Investigation of KLF14 Promoter Methylation in Patients with Type 2 Diabetes. A Preliminary Analysis

Background and Objectives: Kruppel-Like Factor 14 (KLF14) is defined as a master regulator of gene expression in adipose tissue and has been associated with body mass index (BMI) and type 2 diabetes (T2D). Genome-wide association studies aim to find predictive biomarkers for the development of type 2 diabetes, taking into account epigenetic changes affected by environmental factors. DNA methylation plays a decisive role in the regulation and control of gene expression. The objective of this work was to identify the percentage of methylation in the promoter region of the KLF14 gene in patients with type 2 diabetes. and controls and to reveal the contribution of the KLF14 to the development of Type 2 diabetes. Methods: A total of 20 volunteers participated in this study (10 patients with type 2 diabetes and 10 controls), between 35 and 70 years of age, and were evaluated for KLF14 promoter methylation. Genomic DNA was extracted from peripheral blood using the blood DNA extraction kit (Qiagen, Inc., Valencia, CA). Genomic DNA was used for bisulfite conversion by the Epitect Bisulfite kit (Qiagen). The methylation status was assessed by bisulfite sequencing. The calculation of the methylation percentage is performed by measuring the signal (peak height) of cytosine and thymine by applying the formula “a” = [Peak Height C/(Peak Height C Peak Height T)] × 100. Results: By sequencing analysis, we found methylation in 26 of the 35 CpG sites analyzed in the total sample (T2D and controls). Our results show increased methylation levels in the KLF14 gene promoter in diabetic individuals compared to control subjects [54.56% ± 17.89% vs. 35.77% ± 16.67% vs. respectively; RR = 1.9 (1.236 - 2.963), p = 0.001]. Further analysis revealed that specific CpG sites in KLF14 gene display increased methylation in T2D patients. A hierarchical cluster analysis was used to classify a sample in a ranking list, based on relative methylation percentage over or under the mean standardized scores of individual variables. The study identified three groups of dinucleotide CpGs in the total sample, Promoter region analysis identified three GGGCGG boxes of the Sp1 transcription factor. Interpretation and Conclusions: The present investigation, carried out in an Argentine population, demonstrated a higher methylation status in the KLF14 promoter is associated with type 2 diabetes. We postulate that increased methylation in the GGGCGG boxes, could interfere with Sp1 factor binding, and consequently silence/decrease KLF14 transcription in individuals with T2DM. We propose that the methylation status of specific CpG sites of the KLF14 promoter could be used as an epigenetic predictor of type 2 diabetes development.