Methylation patterns of cell-free plasma DNA in relapsing–remitting multiple sclerosis

Abstract

Background

There is growing interest for identification of new targets for biomarker development in multiple sclerosis (MS). The goal of this study was to compare the concentration and the methylation patterns of cell-free plasma DNA (cfpDNA) in patients with relapsing–remitting multiple sclerosis (RRMS) and healthy individuals.

Methods

Three 30-patient cohorts were examined: patients with RRMS, in either remission or exacerbation, and healthy individuals as controls. Concentration of cfpDNA was determined using a standard fluorometric assay. Patterns of methylation in 56 gene promoters were determined by a microarray-based assay (MethDet-56). The data were analyzed to identify statistically relevant differences among the study groups.

Results

The concentration of cfpDNA in patients with RRMS was four to eight-fold higher compared to healthy controls. Significant differences in cfpDNA methylation patterns were detected in all three comparisons: RRMS patients in remission versus healthy controls were recognized with 79.2% sensitivity and 92.9% specificity; RRMS patients in exacerbation versus healthy controls were recognized with 75.9% sensitivity and 91.5% specificity; and RRMS patients in exacerbation versus those in remission were recognized with 70.8% sensitivity and 71.2% specificity.

Conclusion

Based on our findings, we conclude that patients with RRMS display unique disease- and state-specific changes of cfpDNA. Our findings are of clinical significance as they could be used in the development of potentially new biomarkers for MS. This is the first report in our knowledge describing such changes of cfpDNA in patients with MS.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology. The natural course of the disease exhibits several clinical patterns: relapsing–remitting, secondary progressive, primary progressive, and relapsing progressive [1], [2]. The etiologic ambiguity and the clinical variability of the disease create significant diagnostic and prognostic uncertainties and reduce the opportunities for early diagnosis and treatment. Such diagnostic limitations have stimulated growing interest for biomarker development in MS, which has evolved in multiple directions [3]. Protein-based approaches are focused on the detection of altered levels of inflammatory molecules (antibodies, cytokines, etc.) [4], [5], [6], or other cellular proteins found in the bodily fluids of MS patients [7], [8]. Genetic-based approaches concentrate on the identification of genes, related to disease susceptibility or severity (HLA genes, etc.) [9], or on disease-associated genetic variations (single nucleotide polymorphisms, etc.) that may predict risk for disease development and progression [10], [11]. New, high throughput technologies have also been used for biomarker research [12]. However, despite the current research, success at identification of a biomarker for MS remains elusive.

Disease-associated changes in DNA methylation have recently gained interest for biomarker development. DNA methylation is an epigenetic mechanism of long-term regulation of gene expression. It is a process of chemical modification of DNA that adds a methyl group to a cytosine nucleotide when it is located upstream from a guanosine nucleotide (CpG dinucleotides), frequently within and near gene promoters. Abnormal DNA methylation and gene expression are associated with various diseases such as cancers, lymphoproliferative disorders, rheumatoid arthritis, and systemic lupus erythematosus [13], [14], [15], [16]. As for MS, putative epigenetic changes have been suggested to explain the gender bias, the low level concordance in homozygous twins, and the linkage to several genetic loci [17], [18].

Cell-free plasma DNA (cfpDNA) exists as heterogeneous polynucleotides in plasma of all humans. Lately, it has attracted attention as a disease biomarker because of its easy accessibility, established diagnostic value in genetic diseases, and utility for methylation analysis [19]. CfpDNA is believed to be released from proliferating, or dying cells, and to reflect the normal or abnormal turnover of cell populations, although its precise origins are still enigmatic [20]. CfpDNA concentration is found to be elevated in patients with trauma, cancer, inflammation, and stroke [21], [22], [23]. In MS, the utility of cfpDNA for biomarker development has not yet been evaluated. However, given the facts that MS is a disease of immune activation, central nervous system inflammation, oligodendrocyte and neuronal injury, and gliosis, one can hypothesize that the disease can induce changes in cfpDNA.

In this study, we analyzed cfpDNA in patients with relapsing–remitting MS (RRMS) and in healthy individuals. We found that cfpDNA in patients with RRMS displayed unique disease- and state-specific characteristics. Our findings are clinically relevant and suggest that cfpDNA might be useful for biomarker development for MS.