Methylation Profile of miR-9-1 and miR-9-1/-9-3 as Potential Biomarkers of Diabetic Retinopathy.

AIMS: Analysis of the relationship between the methylation profile of miR-9-1 or miRs -9-1 / -9-3 and diabetic retinopathy. BACKGROUND: Diabetic Retinopathy (DR) is a frequent complication of Diabetes mellitus and it has a decisive impact on the quality of life, as it is one of the biggest causes of blindness in the adult population. Levels of microRNA-9 have been shown to be related to diabetes but little is known about its involvement with DR in humans. OBJECTIVE: To analyze the relationship between the methylation profile of miR-9-1 or miRs -9-1/-9-3 and DR. METHODS: 103 patients diagnosed with diabetes for 5 to 10 years were analyzed. The data were categorized according to clinical, biochemical, lifestyle and anthropometric parameters. DNA extracted from leukocyte samples was used to determine the methylation profile of miRs-9-1 and -9-3 using a specific methylation PCR assay. RESULTS: miR-9-1 methylation was related to diabetic retinopathy, indicating that methylation of this miR increases the chances of presenting retinopathy up to 5 times. In our analyses, diabetics with lower levels of creatinine and CRP showed significant reductions (99% and 97%) in presenting DR. Methylation of both miRs-9-1 and 9-3 methylated increases the chances of presenting DR by 8 times; in addition, a sedentary lifestyle can increase the risk for the same complication by up to 6 times. CONCLUSION: Our results suggest that both methylation of miR-9-1 and e miRs-9-1 / 9-3 favors DR in patients with diabetes in a period of 5 to 10 years of diagnosis.

Physical Activity Level Influences MTHFR Gene Methylation Profile in Diabetic Patients.

INTRODUCTION: MTHFR methylation status is associated with microvascular complications in diabetes, but the factors influencing this profile remain unknown. OBJECTIVE: The aim of this study was to evaluate the influence of physical activity level and nutritional status on the methylation profile of the MTHFR gene in patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 111 patients, 43 men and 68 women diagnosed with DM (7.0 ± 2.3 years), answered the International Physical Activity Questionnaire (IPAQ) and underwent blood collection for biochemical analysis, DNA extraction, and MTHFR gene methylation profile determination. RESULT: The comparison of the methylation pattern showed that the partially methylated profile predominates in the insufficiently active group (85%), which does not occur in the sufficiently active group (54%) (p = 0.012). No differences were found in the nutritional status comparison. Logistic regression including overweight, waist circumference, gender, age, time of DM, hypertension, dyslipidemia, smoking, alcoholism, and family DM revealed that the association of the level of physical activity with methylation profile proved to be independent of these confounding variables. Considering the partially methylated profile as a result, being physically inactive favors the partially methylated MTHFR pattern in patients with DM. CONCLUSION: We concluded that insufficient physical activity is associated with partially methylated pattern of MTHFR promoter.

The MTHFR promoter hypermethylation pattern associated with the A1298C polymorphism influences lipid parameters and glycemic control in diabetic patients.

BACKGROUND: Polymorphisms in the gene encoding methylenetetrahydrofolate reductase (MTHFR) have been investigated as risk factors for microvascular complications of diabetes; however, simultaneous analysis of these polymorphisms and the methylation pattern of the gene has never been conducted. The objective of the present study was to evaluate the simultaneous relationship between MTHFR methylation and MTHFR C6TT7 and A1298C polymorphisms with metabolic, inflammatory and oxidative stress parameters related to microvascular complications, diabetic retinopathy (DR) and diabetic nephropathy (DN) in diabetic patients. METHODS: A total of 107 patients who were diagnosed in the previous 5 to 10 years were recruited and divided into groups with complications (DR and/or DN) or without complications. Methylation analysis of the gene promoter was conducted using the MSP technique, and analysis of the A1298C and C677T polymorphisms was conducted using the restriction fragment length polymorphism (RFLP) assay. Microalbuminuria was determined using urine samples, and other analytes of interest were determined in blood samples using commercial kits. The Mann-Whitney and Chi square statistical tests were used with significance considered at p < 0.05. RESULTS: Subjects with a hypermethylated profile and the 1298AA genotype showed the highest levels of blood glucose (p = 0.03), total cholesterol (p = 0.0001) and LDL cholesterol (p = 0.0006). The same profile was associated with higher levels of HbA1c (p = 0.025), glycemia (p = 0.04) and total cholesterol (0.004) in the control group and total cholesterol (p = 0.005) and LDL cholesterol (p = 0.002) in the complications group. Serum creatinine was higher in subjects in the hypermethylated group with the genotype 677CC only in the control group (p = 0.0020). The methylated profile in presence of 677CC + 1298AA and the 677CT/TT +1298AA haplotypes showed higher levels of total cholesterol (p = 0.0024; 0.0031) and LDL cholesterol (p = 0.0060; 0.0125) than 1298AC/CC carriers. The fasting glycemia was higher in hypermethylated profile in the presence of 677CC/1298AA haplotype (p = 0.0077). CONCLUSION: The hypermethylated methylation profile associated with the 1298AA genotype appeared to be connected to higher values of glycemia, total cholesterol and LDL cholesterol.

α-Tocopherol influences glycaemic control and miR-9-3 DNA methylation in overweight and obese women under an energy-restricted diet: a randomized, double-blind, exploratory, controlled clinical trial.

BACKGROUND: Excess weight is a strong risk factor for the development of dysglycaemia. It has been suggested that changes in the metabolism microRNAs, small non-coding RNAs that regulate gene expression, could precede late glycaemic changes. Vitamin E in turn may exert important functions in methylation and gene expression processes. This study aimed to determine the effect of α-tocopherol on glycaemic variables and miR-9-1 and miR-9-3 promoter DNA methylation in overweight women. METHODS: A randomized, double-blind, exploratory, placebo-controlled study was conducted in overweight and obese adult women (n = 44) who ingested synthetic vitamin E (all-rac-α-tocopherol), natural source vitamin E (RRR-rac-α-tocopherol) or placebo capsules and were followed up for a period of 8 weeks. Supplemented groups also received dietary guidance for an energy-restricted diet. An additional group that received no supplementation and did not follow an energy-restricted diet was also followed up. The intervention effect was evaluated by DNA methylation levels (quantitative real-time PCR assay) and anthropometric and biochemical variables (fasting plasma glucose, haemoglobin A1C, insulin, and vitamin E). RESULTS: Increased methylation levels of the miR-9-3 promoter region (P < 0.001) and reduced haemoglobin A1C (P < 0.05) were observed in the natural source vitamin E group after intervention. Increased fasting plasma glucose was observed in the synthetic vitamin E group, despite the significant reduction of anthropometric variables compared to the other groups. CONCLUSIONS: α-Tocopherol from natural sources increased methylation levels of the miR-9-3 promoter region and reduced haemoglobin A1C in overweight women following an energy-restricted diet. These results provide novel information about the influence of vitamin E on DNA methylation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02922491. Registered 4 October, 2016.

Hypermethylation in the promoter of the MTHFR gene is associated with diabetic complications and biochemical indicators.

BACKGROUND: DNA methylation is an epigenetic mechanism for regulating the transcription of many genes and has been linked to the development of various diseases. A promising gene to investigate is methylenetetrahydrofolate reductase (MTHFR), since the enzyme methylenetetrahydrofolate reductase (MTHFR) promotes methyl radical synthesis in the homocysteine cycle and can provide methyl groups for DNA methylation. In addition, several studies have correlated gene polymorphisms of this enzyme with a greater risk of diabetes, but little is known regarding the relationship between epigenetic changes in this gene and diabetes and its complications. The aim of this study was to investigate the relationship between methylation profile in the MTHFR gene promoter and biochemical, inflammatory and oxidative stress markers in individuals with type 2 diabetes (T2DM) who have been diagnosed for 5-10 years with or without diabetic retinopathy (DR) and nephropathy (DN). METHODS: Specific PCR for methylation (MSP) was used to analyze MTHFR methylation profile in leucocytes DNA. Biochemical markers (glycemia, glycated hemoglobin, total cholesterol, LDL, HDL, triglycerides, serum creatinine), inflammatory markers (C-reactive protein and alpha-1 acid glycoprotein) and oxidative stress (total antioxidant and malonaldehyde) were determined in peripheric blood samples and microalbuminuria in 24 h urine samples. The X2 and Mann-Whitney statistical tests were performed and p < 0.05 were considered significant. RESULTS: The hypermethylated profile was most frequently observed in individuals with retinopathy (p < 0.01) and was associated with higher total cholesterol and LDL levels (p = 0.0046, 0.0267, respectively). Individuals with DN and hypermethylated profiles had higher levels of alpha-1 acid glycoprotein (p = 0.0080) and total antioxidant capacity (p = 0.0169) compared to subjects without complications. CONCLUSIONS: Hypermethylation in the promoter of the MTHFR gene is associated with the occurrence of DR and with biochemical, inflammatory and oxidative stress parameters in the context of chronic complications.