Abnormal DNA methylation in CD4⁺ T cells from people with latent autoimmune diabetes in adults

Abstract

Aberrant DNA methylation in T cells has been linked to pathogenesis of autoimmune diseases. To investigate genomic and gene-specific DNA methylation levels in CD4⁺ T cells from patients with latent autoimmune diabetes in adults (LADA), and to investigate changes in the expression of genes that regulate methylation as well as the autoimmune-related gene FOXP3 in these patients. Global CD4⁺ T cell DNA methylation was measured in 15 LADA patients and 11 healthy controls using a methylation quantification kit. mRNA levels of DNA methytransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and FOXP3 were measured by real time PCR. Methylation of a FOXP3 regulatory element region was determined by bisulphite genomic sequencing. Genomic DNA methylation in CD4⁺ T cells from LADA patients was significantly increased compared to controls. DNMT3b mRNA levels were higher in CD4⁺ T cells from LADA patients than in controls. DNMT3b expression positively correlated with global DNA methylation in LADA CD4⁺ T cells. FOXP3 expression was decreased, and the FOXP3 promoter region was hypermethylated in CD4⁺ T cells from LADA patients compared with controls. DNA methylation levels are altered in CD4⁺ T cells from LADA patients, which may contribute to disease onset and progression by affecting the expression of autoimmune-related genes.

Introduction

Epidemiological studies suggest that latent autoimmune diabetes in adults (LADA) may account for 2–12% of all cases of diabetes [1], [2], [3]. The presence of autoantibodies along with islet-reactive T cells in LADA provides strong evidence that the disease process is autoimmune [4], [5], [6]. LADA is thought to be a subgroup of type 1 diabetes, which has a slow procession of autoimmune destruction of β-cells. CD4⁺ regulatory T cells are reduced and the expression of forkhead box P3 (FOXP3) in CD4⁺ T cells is decreased in LADA patients [7], suggesting that defects in immunological tolerance could contribute to the development of LADA. Although there is evidence to show that genetic factors such as human leucocyte antigen (HLA) can confer an increased risk of LADA [8], a low twin concordance for adult-onset type 1diabetes implies that the genetic impact in adult-onset diabetes is limited, favoring a substantial impact of non-genetic factors which may include epigenetics [9]. Epigenetic modifications, including the addition or removal of methyl moieties on cytosines in CpG dinucleotides (5-methylcytosine), cause heritable effects on transcriptional regulation without changing the sequence of the genome. The level and pattern of 5-methylcytosines within regulatory DNA sequences (i.e., the methylation status of the DNA) affects the binding of transcription factors and thus the rate of gene transcription within the region. The regulation of DNA methylation is critical for a diversity of biological events, including embryonic development, X chromosome inactivation, genomic “imprinting”, chromatin condensation and the silencing of endogenous retroviruses [10], [11], [12].

Three enzymes determine the methylation status of DNA in humams: DNA methyltransferase 1 (DNMT1) is involved in maintaining methylation patterns after DNA synthesis and cell division, whereas DNA methyl transferase 3a (DNMT3a) and DNA methyl transferase 3b (DNMT3b) catalyze de novo methylation [13]. In general, methylated DNA results in the down-regulation of gene transcription by recruiting transcriptional co-repressors belonging to the methyl-DNA binding domain family of proteins (MBDs) [14].

In a number of autoimmune disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis, the methylation status of genomic DNA and specific autoimmunity-related genes is altered, increasing expression of genes and leading to autoreactivity. These changes have been shown to be associated with changes in the expression of DNMTs and MBDs [15], [16], [17]. In this study, we investigated global methylation patterns and the expression of DNMTs and MBDs in CD4⁺ T cells of patients with LADA. We also assessed the methylation status of the FOXP3 gene in these cells and correlated FOXP3 expression with methylation status.