and Immunity (2005) 6, 145–152 & 2005 Nature Publishing Group All rights reserved 1466-4879/05 $30.00 www.nature.com/gene

FULL PAPER Two genes encoding immune-regulatory molecules (LAG3 and IL7R) confer susceptibility to

Z Zhang1, K Duvefelt2,5, F Svensson1,2,5, T Masterman2, G Jonasdottir3, H Salter1, T Emahazion1, D Hellgren1, G Falk1, T Olsson4, J Hillert2 and M Anvret1 1Department of Molecular Sciences, Section for Genetics and Bioinformatics, AstraZeneca R&D So¨derta¨lje, So¨derta¨lje, Sweden; 2Neurotec Department, Division of Neurology, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden; 3Department of Medical Epidemiology, Karolinska Institutet, Stockholm, Sweden; 4Center for Molecular Medicine, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden

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. Genes may influence susceptibility to MS, but results of association studies are inconsistent, aside from the identification of HLA class II haplotypes. 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. 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. In stage one, 22 genes contained at least one SNP for which the carriage rate for one allele differed significantly (Po0.08) between patients and controls. After additional genotyping in stage two, two genes—each containing at least three significantly (Po0.05) associated SNPs—conferred susceptibility to MS: LAG3 on 12p13, and IL7R on 5p13. LAG3 inhibits activated T cells, while IL7R is necessary for the maturation of T and B cells. 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. Genes and Immunity (2005) 6, 145–152. doi:10.1038/sj..6364171 Published online 27 January 2005

Keywords: multiple sclerosis; single-nucleotide polymorphism; haplotype

Introduction (reviewed in Oksenberg et al4). At the same time, in a recently published meta-analysis that combined raw Multiple sclerosis (MS [MIM 126200; http://www. genotyping data from all published whole-genome ncbi.nlm.nih.gov/Omim/]) is a chronic inflammatory linkage screens in MS, it was shown that there exist disease of the central nervous system (CNS) with a overlapping regions, which may contain genes predis- prevalence of about 0.2% among northern Europeans.1 posing to MS;5 in addition to the HLA region, regions on The disease is characterized by widespread demyelina- chromosome 17q21 and chromosome 22q13 showed tion and axon damage, caused by an abnormal immune suggestive linkage. Similarly, a comparison of the response, which lead to a range of neurological dis- chromosomal locations of susceptibility loci in the abilities.2 MS is a complex disease; susceptibility is human autoimmune diseases MS, type I diabetes believed to be conferred by the interplay of several mellitus, asthma, Crohn’s disease and familial genetic and environmental factors. The importance of the and their animal models demonstrated additional genetic component is illustrated by the fact that the risk regions of overlap;6 this latter instance of nonrandom of MS in siblings to MS patients is 20- to 40-fold higher clustering supports the notion that susceptibility to than the risk in the general population.3 clinically distinct autoimmune diseases may be influ- Four previous whole-genome linkage screens have enced by a common set of genes. suggested the existence of several chromosomal regions The use of single-nucleotide polymorphisms (SNPs) as containing MS susceptibility genes, yet none of these genetic markers in association studies has been a regions, apart from the HLA region on chromosome successful tool for identifying susceptibility genes in a 6p21, appears to harbor a major MS susceptibility number of complex diseases including type 1 diabetes mellitus, Crohn’s disease and systemic lupus erythema- tosus.7–9 In MS, association studies have investigated Correspondence: Dr F Svensson, Department of Neurology, R54, both positional candidate genes and functional candidate Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. genes, including genes encoding immunoglobulins and E-mail: [email protected] 5These authors contributed equally to this work. T-cell receptors, HLA molecules and myelin antigens, Received 27 September 2004; revised 15 November 2004; accepted and chemokines, interleukins and other cytokines; 15 November 2004; published online 27 January 2005 however, to date, with the exception of various HLA LAG3 and IL7R are associated with MS Z Zhang et al 146 class II haplotypes, no candidate gene has reproducibly genotypes for these SNPs did not conform to Hardy– been shown to be associated with MS.10 Weinberg equilibrium (HWE) in the first control set. We In this study, we have genotyped 123 SNPs, in a total thus genotyped the same SNPs in the second control set of 66 candidate genes, in up to 672 MS patients and 672 (which was used for this purpose only); the SNPs were controls, using the Pyrosequencing technique11 and a found to conform to HWE in this control set, and there two-stage study design. In the first stage, we made use of were no significant differences in carriage counts for a limited number of markers and a limited number of these SNPs between MS patients and controls in the patients and controls; in selecting polymorphisms in this second set. stage, we sought to choose exonic SNPs, giving highest priority to SNPs encoding nonsynonymous nucleotide Linkage-disequilibrium and haplotype analysis changes. In the second stage of the study, for genes In order to elucidate whether the associations of displaying an association to MS in the first stage, we neighboring SNPs were independent of one another, increased both the number of markers in each gene and we performed linkage-disequilibrium (LD) analysis on the number of subjects to be genotyped. the two MS-associated genes in the total data set of 672 Of the 66 genes investigated, 45 were selected on the patients and 672 controls. LD blocks with a |D0| value basis of their location in regions linked to MS or other greater than 0.85 were found within IL7R (Tables 3 and autoimmune diseases on 5, 7, 12, 17 and 4); analysis of LAG3 revealed no such LD blocks within 19,4–6 and the remaining 21 on the basis either of the gene. encoding of potential functional importance in We next analyzed whether the distribution of esti- MS, or of having been the focus of previous association mated IL7R haplotype counts differed between MS studies in MS12–16 (Table 1 and Electronic Supplement). patients and controls (an estimation of haplotype To our knowledge, this is the largest association study frequencies and counts was performed for SNPs belong- ever performed in MS on non-HLA loci. Our results ing to the same LD block). A significant difference in the suggest that two of the 66 candidate genes—lymphocyte- haplotype-count distribution was observed, a result that activation gene-3 (LAG3), on , and the is discussed in more detail below. gene encoding the (IL7R)on —are associated with susceptibility to MS. For both genes, in single-point analysis, carriage MS susceptibility genes: LAG3 and IL7R counts for one allele of at least three SNPs differed In and around LAG3, on chromosome 12p13.32, nine significantly (Po0.05) between MS patients and controls; different SNPs were investigated in the second stage of in addition, in the case of IL7R, predispositional and this study (Figure 1 and Table 2); distributions of carriage protective multi-SNP haplotypes were also identified. counts were found to differ significantly between MS LAG3 and IL7R encode proteins involved, respectively, in patients and controls for three of these SNPs— the activation and maturation of lymphocytes; their rs19922452, rs951818 and rs870849. The C/T transition association with MS suggests that germline allelic encoded by rs870849 in 8 gives rise to an amino- variation in genes involved in immune homeostasis— acid substitution (Thr455Ile); the T/T genotype was and, by extension, derangement of immune homeostasis significantly more common in MS patients than in itself—may influence the risk of this disease. controls (P ¼ 0.026, OR ¼ 1.44; Figure 1 and Table 2). The two remaining associated SNPs are located in a noncoding region, approximately 7.9 kilobases down- Results stream of LAG3 and 2.4 kilobases upstream of the gene encoding CD4; these two SNPs are only 137 base pairs Single-point analysis of candidate-gene SNPs apart and are in LD with one other (data not shown). No Of the 66 candidate genes, 34 were genotyped for a single association was found with six SNPs located down- SNP (on account of the scarcity of known SNPs in or near stream of rs870849; two of these SNPs—rs1882545 and the genes) and 32 for two or more SNPs. In the first stage, rs2365095—are located within in LAG3, while the we found an association, at the 8% significance level, remaining four are located upstream of LAG3.AsnoLD between SNPs in 22 genes and susceptibility to MS. The was found between the associated downstream SNPs power of detecting an odds ratio (OR) of 1.5 in this and the associated intragenic SNP, no haplotype analysis analysis ranged from 44 to 98%, and for the majority was performed for LAG3. (75%) of SNPs, it exceeded 80%. In order to confirm these The five SNPs studied in IL7R are outlined in Figure 2. results, and to increase the power of detection, a second Four of the SNPs are located within the gene, while stage of genotyping was performed on these 22 genes rs1494571 lies in a downstream flanking region; the using a larger number of patient and control samples; intragenic SNPs are both exonic (rs3194051) and intronic further, several additional SNPs, located in or around the (rs987106, rs987107, rs1494554). In an earlier association associated genes, were included in the analysis at this study of IL7R in MS, which included a scan for novel stage. SNPs, only one of these five SNPs (rs987106) was For 80% of the SNPs studied in stage two, the power of identified as polymorphic in the investigated case– detecting an OR of 1.5 was 80% or more, assuming a two- control data set.17 In the present study, three of five sided significance level of 0.05 (see Electronic Supple- SNPs—rs987106 and rs987107 in 6, and rs3194051 ment). For two of the 22 genes, LAG3 and IL7R, three or in exon 8—were associated with MS. The SNP in exon 8 more SNPs were associated with MS at a 5% significance encodes an amino-acid substitution (Val356Ile) in the C- level in single-point analysis of the total case–control terminal domain of the translated . For all of the data set (Table 2). In addition, four SNPs in HAVCR2 associated SNPs, subjects homozygous for the less were initially found to be associated with MS; however, common allele had a significantly higher risk of MS

Genes and Immunity LAG3 and IL7R are associated with MS Z Zhang et al 147 Table 1 Genes studied in up to 672 multiple sclerosis (MS) patients and 672 controls using single-nucleotide polymorphisms (SNPs)

Gene symbol MIM number Genomic location No. of SNPs P (stage 1) SNP with lowest P P (stage 2) MS patients typed Controls typed

TNFRSF1B 191191 1p36.3–p36.2 2 0.03 rs1061622 0.62 663 277 CDW52 114280 1p36 1 0.2 rs17645 190 186 FY 110700 1q21–q22 2 0.16 rs12075 287 273 BCL2L11 603827 2q12–q13 4 0.05 rs724710 0.2 651 650 IL1B 147720 2q14 1 0.69 rs1143633 194 181 IL1RN 147679 2q14.2 2 0.22 rs9005 189 178 MGAT5 601774 2q21 3 0.05 rs1439107 0.66 664 652 ICOS 604558 2q33 2 0.04 rs718183 0.1 638 266 SLC11A1 600266 2q35 2 0.02 rs1059823 0.12 638 269 PDCD1 600244 2q37.3 1 0.05 rs2227981 0.37 626 277 MYD88 602170 3p22–p21.3 1 0.38 rs7744 188 188 TLR9 605474 3p21.3 1 0.62 rs352140 445 269 CCR2 601267 3p21 1 0.04 rs1799864 0.63 633 665 CCR5 601373 3p21 2 0.04 rs1800874 0.53 595 647 CBLB 604491 3q13.11–q13.12 1 0.05 rs1042852 0.38 664 288 CD86 601020 3q21 1 0.29 rs1129055 285 186 CXCL10 147310 4q21 2 0.09 rs8878 285 271 CXCL13 605149 4q21 2 0.9 rs1500498 278 192 SPP1 166490 4q21–q25 2 0.49 rs4754 281 261 FYB 602731 5p13.1 1 0.58 rs379707 186 187 IL7R 146661 5p13 5 0.01 rs3194051 0.004 667 558 IL6ST 600694 5q11 1 0.24 rs1063560 381 191 LY64 602226 5q12 1 0.31 rs2230525 176 175 ITGA2 192974 5q23–q31 2 0.25 rs1062535 191 192 HAVCR2 606652 5q33.2 8 0.001 rs1036199 0.87b 638 457a CCR6 601835 6q27 1 0.08 rs2071171 285 270 IL6 147620 7p21 1 0.73 rs1800795 181 94 AOAH 102593 7p14–p12 1 0.81 rs2228411 183 187 STK17A 604726 7p12–p14 3 0.05 rs1044141 0.52 642 288 TPST1 603125 7q11.21 1 0.63 rs3757417 189 189 SRI 182520 7 q21.1 1 0.04 rs43100 0.6 627 640 SERPINE1 173360 7q21.3–q22 1 0.05 rs1799889 0.53 372 277 IFRD1 603502 7q22–q31 1 0.14 rs7817 188 182 PBEF1 NA 7q22.2 4 0.11 rs2302559 190 182 IL18 600953 11q22.2–q22.3 1 0.83 rs549908 180 187 CASP5 602665 11q22.2–q22.3 2 0.48 rs523104 185 174 CD4 186940 12pter–p12 1 0.39 rs3213427 191 96 LAG3 153337 12p13.32 9 0.005 rs870849 0.03 576 662 TNFRSF1A 191190 12p13.2 1 0.58 rs767455 378 192 CD69 107273 12p13–p12 2 0.1 rs1051065 190 84 C1S 120580 12p13 1 0.09 rs7183 187 190 LTBR 600979 12p13 1 0.16 rs12354 191 96 SLC11A2 600523 12q13 1 0.32 rs1048230 190 192 LNK 605093 12q24 1 0.11 rs739496 190 191 TRA1 191175 12q24.2–q24.3 2 0.14 rs7645 192 192 LGALS3 153619 14q21–q22 1 0.15 rs11125 183 189 SIVA 605567 14q32.33 1 0.26 rs1132975 185 95 TRAF3 601896 14q32.33 1 0.5 rs1131877 182 182 CCL3 182283 17q11–q21 1 0.67 rs1634500 270 237 CCL18 603757 17q11.2 2 0.11 rs14304 152 173 ITGB3 173470 17q21.32 2 0.07 rs4642 0.26 575 672 PECAM1 173445 17q23 2 0.01 rs2812 0.88 576 462 CD79B 147245 17q23 3 0.12 rs7921 184 192 APOH 138700 17q23–qter 1 0.8 rs4581 187 186 PSMC5 601681 17q24–q25 2 0.03 rs13030 0.18 543 564 ICAM1 147840 19p13.3–p13.2 2 0.49 rs5498 188 191 IL12RB1 601604 19p13.1 2 0.08 rs897751 281 265 CAPNS1 114170 19q13.13 3 0.02 rs2075475 0.17 646 665 RPS19 603474 19q13.2 1 0.32 rs873282 190 181 RFX2 142765 19p13.3–p13.2 1 0.05 rs673153 0.59 366 271 IRF3 603734 19q13.3–q13.4 1 0.44 rs7251 284 268 LILRA2 604812 19q13.4 2 0.28 rs1974982 191 181 LILRB2b 604815 19q13.4 2 0.04 rs1974209 0.31 190 183 LILRB3 604820 19q13.4 2 0.57 rs678876 190 183 KIR3DL2 604947 19q13.4 4 0.03 rs592446 0.16 630 628 NKG7 606008 19q13.41 1 0.37 rs3009 191 182

Two-sided probability values for carriage of each allele were calculated by a w2-test; OR ¼ odds ratio; CI ¼ confidence interval; NA ¼ not available. aGenotyped in ‘second control set’ (see text). bNot genotyped in all samples due to problems with unspecific amplification.

Genes and Immunity LAG3 and IL7R are associated with MS Z Zhang et al 148 Table 2 Genotype counts in patients with multiple sclerosis (MS) and controls for single-nucleotide polymorphisms (SNPs) in IL7R and LAG3

Gene SNP Allele MS patients Controls Power Lower P (allele) Àlog P OR (95% CI)

1 2 11 12 22 Total 11 12 22 Total

IL7R rs1494554 A C 195 126 29 350 126 83 19 228 0.867 0.915 (2) 0.04 rs987107 C T 289 186 53 528 301 228 34 563 0.992 0.015 (1) 1.83 0.58 (0.37–0.90) rs987106 T A 147 348 165 660 124 336 197 657 1.000 0.043 (1) 1.37 1.3 (1.0–1.6) rs3194051 A G 378 235 54 667 305 230 23 558 0.996 0.004 (1) 2.36 0.49 (0.30–0.81) rs1494571 G C 57 202 325 584 18 102 135 255 0.92 0.207 (2) 0.68

LAG3 rs1922452 C T 257 296 85 638 231 308 115 654 1.000 0.034 (1) 1.47 1.4 (1.0–1.9) rs951818 T G 250 294 91 635 225 305 119 649 1.000 0.052 (1) 1.28 1.3 (1.0–1.8) rs870849 C T 236 248 92 576 276 309 77 662 1.000 0.027 (1) 1.58 0.69 (0.50–0.96) rs1882545 T C 280 291 68 639 296 280 82 658 1.000 0.305 (1) 0.52 rs2365095 C T 61 247 276 584 68 293 271 632 1.000 0.125 (1) 0.90 rs7488113 C A 263 20 0 283 258 19 0 277 0.453 0.923 (1) 0.03 rs1882551 C G 50 268 319 637 22 116 133 271 0.984 0.783 (1) 0.11 rs11227 T G 248 295 90 633 237 306 107 650 1.000 0.316 (2) 0.50 rs25557a GTTA 27 181 428 636 11 82 173 266 0.952 0.512 (1) 0.29

Two-sided P-values for carriage of each allele were calculated by a w2 test; OR ¼ odds ratio; CI ¼ confidence interval. aBiallelic four-base insertion/deletion.

Table 3 Linkage disequilibrium (|D0|) between pairs of single- these regions would allow a more precise delineation of nucleotide polymorphisms in IL7R the LD block.

rs987107 rs987106 rs3194051 rs1494571

rs1494554 0.97 0.89 0.82 0.96 Discussion rs987107 1.00 1.00 0.98 The field of complex-disease genetics has evolved rs987106 0.95 0.92 rs3194051 0.92 rapidly during the past 10 years; yet, unfortunately, attempts to identify susceptibility genes have for the most part produced disappointing results. Possible Shaded |D0| values are greater than the designated cutoff value for linkage disequilibrium of 0.85. explanations for these failures include population stra- tification, particularly for studies performed on hetero- geneous populations. In MS, many associations of non- HLA candidate genes with disease susceptibility have than carriers of the more common allele (Table 2), been reported; few, if any, however, have been convin- suggesting a recessive mode of action for each. cingly replicated, weakening the evidence for true We investigated three five-SNP haplotypes, which association.18–21 together accounted for 99% of IL7R haplotypes in MS The present study clearly shows the necessity of patients and 96% in controls, with 100% power; the investigating large data sets in association studies in estimated distribution of the haplotypes differed sig- MS. In the first stage of the study—which employed a nificantly between patients and controls, and nine out of liberal, uncorrected significance level of 0.08—22 genes the haplotypes’ 10 possible SNP pairs were found to be were associated with MS; by expanding the data set in in LD with one another (Tables 3 and 4). Two specific the second stage, we were able to exclude 20 of these five-SNP haplotypes were also found to be associated genes. In addition, we increased the number of SNPs with MS: haplotype 1, which was more common in investigated in the two remaining genes, and analyzed patients (Pcorrectedo0.0005); and haplotype 3 multi-SNP haplotypes, in order to localize with greater 22,23 (Pcorrected ¼ 0.0020), which was more common in controls. power and precision the source of the genetic signal, Haplotypes 1 and 3 are identical, apart from at the third and in acknowledgment of the fact that haplotypes often SNP in the haplotype (rs987106), where haplotype 1 encode functional units of protein products. contains T and haplotype 3 A. For many of the SNPs investigated in this study, no Between two of the SNPs included in the investigated difference was observed in the genotype frequencies of IL7R haplotypes, rs1494554 and rs3194051, the |D0| cases and controls; however, on account of unpredictable value was 0.82, that is, below the designated cutoff value variability of LD throughout the genome, to conclude for LD of 0.85; the LD between these two SNPs is in other definitively that the genes in which these SNPs are words strong, but not as strong as that between the other located do not appreciably increase the risk of MS, it pairs of SNPs in the haplotypes. In addition, we have not would be necessary to investigate a greater number of been able to demarcate the upstream and downstream polymorphisms in and around the genes (ideally, in an boundaries of the LD block. Although there are no even larger data set). This study was designed with a known genes in the immediate vicinity of IL7R, there are view to detect genes that increase the risk of MS by a a number of predicted transcripts; analysis of SNPs from factor of at least 1.5. In MS and other complex disorders,

Genes and Immunity LAG3 and IL7R are associated with MS Z Zhang et al 149 Table 4 Estimated frequencies of IL7R haplotypes spanning five single-nucleotide polymorphisms in multiple sclerosis (MS) patients and controls

rs1494554 rs987107 rs987106 rs3194051 rs1494571 MS patientsa Controlsa Odds ratioa Global Pb Haplotype Pc

Haplotype 1 A C T A C 0.495 0.432 1.16 o0.0005 Haplotype 2 C T A G G 0.258 0.234 0.86 0.002 NS Haplotype 3 A C A A C 0.239 0.289 1.00 0.002

NS ¼ not significant. aDetermined using all available genotyping data (for from 350 to 667 MS patients and from 228 to 657 controls) with haplotype 3 as reference. bDetermined by 10 000 simulations of haplotype-count distributions. cBy 1000 permutations of case–control status (see text).

2.0

rs1922452 1.5 rs951818 rs870849 P=0.05 P log − 1.0 rs2365095 rs1882545

0.5

rs7488113 rs1882551 0 7.9 kb LAG3 (6 kb) 1.5 kb PTMS (4 kb)

Figure 1 Schematic overview of the positions and P-values of the seven single-nucleotide polymorphisms (SNPs), located in and around LAG3 and PTMS (MIM 168440), investigated in the full data set 672 multiple sclerosis patients and 672 controls. Arrows indicate transcriptional orientation; kb ¼ kilobases, m ¼ coding SNP, . ¼ noncoding SNP.

2.5 rs3194051

2.0 rs987107

1.5 P rs987106 P =0.05 log −

1.0 rs1494571

0.5

rs1494554 0 IL7R (18 kb)

Figure 2 Schematic overview of the positions and P-values of the five single-nucleotide polymorphisms (SNPs), located in and around IL7R, investigated in the full data set 672 multiple sclerosis patients and 672 controls. Arrow indicates transcriptional orientation; kb ¼ kilobases, m ¼ coding SNP, . ¼ noncoding SNP. it is to be expected that the ORs associated with certain minor-allele frequencies, several of the SNPs in this susceptibility genes will be lower than 1.5; such genes study failed to reach 80% power. will have remained undetected in the present study. The results of the present association study—to our Moreover, on account of genotyping difficulties and low knowledge, the largest ever performed in MS on

Genes and Immunity LAG3 and IL7R are associated with MS Z Zhang et al 150 candidate genes located outside the HLA region— cular polymorphism may underlie the haplotypes’ indicate that two genes, LAG3 and IL7R, are associated positive and negative associations with the risk of MS. with the risk of MS. Both genes contain at least three In a recent array-based study of the expression of over SNPs that are significantly associated with MS, and in 4000 genes in peripheral-blood mononuclear cells from IL7R, predispositional and protective haplotypes were 15 MS patients and 15 age- and sex-matched controls, identified. Although LAG3, on chromosome 12p13, and IL7R was one of only 25 genes that displayed signifi- IL7R, on chromosome 5p13, were originally chosen for cantly higher expression in patients;30 overexpression of study on the basis of their location in regions linked to IL7R, the authors speculate, may result in increased MS and other autoimmune diseases, each gene encodes a numbers of autoantigen-specific T cells. molecule involved in T-cell regulation—a process of self- In conclusion, we found associations between two evident importance in the T-cell-mediated disorder MS. genes and MS. Both genes are involved in regulation of LAG3 (CD223) is an MHC class II ligand evolutiona- the , and both confer a moderate risk for rily related to CD4; it downregulates the activated T cells the development of the disease. These findings are thus on which it is expressed through a high-affinity interac- in accordance with the polygenic disease model pro- tion with its receptor that blocks the binding of CD4.24 posed for MS. Further analysis of these genes in other MS Experiments in knockout mice have shown that LAG3 data sets, as well as functional studies of the proteins negatively regulates both T-cell expansion and the size of they encode, is of utmost importance for the validation of the memory-T-cell pool.25 During inflammation, both our results. LAG3 and MHC class II are strongly upregulated; their interaction may also play a role in the activation of antigen-presenting dendritic cells.26 The SNP rs870849 in exon 8 of LAG3 encodes a Materials and methods substitution of the nonpolar isoleucine for the Samples from patients and controls uncharged polar amino acid threonine—a change that Blood samples for DNA extraction were collected from could possibly alter the conformational and functional 672 Nordic MS patients with a mean age of 45.6 years properties of the protein. We speculate that such an (range, 15–80 years) and a mean disease duration of 14.1 alteration might reduce the binding affinity of LAG3, years (range, 6 months to 63 years). The disease course resulting in an enhanced expansion of T cells and a larger was primary progressive in 12.1% of patients; 71.6% memory-T-cell pool—phenomena that could predispose were female. The mean age of the 672 Nordic controls to the development of MS. At the same time, the recruited for this study was 54.5 years (range, 17–91 differences in the distribution of rs870849 genotypes years); 49.2% were female. MS samples were provided by detected in our study between MS patients and controls the Departments of Neurology of Karolinska University could be due to the effects of a nearby polymorphism in Hospital, in Stockholm, Sweden, which also provided LD with rs870849 but not investigated in this study. samples from a control group consisting of 288 blood Since we were unable to demarcate the downstream donors (Karolinska control group). The remaining 384 boundary of the LAG3 association, and since the controls were randomly selected unrelated members of a appealing candidate gene CD4 lies in the vicinity of the set of mono- and dizygotic twin pairs enrolled in the associated markers, it remains to be determined whether Swedish Twin Registry (Twin Registry control group). the polymorphisms responsible for the association are The combined Karolinska and Twin Registry control sets located in LAG3 itself or in CD4. are referred to here as the first control set; for reasons The interleukin 7 receptor (IL-7R; CD127), a member of explained above, a second control set of 465 Swedish the hematopoietin receptor family, is a type 1 membrane blood donors (mean age, 42.8 years (range, 18–69 years); glycoprotein capable of binding alpha-helical cyto- 42.3% female) was used to evaluate four SNPs in the kines.27 Signalling via IL-7R induces somatic recombina- HAVCR2 gene. tion of the T-cell-receptor and immunoglobulin genes, All patients were diagnosed as having clinically or promoting the proliferation and survival of T and B laboratory-supported definite MS, in accordance with the lymphocytes.28 The IL-7R complex consists of the IL-7R recommendations of Poser and co-workers.31 Informed alpha chain and the common cytokine-receptor gamma consent was obtained from all patients and controls, and chain (CD132); the former molecule transduces trans- the study was approved by the ethical committee of membrane signals through the recruitment of intracel- Karolinska University Hospital. lular messengers to its cytoplasmic tail, while the latter The study was performed in two stages. The first stage activates this transduction.27,29 IL-7Ra is expressed on included between 149 and 288 MS patients and between immature B cells and T cells; in mice, administration of 84 and 288 controls. In the event a P-value below 0.08 (w2 neutralizing antibodies or genetic ablation of IL7-Ra test for carriage of one of the two alleles) was observed blocks lymphocyte development.28 In humans, germline for one or more SNPs within in a gene, a second stage of mutations resulting in defective expression of IL-7R give genotyping was performed on that gene using additional rise to a subtype of the disorder severe combined patients and controls. (The cutoff P-value was originally immunodeficiency. set at 0.10. Further genotyping was not performed on In the present study, we identified three SNPs three genes with P-values between 0.10 and 0.08 (due to associated with MS in IL7R. For a five-SNP LD block the low prior probability of their association with MS). (which also contained two unassociated SNPs), we The de facto cutoff P-value was thus 0.08.) The total identified one predispositional and one protective number of subjects investigated for such genes, during haplotype. Interestingly, these two haplotypes differ at the two stages of the study, was between 356 and 672 MS only one position—at rs987106, in intron 6 of IL7R— patients and between 462 and 672 controls (all from the suggesting that a functional consequence of this parti- first control set). In order to preserve DNA, random sets

Genes and Immunity LAG3 and IL7R are associated with MS Z Zhang et al 151 of 96-well plates containing patient and control samples Statistical analysis were used for different genes during the first stage of For each SNP, P-values for two-by-two contingency genotyping; in this way, we strove to assure access to full tables comparing carriage counts in patients and controls patient and control data sets for SNPs genotyped in the were calculated by a w2 test and were not corrected. A study’s second stage. Despite this approach, for the last single-point power analysis was performed for detecting SNPs added in the two genes displaying association with association in the SNPs using a standard method MS in the second stage—rs1494554 in IL7R and rs7488113 whereby normality in the logarithm of the OR is in LAG3—DNA was not available for all patients and assumed; power was calculated assuming a two-sided controls (Table 2). significance level of 0.05 and one-sided power for detecting an OR of at least 1.5 (or 0.67), in the absence of an adjustment for multiple testing. SNP identification, discovery and genotyping For the genes containing SNPs with single-point P- Information regarding previously characterized SNPs values less than 0.05, haplotype maps were constructed, genotyped in this study was accessed via public (The and haplotype frequencies estimated, in the following SNP Consortium [http://snp.cshl.org/], dbSNP [http:// manner. Pairwise measures of LD were obtained using www.ncbi.nlm.nih.gov/]) and proprietary (AstraZeneca) EH.34 These measures were used to define blocks of databases. In of an initial finding indicating closed gene-centric LD with |D0| values greater than association of one SNP located in LAG3 with MS, all 0.85. Markers within the blocks were then used to coding sequences of the gene, as well as its promoter and estimate haplotype frequencies using the SNPHAP 50- and 30-untranslated regions, were amplified in 96 program (http://www-gene.cimr.cam.ac.uk/clayton/ subjects, by 16 separate polymerase chain reactions software/snphap.txt). Haplotype frequencies were con- (PCRs), resulting in amplicons ranging from 250 to 550 verted into a population contingency table by multi- base pairs. Denaturing high-performance liquid chroma- plying the number of alleles measured by the frequency tography (DHPLC) was then performed using the estimate, and the contingency table was tested for Transgenomic WAVE System (Transgenomic, Omaha, significance using the T1 statistic of CLUMP with Neb, USA). PCR products were then loaded onto a 10 000 simulations.35 preheated reverse-phase column (DNASep; Transge- For the analysis of the distribution of estimated nomic) using an automatic sample injector, and subse- haplotype frequencies, power was calculated, using the quently eluted using an acetonitrile gradient in a 0.1 M EM algorithm36 and a Monte Carlo approach.37,38 The triethylamine-acetate buffer (pH 7.0) at a constant flow analysis was performed for IL7R only, since it was the rate of 0.9 ml/min. Samples detected by DHPLC as being only gene for which an LD block spanning adjacent heterozygous were then sequenced using ABI PRISM Big associated SNPs was detected. To test for association at Dye Terminator (Applied Biosystems, Foster City, CA, the level of single haplotypes, a variation on the USA), and the sequencing products analyzed on an ABI permutation test was used.37,38 The test was performed PRISM 3100 Genetic Analyzer (Applied Biosystems), in after computation of the likelihood ratio for the collapsed order to detect the nature and position of the poly- two-by-two contingency table of one haplotype vs the morphism in the amplified fragment. Two novel SNPs, others in cases and controls. P-values were divided by 3, located in noncoding sequences of LAG3, were discov- in accordance with the Bonferroni method, as a correc- ered in this manner; prior to the submission of the tion for multiple tests. ORs for individual haplotypes present report, these SNPs were registered by others in were calculated using the WHAP program (http:// dbSNP under the identification numbers rs2365095 and www.genome.wi.mit.edu/~shaun/whap/) with haplo- rs7488113. type 3 as reference. PCR primers were designed using Oligo 5.0 software.32 Two primers, ranging from 19 to 21 nucleotides in length, were designed for each PCR, and amplified fragments Acknowledgements ranged from 80 to 150 base pairs in length (see Electronic Supplement for primer sequences). From each subject This study was supported by grants from the European 10 ng of genomic DNA was used for each PCR Commission, the Swedish Medical Research Council, the amplification. PCRs were performed in a total volume Swedish Association of Neurologically Disabled, the of 25 ml, containing the following: PCR primers (Thermo- Swedish Society of Medicine, the Swedish Brain Founda- Hybaid, Ulm, Germany), each at a concentration of tion, the Lars Hierta Foundation, the Swedish Founda- 400 nM; dNTPs, each at a concentration of 80 mM;Mg2 þ ,at tion for Strategic Research, and Karolinska Institutet. We a concentration ranging from 1.5 to 3.5 mM; and 1 U thank Nancy Pedersen of the Swedish Twin Registry and Amplitaq Gold polymerase in 10 Â PCR Gold buffer all the patients and volunteers who participated in the (Applied Biosystems). The solution was subjected to PCR study. conditions consisting of a single cycle at 951C for 3 min (for activation of AmpliTaq Gold), followed by 50 cycles at 951C for 20 s, 55–601C for 20 s, and 721C for 30 s. Amplifications were performed on a PTC-225 DNA References Engine Tetrad (MJ Research Inc., Watertown, MA, USA). 1 Kurtzke JF. Epidemiologic evidence for multiple sclerosis as All SNPs were genotyped using the Pyrosequencing an infection. Clin Microbiol Rev 1993; 6: 382–427. technique, according to the standard protocol provided 2 Lassmann H. Neuropathology in multiple sclerosis: new by the manufacturer (Pyrosequencing AB, Uppsala, concepts. Multiple Sclerosis 1998; 4: 93–98. Sweden).33 Sequencing primers (15 nucleotides) were 3 Ebers GC, Sadovnick AD. The role of genetic factors in designed using Oligo 5.0 software.32 multiple sclerosis susceptibility. J Neuroimmunol 1994; 54: 1–17.

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Genes and Immunity