Genome-wide DNA methylation profile analysis in thoracic ossification of the ligamentum flavum

Thoracic ossification of the ligamentum flavum (TOLF) causes serious spinal canal stenosis. The underlying aetiology may relate to genetic and inflammatory factors. DNA methylation plays a critical role in osteogenesis and inflammation, whereas there is no genome-wide DNA methylation analysis about TOLF. The two subtypes of TOLF (single-level and multiple-level) have distinct clinical features. Using micro-computed tomography (micro-CT), we showed the ossification arose from the joint between two vertebrae at one/both sides of ligament flavum. With Illumina Infinium Human Methylation 850 BeadChip arrays, genome-wide DNA methylation profile was measured in ligament flavum of eight healthy and eight TOLF samples. Only 65 of the differentially methylated cytosine-phosphate-guanine dinucleotides were found in both subtype groups. Principal component analysis and heat map analysis showed a different methylation pattern in TOLF samples, and methylation patterns of two subtypes are also distinct. The Gene Ontology enrichment analysis was significantly enriched in differentiation and inflammation. Pyrosequencing analysis and quantitative real-time polymerase chain reaction were performed to validate the arrays results and expression levels, to test six differentially methylated genes (SLC7A11, HOXA10, HOXA11AS, TNIK, homeobox transcript antisense RNA, IFITM1), using another independent samples (P < 0.05). Our findings first demonstrated an altered Genome-wide DNA methylation profile in TOLF, and implied distinct methylated features in two subtypes.