Investigation of the relationship between MTHFR, IRS and CALCA gene polymorphisms and development of diabetic nephropathy in patients with type 2 diabetes mellitus

ABSTRACT The aim of this study was to investigate the relationship between MTHFR, IRS and CALCA gene polymorphisms and development of diabetic nephropathy (DN) in patients with type 2 diabetes mellitus (DM). Our study included 93 patients with type 2 DM diagnosed as having nephropathy and 95 controls diagnosed with type 2 DM without development of DN. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to determine the genotype distributions of MTHFR, IRS and CALCA gene polymorphisms. The results showed no statistically significant difference between DN patients and type 2 DM controls in terms of genotype distributions of MTHFR (C677T, A1298C), IRS (IRS-1 Gly972Arg, IRS-2 Gly1057Asp) and CALCA T692C gene polymorphisms (p > 0.05). However, in terms of allele frequencies for the MTHFR A1298C gene, the frequency of the C allele was significantly higher in the DN patients compared to the controls (p < 0.05). In the IRS-2 Gly1057Asp gene polymorphism, the G allele frequency was significantly higher in the DN patients than in the type 2 DM controls (p < 0.05). In the DN group, the individuals with one or less mutant alleles were significantly more than in the control group in terms of the IRS-2 Gly1057Asp gene polymorphism (p < 0.05). The C allele frequency for the MTHFR A1298C gene polymorphism and the G allele frequency for the IRS-2 Gly1057Asp gene polymorphism were indicated to be potential a genetic risk factor for the development of DN in patients with type 2 DM who developed DN.


Introduction
Type 2 diabetes mellitus (DM), one of the most common metabolic disorders in the world, is a major cause of high mortality and morbidity. Due to many acute and chronic complications, type 2 DM is a multifactorial disease in which environmental and genetic factors are effective [1][2][3]. Genetic factors play a more important role in type 2 DM, which is characterised by progressive b cell dysfunction and varying rates of insulin resistance. Type 2 DM with incidence of 16% in Turkey and prevalence of 3.5%-5%, accounts for 80%-90% of all diabetes cases [4][5][6]. Factors such as blood glucose level changes, changes in lipid metabolism, platelet dysfunction, which are known to be important for macrovascular and microvascular complications, play an important role in diabetic patients [7,8]. Diabetic nephropathy (DN) is one of the microvascular complications (retinopathy, neuropathy and nephropathy) in diabetic patients (without kidney disease). DN is characterised by a continuous positive urine albumin bar or by albumin secretion at more than 300 mg daily [9].
The methylenetetrahydrofolate reductase (MTHFR) gene is among the genes that are implicated in the development of DN from microvascular complications of type 2 DM [3]. The human MTHFR gene is localised in the telomeric region of the chromosome 1 (1p36.3) and encodes the enzyme MTHFR. The most common polymorphisms of the MTHFR gene are C677T and A1298C. The MTHFR C677T gene polymorphism is characterised by an alanine/valine exchange at position 226 of the protein at the N-terminal catalytic domain of the MTHFR gene at the 4th exon and the cytosine/thymine base translocation at position 677. The MTHFR A1298C gene polymorphism is characterised by a glutamine/alanine exchange in the C-terminal regulatory region of the MTHFR gene as a result of an adenine/cytosine translocation at position 1298 in exon 7 [10].
Changes in these polymorphic regions have been associated with the development of microvascular complications such as DN in DM patients. Although this relationship is known, only a few studies have investigated the relationship between MTHFR gene polymorphisms and DN development [3,[11][12][13].
Insulin receptor substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2) play an important role in glucose metabolism, despite the presence of four genes in the IRS system (IRS-1, IRS-2, IRS-3 and IRS-4) [14]. IRS-1, which encodes one of the members of the IRS protein family, is localised on chromosome 2q36. IRS-1, an endogenous substrate of the insulin receptor, plays an important role in insulin signaling pathways and is expressed in insulin sensitive tissues. Insulin activity is affected by some gene polymorphisms that occur in this gene [15]. The Gly972Arg polymorphism, one of the polymorphisms of the IRS-1 gene, is characterised by the glycine/arginine translocation at codon 972 of the gene. This polymorphism is known to be associated with type 2 DM and gestational diabetes mellitus that lead to insulin resistance and impaired insulin secretion [16,17]. Insulin receptor substrate-2 (IRS-2) also has an important function in the pathogenesis of type 2 DM. The IRS-2 Gly1057Asp gene polymorphism is characterised by the glycine/aspartate exchange in codon 1057 of the gene, which leads to an amino acid change in the IRS-2 protein [18]. These polymorphisms in the IRS-1 and IRS-2 genes are known to be associated with type 2 DM, and are also thought to be potentially related to DN from microvascular complications.
The calcitonin gene-related peptide (CGRP) is also an important member of the calcitonin peptide family and is the most potent vasodilator neuropeptide. CGRP and its receptors are distributed in the cardiovascular system, the peripheral and the central nervous systems [19]. There are two isoforms, calcitonin related polypeptide alpha (CALCA) and calcitonin related polypeptide beta (CALCB) [20]. CALCA is a neurotransmitter found in all tissues of the central and the peripheral nervous systems [21]. The human CALCA gene is localised on cromosome 11p15.2-p15.1. The CALCA gene, which plays an important role in the dilation of the cerebral arteries, consists of 1 promoter and 6 exons. The T692C gene polymorphism, the most common polymorphism in the CALCA gene, is characterised by a timin/cytosine base translocation at position 692 of the 5 'region of the CALCA gene [22,23]. Polymorphisms occurring in the CALCA gene are known to be associated with various disorders. Macrovascular complications of type 2 DM are found among these disorders [23][24][25].
The aim of this study is to investigate the relationship between MTHFR (C677T and A1298C), IRS (IRS-1 Gly972Arg and IRS-2 Gly1057Asp), CALCA T692C polymorphisms and DN development in patients with type 2 DM.

Subjects and methods
This study was carried out with 93 type 2 DM patients diagnosed with DN and 95 type 2 DM controls. Our study was performed with blood samples from the groups of patients and controls. The samples were taken from the Endocrine Polyclinic of Health Research and Application Center (Edirne, Turkey). Clinical Investigations Ethics Committee approval was obtained for this study and informed consent forms were signed by the patients and control subjects after the necessary explanations were made.

DNA isolation
DNA from DN patients and type 2 DM controls was isolated from peripheral blood samples containing ethylenediaminetetraacetic acid (EDTA) using Invitrogen DNA blood kits. The purity and quality of the isolated DNAs was determined by the Nanodrop spectrophotometer method and was checked by 0.8% agarose gel electrophoresis (Supplementary Figure S1).

Polymerase chain reaction (PCR)
PCR (Techne, Techne TC ¡3000 G) and restriction fragment length polymorphism (RFLP) methods were used to determine the MTHFR (C677T and A1298C), IRS (IRS-1 Gly972Arg and IRS-2 Gly1057Asp), CALCA T692C polymorphisms. We prepared 25 mL of PCR mixture containing of 50 ng of isolated DNA, 0.2 mmol/L deoxyribonucleoside triphosphates (dNTP) for each, forward and reverse primers (Invitrogen), 1xPCR buffer, 3 mmol/L MgCl 2 and 1.25 U Taq DNA polymerase (Fermentas-Invitrogen). We used 5'-TGA AGG AGG AGG TGT CTG CGG GA-3' forward primer and 5'-AGG ACG GTG CGG TGA GAG TG-3' reverse primer for the MTHFR C677T gene polymorphism. For the MTHFR A1298C gene polymorphism, 5'-CTT TGG GGA GCT GAA GGA CTA CTAC-3' forward primer and 5'-CAC TTT GTG ACC ATT CCG GTT TG-3' reverse primer were used. 5'-CTT CTG TCA GGT GTC CAT CC-3 ' forward primer and 5'-TGG CGA GGT GTC CAC GTA GC-3' reverse primer were used to determine the IRS-1 Gly972Arg gene polymorphism. 5'-GTC CCC GTC GTC GTC TCT-3' forward and 5'-CTC GAC TCC CGA CAC CTG-3' reverse primers were used to determine the IRS-2 Gly1057Asp gene polymorphism. For the CALCA T692C gene polymorphism; 5'-CGC ATC TGT ACC TTG CAA CT-3' forward and 5'-TCA AAT TCC CGC TCA CTT TA-3' reverse primer sequences were used. For the MTHFR C677T gene polymorphism, amplification was performed with denaturation for 5 min at 94 C, followed by 30 cycles of denaturation for 30 s at 94 C, annealing for 30 s at 62 C and extension for 30 s at 72 C, followed by 5 min of extension at 72 C. For the MTHFR A1298C gene polymorphism, the amplification were performed with denaturation for 5 min 94 C, followed by 35 cycles with denaturation for 30 s at 94 C, annealing for 30 s at 62 C and extension for 30 s at 72 C, followed by 10 min of extension at 72 C. For the IRS-1 Gly972Arg gene polymorphism, the amplification was performed with denaturation for 10 min at 94 C, followed by 32 cycles with denaturation for 45 s at 94 C, annealing for 45 s at 56 C and extension for 45 s at 72 C, followed by 5 min of extension at 72 C. For the IRS-2 Gly1057Asp gene polymorphism, the amplification was performed with denaturation for 10 min at 94 C, followed by 32 cycles with denaturation for 45 s at 94 C, annealing for 45 s at 56 C and extension for 45 s at 72 C, followed by 5 min of extension at 72 C. For the CALCA T692C gene polymorphism, the amplification was performed with denaturation for 5 min at 94 C, followed by 38 cycles with denaturation for 50 s at 94 C, annealing for 50 s at 57 C and extension for 1 min at 72 C, followed by 10 min of extension at 72 C. We used 2% agarose gel electrophoresis to determine the PCR products of the MTHFR and CALCA gene polymorphisms and 3% agarose gel electrophoresis, to determine the PCR products of the IRS gene (Supplementary Figures S2-S6).

Statistical analysis
The variables of age, height and weight were determined for the patients with DN (93) and the controls with type 2 DM but not DN (96) by Independent Samples test. Body surface area (BSA) and diabetes duration variables were analysed by Mann-Whitney test. Chi-square analysis was used to compare variables such as alcohol intake, smoking and family history between the groups of patients and controls. In our study, the genotype and allele frequencies were calculated based on the Hardy-Weinberg equation. The proportions of genotypic distributions of MTHFR (C677T and A1298C), IRS (IRS-1 Gly972Arg and IRS-2 Gly1057Asp), CALCA T692C gene polymorphisms were determined in DN patients and controls with type 2 DM. The relationship between these genotype distributions and DN development was statistically analysed by the chi-square test. For patient and control groups, Kikare analysis was used the in comparison between individuals with two or more mutant alleles and individuals with one or less mutant allele. In addition, the chi-square test was used in the combined genotype analyses between the patient and control groups. The statistical analysis was performed using the SPSS 20.0 package program. The results are presented as mean values with standard deviation ( §). P < 0.05 was considered as statistical significance.

Results and discussion
In our study, there was a significant difference (p < 0.05) in terms of BSA and smoking variables in the comparison between the patient group with DN and the control group with type 2 DM. However, significant difference was not found in terms of age, height, weight, diabetes duration, alcohol intake and family history variables (p > 0.05). (Table 1). There was no statistically significant difference between the genotype distributions of MTHFR (C677T and A1298C), IRS (IRS-1 Gly972Arg and IRS-2 Gly1057Asp), CALCA T692C gene polymorphisms in the patients with DN and controls with type 2 DM (p > 0.05) ( Table 2). In addition, testing for Hardy-Weinberg equilibrium was used to determine the genotype and allele frequencies of the patient and control groups. In terms of allele frequencies, the C allele frequency for genotype distribution of the MTHFR A1298C gene polymorphism was higher in the patients with DN than in the controls, whereas the A allele frequency of the controls was significantly higher than that in patients with DN (p < 0.05).
For the IRS-2 Gly1057Asp gene polymorphism, the D allele frequency was higher in the controls with type 2 diabetes mellitus than in the patients with DN, whereas the G allele frequency was significantly higher in the patients with DN than in the controls (p < 0.05) ( Table 3).
We also compared the patients with two and more mutant alleles and the patients with one and fewer mutant alleles in the group of patients and the control group. There were more individuals (p < 0.05) with one and fewer mutant alleles for the IRS-2 Gly1057Asp gene polymorphism in the DN patient group than in the control group (Table 4). A significant association was not found (p > 0.05) between the group of patients and the control group in terms of combined genotype analysis for MTHFR (C677T and A1298C) gene polymorphisms (Table 5).
There are many studies showing that polymorphisms occurring in certain gene regions in patients with type 2     DM are a risk factor for the development of vascular damage. Some studies indicate that the MTHFR C677T gene polymorphism is associated with microvascular complications such as DN, retinopathy and neuropathy in type 2 DM [26][27][28][29]. There are, however, studies showing no relationship between microvascular complications and the MTHFR C677T gene polymorphism [26][27][28][29].
Ukinc et al. [12] studied the relationship between MTHFR C677T gene polymorphism and the risk of developing diabetic retinopathy, nephropathy and neuropathy in 52 type 2 diabetic patients (32 female and 20 male, mean age of 52.7 § 9.9 years) who had not previously had microvascular complications. The authors reported that this polymorphism is associated with DN from microvascular complications.
In an Egyptian population, the C677T polymorphism in the MTHFR gene reportedly contributes to the development of diabetic nephropathy in type 2 DM patients. The T allele of this polymorphism presumably acts by elevating homocysteine levels and seems to be associated with a faster progression of nephropathy to endstage renal failure [30]. In a Chineese population, the MTHFR C677T genomic polymorphism could influence the development of type 2 diabetic nephropathy. In addition, the T allele of MTHFR C677T significantly elevated the risk of type 2 diabetic nephropathy in comparison to the C allele [31]. In an Emirati population, the MTHFR C677T and A1298C gene polymorphisms were not related to type 2 DM. However, these polymorphisms could be used as risk markers for CVA, nephropathy, high LDL cholesterol and triglycerides in type 2 DM patients [32]. Analysis of many different populations indicated that the MTHFR C677T T allele or TT genotype might be a significant genetic molecular marker to determine the risk of DN in patients with type 2 DM [33]. In an Iraqi population, the TT genotype of the MTHFR gene was suggested to be associated with increased risk of DN in type 2 DM patients [34]. Ramanathan et al. [35] suggested that the C677T and A1298C polymorphism of the MTHFR gene could be associated with diabetic nephropathy in a south Indian population and that the C677T polymorphism could be associated with CKD progression in DN [35]. Another study suggests an association between the C677T polymorphism and nephropathy in patients with type 2 DM, the C allele increasing the risk of nephropathy, and the T allele having a protective role [36].
Although some studies report a significant relationship between the MTHFR A1298C gene polymorphism and the risk of developing DN, there are also studies showing that such a relationship does not exist [37][38][39]. In Japanese patients with type 2 DM, the relationship between DN and the MTHFR 677T allele was found to be significant [51]. In a group of Irish type 1 DM, German type 1 or type 2 DM and Japanese type 2 DM patients, the MTHFR gene polymorphisms was not significantly associated with DN development [40]. Shpichinetsky et al. [41] did not observe any significant differences between the genotypic distributions and allele frequencies of the MTHFR gene polymorphisms in patients with and without DN.
In patients with DM, the MTHFR C677T gene polymorphism is one of the polymorphisms that determine atherosclerosis, endothelial dysfunction, atherothrombosis and microvascular complications of DM [42,43]. DN, which is a microvascular complications of DM, develops after diabetic retinopathy. For example, analysis of the relationship between the risk of developing diabetic retinopathy and MTHFR gene polymorphisms revealed significantly higher levels of microalbuminuria and creatinine levels in the group of patients with diabetic retinopathy than in the control group. This statistical significance indicates that the development of nephropathy will also occur in people who develop diabetic retinopathy [44,45]. While not a single gene has been identified that definitively determines the development of DN, it was thought that multifactorial genetic and environmental factors and multiple genes play an important role in the pathogenesis of DN [46].
Many different genes associated with DM have been predicted. Among these genes, the IRS-1 gene contributes to insulin action in tissues that are sensitive to insulin. Disorders in the IRS-1 gene cause insulin resistance associated with DM [47].
IRS, which plays a very important role in the insulin signalling pathway, is an endogenous substrate and is expressed in insulin-sensitive tissues. After binding to the insulin receptor, the intrinsic tyrosine kinase activity of the insulin receptor beta subunit is activated. Phosphotyrosine residues on IRS proteins become important targets for the p85 subunit of pI3 kinase [48]. Akt is recognised as a downstream target of PI3-kinase. Akt, mediates some metabolic responses induced by insulin, stimulation of glucose transport, translocation of glucose transporters to the cell surface, inactivation of glycogen synthase kinase-3 (GSK-3), and stimulation of glycogen synthesis. Disorders in PI3 kinase activity are expected to obstruct the downward signalling, including the Akt/GSK-3 pathway. However, different effects have been identified in two studies [49,50]. In an in vitro study, a decrease in insulin-stimulated Akt activity was demonstrated [51]. However, in a an in vivo study, normal Akt activity occurred, although there was a decrease in PI3 kinase activity in response to ins€ ulin [52]. The Gly972Arg gene polymorphism near the C-terminus of the IRS-1 gene is located between two potential tyrosine phosphorylation sites. These sites also show the binding sites of the p85 subunit of pI3 kinase [53]. Laura et al. [54] and Fulden and et al. [55] found that the IRS-1 Gly972Arg gene polymorphism was associated with type 2 DM. Kozarova et al. [56] reported a significant relationship between the IRS-1 Gly972Arg gene polymorphism and type 2 DM. Even though the IRS-1 Gly972Arg gene polymorphism does not alter the IRS-1 expression level or the insulin-stimulated tyrosine phosphorylation degree of IRS-1, this polymorphism interferes with the interaction between IRS-1 and pI3 kinase. This impairs the ability of insulin to activate the IRS-1/pI3 kinase/Akt signaling pathway, leading to insulin resistance and DM development [57,58]. The relationship between IRS-1 gene polymorphism and the risk of developing type 2 DM has been investigated in different populations. In some studies, such a relationship was found, whereas in others, it was not. There was not significant relationship between the risk of developing type 2 DM and the IRS-1 Gly972Arg gene polymorphism in the Turkish population [59].
In a South Indian population, the IRS-1 Gly972Arg gene polymorphism was not identified as a genetic risk factor for type 2 DM disease [60]. Pappa et al. [16] found a strong association between the risk of developing gestational DM and the IRS-1 Gly972Arg gene polymorphism. In another study performed to investigate the relationship between IRS gene polymorphisms and DN development risk, it is revealed that the IRS-1 and IRS-2 gene polymorphisms modify the progression of kidney failure in DN patients [61].
There are many studies on the relationship between IRS-2 gene polymorphisms and DM and obesity. In the Pima Indian population, the Asp1057 allele frequency of the IRS-2 Gly1057Asp gene polymorphism was quite high when compared to other populations [62,63]. In another study conducted with caucasians in Italy, compared to obesity IRS-2 Gly1057Asp Asp1057 alleles frequencies were found to be lower in those with diabetes risk [64].
Although the molecular mechanisms associated with the IRS-2 Gly1057Asp gene polymorphism are not entirely known, the exchange of amino acids in this polymorphism is close to the two tyrosine phosphorylation sites of the protein. These are at positions 1042 and 1072. Thereby; changes in the signalling can be due to the IRS-2 gene. In a study conducted by Fritsche et al. [62]. IRS-2 Gly1057Asp gene polymorphism was not associated with beta cell dysfunction. In another study, the DG genotype of the IRS-2 Gly1057Asp gene polymorphism was significantly higher in the Chinese population [47]. A significant relationship was found between the IRS-2 Gly1057Asp gene polymorphism and type 2 DM in studies conducted in Denmark, Germany, Finland, China and Sweden [65]. In another study, the 1057 DD genotype of the IRS-2 gene polymorphism was significantly associated with DM in an Asian Indian population. Some previous studies with Germans show that there is no significant association between this gene polymorphism and insulin sensitivity or insulin secretion function; however, there are some studies that this polymorphism with the same population is associated with obesity by affecting beta-cell function [66,67].
The CALCA T692C gene polymorphism, apart from being associated with DM, coronary heart disease, peripheral vascular disease and cerebrovascular disease, may also be associated with development of DM microvascular complications such as nephropathy [24]. In the CALCA T692C gene polymorphism, which has been associated with diseases such as migraine, Parkinson's disease, schizophrenia and essential hypertension, the C allele has been found to be associated with the risk of essential hypertension [66,67].
In a study conducted by G€ uldiken et al. [24] in migraine patients and healthy control groups, there was no significant difference between the CALCA T692C gene polymorphism and the risk of developing migraine. Luo et al. [23] suggested that the CALCA T692C gene polymorphism could an important genetic risk factor in developing essential hypertension. There are not many studies on the relationship between CALCA gene polymorphisms and various diseases. In addition to this, there is no study on the role of CALCA gene polymorphisms in the development of DN.

Conclusions
In our study, a significant difference was not observed in the genotype distributions of MTHFR (C677T and A1298C), IRS (IRS-1 Gly972Arg and IRS-2 Gly1057Asp) CALCA T692C gene polymorphisms between the DN patient group and the control group with type 2 DM. In addition, the C allele frequency of the MTHFR A1298C gene polymorphism was higher in the patients with DN than in the controls, whereas the A allele frequency was higher in the controls than in the DN patients. For the IRS-2 Gly1057Asp gene polymorphism, the D allele frequency was higher in the controls than in the patients with DN, whereas the G allele frequency was higher in the DN patients than in the controls. The number of individuals with one and fewer mutant alleles was significantly higher in the DN group for the IRS-2 Gly1057Asp gene polymorphism as compared to individuals with two and more mutant alleles. In addition, there was not found a significant relationship between the combined genotype analysis of MTHFR (C677T and A1298C) gene polymorphisms between patient and control groups. Overall, in our study, the C allele frequency for MTHFR A1298C gene polymorphism and the G allele frequency for the IRS-2 Gly1057Asp gene polymorphism were associated with the DN developing risk in the group of patients with DN.