The University of Southampton
University of Southampton Institutional Repository

Can epigenetic changes in DNA methylations explain the altered gene expression in osteoarthritis?

Can epigenetic changes in DNA methylations explain the altered gene expression in osteoarthritis?
Can epigenetic changes in DNA methylations explain the altered gene expression in osteoarthritis?
Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5'-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.
dna, gene expression, gene, expression, osteoarthritis, epigenetic, gene-expression
0301-620X
385-386
Roach, H.I.
1c0cf1f8-15dc-49a8-aad8-9ed3fd13c05b
Inglis, S.
cb950bb9-8b89-41c0-b547-3206580d16b4
Partridge, K.
702fdbd7-aa27-4ae2-adcd-e1a94d9f25e7
Oreffo, R.
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Clarke, N.M.P.
76688c21-d51e-48fa-a84d-deec66baf8ac
Roach, H.I.
1c0cf1f8-15dc-49a8-aad8-9ed3fd13c05b
Inglis, S.
cb950bb9-8b89-41c0-b547-3206580d16b4
Partridge, K.
702fdbd7-aa27-4ae2-adcd-e1a94d9f25e7
Oreffo, R.
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Clarke, N.M.P.
76688c21-d51e-48fa-a84d-deec66baf8ac

Roach, H.I., Inglis, S., Partridge, K., Oreffo, R. and Clarke, N.M.P. (2006) Can epigenetic changes in DNA methylations explain the altered gene expression in osteoarthritis? Journal of Bone and Joint Surgery, 88-B (Supplement III), 385-386.

Record type: Article

Abstract

Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5'-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.

This record has no associated files available for download.

More information

Published date: 2006
Additional Information: British Orthopaedic Research Society: Bristol – 29–30 March, 2004
Keywords: dna, gene expression, gene, expression, osteoarthritis, epigenetic, gene-expression
Organisations: Dev Origins of Health & Disease

Identifiers

Local EPrints ID: 61465
URI: http://eprints.soton.ac.uk/id/eprint/61465
ISSN: 0301-620X
PURE UUID: 3b662507-f532-417b-994c-9cb29d661e72
ORCID for R. Oreffo: ORCID iD orcid.org/0000-0001-5995-6726

Catalogue record

Date deposited: 07 Apr 2009
Last modified: 12 Dec 2021 03:09

Export record

Contributors

Author: H.I. Roach
Author: S. Inglis
Author: K. Partridge
Author: R. Oreffo ORCID iD
Author: N.M.P. Clarke

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×