Genome‐wide profiling of bone reveals differentially methylated regions in osteoporosis and osteoarthritis

Abstract

Objective. To determine genome-wide methylation profiles of bone from patients with hip osteoarthritis (OA) and those with osteoporotic (OP) hip fractures.Methods. Trabecular bone pieces were obtained from the central part of the femoral head of 27 patients with hip fractures and 26 patients with hip OA. DNA was isolated, and methylation was explored with Illumina methylation arrays. RNA was extracted, pooled, and deep-sequenced to obtain the whole transcriptome. Differentially methylated regions were identified, and connections between genes with differentially methylated regions were explored by pathway and text-mining analyses.Results. After quality control, methylation of 23,367 CpG sites (13,463 genes) was analyzed. There was a genome-wide inverse relationship between methylation and gene expression in both patient groups. Comparison of OP and OA bones revealed 241 CpG sites, located in 228 genes, with significant differences in methylation (false discovery rate <0.05). Of them, 217 were less methylated in OP than in OA. The absolute methylation differences were >5% in 128 CpG sites and >10% in 45 CpG sites. The differentially methylated genes were enriched for association with bone traits in the genome-wide association study catalog. Pathway analysis and text-mining analysis with Gene Relationships Across Implicated Loci software revealed enrichment in genes participating in glycoprotein metabolism or cell differentiation, and particularly in the homeobox superfamily of transcription factors.Conclusion. Genome-wide methylation profiling of bone samples revealed differentially methylated regions in OP and OA. These regions were enriched in genes associated with cell differentiation and skeletal embryogenesis, such as those in the homeobox superfamily, suggesting the existence of a developmental component in the predisposition to these disorders.Bone increases in size during the growth period by a modeling process driven by the formation of new bone. Thereafter, it is constantly remodeled by the concerted action of bone-resorbing osteoclasts and bone-forming osteoblasts, originating from hematopoietic and mesenchymal precursors, respectively. When remodeling is to start at a certain site, osteoclast precursors are attracted and committed to differentiate into mature osteoclasts. When the resorption phase ends, surrounding osteoblast precursors proliferate and differentiate into mature osteoblasts that synthesize bone matrix that eventually mineralizes and replaces the old bone resorbed by osteoclasts. Thus, bone remodeling requires the cyclic and sequential proliferation and differentiation of osteoclast and osteoblast precursors. Any disturbance of this process will result in abnormal bone mass. This is the case in osteoporosis (OP), which is characterized by a decrease in bone mass, due to high bone resorption and/or low bone formation, and consequently propensity to fracture. In contrast to OP, in osteoarthritis (OA) bone formation is increased in the

How to interpret epigenetic association studies: a guide for clinicians

“…As an example, Delgado-Calle et al analyzed DNA methylation in bone from patients with hip fractures and with hip osteoarthritis. 30 These between-disease comparisons may produce meaningful results, but it may be difficult to know which disorder is actually different from normal and, consequently, which are the disease-driving changes.…”

“…Thus, as it was the case in some recent studies, bone samples are preferable in principle to study osteoporosis-related epigenetic markers, whereas cartilage samples or synovial samples are preferable in studies of osteoarthritis or rheumatoid arthritis. 30,[32][33][34] Nevertheless, some intriguing similarities in the methylation of some genes in cartilage and bone have been recently reported. 35 There are very scarce data about the correlation of epigenetic marks in blood and the skeleton, but, in view of other studies, caution is recommended before extrapolating blood signatures to bone signatures.…”

“…However, these concerns have not been usually addressed in studies about skeletal epigenetics. 30,32 The analysis of a single type of cells, including immortalized cell lines and primary cell cultures (for example, primary osteoblasts grown from bone pieces), could emerge as an interesting alternative to avoid the problem of heterogeneity. However, the results of those studies must be interpreted with caution, because of the modifications of epigenetic signatures that experience cells in culture.…”

“…Earlier designs interrogated no more than 27 000 CpG, but they produced some useful results including the identification of differentially methylated genes of the HOX family in patients with osteoporosis and osteoarthritis. 30 However, that technology explored just about 1/1000 of the epigenome and consequently missed many regions potentially involved in the pathogenesis of the diseases. Many studies of skeletal and non-skeletal disorders have used more modern array designs that explore the methylation status of more than 450 000 CpGs.…”

“…This was the method for correction used in several epigenome-wide studies in the bone field (Box 2). 30,32,56 Nevertheless, any procedure increasing the statistical stringency to avoid the inflation of type I error implicates increasing the chances of type II error. This is the error occurring when true differences or associations are not identified because they do not appear to be 'significant' in the study.…”