gistfile1.txt

73b488ee Interferon regulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis in three distinct populations
73b4ed02 IRF5 is a transcription factor involved both in the type I interferon and the toll-like receptor signalling pathways.
73b5553a Previously, IRF5 has been found to be associated with systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel diseases.
73b5b4f8 Here we investigated whether polymorphisms in the IRF5 gene would be associated with yet another disease with features of autoimmunity, multiple sclerosis (MS).
73b5c3b2 We genotyped nine single nucleotide polymorphisms and one insertion-deletion polymorphism in the IRF5 gene in a collection of 2337 patients with MS and 2813 controls from three populations: two case–control cohorts from Spain and Sweden, and a set of MS trio families from Finland.
73b5c8d0 Two single nucleotide polymorphism (SNPs) (rs4728142, rs3807306), and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF5 gene, showed association signals with values of p<0.001 when the data from all cohorts were combined.
73b5cd94 The predisposing alleles were present on the same common haplotype in all populations.
73b5d1d6 Using electrophoretic mobility shift assays we observed allele specific differences in protein binding for the SNP rs4728142 and the 5 bp indel, and by a proximity ligation assay we demonstrated increased binding of the transcription factor SP1 to the risk allele of the 5 bp indel.
73b5d5d2 These findings add IRF5 to the short list of genes shown to be associated with MS in more than one population.
73b5da1e Our study adds to the evidence that there might be genes or pathways that are common in multiple autoimmune diseases, and that the type I interferon system is likely to be involved in the development of these diseases.
73b5de1a Multiple sclerosis (MS, OMIM 126200) is an inflammatory disease estimated to affect over two million individuals worldwide.
73b5e1f8 MS is not well recognised as an autoimmune disease, but exhibits features of autoimmunity—that is, activation of the immune system in the absence of apparent ongoing infection.
73b5e5f4 In MS the presumed target for the autoimmune process is the central nervous system, and the disease manifests itself by immune mediated demyelination and damage to axons.
73b5ea0e A spectrum of neurological symptoms are found among MS patients, including sensory or motor pareses, visual disturbances, ataxia, pain, cognitive dysfunction and fatigue.
73b5ede2 MS is a complex disease caused by interaction between environmental and inherited factors.
73b5f1b6 The disease shows familial clustering, and twin studies have revealed that a large portion of this clustering can be attributed to shared genes.
73b5f59e A remarkably low number of susceptibility genes for MS have been identified so far.
73b5f99a Genome-wide linkage studies have indicated several possible susceptibility loci, but the only locus to be identified across most studies is the major histocompatibility complex (MHC) on chromosome 6p21, were the HLA-DRB1*1501 allele is a well established genetic risk factor for MS.
73b5fd46 This locus does not, however, account for the whole genetic component of MS, and multiple loci with smaller contributions to disease susceptibility are likely to exist.
73b6011a Numerous candidate gene studies have also been performed in MS, but findings from one population have been difficult to replicate in other populations.
73b604ee The protein kinase C alpha (PRKCA) gene is one of few genes reported to be associated with MS in more than one population.
73b60890 Recently, the interleukin 7 receptor alpha chain gene (IL7RA) was found to be associated with MS in two independent candidate gene studies and in a genome-wide association study.
73b60c6e The genome-wide association study also identified variants in the interleukin 2 receptor alpha chain gene (IL2RA) as risk factors for MS, which is in accordance with findings in a previous candidate gene study.
73b612a4 It is relatively common that patients affected by an autoimmune disease suffer from another autoimmune disease, and that members of the same family suffer from different autoimmune diseases.
73b6170e For example, in families with systemic lupus erythematosus (SLE, OMIM 152700), MS and rheumatoid arthritis (RA, OMIM 180300) occur more frequently than in the general population.
73b61ac4 Such observations suggest shared genes or involvement of common cellular pathways in these diseases.
73b61ea2 This hypothesis is supported by reports on genes found to be associated with more than one autoimmune disease in experimental models of RA and MS.
73b6224e Shared genes in autoimmune diseases are becoming apparent also in humans, such as PTPN22 in RA and SLE, MHC2TA in RA and MS, and recently the suggested involvement of CD24 in MS and SLE.
73b625f0 The type I interferon (IFN) system has been postulated to play a key role in autoimmune diseases.
73b62992 Increased expression of IFN induced genes has been detected in autoimmune diseases like SLE, RA, Sjogren’s syndrome, and in a subgroup of MS patients.
73b62f8c The interferon regulatory factors (IRFs) are major regulators of genes activated by the type I IFNs,and a role in the regulation of the immune system is well established for the majority of the members of the IRF family of nine genes.
73b634aa The role of IRF5 in the immune response is not as well established as for other IRFs, but IRF5 has recently received attention in studies on autoimmunity.
73b6386a The IRF5 gene displays a complex transcription pattern with three alternative non-coding 5' exons and at least nine alternatively spliced mRNAs.
73b63bf8 IRF5 is expressed in dendritic cells, monocytes and B cells, but its expression can be induced in other cell types by the type I IFNs.
73b63f9a IRF5 regulates the toll-like receptor (TLR) dependent activation of inflammatory cytokines and functions downstream of the TLR-MyD88 pathway where it is activated by MyD88 and TNF receptor associated factor 6 (TRAF6).
73b6438c Our original finding of an association between the IRF5 gene and SLE, which has been replicated in multiple populations, as well as our recent findings of association between IRF5 and RA and inflammatory bowel diseases (IBD), provide additional support for the important role of IRF5 and the type I IFN system in autoimmune diseases.
73b6472e Inspired by these findings, and by the role of recombinant IFN beta as a standard treatment of MS, we investigated whether polymorphisms in the IRF5 gene would also be associated with MS.
73b64ac6 We found that polymorphisms in the IRF5gene displayed associations with MS in three independent patient cohorts from Spain, Sweden and Finland.
73b64e54 The three most strongly associated polymorphisms are located in the promoter region and first intron of IRF5.
73b651ec A functional role is suggested by increased protein binding to the risk alleles of two of these polymorphisms.
73b6593a Ten polymorphisms in the IRF5 gene on chromosome 7q32 were genotyped in MS patient samples collected in three European countries.
73b65cfa The selected polymorphisms include five SNPs and one insertion-deletion polymorphism (indel) in the promoter region or first intron of IRF5, two SNPs in the 3'UTR and two SNPs downstream of IRF5 (fig 1).
73b660a6 The polymorphisms were selected because they have previously been shown to be associated with SLE, RA and IBD, or have been suggested to modulate the expression of IRF5.
73b6647a The polymorphisms were first genotyped in a Spanish cohort of MS patients (n = 660) and controls (n = 833).
73b6681c Seven of the 10 polymorphisms showed nominally significant signals of association with MS (p<0.05) (table 1).
73b66be6 To replicate this finding, we genotyped the same set of polymorphisms in an independent case–control cohort with 1166 MS patients and 1235 matched controls from Sweden.
73b66f88 Two of the SNPs, rs4728142 and rs3807306, and the CGGGG indel polymorphism, showed a nominally significant association (p<0.05) with MS.
73b67474 Each of these three polymorphisms was also associated with MS in the Spanish samples (table 1).
73b6782a In a further attempt to verify these findings from two case–control cohorts of MS patients, we genotyped the same set of polymorphisms in 511 Finnish MS trio families.
73b68a54 Using a transmission disequilibrium test, four polymorphisms showed nominally significant association with MS (table 2).
73b68eaa Two SNPs, rs4728142 and rs3807306, were nominally significantly associated with MS in all three cohorts (tables 1 and 2).
73b69292 Information on HLA-DRB1*15 genotype was available from the Swedish cases and controls, and from the Finnish patients (supplementary table S3).
73b69634 In the Swedish dataset most of the association signal was observed in the DR15-negative stratum, whereas in the Finnish dataset the DR2 stratum provided most of the association signal (supplementary table S4).
73b69a12 When combining the p values from all cohorts, seven of the 10 polymorphisms exhibited association with MS with p values ranging from 0.0002 to 0.04.
73b69dc8 The strongest association signals in this combined analysis were observed for the SNP rs4728142 (p = 0.0002) located ~5 kb upstream of IRF5, the CGGGG indel located 64 bp upstream of exon 1a of IRF5 (p = 0.0005), and the SNP rs3807306 in the first intron of IRF5 (p = 0.0002) (table 3, fig 1).
73b6a1b0 The linkage disequilibrium (LD) pattern of the polymorphisms was similar between the three populations (figure 2), and displayed relatively high LD with pair-wise r2 values of 0.61–0.88 for the three associated polymorphisms in the Spanish, Swedish and Finnish unaffected subjects.
73b6a5a2 We performed haplotype association tests in the three populations to investigate whether haplotypes could capture the association signal at a higher significance than the individual polymorphisms, and whether all three populations harbour the same disease associated haplotype(s).
73b6a96c The haplotype analysis was performed using a five marker sliding window approach.
73b6ad0e The associated risk alleles of the polymorphisms were all present on the most common haplotype, which had a frequency of 0.43–0.51 and was the same in all three cohorts (table 4).
73b6b09c The association signals were comparable to those in the analysis of individual polymorphisms.
73b6b434 We used electrophoretic mobility shift assays (EMSA) to test for differential protein binding to the alleles of the three polymorphisms rs4728142, rs3807306 and the CGGGG indel, which displayed association signals with values of p<0.001 in the combined analysis of the three MS cohorts.
73b6b7d6 This analysis revealed a stronger binding of protein to the risk alleles of the SNP rs4728142 (the A allele) and of the CGGGG indel polymorphism (the 4×CGGGG allele) (fig 3).
73b6bb64 For the CGGGG indel polymorphisms the insertion of one CGGGG repeat in the longer (4×CGGGG) allele is predicted to create an additional and third binding site for the transcription factor SP1, while the shorter (3×CGGGG) allele has two SP1 binding sites (TFSEARCH:http://www.cbrc.jp/research/db/TFSEARCH.html).
73b6bf06 By using the proximity ligation assay we confirmed that SP1 protein binds to the alleles of the CGGGG indel, and that an increased amount of SP1 is bound to the 4×CGGGG allele than to the 3×CGGGG allele of the indel polymorphism (fig 4).
73b6c672 Here we describe the association of three polymorphisms in the IRF5 gene with MS in three independent patient cohorts from Spain, Sweden and Finland.
73b6cc6c The SNPs rs4728142 and rs3807306 reached nominal significance for association in all cohorts and revealed strong signals of association with MS (p = 0.0002) when the data from all three cohorts was combined.
73b6d0a4 The SNP rs4728142 is located ~5 kb upstream of the alternative exon 1a of IRF5, and rs3807306 is located in the first intron of the gene, 96 bp upstream of alternative exon 1c (fig 1).
73b6d57c The SNP rs3807306 was the only IRF5 polymorphism reported in the recently performed genome-wide association study on MS.
73b6d932 In that study the SNP rs3807306 exhibited association with MS with a TDT p value of 0.014 (https://imsgc.org/), but it did not pass the threshold for inclusion in the confirmatory phase of the study.
73b6dcc0 This study also identified the T allele as the risk allele.
73b6e0b2 It is notable that the initial TDT screening phase of this genome-wide study only had about 6% power to detect each locus at the chosen cut off and with the observed modest risk ratios.
73b6e45e The effect of this low power becomes apparent when looking at the SNPs in IL2RA and IL7RA, which did not pass the initial p value cut off for the TDT, but turned out to be the most strongly associated markers after the replication phase.
73b6e800 In our study the CGGGG indel polymorphisms, located 64 nucleotides upstream of the alternative exon 1a of IRF5, also showed evidence of association with MS.
73b6ec56 Although the CGGGG indel did not reach statistical significance independently in all three cohorts, the combined analysis revealed a clear association signal between the CGGGG indel and MS (p = 0.0005), with the longer allele (4×CGGGG) as the risk allele.
73b6f1ba The risk alleles of the SNPs rs4728142 and rs3807306 and of the CGGGG indel are present on the same common haplotype in each of the three populations (table 4).
73b6f5b6 In a recent study on IBD we analysed the same set of polymorphisms as in the current study on MS, and found that the same three polymorphisms were associated with IBDs, with the strongest signal of association for the CGGGG indel.
73b6f94e We have recently performed an association study of a comprehensive set of polymorphisms in IRF5 that were identified by sequencing the introns and exons of IRF5 in SLE patients.
73b6fcdc This analysis identified a set of correlated polymorphisms in IRF5 that gave strong signals of association with SLE (p < 10 -6), including the SNPs rs4728142, and rs3807306 and the CGGGG indel.
73b7006a In SLE logistic regression analysis conditional only on the CGGGG indel abolished all the other association signals from this set of correlated SNPs.
73b7045c Because the effect size of the polymorphisms are lower in MS than in SLE we cannot distinguish which one of them would be the most likely causal variant in MS.
73b709de In an earlier association study on RA we analysed five SNPs in IRF5, including the SNPs rs729302, rs375385, rs2004640 and rs3807306, but not the CGGGG indel, and found the strongest signal of association with RA for the SNP rs3807306.
73b70db2 Taken together, the association results from the current study on MS and previous studies on IBD, SLE and RA indicate that one or more of these correlated polymorphisms in the promoter and first intron of the IRF5 gene could be a universal risk factor for chronic inflammatory disorders, but further studies are required to dissect if the mechanism is the same in all of these disorders.
73b711f4 Using EMSA, as a preliminary functional test for the three polymorphisms in IRF5 that gave the strongest association signals with MS, we observed stronger protein binding to the risk alleles of the SNP rs4728142 (the A allele) and of the CGGGG indel polymorphisms (the 4×CGGGG allele), whereas both alleles of the SNP rs3807306 appear to bind an equal amount of protein.
73b71668 We confirmed experimentally using an antibody against SP1 in the proximity ligation assay31that an increased amount of transcription factor SP1 binds to the risk allele (the 4×CGGGG allele) of the CGGGG indel polymorphism.
73b71a1e This result is also supported indirectly by a study in which they demonstrated that SP1 binds to a similar sequence motif in the IRF1 and IRF4 genes.
73b71dac It is notable that the SNP rs12539741 located in the 3'-end of the IRF5 gene does not show any association with MS in our study.
73b7213a In SLE this SNP and two other linked SNPs, the SNPs rs2070197 in the 3'-UTR of IRF5 and rs10488631 located ~5 kb downstream of IRF5, give particularly strong association signals.
73b72608 Two recent genome-wide association studies failed to detected an association with RA for the SNP rs10488631 or its proxies and in our study on IBD we did not detect an association with the SNP rs10488631 either.
73b729d2 Thus it appears that in SLE there are two groups of independently associated polymorphisms in the IRF5 gene region, whereas in MS, RA and IBD association from only one of these groups is observed.
73b72d60 According to data from the HapMap project (www.hapmap.org), the SNP rs10488631 is in complete LD with multiple SNPs located in a 100 kb region downstream of IRF5, which also contains the transportin 3 (TNPO3) gene.
73b730f8 The polymorphisms in the IRF5 gene that we found to be associated with MS in our study are not strongly correlated (r2 0.1–0.2) with the SNP rs10488631 or its proxies in the TNPO3 gene, indicating that it actually is IRF5, and not TNPO3, that is primarily responsible for the association with MS that we observe.
73b73486 Three different European populations were included in the present study, and the observed allele and haplotype frequencies did not show major inter-population differences.
73b7381e The disease predisposing alleles are the same and occur on the same major haplotype in the Spanish, Swedish and Finnish populations.
73b73bac These results indicate that the predisposing alleles are widely distributed in the Caucasian population.
73b74110 The effect sizes of the risk alleles are relatively small, with odds ratios of about 1.2, indicating that large datasets are needed to replicate these findings.
73b74516 The strength of our study is that we used both case–controls and family based association testing.
73b748b8 Population stratification artefacts are common in case–control settings, but unlikely in family based studies.
73b74c50 On the other hand, the TDT may be biased by erroneous detection of association to alleles with high frequency in the analysed populations.
73b74fe8 MS is a common disease, and most likely caused by interaction between multiple common allelic variants of genes.
73b75376 The association of IRF5 polymorphisms with MS in the cohorts studied here suggests that IRF5 is one of these genes that contribute to the disease.
73b7570e Interestingly, in the animal models for MS, experimental autoimmune encephalomyelitis, deletion of the IFN beta gene leads to more severe disease, suggesting that the inherent type I IFN function contributes to the autoimmune disease.
73b75c86 Currently, the most common therapy in MS is IFN beta, which has been shown to reduce the magnetic resonance imaging activity and relapse rate in MS.
73b7603c This therapeutic effect is consistent with an immunoregulatory role of the type I IFN pathway in MS.
73b763c0 The findings from our study add IRF5 to the short list of genes with confirmed association with MS.
73b76ce4 Our study also contributes to the evidence that there might be genes or pathways that are common between multiple autoimmune diseases, and that the type I IFN signalling system, to which the IRF5 gene belongs, is likely to be one of these pathways.
73b77824 EVI5 is a risk gene for multiple sclerosis
73b77bbc HLA-DRB1 is the major locus associated with risk for multiple sclerosis (MS).
73b78116 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.
73b786fc Consistent association but lower significance was found for 13 other SNPs.
73b78ab2 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.
73b78f58 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).
73b79426 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.
73b79912 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).
73b79cdc This study confirms EVI5 as another risk locus for MS; however, much of the genetic basis of MS remains unidentified.
73b7a07e Multiple sclerosis (MS) is a complex disease, resulting from genetic as well as environmental factors.
73b7a420 For long, HLA-DRB1 has been the only locus consistently involved with higher risk for MS.
73b7a7c2 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.
73b7abaa Thirteen other SNPs, although less significant, also showed evidence for association with MS.
73b7af7e 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.
73b7b352 In this study, we assessed the risk contribution of these 17 SNPs in MS patients from a Dutch genetically isolated population.
73b7b6f4 Apart from the HLA locus, a novel risk gene was confirmed.
73b7ba82 This finding is further validated for the general MS population in an independent large set of Canadian MS patients.
73b7c1a8 We tested the 17 MS SNPs that were reported to be associated in the collaborative genome-wide MS study (Table 1).
73b7c54a 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).
73b7c8f6 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).
73b7cca2 These ORs are considerably higher than those reported by the International MS Consortium; the confidence intervals were not overlapping.
73b7d026 The two SNPs in the EVI5 gene were in nearly complete linkage disequilibrium (D'=0.99).
73b7d3b4 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.
73b7d77e 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).
73b7dea4 This study replicates in a Dutch genetically isolated population, the recent indication that EVI5 is a risk gene for MS.
73b7e232 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.
73b7e5ca A possible explanation for this may have been the lack of power in the initial study on this small population.
73b7e962 The study on the genetic isolate had limited power to verify the low ORs of the recent genome-wide study.
73b7ecf0 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.
73b7f07e It is striking that despite this relatively low power, our study does show convincing evidence for EVI5 at chromosome 1p22 as a risk allele.
73b7f402 This underlines the strength of studying genetically isolated populations in complex diseases.
73b7f862 The ORs observed for EVI5 are higher than the ORs reported by the International MS Consortium.
73b7fc18 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.
73b7ffe2 This strengthens the evidence that EVI5 is associated with MS risk.
73b8067c It is still not clear whether the causal allele acts through EVI5 itself.
73b80aaa EVI5 is a common site of retroviral integration and has been linked to lymphomagenesis.
73b80e42 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.
73b811e4 Identification of the exact causal allele will require the sequencing and genotyping of additional samples.
73b81572 In addition, functional immunological studies stratified according to EVI5 genotype are planned.
73b82738 A sequence variation in the MOG gene is involved in multiple sclerosis susceptibility in Italy
73b82ad0 Several studies suggest that the histocompatibility complex (HLA) class I region harbours genes modulating multiple sclerosis (MS) susceptibility independently from the effect of class II alleles.
73b82e68 A candidate gene in this region is MOG, encoding the myelin oligodendrocyte glycoprotein.
73b831ec A significant association with the missense variation V142L (rs2857766) was previously reported in a small sample of 50 Italian MS patients.
73b8357a We confirmed this result in two independent Italian sample sets consisting of 878 MS patients and 890 matched controls (P=6.6 × 10−4) and 246 trio families (P=1.5 × 10−3).
73b838fe The comparison of genotype frequencies suggested a dominant-protective effect of L142.
73b83c8c In the combined sample sets L142 conferred an odds ratio (OR)=0.70 (95% confidence interval (CI): 0.60–0.82) that remained similar after accounting for HLA-DRB1*15 carrier status.
73b8401a The association with MOG V142L was still significant after conditioning for all DRB1 alleles (P=0.035).
73b84402 Eleven additional single nucleotide polymorphisms in the MOG gene (namely −1077T/C, −910T/C, −875A/G, −93T/C, S5S, Indel L22, V145I, +814C/T, +900A/G, +1024A/T, +1059C/T), two microsatellites in the MOG 5' flanking (MOGCA) and 3' untranslated (MOGTAAA) regions and four microsatellites in the HLA-class I region, from HLA-B to HFE, (namely MIB, D6S265, D6S1683 and D6S2239) were tested by transmission disequilibrium test in 199 trio families.
73b847a4 None of these polymorphisms or of their haplotypic combinations showed a significant transmission distortion, in the absence of V142L.
73b84b32 In conclusion, MOG V142L, or an untested variant in tight-linkage disequilibrium with it, is an independent MS susceptibility-modulating factor in the HLA class I region.
73b8528a Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system1 caused by an interplay of environmental and genetic factors.
73b85618 The only genetic factor that has been clearly demonstrated by linkage and association studies maps in the human major histocompatibility complex (HLA) region. In particular, the class II allele HLA-DRB1*1501 (DR15) is positively associated in all tested Caucasoid populations.
73b859a6 The only exception is represented by Sardinia where the frequency of DR15 is low both in patients and controls, while MS is principally associated with DR4 and DR3.
73b85d2a Although a weaker positive association with DR4 and DR3 has been reported also in other populations, this was not detected in a large case-control study in continental Italy.
73b860ae Negative associations have also been reported.
73b86432 More recently, several studies suggested that genes located in the HLA class I region modulate susceptibility to MS independently of class II.
73b867ac Fogdell-Hahn et al. and Harbo et al. found a significantly increased frequency of the class I HLA-A*03 allele in northern Europe MS patients.
73b86c3e The A*03 allele is part of the extended HLA haplotype (hp) characterized by DR15.
73b86ff4 However, the effect of this association was additive to the risk conferred by DR15 and not a consequence of linkage disequilibrium (LD) between the two alleles.
73b8738c In addition Fogdell-Hahn et al. found a protective effect of HLA-A*02.
73b87710 This was confirmed in a larger Swedish panel.
73b87a94 A protective effect independent of HLA class II was recently detected for HLA-Cw*05 in a large UK and US cohort of simplex families and sporadic cases.
73b87e22 Rubio et al. reported in Tasmanian patients with European ancestry a significant association with D6S265 and MOGCA microsatellites mapping in the HLA class I region 100 kb centromeric and 500 kb telomeric to HLA-A, respectively.
73b881c4 In addition they found that having class I and II susceptibility variants on the same hp provides an additive effect on MS risk.
73b88548 In the Sardinian population the D6S1683 microsatellite, located about 980 kb telomeric to HLA-A, was significantly associated20 with MS independently of class II.
73b888cc Altogether these data point to the presence of at least one MS risk gene, either additive to or independent of class II, in a region of about 1 MB in the HLA class I region.
73b88cfa A very interesting candidate gene mapping in this region is MOG, encoding the myelin oligodendrocyte glycoprotein, a quantitatively minor component of myelin which is specific of the central nervous system and is located exclusively on the surface of myelin sheaths and olygodendrocytes.
73b890a6 MOG is a potential target antigen of the central nervous system, known to induce an autoreactive T-cell response and pathogenic demyelinating anti-MOG antibodies in MS patients and in EAE (experimental autoimmune encephalomyelitis) mice, the animal model of MS.
73b89434 So far, association studies of MOG markers with MS susceptibility yielded conflicting results.
73b897c2 No association was found in 169 French MS patients with three MOG dinucleotide repeats, whereas a significant association was reported for one of these markers (MOGCA) in the above mentioned Tasmanian patients and by Barcellos et al.
73b89c04 Single nucleotide polymorphisms (SNPs) located in the MOG 5′ flanking and coding region were not associated with MS susceptibility in 100 German paediatric patients, 82 French adult patients and in 397 Sardinian trio families.
73b8a2da Conversely, in the continental Italian population a significant association with the missense variation V142L in the MOG transmembrane region was reported in a small sample of 50 MS patients.
73b8a6d6 Possible explanations of the inconsistency among the different studies might be population differences and/or the small sample sizes.
73b8aa64 The aim of the present study was to confirm the Italian finding in a larger independent sample.
73b8adde To this purpose an association study with the V142L SNP was performed by both a case-control and an intrafamilial association approach.
73b8b162 We confirmed the result previously reported in the continental Italian population.
73b8b4f0 Consequent steps were to examine whether this association was independent of known MS susceptibility factors in the HLA region and if other sequence variations in the MOG gene and in the class I region were more significantly associated with MS.
73b8bbee Association with MOG—V142L polymorphism.
73b8bf86 The MS association with the V142L MOG missense variation was tested in two independent Italian sample sets consisting of 878 MS patients and 890 controls (sample set 1) and 246 simplex families including the MS patient and both parents (sample set 2).
73b8c314 Significantly different case and control allele frequencies were observed in both sample sets (P=6.6 × 10−4, P=1.5 × 10−3) and in the combined sample (P=6.7 × 10−6). In particular, the minor allele (L142) was under-represented in the patients (Table 1).
73b8c7c4 Transmission disequilibrium test (TDT) analysis in the 246 families showed a preferential non-transmission of L142 (transmitted:non-transmitted allele 45:82, P=0.001).
73b8cb7a The comparison of genotype frequencies in all MS patients (N=1124) and in the controls of sample set 1 (N=890) suggested a dominant-protective effect of L142 since both L142 homozygotes and heterozygotes were significantly decreased among the patients (Table 2).
73b8cf1c To eliminate the possible confounding effects of LD with HLA-DRB1*15, we conditioned the analysis of MOG 142 association on DRB1*15.
73b8d2b4 L142 remained significantly less frequent in DRB1*15-negative patients than in DRB1*15-negative controls (0.163 vs 0.213, P=9.7 × 10−4; Table 1).
73b8d64c This result was in line with the very weak LD between L142 and DRB1*15 detected in this panel (D'=−0.011; r2=0. 059; P=0.02). The effect of L142 and DRB1*15 alleles on MS risk was further evaluated by logistic regression modelling (Table 3).
73b8d9d0 All individuals were categorized according to the DRB1*15 and L142 status (grouped as positive/negative for each of them). The OR conferred by L142 (0.70) remained similar after accounting for DRB1*15 carrier status (0.72).
73b8dd9a Moreover, the MS risk conferred by DRB1*15 (OR=2.81) did not change when adjusted for the presence of L142 (OR=2.77).
73b8e132 The interaction between the two markers was also tested in the model: no evidence of interaction was found (P=0.40).
73b8e4ac These data demonstrate that the involvement of MOG L142 in MS susceptibility was not secondary to the well-known major effect of DRB*15.
73b8e83a Moreover, the effect of these two MS-associated alleles was not reciprocally modified.
73b8ebf0 Although the majority of the DRB1 association with MS is attributable to DRB1*15, other DRB1 alleles are also involved in MS susceptibility.
73b8ef88 We therefore considered the relationship between the V142L association and all DRB1 alleles in a random subset of 540 MS cases and 558 controls fully typed for DRB1.
73b8f316 A weak LD was detected between MOG V142L and DRB1 (global D'=0.19, Cramer's V=0.17 in the patients; D'=0.17, Cramer's V=0.17 in the controls).
73b8f6d6 V142L was still significantly associated in this panel after conditioning on the DRB1 locus by the COCAPHASE program (unconditioned P=0.0031; conditioned P=0.035).
73b8fa6e Moreover, both in the total panel and in DRB1*15-negative individuals, the OR conferred by L142 remained similar after accounting for each DRB1 allele separately by logistic regression modelling (data not shown).
73b8fdfc Having proved that the V142L association is independent of HLA-DR, we then tested its relationship with other polymorphisms in the MOG gene and in the HLA class I region.
73b90176 This analysis was mainly performed in the trio families in order to be able to directly deduce haplotypic combinations from family segregation.
73b90504 Test of association with other MOG sequence variationsThe involvement of MOG in MS susceptibility was further tested by analysing the association with 11 SNPs, of which 4 in the 5' flanking, 3 in the coding and 4 in the 3' untranslated regions of the gene (Figure 1b).
73b9089c All the selected SNPs were detected directly in MS patients.
73b90c16 SNPs in MOG-5' flanking and coding regions were selected from the literature and were detected by extensive sequencing of these regions in Italian and German patients.
73b90f90 Since no sequencing scan was available for the 3'-UTR in MS patients, we screened MOG 3'-UTR (1195 bp) in 25 patients by DHPLC and identified 4 variations.
73b9131e Three of these had been previously detected in normal individuals and are included in the SNP database (http://www.ncbi.nlm.nih.gov/SNP/).
73b916b6 The association was first tested by TDT in the trio families already analysed for position 142.
73b91a30 Complete genotyping results for all the tested SNPs were obtained for 199 families.
73b91daa In this random subset of families L142 was still preferentially non-transmitted (T:NT=31:63; P=0.001).
73b92200 Conversely, none of the other SNPs showed a significant transmission distortion.
73b925ac To increase the power of our analysis we further tested the association of MOG SNPs by a case-control analysis in 290 controls and 343 patients.
73b92930 Combining the two sets (trios and cases/controls) we were able to compare allele frequencies of 1084 case and 978 control chromosomes.
73b92cbe Considering a range from 0.05 to 0.20 of the minor allele frequency of the tested SNPs, this sample size has an 80% power to detect a risk factor conferring an OR ranging from 1.35 to 1.6 at an alpha level of 0.05 (for comparison, the V142 allele in this panel conferred an OR=1.5).
73b9304c Thus, this study had sufficient power to detect a risk factor with an OR~V142L for the majority of tested SNPs with the exception of the rare –1077T/C, –910T/C and Indel L22 variations.
73b9343e None of the tested MOG SNPs, besides MOG V142L, was significantly associated with MS (Table 4).
73b937f4 The same result was obtained when including only DRB1*15-negative individuals and families (data not shown).
73b93f10 Linkage disequilibria between V142L and the tested MOG SNPs are shown in Table 5. V142L showed a modestly significant LD only with S5S and V145I.
73b942ee TDT results on the hps including all the analysed SNPs are shown in Table 6.
73b9467c Ten hps, accounting for 89% of the total, had a frequency higher than 0.01.
73b94a00 The remaining hps showed a frequency 0.01 both in the patients and in controls.
73b94d84 L142 was mainly included in one hp (hp no. 3) which was preferentially non-transmitted (P=0.018).
73b95162 With the only exception of position 142, this hp shared the same markers with hp no. 1 which, as opposed to hp no. 3, was significantly over-transmitted (P=0.002).
73b95504 When V142L was removed from the analysis, the transmission of this hp combination was no longer distorted.
73b95888 One further rare hp (no. 7) showed a transmission distortion (P=0.03) either including or excluding position 142 in the analysis.
73b95c0c This association was no longer significant when considering only DRB1*15-negative families (Table 6).
73b95f90 Conversely, hp no. 3 carrying L142 remained significantly under-transmitted also in DRB1*15-negative families (P=0.007).
73b96314 Besides the above 11 SNPs, we tested in the same MS families the association of an MOG intragenic microsatellite, MOGTAAA, located in the 3'-UTR (Figure 1).
73b9668e None of the microsatellite alleles or of their hp combinations with the tested SNPs was significantly associated.
73b96a08 In conclusion, none of the tested variations in the MOG gene showed an association with MSV142L.
73b96d8c Thus the association with V142L was not secondary to any other tested MOG variation.
73b97110 Test of association with other markers of the HLA class I regionTo test whether the association observed for V142L extended outside the MOG gene, we analysed the MS association of six microsatellites (five in the class I region and tumour-necrosis factor alpha (TNFalpha); Figure 1a) in the 199 families already tested for all MOG SNPs.
73b9749e None of the microsatellite alleles showed a significant transmission distortion.
73b9782c Among those previously reported to be significantly associated, a non-significant trend was observed only for the MOGCA allele 5 (T:NT 72:52, P=0.073).
73b97ba6 L142 showed a significant LD with the microsatellite alleles MOGCA-6 and D6S265-5 (Table 5).
73b97f48 The transmission of the three allele hps (L142, MOGCA-6, D6S265-5) was distorted (T:NT 12:20) but not significantly (P=0.16), while a significantly lower transmission (T:NT 13:28, P=0.019) was seen for the two allele hps (L142, D6S265-5).
73b982e0 When considering only L142-negative chromosomes, no transmission distortion was observed either for D6S265-5 (T:NT 18:17) or for MOGCA-6 (T:NT 2:3).
73b98a42 Our results strongly support the presence of an additional MS risk-modulating element in the HLA class I region, independent of DRB1 alleles, and point to MOG V142L as the possible involved factor in the Italian population.
73b98f42 The association of MOG V142L with MS susceptibility was detected by two independent, population- and family-based, association studies, thus confirming a previously reported result in a small sample of 50 Italian MS patients.
73b99302 Notably V142L was the only SNP in the MOG gene significantly associated to MS, in accordance with previous negative results obtained for MOG markers not including V142L.
73b9969a Besides the continental Italian population, the association of V142L was previously tested in one study performed in German paediatric MS patients and in a recent study in the Sardinian population.
73b99a14 In both studies it was not significantly associated with MS.
73b99da2 This discrepant result may be related to the different population, to the different age of onset of the disease or to the sample size. The MOG142 association with MS was not modified when conditioning the analysis on HLA-DRB1 alleles.
73b9a126 Thus the protective effect of L142 is independent of HLA-DRB1.
73b9a4b4 While it is possible that MOG142 is only a marker in LD with a causative sequence variation mapping in an unexplored sequence inside or outside the MOG gene, the possibility of a primary involvement of MOG142 is, however, indicated by the following circumstantial evidence:
73b9a842 a V at position 142 is conserved among all tested mammalian species (mouse, rat, cow, macaca, orangutan), suggesting that it has a non-dispensable functional role;V142L is a conservative substitution located in the first MOG transmembrane domain.
73b9ac16 There are several instances of diseases in which V/L variations in transmembrane domains (for example in proteins encoded by CFTR, ABC7, PSEN1, TSHR, ACHR, VKORC1) were shown to be causative mutations (references and www.genet.sickkids.on.ca/cftr).
73b9afae What could be the role in MS susceptibility of a conservative V > L substitution in an MOG transmembrane domain.
73b9b328 One possible mechanism could be related to the role of MOG as an autoantigen.
73b9b6a2 Immunization of experimental animals with MOG peptides induces EAE.
73b9ba26 Interestingly, there is a high similarity between MOG-induced animal models and biopsies from MS patients, both showing large demyelinating lesions and axonal loss.
73b9bda0 MOG has an extracellular part including aa 1–122 with an immunnoglobulin-like domain, a transmembrane part from aa 123 to 152 and an intracellular and a second transmembrane part comprising aa 123–218.
73b9c124 Up to now, T-cell responses against the extracellular part of MOG have been mainly looked at.
73b9c49e However more recent data show that the intracellular part of MOG is much more immunogenic than the extracellular part.
73b9c82c In particular, there is a dominant MOG epitope recognized by CD4+ T cells within the intracellular part of MOG comprising amino acids immediately flanking the 142 residue.
73b9cbba The reported experiments were performed only with the more frequent V142 sequence.
73b9cf48 As an attractive hypothesis, the L residue could confer protection for MS by decreasing the immunogenicity of this epitope.
73b9d308 Notably, analysis by computer programs (http://www.immuneepitope.org, http://www.syfpeithi.de/) showed that the presence of either V or L at position 142 changes the affinity score for class I and class II HLA molecules.
73b9dc18 A second proposed mechanism refers to the possible influence of transmembrane variations on signal transmission.
73b9dfec In fact, mutations in transmembrane sequences of different plasma membrane receptors alter the signalling cascade by affecting the correct repartition into the glycosphingolipid-cholesterol membrane microdomains ('lipid rafts').
73b9e398 According to current models, upon ligand or antibody cross-linking, different plasma membrane receptors undergo enhanced repartitioning into lipid rafts as an obligatory first step towards participation in early signal-transduction events.
73b9e730 Lipid rafts provide a specialized environment for novel molecular interactions that can activate signal-transduction pathways.
73b9ebcc Recent data demonstrated that the signalling cascade activated by antibody cross-linking of MOG is dependent on MOG repartition into lipid rafts.
73b9ef78 It is tempting to speculate that the V142L substitution could influence MOG repartition into lipid rafts and hence signal transduction.
73b9f2fc Finally, missense variations modifying exonic splicing enhancer (ESE) or silencer sites have been reported as causative mutations of genetic diseases.
73b9f69e However, the involvement of MOG exon 3 missense SNP in this mechanism seems unlikely since (1) the known alternatively spliced isoforms of human MOG do not involve exon 3; (2) ESE prediction programs (ESEfinder, http://rulai.cshl.edu/tools/ESE/ and RESCUE ESE, http://genes.mit.edu/burgelab/rescue-ese/) did not predict a different score for candidate ESE motifs for the two MOG142 alleles and (3) no different splicing isoforms were observed in V142L heterozygous individuals when cDNA from T cells was amplified in nested PCR conditions (data not shown).
73b9fa36 In conclusion, our data confirm the presence of an MS risk factor, independent of HLA-DR, in the HLA class I region and propose that it might be located in the MOG gene.
73b9fdba The relationship between MOG L142 and the other recently identified HLA class I protective markers (HLA-A2 and –Cw5) remains to be tested.
73ba013e In addition, its possible pathogenetic role should be clarified by functional analysis.
73ba0bc0 Two genes encoding immune-regulatory molecules (LAG3 and IL7R) confer susceptibility to multiple sclerosis
73ba0f44 Multiple sclerosis (MS) is a T-cell-mediated disease of the central nervous system, characterized by damage to myelin and axons, resulting in progressive neurological disability.
73ba12d2 Genes may influence susceptibility to MS, but results of association studies are inconsistent, aside from the identification of HLA class II haplotypes.
73ba1656 Whole-genome linkage screens in MS have both confirmed the importance of the HLA region and uncovered non-HLA loci that may harbor susceptibility genes.
73ba19e4 In this two-stage analysis, we determined genotypes, in up to 672 MS patients and 672 controls, for 123 single-nucleotide polymorphisms (SNPs) in 66 genes. Genes were chosen based on their chromosomal positions or biological functions.
73ba1d7c In stage one, 22 genes contained at least one SNP for which the carriage rate for one allele differed significantly (P<0.08) between patients and controls. After additional genotyping in stage two, two genes—each containing at least three significantly (P<0.05) associated SNPs—conferred susceptibility to MS: LAG3 on chromosome 12p13, and IL7R on 5p13.
73ba2114 LAG3 inhibits activated T cells, while IL7R is necessary for the maturation of T and B cells.
73ba24ca These results imply that germline allelic variation in genes involved in immune homeostasis—and, by extension, derangement of immune homeostasis—influence the risk of MS.
73ba2e66 Multiple sclerosis (MS [MIM 126200; http://www.ncbi.nlm.nih.gov/Omim/]) is a chronic inflammatory disease of the central nervous system (CNS) with a prevalence of about 0.2% among northern Europeans. 
73ba31f4 The disease is characterized by widespread demyelination and axon damage, caused by an abnormal immune response, which lead to a range of neurological disabilities.
73ba3578 MS is a complex disease; susceptibility is believed to be conferred by the interplay of several genetic and environmental factors. 
73ba38fc The importance of the genetic component is illustrated by the fact that the risk of MS in siblings to MS patients is 20- to 40-fold higher than the risk in the general population.
73ba3c94 Four previous whole-genome linkage screens have suggested the existence of several chromosomal regions containing MS susceptibility genes, yet none of these regions, apart from the HLA region on chromosome 6p21, appears to harbor a major MS susceptibility locus (reviewed in Oksenberg et alt).
73ba4112 At the same time, in a recently published meta-analysis that combined raw genotyping data from all published whole-genome linkage screens in MS, it was shown that there exist overlapping regions, which may contain genes predisposing to MS; in addition to the HLA region, regions on chromosome 17q21 and chromosome 22q13 showed suggestive linkage.
73ba4518 Similarly, a comparison of the chromosomal locations of susceptibility loci in the human autoimmune diseases MS, type I diabetes mellitus, asthma, Crohn's disease and familial psoriasis and their animal models demonstrated additional regions of overlap; this latter instance of nonrandom clustering supports the notion that susceptibility to clinically distinct autoimmune diseases may be influenced by a common set of genes.
73ba48c4 The use of single-nucleotide polymorphisms (SNPs) as genetic markers in association studies has been a successful tool for identifying susceptibility genes in a number of complex diseases including type 1 diabetes mellitus, Crohn's disease and systemic lupus erythematosus.
73ba4c66 In MS, association studies have investigated both positional candidate genes and functional candidate genes, including genes encoding immunoglobulins and T-cell receptors, HLA molecules and myelin antigens, and chemokines, interleukins and other cytokines; however, to date, with the exception of various HLA class II haplotypes, no candidate gene has reproducibly been shown to be associated with MS.
73ba4ffe In this study, we have genotyped 123 SNPs, in a total of 66 candidate genes, in up to 672 MS patients and 672 controls, using the Pyrosequencing technique and a two-stage study design.
73ba5396 In the first stage, we made use of a limited number of markers and a limited number of patients and controls; in selecting polymorphisms in this stage, we sought to choose exonic SNPs, giving highest priority to SNPs encoding nonsynonymous nucleotide changes.
73ba572e In the second stage of the study, for genes displaying an association to MS in the first stage, we increased both the number of markers in each gene and the number of subjects to be genotyped.
73ba5ac6 Of the 66 genes investigated, 45 were selected on the basis of their location in regions linked to MS or other autoimmune diseases on chromosomes 5, 7, 12, 17 and 19 and the remaining 21 on the basis either of encoding proteins of potential functional importance in MS, or of having been the focus of previous association studies in MS (Table 1 and Electronic Supplement).
73ba5e5e To our knowledge, this is the largest association study ever performed in MS on non-HLA loci.
73ba6282 Our results suggest that two of the 66 candidate genes—lymphocyte-activation gene-3 (LAG3), on chromosome 12, and the gene encoding the interleukin 7 receptor (IL7R) on chromosome 5 — are associated with susceptibility to MS.
73ba6624 For both genes, in single-point analysis, carriage counts for one allele of at least three SNPs differed significantly (P0.05) between MS patients and controls; in addition, in the case of IL7R, predispositional and protective multi-SNP haplotypes were also identified.
73ba69c6 LAG3 and IL7R encode proteins involved, respectively, in the activation and maturation of lymphocytes; their association with MS suggests that germline allelic variation in genes involved in immune homeostasis—and, by extension, derangement of immune homeostasis itself—may influence the risk of this disease.
73ba70e2 Single-point analysis of candidate-gene SNPsOf the 66 candidate genes, 34 were genotyped for a single SNP (on account of the scarcity of known SNPs in or near the genes) and 32 for two or more SNPs.
73ba78da In the first stage, we found an association, at the 8% significance level, between SNPs in 22 genes and susceptibility to MS.
73ba7c86 The power of detecting an odds ratio (OR) of 1.5 in this analysis ranged from 44 to 98%, and for the majority (75%) of SNPs, it exceeded 80%.
73ba8014 In order to confirm these results, and to increase the power of detection, a second stage of genotyping was performed on these 22 genes using a larger number of patient and control samples; further, several additional SNPs, located in or around the associated genes, were included in the analysis at this stage.
73ba84b0 For 80% of the SNPs studied in stage two, the power of detecting an OR of 1.5 was 80% or more, assuming a two-sided significance level of 0.05 (see Electronic Supplement).
73ba8870 For two of the 22 genes, LAG3 and IL7R, three or more SNPs were associated with MS at a 5% significance level in single-point analysis of the total case–control data set (Table 2).
73ba8bf4 In addition, four SNPs in HAVCR2 were initially found to be associated with MS; however, genotypes for these SNPs did not conform to Hardy–Weinberg equilibrium (HWE) in the first control set.
73ba8f8c We thus genotyped the same SNPs in the second control set (which was used for this purpose only); the SNPs were found to conform to HWE in this control set, and there were no significant differences in carriage counts for these SNPs between MS patients and controls in the second set.
73ba9324 Linkage-disequilibrium and haplotype analysisIn order to elucidate whether the associations of neighboring SNPs were independent of one another, we performed linkage-disequilibrium (LD) analysis on the two MS-associated genes in the total data set of 672 patients and 672 controls.
73ba96b2 LD blocks with a |D'| value greater than 0.85 were found within IL7R (Tables 3 and 4); analysis of LAG3 revealed no such LD blocks within the gene.
73ba9a36 We next analyzed whether the distribution of estimated IL7R haplotype counts differed between MS patients and controls (an estimation of haplotype frequencies and counts was performed for SNPs belonging to the same LD block).
73ba9dba A significant difference in the haplotype-count distribution was observed, a result that is discussed in more detail below.
73baa13e MS susceptibility genes: LAG3 and IL7RIn and around LAG3, on chromosome 12p13.32 nine different SNPs were investigated in the second stage of this study (Figure 1 and Table 2); distributions of carriage counts were found to differ significantly between MS patients and controls for three of these SNPs — rs19922452, rs951818 and rs870849.
73baa4cc The C/T transition encoded by rs870849 in exon 8 gives rise to an amino-acid substitution (Thr455Ile); the T/T genotype was significantly more common in MS patients than in controls (P=0.026, OR=1.44; Figure 1 and Table 2).
73baa864 The two remaining associated SNPs are located in a noncoding region, approximately 7.9 kilobases downstream of LAG3 and 2.4 kilobases upstream of the gene encoding CD4; these two SNPs are only 137 base pairs apart and are in LD with one other (data not shown).
73baabe8 No association was found with six SNPs located downstream of rs870849; two of these SNPs—rs1882545 and rs2365095 — are located within introns in LAG3, while the remaining four are located upstream of LAG3.
73baaf6c As no LD was found between the associated downstream SNPs and the associated intragenic SNP, no haplotype analysis was performed for LAG3. The five SNPs studied in IL7R are outlined in Figure 2.
73bab2fa Four of the SNPs are located within the gene, while rs1494571 lies in a downstream flanking region; the intragenic SNPs are both exonic (rs3194051) and intronic (rs987106, rs987107, rs1494554).
73bab674 In an earlier association study of IL7R in MS, which included a scan for novel SNPs, only one of these five SNPs (rs987106) was identified as polymorphic in the investigated case–control data set.
73bab9f8 In the present study, three of five SNPs — rs987106 and rs987107 in intron 6, and rs3194051 in exon 8 — were associated with MS.
73babd72 The SNP in exon 8 encodes an amino-acid substitution (Val356Ile) in the C-terminal domain of the translated protein.
73bac100 For all of the associated SNPs, subjects homozygous for the less common allele had a significantly higher risk of MS than carriers of the more common allele (Table 2), suggesting a recessive mode of action for each.
73bac4a2 We investigated three five-SNP haplotypes, which together accounted for 99% of IL7R haplotypes in MS patients and 96% in controls, with 100% power; the estimated distribution of the haplotypes differed significantly between patients and controls, and nine out of the haplotypes' 10 possible SNP pairs were found to be in LD with one another (Tables 3 and 4).
73bac92a Two specific five-SNP haplotypes were also found to be associated with MS: haplotype 1, which was more common in patients (Pcorrected0.0005); and haplotype 3 (Pcorrected=0.0020), which was more common in controls.
73bacce0 Haplotypes 1 and 3 are identical, apart from at the third SNP in the haplotype (rs987106), where haplotype 1 contains T and haplotype 3 A.
73bad06e Between two of the SNPs included in the investigated IL7R haplotypes, rs1494554 and rs3194051, the |D'| value was 0.82, that is, below the designated cutoff value for LD of 0.85; the LD between these two SNPs is in other words strong, but not as strong as that between the other pairs of SNPs in the haplotypes.
73bad3fc In addition, we have not been able to demarcate the upstream and downstream boundaries of the LD block. Although there are no known genes in the immediate vicinity of IL7R, there are a number of predicted transcripts; analysis of SNPs from these regions would allow a more precise delineation of the LD block.
73badafa The field of complex-disease genetics has evolved rapidly during the past 10 years; yet, unfortunately, attempts to identify susceptibility genes have for the most part produced disappointing results.
73bade88 Possible explanations for these failures include population stratification, particularly for studies performed on heterogeneous populations.
73bae20c In MS, many associations of non-HLA candidate genes with disease susceptibility have been reported; few, if any, however, have been convincingly replicated, weakening the evidence for true association.
73bae586 The present study clearly shows the necessity of investigating large data sets in association studies in MS.
73bae90a In the first stage of the study—which employed a liberal, uncorrected significance level of 0.08 — 22 genes were associated with MS; by expanding the data set in the second stage, we were able to exclude 20 of these genes.
73baec98 In addition, we increased the number of SNPs investigated in the two remaining genes, and analyzed multi-SNP haplotypes, in order to localize with greater power and precision the source of the genetic signal, and in acknowledgment of the fact that haplotypes often encode functional units of protein products.
73baf026 For many of the SNPs investigated in this study, no difference was observed in the genotype frequencies of cases and controls; however, on account of unpredictable variability of LD throughout the genome, to conclude definitively that the genes in which these SNPs are located do not appreciably increase the risk of MS, it would be necessary to investigate a greater number of polymorphisms in and around the genes (ideally, in an even larger data set).
73baf3b4 This study was designed with a view to detect genes that increase the risk of MS by a factor of at least 1.5. In MS and other complex disorders, it is to be expected that the ORs associated with certain susceptibility genes will be lower than 1.5; such genes will have remained undetected in the present study.
73baf742 Moreover, on account of genotyping difficulties and low minor-allele frequencies, several of the SNPs in this study failed to reach 80% power.
73bafada The results of the present association study — to our knowledge, the largest ever performed in MS on candidate genes located outside the HLA region — indicate that two genes, LAG3 and IL7R, are associated with the risk of MS.
73bafe72 Both genes contain at least three SNPs that are significantly associated with MS, and in IL7R, predispositional and protective haplotypes were identified. Although LAG3, on chromosome 12p13, and IL7R, on chromosome 5p13, were originally chosen for study on the basis of their location in regions linked to MS and other autoimmune diseases, each gene encodes a molecule involved in T-cell regulation — a process of self-evident importance in the T-cell-mediated disorder MS. LAG3 (CD223) is an MHC class II ligand evolutionarily related to CD4; it downregulates the activated T cells on which it is expressed through a high-affinity interaction with its receptor that blocks the binding of CD4.
73bb02c8 Experiments in knockout mice have shown that LAG3 negatively regulates both T-cell expansion and the size of the memory-T-cell pool.
73bb0674 During inflammation, both LAG3 and MHC class II are strongly upregulated; their interaction may also play a role in the activation of antigen-presenting dendritic cells.
73bb0a0c The SNP rs870849 in exon 8 of LAG3 encodes a substitution of the nonpolar amino acid isoleucine for the uncharged polar amino acid threonine — a change that could possibly alter the conformational and functional properties of the protein.
73bb0f2a We speculate that such an alteration might reduce the binding affinity of LAG3, resulting in an enhanced expansion of T cells and a larger memory-T-cell pool — phenomena that could predispose to the development of MS.
73bb1330 At the same time, the differences in the distribution of rs870849 genotypes detected in our study between MS patients and controls could be due to the effects of a nearby polymorphism in LD with rs870849 but not investigated in this study.
73bb16dc Since we were unable to demarcate the downstream boundary of the LAG3 association, and since the appealing candidate gene CD4 lies in the vicinity of the associated markers, it remains to be determined whether the polymorphisms responsible for the association are located in LAG3 itself or in CD4.
73bb1a6a The interleukin 7 receptor (IL-7R; CD127), a member of the hematopoietin receptor family, is a type 1 membrane glycoprotein capable of binding alpha-helical cytokines.
73bb1df8 Signalling via IL-7R induces somatic recombination of the T-cell-receptor and immunoglobulin genes, promoting the proliferation and survival of T and B lymphocytes. The IL-7R complex consists of the IL-7R alpha chain and the common cytokine-receptor gamma chain (CD132); the former molecule transduces transmembrane signals through the recruitment of intracellular messengers to its cytoplasmic tail, while the latter activates this transduction.
73bb2186 IL-7R is expressed on immature B cells and T cells; in mice, administration of neutralizing antibodies or genetic ablation of IL7-R blocks lymphocyte development.
73bb2514 In humans, germline mutations resulting in defective expression of IL-7R give rise to a subtype of the disorder severe combined immunodeficiency.
73bb28a2 In the present study, we identified three SNPs associated with MS in IL7R.
73bb2c30 For a five-SNP LD block (which also contained two unassociated SNPs), we identified one predispositional and one protective haplotype.
73bb2fbe Interestingly, these two haplotypes differ at only one position — at rs987106, in intron 6 of IL7R — suggesting that a functional consequence of this particular polymorphism may underlie the haplotypes' positive and negative associations with the risk of MS.
73bb3356 In a recent array-based study of the expression of over 4000 genes in peripheral-blood mononuclear cells from 15 MS patients and 15 age- and sex-matched controls, IL7R was one of only 25 genes that displayed significantly higher expression in patients; overexpression of IL7R, the authors speculate, may result in increased numbers of autoantigen-specific T cells.
73bb36da In conclusion, we found associations between two genes and MS.
73bb3a5e Both genes are involved in regulation of the immune system, and both confer a moderate risk for the development of the disease.
73bb3de2 These findings are thus in accordance with the polygenic disease model proposed for MS.
73bb415c Further analysis of these genes in other MS data sets, as well as functional studies of the proteins they encode, is of utmost importance for the validation of our results.
73bb4cf6 IL2RA and IL7RA genes confer susceptibility for multiple sclerosis in two independent European populations
73bb507a Multiple sclerosis (MS) is the most common chronic inflammatory neurologic disorder diagnosed in young adults and, due to its chronic course, is responsible for a substantial economic burden.
73bb53fe MS is considered to be a multifactorial disease in which both genetic and environmental factors intervene.
73bb578c The well-established human leukocyte antigen (HLA) association does not completely explain the genetic impact on disease susceptibility.
73bb5b74 However, identification and validation of non-HLA-genes conferring susceptibility to MS has proven to be difficult probably because of the small individual contribution of each of these genes.
73bb6164 Recently, associations with two single nucleotide polymorphisms (SNPs) in the IL2RA gene (rs12722489, rs2104286) and one SNP in the IL7RA gene (rs6897932) have been reported by several groups.
73bb67ae These three SNPs were genotyped in a French and a German population of MS patients using the hME assay by the matrix-assisted laser desorption/ionization time of flight technology (Sequenom, San Diego, CA, USA).
73bb6dd0 We show that these SNPs do contribute to the risk of MS in these two unrelated European MS patient populations with odds ratios varying from 1.1 to 1.5. The discovery and validation of new genetic risk factors in independent populations may help toward the understanding of MS pathogenesis by providing valuable information on biological pathways to be investigated.
73bb79ec Multiple sclerosis (MS)—the most common cause of chronic neurologic disability beginning in early to middle adult life—is considered to be a multifactorial disease involving both genetic and environmental factors.
73bb7ff0 Evidence of genetic contribution to MS susceptibility arose from familial aggregation, twin studies and high incidence in populations of northern European origin, when compared with African and Asian groups.
73bb85f4 The prevalence pattern may be explained, at least in part, by migrations of northern European groups and their descendants which strengthens the hypothesis of genetic factors contributing to MS.
73bb89d2 Epidemiological studies performed in twins and siblings clearly indicate the impact of genetic factors.
73bb8d74 In addition, the geographical distribution of MS and studies of migrants also suggest a considerable contribution of environmental factors that may not be evenly distributed over the world.
73bb90f8 Current evidence indicates that there is not only one gene conferring susceptibility, but rather a number of genes — each contributing only a small amount.
73bb947c A large number of studies performed so far confirmed HLA-DRB1 on chromosome 6p21 to represent a major risk factor for MS in all populations tested.
73bb9800 However, it does not fully explain the genetic basis of the disease.
73bb9b84 Other loci outside the human leukocyte antigen (HLA) region were suspected by the results of several candidate genes or genome-wide approaches, but could not be validated in other samples — until recently.
73bb9f62 Variations in the interleukin-2 receptor-alpha gene (IL2RA) and the interleukin-7 receptor-alpha chain (IL7RA) were demonstrated to confer a risk for MS in candidate gene approaches and a whole genome screen.
73bba30e The association of MS with IL7RA was originally reported in a candidate gene approach by Zhang et al. 2 years ago, and is now replicated on large additional samples.
73bba69c Interestingly, linkage to chromosome 5p13, precisely where IL7RA is located, was also reported in a Canadian MS cohort with a maximum logarithm (base 10) of odds score of 4.2413 and supported later by others.
73bbaab6 With respect to IL2RA, genome-wide linkage analysis has shown potential linkage on chromosome 10p15, which contains the IL2RA gene.
73bbae9e Subsequently, a candidate approach on this gene first suggested an association between MS and IL2RA.
73bbb452 This result was strongly supported by the whole genome approach on extremely large samples.
73bbb81c This study reports the results obtained in two independent European MS samples (French and German) by genotyping the three single nucleotide polymorphisms (SNPs) showing the strongest association with MS in the study of the International Multiple Sclerosis Genetics Consortium.
73bbbbbe Two SNPs are within the IL2RA gene (rs12722489, rs2104286) and one in the IL7RA gene (rs6897932).
73bbc2c6 The three SNPs (rs12722489, rs2104286 and rs6897932) were typed in the German sample, consisting of 206 MS patients and 605 age and sex-matched controls, and in the French sample, consisting of 540 MS trios.
73bbc640 Basic demographics of both samples are given in Table 1.
73bbc9c4 A statistical comparison showed significant difference between the samples for age at time of analysis, disease course and Expanded Disability Status Scale (EDSS).
73bbcd3e However, these differences do not alter the overall genetic findings discussed below.
73bbd0cc Risk allele frequency (RAF) for the three SNPs, given in Table 2, were estimated from the German control sample and from the French untransmitted allele sample, respectively.
73bbd450 No significant difference was found between our two populations, and the RAFs are very similar to that of the original report.
73bbd7d4 We first estimated the expected power of our two samples, under two scenarios (see Materials and methods section) represented by the allele frequencies (0.75 for the two first SNPs and 0.85 for the third one) and the corresponding odds ratio (1.2 and 1.25 respectively) given in the original report.
73bbdb62 In both scenarios, the expected power for the French trio sample and the German sample was 56 and 38%, respectively.
73bbdef0 This clearly demonstrates the interest in the joint analysis of samples.
73bbe274 Since our aim is to replicate the signals found in other populations, all the P-values reported here are based on a one-sided test.
73bbe5f8 In the trios, 213 transmissions were informative for rs12722489, 355 were informative for rs2104286 and 389 were informative for rs6897932, respectively.
73bbe972 The SNP rs2104286 within the IL2RA gene demonstrated a significant association in the French MS trios and the German sample—whether analysed separately or combined in both samples (Table 2).
73bbecf6 The SNP rs12722489, also located within the IL2RA gene, shows significant association in the German sample and a positive trend in the French trios (Table 2).
73bbf08e Linkage disequilibrium between these two SNPs is strong (D'=1 in the German cases, German controls, and French case chromosomes, with D' estimated at 0.989 in the French control chromosomes), but far from perfect (r2=0.500, 0.552, 0.555 and 0.417, for the above-mentioned samples, respectively).
73bbf41c The SNP rs6897932 located within the IL7RA gene exhibits a significant association in the French trios and a positive trend in the German sample.
73bbf7aa Combining the results of both samples a significant P-value is obtained (Table 2).
73bc0042 The identification of non-HLA genes conferring susceptibility for MS is difficult.
73bc056a Linkage analysis in nuclear families failed to identify additional loci with genome-wide significance, but highlighted the IL2R and IL7R regions and the involvement of these two genes were suggested by candidate gene approaches.
73bc0920 Recent work on extremely large samples rendered IL2RA and IL7RA highly attractive and motivated further investigations.
73bc0d1c The risks associated to the SNPs of IL2RA and IL7RA however are modest at best (odds ratios between 1.1 and 1.5), so that only replications in a large number of different samples will make their involvement in MS susceptibility convincing.
73bc10dc We confirm here the positive association with the same risk alleles within the IL2RA and the IL7RA genes in two independent European samples. The odds ratios are consistent with those previously published.
73bc1460 The two SNPs typed in the IL2RA gene are in strong, but not perfect, linkage disequilibrium, which could explain the observed difference in P-values and odds ratios obtained.
73bc18de Therefore, our findings convincingly corroborate the recently published results.
73bc1c8a In the meantime, another independent replication on a Canadian sample published similar results.
73bc202c In addition, association of IL2RA with MS susceptibility was also recently concluded by Matesanz et al. with other SNPs located within the IL2RA gene. Further studies on this gene are needed to make a link between those different signals.
73bc23c4 In addition, it will be very important to take advantage of both the linkage and association information for a better understanding and a refined modelization of the effects of both the IL2RA and IL7RA genes.
73bc2748 Together with the well-known HLA association these results fit very well the assumption that MS is an autoimmune disease caused by lymphocytes and monocytes infiltrating the central nervous system.
73bc2acc Both cytokines are of central importance in promoting the growth and differentiation of T and B cells.
73bc2e50 IL-2 is an essential T-cell growth factor required for division of antigen-activated T cells.
73bc31d4 Activation of T cells induces the synthesis of IL2RA and the formation of the high-affinity IL-2 receptor, allowing the cell to respond to very low concentrations of IL-2.
73bc3558 IL-7 is important for memory T cells and the development of γ/Δ T cells, which are present in inflammatory lesions of MS patients.
73bc38e6 The alpha-chain of its receptor expresses differently according to the IL7RA genotypes.
73bc3c7e Interestingly, the likely causal SNP in the IL7RA gene has been shown to be responsible for increased exon skipping, which results in the production of more soluble IL-7 receptor than those of the membrane-bound isoform.
73bc4016 Only marginal effect of these genes has been tested.
73bc4390 They may interact with other genes.
73bc47b4 Even if, as underlined in the elegant editorial of Peltonen, these two genes do not provide clues for unsuspected pathways, focusing on genes which are biologically interacting with IL2RA and IL7RA will be the next step.
73bc4b88 As stated by Marrosu, 'the implication of interleukins in susceptibility to MS might lead to new avenues of investigation'.
73bc4f20 Taking into account the complexity of MS including the participation of other immune cells, for example monocytes, other cytokines, adhesion molecules, growth factors and neurodegenerative processes, there is a high probability that additional pathways are involved in pathogenesis.
73bc52ae Unravelling the MS mystery is just beginning.