The significance of interleukin 4 (IL-4) (590-C/T) gene polymorphism in Iraqi patients with type 2 diabetes mellitus: A case-control study

 

Maysoon Mohammed Hassan

College of Dentistry, Wasit University, Iraq

 

ABSTRACT:

One of well knows ant-inflammatory cytokines is Interleukin 4 (IL-4). It plays major roles in the control of immune responses at various levels. It is involved in the proliferation of activated B and T cells, inhibition of type 1 T helper differentiation, and promotion of type 2 T helper cell differentiation, control of B cell maturation (10-13). Moreover, it is involved in the growth, apoptosis, proliferation, and maturation of other hemopoietic and nonhemopoietic cells (14). Previous studies have investigated its role in allergic and autoimmune disorders (14-17). The gene for IL-4 is located at chromosome 5 within the long arm and possesses four exons (18). Several authors have shown a possible association of IL-4 gene polymorphism to a number of clinical disorders such as multiple sclerosis, atopic dermatitis, rheumatoid arthritis, and asthma (19-21). IL- 4 gene polymorphism has been linked to type 1 diabetes mellitus, but we in the current study aimed at investigating the significance of IL-4 590C/T (rs2243250) gene polymorphism in relation to type 2 diabetes and its long term complications. This study was aimed to assess the significance of IL-4 590C/T (rs2243250) gene polymorphism in relation to type 2 diabetes and its long term complications. The present case-control study was carried out at the diabetes center in Al-Diwaniayh Teaching Hospital, Al-Diwaniyah Province, Iraq. The study started in January 2019 and ended on July 2019. The study included 32 types 2 diabetes mellitus patients and 47 apparently healthy control subjects. Five ml sample of venous blood (antecubital vein) was obtained for the purpose of PCR analysis of IL-4 (590-C/T) gene polymorphism. The results of currents study revealed that the distribution of patients with type 2 diabetes was 21 (65.6 %), 9 (28.1 %) and 2 (6.3 %) as CC, CT, and TT genotypes; whereas, the distribution of control subjects was 33 (70.2 %), 10 (21.3 %) and 4 (8.4 %), as CC, CT, and TT genotypes. There was no significant difference in the frequency distribution of diabetic patients and control subjects according to IL- 4rs2243250 genotype (P = 0.757). IL- 4rs2243250 genotype polymorphism was not significantly correlated to retinopathy, nephropathy, neuropathy, CVA, IHD and dyslipidemia (P > 0.05), IL-4 IL- 4rs2243250 genotype polymorphism is neither associated with type 2 diabetes mellitus nor is linked to its long term microvascular and or macrovascular complications.

 

 

INTRODUCTION:

Chronic hyperglycemia is a characteristic that is shared by a number of heterogeneous disorders which are collectively referred to as diabetes mellitus ¹. Diabetes mellitusis, therefore, formed etiological perspective is diverse, and a lot of medical conditions may lead to common manifestations that are pathognomonic of diabetes mellitus¹. However, the classification of diabetic disorders exists, and many basic forms of diabetes mellitus are well known by doctors and people². Type 2 diabetes mellitus is the most common form, and previously it was known as non-insulin dependent diabetes³. Nevertheless, and because a significant number of patients with “non-insulin dependent diabetes” will develop profound insulin deficiency at some point of their life with the progression of the disease, this nomenclature proved obsolete, and nowadays it is called type 2 diabetes. Another well-known nomenclature of type 2 diabetes mellitus was adult-onset diabetes mellitus; however, it becomes well known that at least small proportion of patients with diabetes mellitus are young and even within the pediatric age group, for that reason the new name “type 2” becomes the preferred term to describe this common medical disorder ⁴.

 

Other forms of diabetes included type 1 diabetes mellitus⁵, gestational diabetes⁶, diabetes mellitus in association with endocrine disorders ⁷, drug-induced diabetes mellitus ⁸, monogenic forms of diabetes mellitus (also called maturity-onset diabetes of the young) ⁹ and other less common forms such as those complicating exocrine pancreatic disorders and pancreatic surgical resection⁷. Type 1 diabetes is mostly autoimmune disease; however, idiopathic forms exist⁵, gestational diabetes usually accompany pregnancy⁶, endocrine abnormalities that are characterized by overproduction and secretion of insulin counter-regulatory hormones such as acromegaly and Cushing syndrome will lead to chronic hyperglycemia ⁷, drugs such as corticosteroids leads to chronic elevation of blood glucose ⁸.

 

Form pathophysiological perspective, type 2 diabetes mellitus is due to resistance to insulin action and some reduction of insulin secretion; although, there is transient hyperinsulinemia in early stages of disease⁴. The etiology of type 2 diabetes mellitus is multifactorial and till now is poorly understood despite the bulk of literature describing enormous amount of researches with this regard ⁴.    

 

One of well knows anti-inflammatory cytokines is Interleukin 4 (IL-4). It plays major roles in the control of immune responses at various levels. It is involved in the proliferation of activated B and T cells, inhibition of type 1 T helper differentiation, and promotion of type 2 T helper cell differentiation, control of B cell            maturation^10-13. Moreover, it is involved in the growth, apoptosis, proliferation, and maturation of other hemopoietic and nonhemopoietic cells ¹⁴. Previous studies have investigated its role in allergic and autoimmune disorders ^14-17. The gene for IL-4 is located at chromosome 5 within the long arm and possesses four exons ¹⁸. Several authors have shown a possible association of IL-4 gene polymorphism to a number of clinical disorders such as multiple sclerosis, atopic dermatitis, rheumatoid arthritis, and asthma ^19-21. IL- 4 gene polymorphism has been linked to type 1 diabetes mellitus, but we in the current study aimed at investigating the significance of IL-4 590C/T (rs2243250) gene polymorphism in relation to type 2 diabetes and its long term complications.    

 

PATIENTS AND METHODS:

The present case-control study was carried out at the diabetes center in Al-Diwaniayh Teaching Hospital, Al-Diwaniyah Province, Iraq. The study started in January 2019 and ended on July 2019. The study included 32 patients with an established diagnosis of type 2 diabetes mellitus according to records of diabetes center. Those patients were selected randomly from the pool of patients already registered in that center. Those diabetic patients were considered a study group; whereas, 47 apparently healthy subjects were selected to serve as a control group.

 

Variables included in the questionnaire form included: age, gender, residency, education, smoking, ethanol intake, duration of diabetes mellitus, history of ischemic heart disease, and cerebrovascular accidents (CVA). Wight in kilogram and height in centimeter were estimated for each participant in order to calculate body mass index (BMI) according to the following formula: BMI= weight (kg)/ (height in meter)^². Five ml sample of venous blood (antecubital vein) was obtained for the purpose of PCR analysis of IL-4 (590-C/T) gene polymorphism.  

 

The step of genomic DNA extraction was carried out using 100 μl of venous blood, and the kit used for this step was the DNeasy Blood and Tissue Kit (Qiagen, Germany). A TaqMan single nucleotide polymorphism (SNP) genotyping assay and 48-well StepOne Real-Time PCR System were used for the purpose of analysis of IL4 rs2243250 gene polymorphism, taking into consideration the detailed manufacturer’s instructions (Applied Biosystems, USA).

 

The rest of the venous blood sample was used to do the following biochemical tests: fasting blood sugar, HbA1c, and serum lipid profile. Patients were examined by two physicians for identification of microvascular complications, namely retinopathy, neuropathy, and nephropathy. The study was approved by ethical approval committee, and verbal consent was obtained from each participant following full illustration of the aim and the procedures of the current study.

 

STATISTICAL ANALYSIS

Data were then transformed into an SPSS (version 23) spreadsheet for statistical analysis. Quantitative data were expressed as mean, standard deviation, and range, whereas, categorical data were expressed as number and percentage. Independent samples t-test was used to compare mean values between two groups, whereas, Chi-square test and Fischer exact test was used to study the association between any two categorical variables provided that the assumption required for that test is fulfilled. The level of significance was considered at P ≤ 0.05.

 

RESULTS:

The demographic characteristics of patients and control subjects enrolled in the present study were outlined in table 1. There was no significant difference in mean age between diabetic patients and control subjects (P = 0.631), 57.81 ±7.83 years versus 56.91 ±8.31 years, respectively. There was also an insignificant difference in the frequency distribution of patients and control subjects according to gender (P = 0.098). In addition, there was no significant difference in the distribution of diabetic patients and control subjects according to body mass index (P = 0.947). Added to that, there was no significant difference in the distribution of diabetic patients and control subjects according to residency (P = 0.562).  According to the level of education, control subjects showed a significantly higher number of individuals with university-level than diabetic patients (P = 0.016). There was no significant difference in the distribution of diabetic patients and control subjects according to economic status (P = 0.888). Moreover, there was no significant difference in the distribution of diabetic patients and control subjects according to the occupation (P = 0.156). Furthermore, there was no significant difference in the distribution of diabetic patients and control subjects according to smoking (P = 0.935). Neither patients nor control subjects admitted to being alcoholic.

 

The mean duration of diabetes was 4.0 ±1.8 years. Mean fasting blood sugar of diabetic patients was 153.44 ±18.94 mg/dl, and the mean HbA1c was 8.69 ±1.26 %.

 

The frequency distribution of diabetic patients and control subjects according to IL- 4rs2243250 genotype was shown in table 2.  The distribution of patients with type 2 diabetes was 21 (65.6 %), 9 (28.1 %) and 2 (6.3 %) as CC, CT and TT genotypes; whereas, the distribution of control subjects was 33 (70.2 %), 10 (21.3 %) and 4 (8.4 %), as CC, CT and TT genotypes. There was no significant difference in the frequency distribution of diabetic patients and control subjects according to IL- 4rs2243250 genotype (P = 0.757), as shown in table 2.  

 

Table 1: Demographic characteristics of diabetic patients and control subjects

Characteristic	Type 2 DM n = 32	Control group n = 47	P
Age (years)
Range	43 - 71	42 - 71	0.631 † NS
Mean ±SD	57.81 ±7.83	56.91 ±8.31
Gender	2 (6.3 %)	1 (2.1 %)	0.098 ¥ NS
Male, n (%)	13 (40.6 %)	28 (59.6 %)
Female, n (%)	19 (59.4 %)	19 (40.4 %)
BMI
Normal (18.5 - 24.9 kg/m2), n (%)	6 (18.8 %)	10 (21.3 %)	0.947 ¥ NS
Overweight (25 - 29.9 kg/m2), n (%)	17 (53.1 %)	25 (53.2 %)
Obese (≥ 30 kg/m2), n (%)	9 (28.1 %)	12 (25.5 %)
Residency
Urban, n (%)	25 (78.1 %)	34 (72.3 %)	0.562 ¥ NS
Rural, n (%)	7 (21.9 %)	13 (27.7 %)
Education
Illiterate, n (%)	6 (18.8 %)	4 (8.5 %)	0.016 ¥ S
Primary, n (%)	16 (50.0 %)	16 (34.0 %)
Secondary, n (%)	10 (31.3 %)	16 (34.0 %)
University, n (%)	0 (0.0 %)	11 (23.4 %)
Economic status
Poor, n (%)	8 (25.0 %)	10 (21.3 %)	0.888 ¥ NS
Moderate, n (%)	16 (50.0 %)	26 (55.3 %)
Good, n (%)	8 (25.0 %)	11 (23.4 %)
Occupation
Employed, n (%)	16 (50.0 %)	31 (66.0 %)	0.156 ¥ NS
Unemployed, n (%)	16 (50.0 %)	16 (34.0 %)
Smoking
Positive, n (%)	14 (43.8 %)	21 (44.7 %)	0.935 ¥ NS
Negative, n (%)	18 (56.3 %)	26 (55.3 %)
Ethanol
Positive, n (%)	0 (0.0 %)	0 (0.0 %)
Negative, n (%)	32 (100.0 %)	47 (100.0 %)
Duration of diabetes (years)
Range	1- 8
Mean ±SD	4.0 ±1.8

n: number of cases; †: independent sample test; ¥: Chi-square test; NS: not significant at P ≤ 0.05; S: significant at P ≤ 0.05

 

 Table 2: Frequency distribution of patients and control subjects according to IL- 4rs2243250 genotype

IL- 4rs2243250 genotype	Type 2 DM n = 32		Control group n = 47		P
	n	%	n	%
CC	21	65.6	33	70.2	0.757 † NS
CT	9	28.1	10	21.3
TT	2	6.3	4	8.5

n: number of cases; †: Chi-square test; NS: not significant at P ≤ 0.05

 

  

 

Table 3: Association between IL- 4rs2243250 genotype polymorphism and microvascular and macrovascular complications of diabetes mellitus

Complication	rs2243250 genotype	Total	Positive		P
			n	%
Retinopathy	CC	21	6	28.6	reference
	CT	9	2	22.2	1.000 F NS
	TT	2	1	50.0	0.526 F NS
Nephropathy	CC	21	9	42.9	reference
	CT	9	6	66.7	0.393 F NS
	TT	2	1	50.0	1.000 F NS
Neuropathy	CC	21	4	19.0	reference
	CT	9	2	22.2	1.000 F NS
	TT	2	0	0.0	1.000 F NS
Dyslipidemia	CC	21	14	66.7	reference
	CT	9	5	55.6	0.687 F NS
	TT	2	1	50.0	1.000 F NS
IHD	CC	21	6	28.6	reference
	CT	9	1	11.1	0.393 F NS
	TT	2	1	50.0	0.526 F NS
CVA	CC	21	2	9.5	reference
	CT	9	1	11.1	1.000 F NS
	TT	2	1	50.0	0.249 F NS

  n: number of cases; F: Fischer exact test; NS: not significant at P ≤ 0.05

 

 

Table 3 outlined the association of IL- 4rs2243250 genotype polymorphism in diabetic patients to microvascular and macrovascular complications. IL- 4rs2243250 genotype polymorphism was not significantly correlated to retinopathy, nephropathy, neuropathy, CVA, IHD, and dyslipidemia (P > 0.05), as shown in table 3.

 

DISCUSSION

The role of immunology in clinical disorders is well established, and a lot of disorders, such as autoimmune diseases and allergic disorders are attributed to immunologic disturbances. The list of these disorders is out of the scope of this brief discussion. IL-4 is among well-known cytokines that have both anti-inflammatory and immune regulatory properties. Gene polymorphism may result in abnormalities that may favor increased or reduced production and secretion of cytokines and among these is IL-4 IL- 4rs2243250 genotype polymorphism. In the current study, we found no significant association between frequency distribution of patients and control subjects according to IL-4 IL- 4rs2243250 genotype polymorphism a finding that is in accordance with other authors ²². However, Neelofar et al. ²² found a significant association between IL-4 IL- 4rs2243250 genotype polymorphism and nephropathy in diabetic patients a finding that is on the contrary to that of the current study. Other authors have studied the association between IL- 4 rs2243250 genotype polymorphism and diabetic nephropathy ²³ and found no significant association in support of the findings of the current study. Other authors have found contradictory results also ^{24, 25}.

 

In addition, we tried to link IL- 4 rs2243250 genotype polymorphism to other microvascular complications such as retinopathy and neuropathy as well as macrovascular complications such as CVA and IHD. However, we found no significant association between these complications and IL- 4 rs2243250 genotype polymorphism.

 

CONCLUSION

In conclusion, IL-4 IL- 4rs2243250 genotype polymorphism is neither associated with type 2 diabetes mellitus nor is linked to its long term microvascular and or macrovascular complications.  

 

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Received on 15.07.2019           Modified on 18.08.2019

Accepted on 16.09.2019         © RJPT All right reserved