Clinical and biochemical characteristics of the studied population
The DN group had a higher frequency of males with systemic arterial hypertension and dyslipidemia. Many diabetic patients have associated comorbidities, such as hypertension, and they may also present dyslipidemia, which is a risk factor for other chronic diseases. There is evidence that changes in blood pressure lead to urinary albumin excretion and, consequently, severe renal damage [
27]. Evidence shows that dyslipidemia has a strong relationship with the microvascular complications of diabetes, especially nephropathy [
28].
The comparison of biochemical parameters showed found significantly higher levels of HbA
1C in the DR group. This finding is in agreement with previous studies that relate blood glucose control measured by the HbA
1C levels and the risk of being affected by this complication [
29]. It is not surprising that the relationship between DN, creatinine and microalbuminuria levels, was also observed patients with complications and without complications were compared, possibly because of the significance identified in the DN group. However, in this study, we did not observe a difference between microalbuminuria and DR, contrary to previous studies. We did observe a difference in creatinine levels [
30].
Microalbuminuria was higher in the retinopathy and/or nephropathy and nephropathy groups, while creatinine was higher in the retinopathy and/or nephropathy, retinopathy and nephropathy groups. Although they are biomarkers of nephropathy, some studies also associate them with retinopathy [
31]. It was found that glomerular filtration rates, even at low levels, in subjects with renal impairment were associated with the presence and severity of diabetic retinopathy compared to those with normal renal function [
32]. Another study found that the progression of retinopathy is influenced by total cholesterol and creatinine.
CRP is an important marker of systemic inflammation, which has been physiologically related to atherosclerosis [
33], pre-diabetes, is observed at high concentrations in neuropathic individuals [
34] and correlates with increased macular thickness in patients with diabetic retinopathy [
35]. In this study, we did not find a relationship between CRP, DN and DR, although in the second case, the values were close to the statistical limits of significance. The relationship with DR has been controversial. A study of diabetic Chinese population showed an inverse relationship between CRP values and DR [
36], while a meta-analysis showed a relationship between the variables, suggesting that CRP can be used as a severity biomarker of retinal complications [
37].
DNA methylation and diabetic complications
The present study showed that the hypermethylated profile in the promoter of the
MTHFR gene is associated with diabetic complications, particularly with retinopathy (Fig.
1). The group with DN showed a higher frequency of individuals with a partially methylated profile, similar to individuals with no complications. Partially methylated DNA indicates that methylation is not occurring in all alleles and/or leukocytes. A quantitative analysis could elucidate the percentage of methylation in these samples and may reveal differences between the group with no complications and the ND group.
A study of the Egyptian population identified
MTHFR hypermethylation in the peripheral blood of patients with end-stage renal disease [
38]. It is important to emphasize that the hypermethylation studied in the cited work was only samples that presented the partially methylated profile, since no fully methylated samples were found, unlike in the present study. On the other hand, the methylation that was found in our samples was not found in another study. The patient group in that consisted of individuals with renal disease, only 23% were diabetic; the control group consisted of healthy individuals, which changes the parameters of comparison compared to this study, because all patients in this study were diabetic.
Contrary to the methylation profile found in both the present work and in the Egyptian population, a recent study on a Chinese population showed a trend to hypomethylation of the
MTHFR promoter in diabetic individuals with DN compared to healthy individuals [
19]. In this same study, it was shown that diabetic patients without DN presented approximately 66.6% of methylated profile, although it is not clear whether it was total or partial methylation. Additionally, in this study, 36.6% of total methylation was found in diabetic subjects without complications. It is important to mention that the technique used and the CpG sites in all of these studies were the same, suggesting that these differences may be, primarily, the result of the demographic and clinical characteristics of the study population, which differed significantly between the studies. In addition, environmental factors that may be related to ethnicity-related habits, such as diet, may also influence the methylation profile of DNA.
Hypermethylation has also been identified in patients with ischemic stroke [
39], preeclampsia [
40], progenitors of people with Down syndrome [
41], but it was not detected in patients with Non-Hodgkin’s Lymphoma. In male infertile patients with non-obstructive azoospermia, hypermethylation was identified in testicular samples, but not in peripheral blood [
25].
As previously mentioned the CpG sites studied in the present work are located in a CpG island and are close to the binding sites for various transcription factors [
20,
22]. Thus, hypermethylation of the gene could lead to decreased gene expression and consequently deregulate homocysteine reactions, leading to a decrease in the methyl radical and consequent epigenetic dysregulation due to the possible DNA hypomethylation. It has already been shown in cultured smooth muscle cells that demethylation was associated with increased levels of
MTHFR transcripts [
42].
Correlation of the methylation profile at the MTHFR gene promoter with biochemical indicators of patients with diabetic complications
The hypermethylated profile is associated with a higher level of total cholesterol (p = 0.0103) and LDL cholesterol (p = 0.0415) in individuals with DR. The same association was identified in patients with end-stage renal disease. One possible explanation for this effect is that the increase in homocysteine resulting from low
MTHFR expression (caused by hypermethylation in the gene promoter) would positively modulate cholesterol synthesis [
43] since carbon metabolism has been described as the main donor of methyl groups [
39].
In the present work, relationships between both total cholesterol and LDL with hypermethylated profile of MTHFR were obtained in patients with well-defined clinical profiles. It was not identified in patients with ND, and in diabetics without complications. These results suggest that the relationship is not simply be a consequence of Hcy metabolism but that other mechanisms that affect the synthesis or processing of cholesterol may be implicated and are present in patients with DR and those with end-stage renal disease.
In a review of lipid markers of DR that high levels of cholesterol and LDL were found to be associated with the development and severity of DR, especially in patients with formation of hard exudates in the retina [
44]. This is probably due to the hyperglycemia that leads to oxidative stress and activates metabolic pathways of cell injury, especially AGE pathway, which generally increases with diabetes and is generated by non-enzymatic reactions of reducing sugars and lipoproteins. The effect of oxidative stress results in lipid peroxidation, production of malonaldehyde, damage to cell membranes and the accumulation of lipids [
44‐
47].
The hypermethylated profile of the diabetic patients is also associated with serum AGP values and TAC. However, for AGP, the relationship was only observed in patients with DN and with complications with significantly higher levels in patients who have the methylated gene profile. AGP, also called orosomucoid is an acute phase protein produced in the liver under cytokine stimulation [
48]. Levels of AGP have been related to parameters indicative of renal damage and the excretion of urinary albumin in diabetic patients [
49] suggesting that it may be a predictor of the risk of this complication [
50]. The relationship between
MTHFR hypermethylation and increased levels of AGP may refine the screening of patients with potential renal complications.
For TAC, surprisingly, the relationship between hypermethylation and increased antioxidant capacity was evidenced in all groups except diabetic retinopathy. The same effect was not found in the MDA, a measure of lipid peroxidation. The relationship between the methylated profile of MTHFR and the increase in the levels of AGP and TAC remains to be clarified. It is possible that MTHFR hypermethylation leads to a decrease in gene expression, which would lead to global and/or site-specific hypomethylation, culminating in increased expression of several genes, including genes related to AGP synthesis and oxidative stress (TAC).
On the other hand, HbA1C, serum creatinine and urinary albumin, although altered in individuals with complications (Table
1), do not seem to be related to the methylation of the
MTHFR gene, since after the correlation analysis between the methylation profile × biochemical indicators, they remained at similar levels in all groups (Table
2). This is easily explained, since diabetes is a multifactorial disease and, therefore, there is a combination of factors involved, in addition to the hypermethylation of
MTHFR.
In summary, the present study analyzed the relationship between the methylation profile of the MTHFR gene with chronic complications of diabetes and the relation of this epigenetic mark with biochemical, inflammatory and oxidative stress parameters. It is the first analysis described in the literature with this approach that studies the gene in question for epigenetic aspects. It was performed in patients with time of finally managed diabetes in the phase in which the chronic complications usually start. We observed that the hypermethylated profile was the most frequent in individuals with DR and that this profile was associated with higher levels of total cholesterol and LDL. For patients with DN, although the hypermethylated profile did not predominate, it was associated with higher levels of AGP and TAC.
The primary weakness of the study is the reduced sample number, which is a consequence of the difficulty of recruiting individuals who fit within the inclusion criteria, especially regarding how long they have had diabetes. In addition, a quantitative analysis of the methylation profile could elucidate which proportion of the DNA from the partially methylated samples is methylated.