Genome-wide analysis of DNA methylation profile identifies differentially methylated loci associated with human intervertebral disc degeneration

Abstract

Background: Environmental and endogenous factors under genetic predisposition are considered to initiate the human intervertebral disc (IVD) degeneration. DNA methylation is an essential mechanism to ensure cell-specific gene expression for normal development and tissue stability. Aberrant epigenetic alterations play a pivotal role in several diseases, including osteoarthritis. However, epigenetic alternations, including DNA methylation, in IVD degeneration have not been evaluated. The purpose of this study was to comprehensively compare the genome-wide DNA methylation profiles of human IVD tissues, specifically nucleus pulpous (NP) tissues, with early and advanced stages of disc degeneration.

Methods: Human NP tissues were used in this study. The samples were divided into two groups: early stage degeneration (n = 8, Pfirrmann's MRI grade: I-III) and advanced stage degeneration (n = 8, grade: IV). Genomic DNA was processed for genome-wide DNA methylation profiling using the Infinium MethylationEPIC BeadChip array. Extraction of raw methylation data, clustering and scatter plot of each group values of each sample were performed using a methylation module in GenomeStudio software. The identification of differentially methylated loci (DMLs) and the Gene Ontology (GO) analysis were performed using R software with the ChAMP package.

Results: Unsupervised hierarchical clustering revealed that early and advanced stage degenerated IVD samples segregated into two main clusters by their DNA methylome. A total of 220 DMLs were identified between early and advanced disc degeneration stages. Among these, four loci were hypomethylated and 216 loci were hypermethylated in the advanced disc degeneration stage. The GO enrichment analysis of genes containing DMLs identified two significant GO terms for biological processes, hemophilic cell adhesion and cell-cell adhesion.

Conclusions: We conducted a genome-wide DNA methylation profile comparative study and observed significant differences in DNA methylation profiles between early and advanced stages of human IVD degeneration. These results implicate DNA methylation in the process of human IVD degeneration.

Fig 1. Magnetic resonance imaging (MRI) of human intervertebral disc classified according to the Pfirrmann grading system[22].

Grades I to III were classified as early stage degeneration (ED). Grade IV and V were classified as advanced stage degeneration (AD).

Fig 2. Unsupervised hierarchical clustering of DNA methylation values of human nucleus pulposus (NP) tissues in eight early stage (ED) and eight advanced stage (AD) of disc degeneration.

Fig 3. Scatter plot of average DNA methylation level of early stage (ED) and advanced stage (AD) of degeneration.

Fig 4. Differences in β value of four highest hypomethylated and hypermethylated loci between early stage (ED) and advanced stage (AD) of degeneration.

* = adjust. P. Val < 0.05; ** = adjust. P. Val < 0.01; *** = adjust. P. Val < 0.001. A: CARD14 (Caspase Recruitment Domain Family Member 14), B: CRHR1 (Corticotropin Releasing Hormone Receptor 1), C: C14orf139 (Chromosome 14 Open Reading Frame 139), D: ZBTB47 (Zinc Finger And BTB Domain Containing 47), E: GNL3 (G Protein Nucleolar 3), F: SNORA52 (Small Nucleolar RNA, H/ACA Box 52), G: XKR5 (X Kell Blood Group Precursor-Related Family, Member 5), H: MED23 (Mediator Complex Subunit 23).

Fig 5. Scatter plots of the β value of four highest hypomethylated and hypermethylated loci between early stage (ED) and advanced stage (AD) of degeneration according to age.

Blue dots indicate the β values of the early stage of degenerated (ED) samples, and orange dots indicate β values of the advanced stage of degenerated (AD) samples. R2: correlation coefficient. *P<0.05, **P<0.01. A: CARD14 (Caspase Recruitment Domain Family Member 14), B: CRHR1 (Corticotropin Releasing Hormone Receptor 1), C: C14orf139 (Chromosome 14 Open Reading Frame 139), D: ZBTB47 (Zinc Finger And BTB Domain Containing 47), E: GNL3 (G Protein Nucleolar 3), F: SNORA52 (Small Nucleolar RNA, H/ACA Box 52), G: XKR5 (X Kell Blood Group Precursor-Related Family, Member 5), H: MED23 (Mediator Complex Subunit 23).