Original Article

Genes and Immunity (2008) 9, 334–337; doi:10.1038/gene.2008.22; published online 10 April 2008

EVI5 is a risk gene for multiple sclerosis

I A Hoppenbrouwers1, Y S Aulchenko2, G C Ebers3, S V Ramagopalan3, B A Oostra4, C M van Duijn2 and R Q Hintzen1

  1. 1Department of Neurology, MS Centre ErasMS, Erasmus MC, Rotterdam, The Netherlands
  2. 2Genetic Epidemiology Unit, Departments of Epidemiology & Biostatistics, Erasmus MC, Rotterdam, The Netherlands
  3. 3Wellcome Trust Centre for Human Genetics and Department of Clinical Neurology, University of Oxford, Oxford, UK
  4. 4Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands

Correspondence: Dr RQ Hintzen, Department of Neurology, Erasmus MC Rotterdam, Postbox 2040, 3000 CA, Rotterdam, The Netherlands. E-mail: rhintzen@xs4all.nl

Received 6 February 2008; Revised 27 February 2008; Accepted 27 February 2008; Published online 10 April 2008.

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Abstract

HLA-DRB1 is the major locus associated with risk for multiple sclerosis (MS). A recent genome-wide study showed three additional single-nucleotide polymorphisms (SNPs), within the IL2RA and IL7RA genes respectively, also to be associated with MS. Consistent association but lower significance was found for 13 other SNPs. In this study, we aimed to verify association of these SNPs with MS in 46 MS patients and 194 controls from a Dutch genetically isolated population. Apart from the human leukocyte antigen locus, the EVI5 gene on chromosome 1 was confirmed as a novel risk gene, with odds ratios (ORs) even higher than those from the MS Consortium (ORs 2.01 and 1.9; P=0.01). The risk effect of EVI5 was further validated for the general MS population in an independent set of 1318 MS patients from the Canadian Collaborative Project on the Genetic Susceptibility to MS. On the basis of the transmission disequilibrium testing, a weak but significant risk effect was observed (OR 1.15; P=0.03 and OR 1.15; P=0.04). This study confirms EVI5 as another risk locus for MS; however, much of the genetic basis of MS remains unidentified.

Keywords:

multiple sclerosis, genetic risk variants, EVI5

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Introduction

Multiple sclerosis (MS) is a complex disease, resulting from genetic as well as environmental factors. For long, HLA-DRB1 has been the only locus consistently involved with higher risk for MS. Next to the human leukocyte antigen (HLA) region, a recent genome-wide study also showed two single-nucleotide polymorphisms (SNPs) within the IL2RA gene and one SNP within the IL7RA gene to be strongly associated with MS susceptibility. Thirteen other SNPs, although less significant, also showed evidence for association with MS. In total, 17 SNPs were found to be associated with MS both in the screening phase and in the replication phase of the study, of which the SNP in the HLA region again showed the strongest association.1

In this study, we assessed the risk contribution of these 17 SNPs in MS patients from a Dutch genetically isolated population. Apart from the HLA locus, a novel risk gene was confirmed. This finding is further validated for the general MS population in an independent large set of Canadian MS patients.

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Results

We tested the 17 MS SNPs that were reported to be associated in the collaborative genome-wide MS study (Table 1).1 The HLA-DRB1 surrogate SNP (rs3135388) was significantly associated with MS in this study (P=0.001, odds ratio (OR) 2.99, 95% confidence interval (CI) 1.56–5.74). Furthermore, two SNPs, both on chromosome 1, located in the EVI5 (ecotropic viral integration site 5) gene gave significant P-values in our replication study: rs10735781 (P=0.01, OR=2.01, 95% CI 1.19–3.39) and rs6680578 (P=0.01, OR=1.9, 95% CI 1.16–3.11). These ORs are considerably higher than those reported by the International MS Consortium; the confidence intervals were not overlapping. The two SNPs in the EVI5 gene were in nearly complete linkage disequilibrium (D=0.99). The IL7RA rs689732 and for IL2RA rs12722489 and rs2104286, SNPs were not significantly associated with MS (Table 1).


The two SNPs located in the EVI5 gene were subsequently tested in an independent set of 756 Canadian families containing 1318 MS patients. Both SNPs had a weak but significant contribution in this population (rs10735781: P=0.03, OR=1.15, 95% CI 1.01–1.30; rs6680578: P=0.04, OR=1.15, 95% CI 1.01–1.30) (Table 2) and were in nearly complete linkage disequilibrium (D′=0.98).


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Discussion

This study replicates in a Dutch genetically isolated population, the recent indication that EVI5 is a risk gene for MS. Another interesting observation in the genetic isolate is that we found a very significant association of the HLA-DRB1*15 tagging SNP rs3135388 with MS, whereas previous extensive HLA-DRB1 typing in this same population showed no significant association with the known HLA-DRB1*15 allele or other alleles at the two-digit level. A possible explanation for this may have been the lack of power in the initial study on this small population.2

The study on the genetic isolate had limited power to verify the low ORs of the recent genome-wide study.1 The fact that we did not find statistical evidence for 14 of 17 SNPs that were earlier reported to be associated with MS does not exclude an effect of these SNPs/genes, as the probability of false-negative findings is high in this study. It is striking that despite this relatively low power, our study does show convincing evidence for EVI5 at chromosome 1p22 as a risk allele. This underlines the strength of studying genetically isolated populations in complex diseases.3, 4 The ORs observed for EVI5 are higher than the ORs reported by the International MS Consortium.1

EVI5 was also found to be significantly associated with MS in a separate group ascertained as part of the Canadian Collaborative Project on the Genetic Susceptibility to MS. This strengthens the evidence that EVI5 is associated with MS risk. It is still not clear whether the causal allele acts through EVI5 itself. EVI5 is a common site of retroviral integration and has been linked to lymphomagenesis.5 It remains to be seen whether and to what extent it could influence T-cell function and also if it could be related to retroviral elements associated with MS.6 Identification of the exact causal allele will require the sequencing and genotyping of additional samples. In addition, functional immunological studies stratified according to EVI5 genotype are planned.

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Patients and methods

Participants from the Dutch genetically isolated population

This study was performed within the framework of the previously described Genetic Research in Isolated Populations (GRIP) programme.3, 7 The Medical Ethics Committee of the Erasmus MC approved GRIP protocols. The GRIP population is a genetically isolated community in the southwest of The Netherlands. The isolate was founded by less than 400 individuals around the middle of the eighteenth century. Minimal inward migration and considerable population growth subsequently occurred. An estimated 20000 descendants of this population are now scattered over eight adjacent communities. The genealogical database currently contains information on more than 90000 people spanning 23 generations. Residents in the GRIP area are generally related via multiple lines of descent.

The ascertainment and clinical characteristics of MS patients in the GRIP population have been described in detail previously.2 Originally, 48 MS patients (13 male, 35 female) were included. Later on, another female MS patient was identified and included.

All 49 MS patients (13 male, 36 female) were diagnosed according to standard diagnostic criteria, with regular clinical phenotypes. Although most were originally diagnosed as sporadic MS patients, a total of 25 (51%) could be linked to an extended pedigree.2

Three of the 49 MS patients were excluded from analysis because they were sisters of one MS patient. Clinical characteristics from the 46 MS patients who were included for analysis are described in Table 3a.


In this population, there was a trend towards a higher prevalence of the HLA-DRB1*15 allele in patients compared to controls, but no significant differences could be found (OR 1.79; P=0.12).2

For the control group, we included 194 healthy controls from the same area who were all distantly related (greater than or equal to5 meioses).

All patients and controls gave written informed consent to participate in this study.

Participants from the Canadian Collaborative Project on the Genetic Susceptibility to MS

A total of 2825 individuals from 756 families were typed as part of the Canadian Collaborative Project on the Genetic Susceptibility to MS for which the methodology has been described.8, 9 This includes 1318 individuals with definite MS and 1507 of their unaffected first-degree relatives. Clinical characteristics from the 1318 patients are described in Table 3b.


The Canadian families consisted of 456 multicase families (that is, parents with two or more affected offspring) and 300 parent–child trios.

Genotyping of individuals from the Dutch genetically isolated population

We genotyped all participants using the Affymetrix GeneChip Mapping 250K according to the protocol. This array set contains the Nsp array and includes approximately 262000 SNPs.

Genotyping of participants from the Canadian Collaborative Project on the Genetic Susceptibility to MS

All genotypes were generated blind to pedigree structure and disease status of the individual. Genotyping of SNPs was performed using the Sequenom MassEXTEND protocol (www.sequenom.com). Only conservative and moderate genotyping calls were accepted in this study. Samples having aggressive or low-probability quality genotypes were re-analysed. The concordance of genotyping between Affymetrix and Sequenom platforms has previously been validated.1

Statistical analyses of data from the Dutch genetically isolated population

For analysis, we used the R library GenABEL version 1.1–8.10 We used the Armitage's test to estimate P-values. We used the genomic control method11 to adjust for the relationship between GRIP participants.12 The inflation factor was estimated to be 1.28. We imputed the SNPs that were not available from the Affymetrix GeneChipMapping 250K Array using HAPMAP CEU haplotypes as a reference. Mach software was used for imputations.13, 14

Statistical analysis of data from the Canadian Collaborative Project on the Genetic Susceptibility to MS

Transmission dis-equilibrium test was performed using the PLINK analysis package.15 PLINK implements sib-transmission dis-equilibrium test, which calculates empirical probabilities for χ2 statistics, accurately reflecting association independent of linkage within families. This calculation is done by permuting parent alleles while fixing the identical by descent status of sibs within a family.

The transmission dis-equilibrium test counts the number of times an allele is transmitted to affected offspring from heterozygous parents. For transmission dis-equilibrium tests, the χ2 distribution was used to assess significance. The OR was calculated as described in Kazeem and Farrall.16

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Notes

Conflict of interest

All authors did not have a conflict of interest.

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References

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Acknowledgements

This study was supported by grants from MS Research Netherlands (RQH and CvD), the Netherlands Organisation for Scientific research (ZON-MW, RQH), Erasmus MC and the Multiple Sclerosis Society of the United Kingdom (GCE). The GRIP study is supported by Centre for Medical Systems Biology (CMSB). We are grateful to all patients and their relatives, general practitioners and neurologists for their contributions and to P Veraart for her help in genealogy and E Croes (MD, PhD) for help in data collection. IH and YA contributed equally to this study. There were no sources of funding that have supported the paper. The study sponsors had no role in the study design, data collection, data analysis, data interpretation, writing of the report or decision to submit the paper for publication.

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