values≤0.0001).

Table 1.

Characteristics of the Participants

	Groups
Characteristics	T2DM	Control	p-value
Number of individuals (male/female)	336 (166/170)	341 (173/168)	0.56
Age (year)	53.40±8.65	54.10±9.22	0.31
BMI (kg/m²)	26.10±3.85	23.80±3.05	0.0001
Waist to hip ratio	0.92±0.05	0.87±0.09	0.0001
Fasting plasma glucose (mM)	9.32±2.34	5.16±0.67	0.0001
HbA1c (%)	8.80±0.91	5.60±0.65	0.0001
TG (mM)	2.1±1.6	1.6±0.28	0.0001
TC (mM)	5.73±0.33	4.50±0.27	0.0001
HDL-C (mM)	1.17±0.25	1.36±0.31	0.0001
LDL-C (mM)	3.39±0.88	3.11±0.85	0.0001
Systolic blood pressure (mm Hg)	129.14±16.25	116.36±15.36	0.0001
Diastolic blood pressure (mm Hg)	86.27±10.05	77.94±8.63	0.0001
Fasting serum insulin (μIU/mL)	9.36±0.40	7.16±0.28	0.0001
HOMA-IR	3.24±0.54	1.72±0.36	0.0001

Data are presented as mean±SD.

p values<0.05 are considered as significant.

BMI, body mass index; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HbA1c, hemoglobin A1c; HOMA-IR, homeostasis model assessment-insulin resistance.

The distribution of IRS-1 Gly972Arg and IRS-2 Gly1057Asp variants between T2DM and control subjects as well as the combined data set is given in Table 2. Both variants were not in Hardy-Weinberg equilibrium in T2DM and control subjects. The observed genotypic frequencies of IRS-1 Gly972Arg variant were not in Hardy-Weinberg equilibrium in T2DM subjects (χ²=28.3, df=1, p=0.00) nor in control subjects (χ²=47.4, df=1, p=0.00). IRS-2 Gly1057Asp genotypic frequencies were also not in Hardy-Weinberg equilibrium in T2DM subjects (χ²=28.9, df=1, p=0.00) nor in control subjects (χ²=32.1, df=1, p=0.00). The allelic frequency of IRS-1 Gly972Arg variation was significantly different for the R allele between 33.3% of cases and 25.4% of controls (p=0.021). A significant difference was also observed in the allelic frequency distribution of the IRS-2 Gly1057Asp variation between cases and controls with a higher occurrence of the D allele in 10.2% of cases and 5.7% of controls (p=0.001).

Table 2.

The Genotype/Haplotype Status and Associated Risk for Type 2 Diabetes Mellitus in the Studied Groups

			Groups
Gene	SNP	Genotype/allele	T2DM (336)	Control (341)	p-value	OR (95% CI)
IRS-1	Gly972Arg (G>R)	GG	171 (53.6%)	214 (69.8%)	Reference	1.0
		GR	106 (36%)	81 (26.7%)	0.001^a	1.76 (1.26-2.45)
		RR	59 (10.4%)	46 (3.5%)	0.000^a	3.86 (1.95-7.64)
		GG vs. GR+RR			0.002^a	1.63 (1.2-2.21)
		Allele-G frequency	448 (66.7%)	509 (74.6%)
		Allele-R frequency	224 (33.3%)	173 (25.4%)	0.002^a	1.47 (1.16-1.86)
IRS-2	Gly1057Asp (G>D)	GG	239 (71.1%)	275 (80.6%)	Reference	1.0
		GD	71 (21.1%)	50 (14.7%)	0. 016	1.63 (1.09-2.44)
		DD	26 (7.8%)	16 (4.7%)	0.058	1.87 (0.98-3.58)
		GG vs. GD+DD			0.004^a	1.69 (1.18-2.42)
		Allele-G frequency	549 (82%)	600 (88%)
		Allele-D frequency	123 (18%)	82 (12%)	0.001^a	1.64 (1.21-2.22)
IRS-1+IRS-2	G>R+G>D	GG/GG	108 (32.1%)	165 (48.4%)	Reference	1.0
		GR/GG	85 (25.3%)	70 (20.5%)	0.002^a	1.86 (1.25-2.76)
		RR/GG	46 (13.7%)	40 (11.7%)	0.024^a	1.76 (1.08-2.86)
		GG/GD	43 (12.8%)	36 (10.6%)	0.020^a	1.83 (1.10-3.02)
		GG/DD	20 (6.0%)	13 (3.8%)	0.023^a	2.35 (1.12-4.92)
		GR/GD	18 (5.4%)	9 (2.6%)	0.009^a	3.1 (1.32-7.05)
		GR/DD	3 (0.9%)	2 (0.6%)	0.368	2.29 (0.38-13.94)
		RR/GD	10 (3.0%)	5 (1.5%)	0.052	3.05 (1.02-9.19)
		RR/DD	3 (0.9%)	1 (0.3%)	0.190	4.58 (0.47-44.64)

p values, p Chi Square; OR (95% CI), odds ratio with 95% confidence interval.

p values<0.05 are considered as significant.

The corresponding risk associated with different genotypes is also shown in Table 2. Logistic regression analysis of IRS-1 Gly972Arg GR and RR genotypes gave a higher risk for T2DM (OR=1.76, 95% CI=1.26-2.45, p=0.001 and OR=3.86, 95% CI=1.95-7.64, p=0.0001, respectively, for the two genotypes). For the dominant model analysis, we combined genotypes GA and AA and compared to genotype GG (GG vs. GR+RR). IRS-1 Gly972Arg polymorphism was found to be significantly associated with T2DM (ORs=1.63, 95% CI=1.2-2.21), with p<0.002 for the dominant model.

GD genotypes of IRS-2 Gly1057Asp gave a higher risk for T2DM (OR=1.63, 95% CI=1.09-2.44, p=0.016). However, the frequency of DD genotype of this variant showed no significant difference between T2DM and control subjects (OR=1.33, 95% CI=0.98-3.58, p=0.058). Furthermore, the dominant model analysis of the IRS-2 Gly1057Asp genotypes (GG vs. GD+DD) showed enhanced association with T2DM (OR=1.69, 95% CI=1.18-2.42, p=0.004). Logistic regression models were also used to calculate the ORs for the combinations of the IRS-1 Gly972Arg and IRS-2 Gly1057Asp genotypes and the clinical risk factors (Table 2). Among several combinations, GR/GD gives the highest risk for T2DM (OR=3.1, 95% CI=1.32-7.05, p=0.009). Several other combinations were also significantly associated with T2DM, including, GR/GG (OR=1.86, 95% CI=1.25-2.76, p=0.002), RR/GG (OR=1.76, 95% CI=1.08-2.86, p=0.024), GG/GD (OR=1.83, 95% CI=1.10-3.02, p=0.020), and GG/DD (OR=2.35, 95% CI=1.12-4.92, p=0.023). The frequencies of GR/DD, RR/GD, and RR/DD were not significantly different between study groups (p=0.368, p=0.052, and p=0.190, respectively).

We classified the clinical and biochemical characteristics of the study subjects according to the genotypes of Gly972Arg polymorphism in the IRS-1 gene (Table 3). As shown in this table, hemoglobin A1c (GG; 5.61±0.36 vs. GR+RR; 5.78±0, p=0.002), serum triglyceride (GG; 1.61±0.24 vs. GR+RR; 1.67±0.15 mM, p=0.002), and systolic blood pressure (GG; 128.27±17.16 vs. GR+RR; 129.36±18.37 mmHg, p=0.001) were higher in the control subjects with the GR+RR genotype when compared with the GG genotype. Among the T2DM subjects, there was no significant difference in any of the clinical or biochemical parameters between the GG and GR+RR genotypes except for fasting plasma glucose, which was lower in those with the GG genotype (GG; 9.11±0.67 vs. GR+RR; 9.32±0.94 mM, p=0.02).

Table 3.

Clinical and Biochemical Characteristics of the Study Subjects Classified According to the Genotypes of Gly972Arg Polymorphism in IRS-1 Gene

	Type 2 diabetic subjects			Control subjects
Characteristics	GG (171)	GR+RR (165)	p-value	GG (214)	GR+RR (127)	p-value
BMI (kg/m²)	26.30±4.21	26.25±3.15	0.90	23.44±3.52	23.80±3.27	0.35
Waist to hip ratio	0.90±0.05	0.91±0.11	0.28	0.87±0.10	0.88±0.07	0.32
Fasting plasma glucose (mM)	9.11±0.67	9.32±0.94	0.02^a	5.21±0.64	5.18±1.01	0.76
HbA1c (%)	8.80±2.1	8.70±1.9	0.65	5.61±0.36	5.78±0.65	0.002^a
TG (mM)	2.13±0.95	2.08±0.77	0.6	1.61±0.24	1.67±0.15	0.005^a
TC (mM)	5.73±0.41	5.78±0.38	0.25	4.53±0.33	4.50±0.30	0.40
HDL-C (mM)	1.17±0.19	1.2±0.21	0.17	1.33±0.22	1.36±0.25	0.25
LDL-C (mM)	3.39±0.65	3.40±0.74	0.89	3.12±0.51	3.11±0.48	0.86
Systolic blood pressure (mm Hg)	128.27±17.16	129.36±18.37	0.57	116.12±13.73	119.81±12.33	0.01^a
Diastolic blood pressure (mm Hg)	86.23±10.75	85.91±9.86	0.78	78.55±10.22	77.64±9.03	0.39
Fasting serum insulin (μIU/mL)	9.36±2.40	9.47±3.88	0.75	7.16±0.40	7.18±0.30	0.60
HOMA-IR	3.87±0.36	3.93±0.54	0.23	1.73±0.38	1.71±0.49	0.69

Data are presented as mean±SD.

p values<0.05 are considered as significant.

Table 4 presents the clinical and biochemical characteristics of the study subjects categorized according to the genotypes of IRS-2 Gly1057Asp polymorphism. As shown in this table, normoglycemic subjects carrying the GD+DD genotypes of IRS-2 Gly1057Asp variation had significantly higher fasting plasma glucose (GG; 5.59±1.27 vs. GR+RR; 5.73±0.35 mM, p=0.03) and total cholesterol (GG; 4.48±0.55 vs. GR+RR; 4.67±0.39 mM, p=0.01), as compared with those with the GG genotype. In T2DM subjects, no significant difference was found in any of the clinical or biochemical parameters between GG and GD+DD genotypes.

Table 4.

Clinical and Biochemical Characteristics of the Study Subjects Classified According to the Genotypes of Gly1057Asp Polymorphism in IRS-2 Gene

	Type 2 diabetic subjects			Control subjects
Characteristics	GG (283)	GD+DD (53)	p-value	GG (306)	GD+DD (35)	p-value
BMI (kg/m²)	25.940±4.16	26.21±4.23	0.67	22.97±3.35	23.55±3.87	0.40
Waist to hip ratio	0.90±0.12	0.92±0.09	0.16	0.88±0.32	0.91±0.72	0.81
Fasting plasma glucose (mM)	9.23±0.73	9.26±0.81	0.80	5.59±1.27	5.73±0.35	0.03^a
HbA1c (%)	8.87±1.96	8.61±1.89	0.37	5.59±0.27	5.80±0.61	0.07
TG (mM)	2.10±0.87	2.12±0.90	0.88	1.66±0.18	1.64±0.21	0.59
TC (mM)	5.73±0.43	5.74±0.35	0.86	4.48±0.55	4.67±0.39	0.01^a
HDL-C (mM)	1.19±0.20	1.18±0.25	0.78	1.40±0.26	1.35±0.19	0.16
LDL-C (mM)	3.41±0.49	3.42±0.55	0.90	3.08±0.44	3.12±0.51	0.66
Systolic blood pressure (mm Hg)	128.30±18.33	128.19±17.52	0.97	117.43±14.78	118.81±15.34	0.61
Diastolic blood pressure (mm Hg)	86.05±11.64	85.89±10.79	0.92	79.55±9.45	77.37±10.56	0.25
Fasting serum insulin (μIU/mL)	9.41±3.14	9.40±3.46	0.98	7.21±0.94	7.19±0.29	0.78
HOMA-IR	3.86±0.39	3.88±0.62	0.82	1.80±0.43	1.72±0.62	0.46

Data are presented as mean±SD.

p values<0.05 are considered as significant.

Discussion

One commonly used approach to define the genetic predisposition of T2DM is the candidate gene approach. This approach focuses on the search for an association between T2DM and sequence variants in or near biologically defined candidate genes which have been chosen based on their known physiological function. The importance of these variants or other nearby variants is tested by comparing the frequency in T2DM patients and normal glucose-tolerant subjects (Whittemore, 1996). Among the few T2DM susceptibility gene variants are Gly972Arg polymorphism of IRS-1 and Gly1057Asp polymorphism of IRS-2 gene (Hribal et al., 2000; Jellema et al., 2003; Stefan et al., 2003; Okazawa et al., 2005; Bodhini et al., 2007).

Many studies in different populations have been conducted to find an association between the IRS-1 Gly972Arg and IRS-2 Gly1057Asp variants and T2DM. Contradictory results have been reported about the effect of these variants on the risk of developing T2DM and its related metabolic traits (Almind et al., 1993, 1996; D'Alfonso et al., 2003; Jellema et al., 2003; Florez et al., 2004; Okazawa et al., 2005; Ouederni et al., 2009).

According to the results obtained from this study, it clear that there is an association between some genotype of IRS-1 Gly972Arg and IRS-2 Gly1057Asp variants and T2DM. The frequency of the R allele of IRS-1 Gly972Arg and the D allele of IRS-2 Gly1057Asp variants significantly were higher in T2DM subjects compared with normoglycemic subjects. The association of R allele of IRS-1 Gly972Arg and D allele of IRS-2 Gly1057Asp variants with T2DM was reported in previously published studies (Almind et al., 1993, 1996; El Mkadem et al., 2001, McCarthy and Hattersley, 2001; Jellema et al., 2003; Stefan et al., 2003; Okazawa et al., 2005; Bodhini et al., 2007), while several studies failed to show any association of these polymorphisms with T2DM (Ito et al., 1999; Wang et al., 2001; D'Alfonso et al., 2003; van Dam et al., 2004; Ouederni et al., 2009).

We also found that normoglycemic subjects carrying the GR+RR genotypes of IRS-1 Gly972Arg polymorphism had significantly higher levels of HbA1c (p=0.002), serum triglyceride (p=0.005), and systolic blood pressure (p=0.01) in comparison with normal subjects with the GG genotype taken as reference. Significant elevation of the levels of HbA1c and triglyceride in normal subjects with GR+RR genotypes compared with those with the GG genotype may be due to the impairment of IRS-1 ability to dock to the p85 regulatory subunit of the phosphoinositide (PI) 3-kinase, thus provoking a reduced activation of the IRS-1/PI 3-kinase/Akt signal transduction pathway, which regulates insulin metabolic effects, such as stimulation of glucose uptake and inhibition of lipolysis (Sesti et al., 2001). Systolic blood pressure was significantly higher in normoglycemic subjects with GR+RR genotypes as compared with those with the GG genotype, which is consistent with linkage studies showing an association of IRS-1 Gly972Arg variant with systolic blood pressure (Rosskopf et al., 2000; Marini et al., 2003). On the other hand, T2DM patients carrying the GR+RR genotypes of this polymorphism had higher fasting plasma glucose levels in relation to the patients with the GG genotype. This may be because of further disturbance of the insulin-signaling pathway, which leads to more reduction of glucose uptake in these patients.

Normoglycemic subjects carrying the GD+DD genotypes of IRS-2 Gly1057Asp variation had significantly higher fasting plasma glucose and total cholesterol, as compared with those with the GG genotype. In T2DM subjects, no significant difference was found in any of the clinical or biochemical parameters between GG and GD+DD genotypes. These results were obtained from the first study that examined the association between IRS-2 Gly1057Asp and T2DM in the Kurdish ethnic group.

Yet another highlight of our study was the correlation between the combinations of the genotypes of IRS-1 Gly972Arg and IRS-2 Gly1057Asp variants and T2DM. Logistic regression analysis also showed significantly increased risk of T2DM for several genotypic combinations of IRS-1 Gly972Arg and IRS-2 Gly1057Asp variants, including, GR/GG, RR/GG, GG/GD, and GG/DD. The combinations found in the present study can serve as genetic markers for association studies with susceptibility to T2DM in the Kurdish ethnic group.

In conclusion, the findings of our study revealed that the IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphism is associated with Type 2 diabetes in the Kurdish ethnic group. However, drawing any definitive conclusion on the effect of these variants on T2DM requires study of the association in larger groups in other populations.

Footnotes

Acknowledgments

This project was supported financially by deputy for research and technology of the Ilam University of Medical Sciences.

Author Disclosure Statement

No competing financial interests exist.

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