No evidence for an effect of DNA methylation on multiple sclerosis severity at HLA-DRB1*15 or HLA-DRB5

https://doi.org/10.1016/j.jneuroim.2010.03.002Get rights and content

Abstract

Multiple sclerosis (MS) is a complex neurological disease with huge variability in disease outcome. The majority of MS genetic susceptibility is determined by major histocompatibility complex (MHC) alleles, in particular haplotypes carrying HLA-DRB1*1501. HLA-DRB1*1501 also affects the clinical outcome of the disease and animal research has suggested that HLA-DRB5 interacts with HLA-DRB1*1501 to influence disease severity. We used an extremes-of-outcome design with 48 benign and 20 malignant MS patients to assess whether or not DNA methylation at HLA-DRB1*1501 and/or HLA-DRB5 also contributes to MS phenotypic heterogeneity. We found no significant effect of DNA methylation across HLA-DRB1*1501 and HLA-DRB5 on severity, although we cannot rule out time- or tissue-specific effects of DNA methylation.

Introduction

Multiple sclerosis (MS) is a complex neurological disease resulting from demyelination and axonal loss (Noseworthy et al., 2000). The major genetic risk factor for MS is the major histocompatibility complex (MHC) class II region, in particular HLA-DRB1*1501-bearing haplotypes (Dyment et al., 2005), although recent research has shown that MS susceptibility is actually determined by MHC class II diplotypes (Lincoln et al., 2009). The course of MS varies widely, as does the rate at which disability accumulates. Most patients initially manifest a relapsing–remitting form of disease, whereby periods of disability are interspersed with neurological recovery and remission. Others have primary progressive MS, in which there is a steady decline in disability from baseline. Patients with relapsing–remitting MS mostly later convert to secondary progressive MS as a background decline in disability occurs and so remission becomes incomplete. Although there are some markers of severity, none of these are highly specific for different forms of MS (Degenhardt et al., 2009).

HLA-DRB1*1501 has been shown to influence the clinical course of MS (DeLuca et al., 2007, Okuda et al., 2009). Furthermore, in transgenic mice manifesting a spontaneous form of experimental autoimmune encephalitis (EAE), the severity of disease was increased in animals carrying HLA-DRB1*1501 when present without HLA-DRB5 (Gregersen et al., 2006). Normally HLA-DRB1*1501 is in complete linkage disequilibrium with HLA-DRB5 and so the results of this EAE study are not directly applicable in humans unless epigenetic mechanisms are at play.

Epigenetics refers to the alteration of genomic expression through mechanisms other than changes in the DNA sequence, and includes DNA methylation (Handel et al., 2009). Most DNA methylation occurs at CpG dinucleotides across the genome; these are points at which a cytosine base is linked via a phosphate group to an adjacent guanine base. MS shows maternal parent-of-origin and sex-specific effects with a rapid alteration in the sex ratio of patients over the past century (Ebers et al., 2004, Herrera et al., 2008, Hoppenbrouwers et al., 2008, Orton et al., 2006). These epidemiological data are highly suggestive of heritable epigenetic marks, and indeed the epigenetic effects have been localised to the MHC (Chao et al., 2009, Ramagopalan et al., 2008).

We hypothesised that patients with particularly severe forms of MS (malignant MS) might show relative epigenetic inactivation of HLA-DRB5 compared with patients with a very mild disease course (benign MS) through DNA methylation and vice versa for HLA-DRB1. This extremes-of-outcome design comparing benign and malignant patients has previously been fruitful in identifying HLA-DRB1 alleles involved in MS outcome (DeLuca et al., 2007).

Section snippets

Subjects and ethics

Subjects were ascertained through the Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS) for which the methodology has been previously described (Sadovnick et al., 1998). Each participating clinic in the CCPGSMS obtained ethical approval from the relevant institutional review board, and the entire project was reviewed and approved by the University of British Columbia and the University of Western Ontario and informed consent was obtained from all patients.

The

Benign vs. malignant MS

We compared DNA methylation at CpG dinucleotides across HLA-DRB1*1501 and HLA-DRB5 in 48 HLA-DRB1*1501 positive benign patients and 20 HLA-DRB1*1501 positive malignant patients. There were no significant differences between the groups when compared across individual CpG dinucleotides or across the entire regions of HLA-DRB1*1501 and HLA-DRB5 (Fig. 1; p > 0.05). There was a non-significant trend towards more HLA-DRB5 DNA methylation in the malignant than the benign group (malignant 60.3% vs.

Discussion

Our findings suggest that DNA methylation of CpG dinucleotides across HLA-DRB1*1501 and HLA-DRB5 does not determine whether patients manifest benign or malignant MS. We did find significant differences in overall DNA methylation across HLA-DRB1*1501 when comparing benign and malignant HLA-DRB1*1501 hetero- and homozygotes. However, the fact that in heterozygotes there is more DNA methylation in malignant patients than benign whereas the reverse is true in homozygotes argues against this being a

Competing interests

The authors have no conflicts of interest to declare.

Author contributions

SVR and GCE were responsible for the experimental design. SVR, GCD, JM, LH and ADS carried out the experiments. AEH and SVR analysed the data. AEH, SVR and GCE wrote the manuscript.

Acknowledgements

This work was funded by the Multiple Sclerosis Society of the United Kingdom and the Multiple Sclerosis Society of Canada Scientific Research Foundation. We would like to thank all contributing physicians and scientists from the CCPGMS. SVR is funded by a Goodger Scholarship (University of Oxford).

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