Genome-wide DNA methylation profiling of hip articular cartilage identifies differentially methylated loci associated with osteonecrosis of the femoral head

Abstract

Objective: Recent studies demonstrated a critical role of hip articular cartilage destruction in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to characterize the genome-wide DNA methylation profile of hip cartilage obtained from patients with ONFH and healthy subjects.

Methods: Hip articular cartilage specimens were collected from 15 ONFH patients (including 11 males and 4 females) and 15 control subjects (including 11 males and 4 females) with femoral neck fracture. The average ages of the ONFH patients and control subjects were 50.27 ± 5.27 years and 61.67 ± 3.38 years, respectively. Genome-wide DNA methylation profiles of 5 ONFH and 5 control cartilages were determined by Illumina HumanMethylation850 array. Differential methylation analysis of DNA methylation profiles were performed by the empirical Bayes moderated t-test of the limma package. Mass spectrograph (MS) analysis of 10 ONFH cartilages and 10 normal cartilages were performed to validate the results of genome-wide DNA methylation profiling. Immunohistochemistry (IHC) of 4 ONFH cartilages and 4 control cartilages were conducted to evaluate the expression levels of proteins encoded by identified differentially methylated genes. t-test was used to assess the significance of protein expression differences between ONFH patients and controls in IHC.

Results: We identified a total of 2872 differentially methylated CpG sites, annotated to 480 hypermethylated genes and 1335 hypomethylated genes for ONFH. The results of MS validation were consistent with that of genome-wide DNA methylation profiling. IHC further confirmed the increased protein expression of CARS (mean and 95%CI of superficial zone 59.67% [48.46, 56.14], and deep zone 31% [25.85, 30.61]), PDE4D (superficial zone 50.33% [33.64, 40.68] and deep zone 28.67% [10.81, 36.47]), ADAMTS12 (superficial zone 53.67% [36.01, 40.93] and deep zone 34.67% [22.56, 37.18]), LRP5 (superficial zone 59.63% [27.32, 39.61] and deep zone 22.95% [5.28, 19.29]), RUNX2 (superficial zone 52.58% [11.64, 31.33] and deep zone 35.01% [10.03, 27.44]) in ONFH articular cartilage.

Conclusion: Our results suggest the implication of DNA methylation alterations in the development of ONFH, and provide novel clues for pathogenetic and therapeutic studies of ONFH.

Keywords: Genome-wide DNA methylation; Hip articular cartilage; Osteonecrosis of the femoral head; Pathway enrichment analysis.