Identification of differentially methylated regions in new genes associated with knee osteoarthritis.
Epigenetic changes in articular chondrocytes are associated with osteoarthritis (OA) disease progression. Numerous studies have identified differentially methylated cytosines in OA tissues; however, the consequences of altered CpG methylation at single nucleotides on gene expression and phenotypes are difficult to predict. With the objective of detecting novel genes relevant to OA, we conducted a genome-wide assessment of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). DNA was extracted from visually damaged and normal appearing, non-damaged human knee articular cartilage from the same joint and then subjected to reduced representation bisulfite sequencing. DMRs were identified using a genome-wide systematic bioinformatics approach. A sliding-window of 500 bp was used for screening the genome for regions with clusters of DMSs. Gene expression levels were assessed and cell culture demethylation experiments were performed to further examine top candidate genes associated with damaged articular cartilage. More than 1000 DMRs were detected in damaged osteoarthritic cartilage. Nineteen of these contained five or more DMSs and were located in gene promoters or first introns and exons. Gene expression assessment revealed that hypermethylated DMRs in damaged samples were more consistently associated with gene repression than hypomethylated DMRs were with gene activation. Accordingly, a demethylation agent induced expression of most hypermethylated genes in chondrocytes. Our study revealed the utility of a systematic DMR search as an alternative to focusing on single nucleotide data. In particular, this approach uncovered promising candidates for functional studies such as the hypermethylated protein-coding genes FOXP4 and SHROOM1, which appear to be linked to OA pathology in humans and warrant further investigation.
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.
Carolina A
CA
Bonin
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.
Eric A
EA
Lewallen
Department of Biomedical Statistics and Informatics, Rochester, MN, United States.
Saurabh
S
Baheti
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.
Elizabeth W
EW
Bradley
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.
Michael J
MJ
Stuart
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.
Daniel J
DJ
Berry
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, United States.
Andre J
AJ
van Wijnen
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, United States. Electronic address: westendorf.jennifer@mayo.edu.
Jennifer J
JJ
Westendorf
D001374:Azacitidine / Q000494:pharmacology; D002358:Cartilage, Articular / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects; D018899:CpG Islands; D019175:DNA Methylation; D051858:Forkhead Transcription Factors / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D008840:Microfilament Proteins / Q000235:genetics*; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012016:Reference Values; D015203:Reproducibility of Results
C576732:FOXP4 protein, human; D051858:Forkhead Transcription Factors; D008565:Membrane Proteins; D008840:Microfilament Proteins; C539270:Shroom1 protein, human; D001374:Azacitidine
Articular cartilage; DNA methylation; Differentially methylated regions; FOXP4; SHROOM1