The Babraham Institute

Author Of 1 Presentation

Pathogenesis – Immunology Poster Presentation

P0956 - DNA methylation of cerebrospinal fluid cells in Multiple Sclerosis (ID 1193)

Speakers
Presentation Number
P0956
Presentation Topic
Pathogenesis – Immunology

Abstract

Background

Multiple Sclerosis (MS) is a chronic inflammatory disease of a yet unknown cause characterized by autoimmune destruction of myelin and neurons in the central nervous system. Immune cells from the cerebrospinal fluid (CSF) can provide valuable insight into the pathogenic processes occurring in the typically inaccessible target organ; however, they have not been sufficiently utilized due to low numbers. DNA methylation is an epigenetic modification that without altering the genetic code can stably change the expression of genes and thus may play an important role in MS development. Moreover, due to stability, high specificity and the ability to be measured in limited amounts of material genome-wide, methylation changes may assist in studying MS pathogenesis.

Objectives

We aimed to establish and optimize a genome-wide methylation approach to investigate methylation changes that render immune cells pathogenic in MS with the prospect of better understanding disease pathogenesis.

Methods

Cells from the CSF of relapsing-remitting MS (RRMS n=5) and age- and sex-matched non-inflammatory controls (NINDC n=5) were extracted. Whole-genome bisulfite sequencing (WGBS) libraries were generated using the post-bisulfite adaptor tagging (PBAT) protocol, which originally was designed for single-cell and adapted to the limited cell amount as previously described (1). Results were replicated in a larger cohort (RRMS n=18 and NINDC n=7), while the reliability of the methodology was confirmed with technical replication of the original cohort.

Results

We first explored the global landscape of CpG methylation levels, as well as specific genomic features including promoters, enhancers and CTCF binding sites. We conducted statistical analysis based on three distinct methods, which identified differentially methylated CpG (DMCs). Methylation levels calculated in the sequencing data were well correlated with the independent technical replicates where the sequencing depths were comparatively lower which gave more evidence the less sequenced data can reflect the true values to some degree. Besides, in the replicate sample groups, a large proportion of DMCs changed in the same direction when applying a minimal 0.2 difference. Moreover, NFATC1, NFKBID, MAPK1, MAP2K2 and other genes participated in the regulation of the immune response were differentially modified. In addition, the differentially modified genes were significantly enriched in pathways including integrin signaling pathway which is important for cell movement and activation, CD40 signaling pathway which is related with the lymphocyte activation, as well as various cytokine signaling pathways such as IL-1, IL-6, IL-17.

Conclusions

Genome-wide methylation analysis, based on a low cell-number library preparation, will enable us to study MS pathogenesis using samples that are difficult to comprehensively study by many other methods.

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