DNA Methylation in Degenerating Human Intervertebral Discs

Abstract

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) and is associated with widespread changes in gene expression, extracellular matrix (ECM) degradation, inflammation, and innervation. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences. To date, little is known about epigenetic dysregulation in degenerated IVDs. The objective of this study was to investigate changes in DNA methylation in degenerating human IVDs from chronic LBP patients. Human lumbar IVD segments at L4/5 level including control (n=4) and degenerating (n=6) were homogenized in liquid nitrogen with a mortar and pestle and genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. Epigenome-wide 5mC mapping was performed following bisulfite sequencing using the Infinium MethylationEPIC BeadChip 850k Arrays. All analysis was performed in the R package RnBeads 2.0. Principal component analysis and hierarchical clustering analysis revealed group differences between healthy and degenerating IVDs. Pathway analysis revealed enrichment in genes related to ECM organization, signaling by receptor tyrosine kinases and signaling by interleukins. Further analysis at the single gene promoter level showed hypermethylation in ECM-related genes linked to IVD degeneration including sparc and col2a1 and hypomethylation of pro-inflammatory cytokines associated with IVD degeneration including IL-1b and IL-8. These changes in methylation would be expected to result in loss of ECM and increased inflammation. Persistent reprogramming of gene expression by DNA methylation could drive IVD degeneration and inflammation. Understanding the role of DNA methylation in IVD pathology may provide a scientific framework for the development of new therapeutic approaches.