and Immunity (2003) 4, 518–523 & 2003 Nature Publishing Group All rights reserved 1466-4879/03 $25.00 www.nature.com/gene

BRIEF COMMUNICATION Intercellular adhesion molecule-1: a protective haplotype against multiple sclerosis

I Cournu-Rebeix1,EGe´nin2, G Lesca1, A Azoulay-Cayla3, N Tubridy1, E Noe´1, M Clanet4, G Edan5, F Clerget-Darpoux2,GSe´mana6 and B Fontaine1,3 1Laboratoire des affections de la mye´line et des canaux ioniques musculaires-INSERM U546, Faculte´ de Me´decine Pitie´-Salpeˆtrie`re, Paris, France; 2INSERM U535, Centre Hospitalier Universitaire, Le Kremlin Biceˆtre, France; 3Fe´de´ration de Neurologie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris, France; 4Service de Neurologie, Centre Re´gional Hospitalier Universitaire Purpan, Toulouse, France; 5Service de Neurologie, Centre Re´gional Hospitalier Universitaire Pontchaillou, Rennes, France; 6Laboratoire d’Immunoge´ne´tique, Etablissement Franc¸ais du Sang Bretagne et Laboratoire d’Immunologie (UPRES EA 1257), Centre Hospitalier Re´gional Universitaire et Faculte´ de Me´decine, Rennes, France

Intercellular adhesion molecule-1 (ICAM-1) and its receptors are adhesion molecules that play a key role in the transmigration of inflammatory cells through the blood–brain barrier, one of the earliest events in multiple sclerosis (MS), which leads to demyelination in the central nervous system. To investigate the role of genes encoding ICAM-1 and its receptors, we used a strategy of genetic linkage and association in 439 case–parent MS families of French origin, well characterized according to HLA status and severity. We demonstrate that the genes encoding ICAM-1 receptors do not influence MS susceptibility or severity. ICAM-1 had a modest, but significant effect on MS genetic susceptibility, independent of HLA and disease severity. We observed a rare, and an as yet unreported, ICAM-1 haplotype defined by amino acids K469 and R241 that was never transmitted to patients suggesting a protective effect against MS in our population. Genes and Immunity (2003) 4, 518–523. doi:10.1038/sj.gene.6364009

Keywords: multiple sclerosis; intercellular adhesion molecule-1; haplotypes; association; transmission disequilibrium test; receptors

Multiple sclerosis (MS) is the most common neurolo- characterized.6,7 Several reports have shown that inter- gical disease in young adults and is characterized by a cellular adhesion molecule 1 (ICAM-1), which is complex and multifactorial pathogenesis. Numerous expressed at the surface of endothelial cells, has a studies have shown that both genetic and environmental prominent role in facilitating the entrance of inflamma- factors contribute to an increased risk of developing tory cells into the CNS.8–10 ICAM-1 is a ligand of MS.1,2 Little is known, however, about the exact nature of lymphocyte function-associated antigen-1 (LFA-1 the involved genetic factors, except for the role of HLA or CD11a/CD18 or aLb2) and Mac-1 (CD11b/CD18 or

Class II with, in most populations, an association found aMb2), two receptors localized to the membrane of with the DR2 allele. The role of environmental exposures, inflammatory cells.11,12 Studies of the interaction between hypothetically viral infection, which causes inflamma- ICAM-1 and its receptors have demonstrated that tion and demyelination in the central nervous system both LFA-1 and Mac-1 contribute equally to (CNS) of MS patients has also been suggested but adhesion efficiency during transendothelial migration remains unclear. of neutrophils.13,14 ICAM-1 has also been described as The infiltration of inflammatory cells into the CNS is the major receptor for rhinovirus,15,16 strengthening the considered to be one of the earliest events in the interest in this gene, according to the viral hypothesis formation of a plaque, the fundamental pathological of MS. lesion in MS. Inflammatory cells, activated by poorly Because of the key role of ICAM-1 in BBB physiology, understood mechanisms, enter the brain by crossing the genetic variations in its gene or in the genes coding for its blood–brain barrier (BBB) through a complex cascade of receptors may be involved in MS susceptibility and/or molecular events in which adhesion molecules are key- severity. Conflicting results on the role of ICAM-1 in MS players.3,4 Adhesion molecules, induced at the surface of susceptibility have been reported. An association was endothelial cells by released by T lymphocytes first reported with the allele K469 of an intragenic SNP of and macrophages, allow transmigration of inflammatory ICAM-1 on a Polish sample of cases and controls.17 This cells across the BBB, leading to demyelination.5 association was not replicated in subsequent studies Molecules that contribute to this mechanism are well using different samples of cases and controls18,19 and in a meta-analysis combining the sample of Luomala and co-workers and the initial sample.20 Moreover, no Correspondence: Dr B Fontaine, INSERM U546, 105 bd de l’hoˆpital, association was found with another intragenic SNP (SNP 75013 Paris, France. E-mail: [email protected] G/R 241). Intercellular adhesion molecule-1 I Cournu-Rebeix et al 519 Table 1 Primers for amplification by polymerase chain reaction of adhesion molecule genes

Genes Base change AA change Primers for amplification Primer or probes for genotyping TM (1C)

ICAM1 CA repeat None 50-(6-Fam)GCAAGCAGTGGGGAAGGG-30 None 50-TGGGTGACAGAGCGAGAG-30 ICAM1 C/T12957 G/R241 50-CGTCCATCCCTGTCTGCT-30 50-CCGAGACTGGGAACAGCC-30 55 50-GTCCTCTGCGGTCACACT-30 ICAM1 T/C13858 K/E469 50-GCCCCCGACTGGACGAGA-30 50-GCACATTCACGGTCACCT-30 55 50-GGATACAACAGGCGGTGA-30 CD18 C/G10385 G/G273 50-CTCTGCATCGTCTCCTCT-30 50-GTCAGGATGGCGCCCAGCTT-30 55 50-ACCCCACCCTTGTCTCCT-30 CD18 C/A23837 P/Q819 50-GGAGCACTTGGTGAAGACAA-30 50-CCCAGAAGCACCCGGCC-30 58 50-GGGGAATACAGCGGACACA-30 Cd11a C/T21383 None 50-AGCACATGGATGCTTTTCTC-30 50-CTGAGGAAAGTGTGTGGGGC-30 55 50-CAGTGTCCTCCGCCCTGT-30 Cd11a T/C26442 None 50-CCCTGATGTCCTTCTCTCC A-30 50-CCAAGGTCAGAGCTCTCCTC-30 55 50-GGAACAACCCCCGTCTTC-30 Cd11a C/T35579 None 50-CAATCTCGGCTCACTGCAAC-30 50-AATTAGCTGGGTGTGGTGGC-30 55 50-GAGGACAGGAGTTCAAGACCA-30 CD11b C/T2573a A/V858 50-GCCTCCTCCACCGAAGTGT-30 50-(6-fam)CTCTTCAAGGCCC-30 60 50-(Vic)CTCTTCAAAGCCC-30 50-CTCTGAGTTTTCCGGGAAGATG-30 aPosition in mRNA. DNA samples were amplified by polymerase chain reaction (PCR) according to standard protocols. Genotyping of the CA repeat was performed by electrophoresis of a PCR fragment amplified with fluorescent primers on an ABI PRISM 377 sequencer (Applied Biosystems, USA). Genotypes of the SNP were assessed using either a single labeled base extension method for an ABI PRISM 377 sequencer (SnaPshot) (ICAM1, CD18 and CD11a) or a 50 nuclease assay on an ABI 7000 sequence detection system (CD11b) (Applied Biosystems, USA).

In this study, we investigate the association between relating to biomedical research. Geographic and ethnic ICAM-1 and MS in a large population of French MS origin, sex, age at onset, duration of the disease, disease case–parent trios, using two single nucleotide poly- course and disease severity as measured by the morphisms and one microsatellite (Table 1). We also Expanded Disability Status Scale (EDSS)31 were recorded study the role of genes coding for ICAM-1 receptors for each patient. Since we have previously demonstrated LFA-1 and MAC-1 that have never been studied before that familial factors contribute to MS severity,32 we in relation to MS susceptibility or severity (six SNPs, stratified our sample based on severity. We used the Table 1). Using a strategy of multilocus genetic analysis, progression index (PI) defined as the ratio of the EDSS vs we report the association of a protective ICAM-1 disease duration (in years) for patients with a disease haplotype against MS in this population. duration of 5 years or more.32 Patients with a PI less than To test for genetic association with the disease, the 0.4 and over 0.4 were classified as mild and severe, transmission disequilibrium test (TDT) was performed at respectively. Among the 430 patients with information each .21 This test focuses on heterozygous parents on PI, 250 were classified as mild and 180 as severe. and compares alleles transmitted and nontransmitted to HLA Class II DR alleles were determined for 429 out of affected offspring. For ICAM-1, each marker was tested the 439 patients. Since HLA-DR2 allele is associated with individually but also in relation with the other markers MS,33 we have also stratified our sample according to by looking at the haplotype distribution. It has been whether or not the proband was a carrier of the HLA shown that haplotype tests may be more powerful at Class II DR2 allele (among the 429 patients genotyped for detecting the role of a given genomic region in disease HLA, 223 (52%) were carriers). The demographic susceptibility than single-locus methods.22–25 Indeed, characteristics of our population are similar to published disease susceptibility may be due to the combined effects series. There was a female preponderance (68%) and the of multiple sequence variants. mean age at onset was 26 years (standard deviation To control for the numerous tests performed, we (s.d.) ¼ 7). The mean duration of the disease was 9 years adopted a strategy in two steps. All the markers were (s.d. ¼ 7) and the mean EDSS was 3.5 (s.d. ¼ 2). tested in a first subset of 150 families referred to as batch Parental transmitted and nontransmitted allele fre- 1. For such a sample size, the probability to detect a locus quencies of CD18, CD11a, CD11b and ICAM-1 gene with a genotype relative risk of 2 (assuming a multi- polymorphisms are presented in Table 2. With regard to plicative model) for a nominal type I error of 5% is the frequencies of the ICAM-1 microsatellite alleles, there greater than 80%.26 When for a marker, the uncorrected were 7, 85 and 8% of alleles 1, 2 and 3 respectively, P-value was below 5% in batch 1, the remaining 289 considering the nontransmitted parental alleles. There case–parent families (batch 2) were tested. were 5, 86 and 9 of alleles 1, 2 and 3, respectively, A total of 439 case–parent MS families, all comprising considering patient alleles. one affected child with definite MS27,28 and his two Performing single-locus TDT on batch 1 for CD18, parents, were recruited throughout France by the French CD11a and CD11b genes, no association was found with Multiple Sclerosis Genetic Group as previously de- MS susceptibility with or without stratification on HLA scribed.29,30 Informed written consent was obtained from status and on disease severity (data not shown). For the each individual participating in the study in accordance ICAM-1 gene, there was a difference in batch 1 in the with the Helsinki Convention and the French law distribution between transmitted and nontransmitted

Genes and Immunity Intercellular adhesion molecule-1 I Cournu-Rebeix et al 520 Table 2 Allele frequencies of CD18, Cd11a and CD11b genes (in %)

Gene Polymorphism Number of Rarest allele a Frequency of the rarest allele

(a) Nontransmitted parental alleles CD18 G/G273 292 G273 24 P/Q819 280 Q819 21 CD11a Intron 11 SNP 292 T21383 46 Intron 16 SNP 250 C26442 44 Intron 21 SNP 292 T35579 17 CD11b A/V858 292 A858 14 ICAM-1 G/R241 702 R241 11 K/E469 442 E469 46

(b) Patients CD18 G/G273 292 G273 27 P/Q819 280 Q819 18 CD11a Intron 11 SNP 292 T21383 40 Intron 16 SNP 250 C26442 43 Intron 21 SNP 292 T35579 13 CD11b A/V858 292 A858 14 ICAM-1 G/R241 702 R241 9 K/E469 442 E469 48

aAmino-acid position for CD18 and CD11b and ICAM-1 polymorphisms, base position for CD11a SNPs. Allele frequencies for each polymorphism were calculated using, as controls, parental alleles not transmitted to the affected child in each case–parent family. These frequencies provide unbiased estimates of alleles frequencies in the general population.41

a

G/R241 K/E469 CA repeat * * 5’...CCCTGGACGGGCTGTTC...3’ 5’...TCACCCGCGAGGTGACC...3’ TTCCCAGTAGGCG(CA)nTACACATCTGTT 5’... CCCTGGACTGGCTGTTC …3’ 5’...TCACCCGCTAGGTGACC...3’

ICAM-1 mRNA ′ 5 3′

120 bp

ICAM-1 N b C Rhinoviruses Ig-1 Ig-2 Ig-3 Ig-4 Ig-5 K/E469

8 p=0.0156* LFA-1 G/R241 7 Mac-1 6 5 4 3

TDT value 2 1 0 469E/241G 469E/241R 469K/241G 469K/241R

Haplotypes ICAM-1

Figure 1 Genetic analysis of ICAM-1 gene in MS. (a) Analysis of a microsatellite and two single nucleotide polymorphisms in the human ICAM-1 gene: The exon distribution and the protein structural domains (indicated by black boxes) of ICAM-1 are illustrated. Locations of DNA polymorphisms are indicated by stars. Locations of binding sites of LFA-1, Mac-1 and human rhinoviruses are shown in a diagram of an ICAM-1 molecule. (b) TDT results for ICAM-1 haplotypes. *Statistically significant: To test for transmission of haplotypes in the ICAM-1 gene, the multilocus TDT implemented in Genehunter was used (Genehunter version 2.0 beta).

Genes and Immunity Intercellular adhesion molecule-1 I Cournu-Rebeix et al 521 Table 3 Analysis of ICAM-1 gene in batch 1 and the entire population (batch 1+batch 2) with and without stratification for severity and HLA-DR2 status: number of allele transmissions and nontransmissions from parents to patients

Families Allele Heterozygous parents Transmitted alleles Nontransmitted alleles w2a P

Batch 1 G/R241 1 38 21 17 0.421 >0.05 K/E469 1 130 61 69 0.492 >0.05 CA repeat 1 35 11 24 4.829 0.03 282 52 30 5.902 0.01 3 51 21 30 1.588 >0.05

Entire population G/R241 1 145 81 64 1.993 >0.05 K/E469 1 202 101 101 0.000 >0.05 CA repeat 1 96 40 56 2.667 >0.05 2 207 110 97 0.816 >0.05 3 123 63 60 0.073 >0.05

HLA DR2+ G/R241 1 68 39 29 1.471 >0.05 K/E469 1 96 53 43 1.042 >0.05 CA repeat 1 46 16 30 4.261 0.04 2 94 52 42 1.064 >0.05 3 52 28 24 0.308 >0.05

HLA DR2À G/R241 1 71 39 32 0.690 >0.05 K/E469 1 102 46 56 0.980 >0.05 CA repeat 1 35 18 17 0.029 >0.05 2 70 35 35 0.000 >0.05 3 39 19 20 0.026 >0.05 Mild group G/R241 1 43 24 19 0.581 >0.05 K/E469 1 68 37 31 0.529 >0.05 CA repeat 1 23 8 15 2.130 >0.05 2 49 28 21 1.000 >0.05 3 30 15 15 0.000 >0.05 Severe group G/R241 1 48 26 22 0.333 >0.05 K/E469 1 72 37 35 0.056 >0.05 CA repeat 1 24 9 15 1.500 >0.05 2 55 31 24 0.891 >0.05 3 35 17 18 0.029 >0.05 aResults are shown only for allele 1 of each tested SNP. alleles at the microsatellite marker. One of the alleles of missions and nontransmissions from heterozygous par- the microsatellite (allele 1 that has never been reported ents to affected offspring (data not shown). For rarer before) was significantly less transmitted and, conver- haplotypes, however, we observed a lack of transmission sely, one of the alleles (allele 2) was more transmitted to of one haplotype, R241/K469/2. This particular haplo- affected offspring (Table 3). type was indeed found in four families and never According to our decision criteria for the two-step transmitted to affected offspring. This same combination analysis, only the microsatellite should then be tested on R241 and K469 was also observed in three other families batch 2 (289 families). However, we decided to also in which it was never transmitted to the affected child. In genotype the different individuals at the two ICAM-1 one family, it was associated to allele 3 at the CA repeat intragenic SNPs, in order to obtain some haplotypic and in the two other families, no typing was available for information. The results are presented in Table 3. When the CA repeat. Thus, we show that the ICAM-1 the loci were studied individually, no difference was haplotype defined by R241 and K469 is found in seven found on the allele transmissions from heterozy informative families and is never transmitted from gous parents to affected offspring in batch 2 and parents to affected children (TDT ¼ 7, binomial exact test in the entire population (batch 1+batch 2) at any of the P ¼ 0.01) (Figure 1). Linkage disequilibrium between the tested markers. However, after stratification on HLA different ICAM-1 markers was evaluated on the trans- status, a lack of transmission of the CA-repeat allele 1 mitted parental haplotypes. Linkage disequilibrium is could be observed in DR2+families (uncorrected strong between ICAM-1 G/R241 and ICAM-1 K/E469 P ¼ 0.04) but there was no significant difference in the SNPs (D ¼À0.0359, Po1.5  10À5) as well as between the transmissions of this allele to DR2+ and DR2À patients microsatellite and ICAM-1 K/E469 SNP (D ¼À0.028, (w2 tests). P44.2  10À7). However, there is no evidence for linkage For the frequent three-locus haplotypes, no significant disequilibrium between the microsatellite and ICAM-1 difference was observed between the number of trans- G/R241 alleles (D ¼À0.0001, P40.99).

Genes and Immunity Intercellular adhesion molecule-1 I Cournu-Rebeix et al 522 In this article, we report a genetic study of ICAM-1 and Group, Genethon, the CIC Pitie´-Salpeˆtrie`re, INSERM and its receptors as candidates for MS genes. We took the French Ministry of Research (CRB) provided help for advantage of a large cohort of case–parent MS families the DNA bank. This work was supported by ARSEP, of French origin to look for both linkage and association INSERM and FRM (Action 2000). between markers of these candidate genes and MS. No association between MS and genes encoding ICAM-1 receptors was detected. However, we found that one haplotype of the ICAM-1 gene formed by the combina- tion of allele R at position 241 in exon 4 and allele K at References position 469 in exon 6 was never transmitted to affected offspring, suggesting that this haplotype may be protec- 1 Alperovitch A. Epidemiologie de la scle`rose en plaques. Med tive against MS. Therap 1995; 1: 545–548. 2 Poser CM. The epidemiology of multiple sclerosis: a general ICAM-1 gene is located on 19 p13, a overview. Ann Neurol 1994; 36: 180–193. region identified as of possible significance in two 3 Cannella B, Cross AH, Raine CS. Upregulation and coexpres- 34,35 independent screenings in MS families. Previous sion of adhesion molecules correlate with relapsing auto- studies have addressed the role of ICAM-1 in MS immune demyelination in the central nervous system. J Exp genetics with conflicting results. One study, performed Med 1990; 172: 1521–1524. on case–controls of Polish origin, found a genetic 4 Springer TA. Adhesion receptors of the immune system. association with ICAM-1 K469.17 This result was not Nature 1990; 346: 425–434. confirmed in three subsequent studies.18–20 However, 5 Raine CS, Cannella B. Adhesion molecules and central except for the study of Marrosu et al,19 no multilocus nervous system inflammation. Semin Neurosci 1992; 4: 201–219. analysis of the two SNPs was performed. In the study 6 Osborn L. Leukocyte adhesion to in inflamma- 19 tion. Cell 1990; 62: 3–6. by Marrosu et al, haplotypes were studied but the 7 Springer TA. Traffic signals for lymphocyte recirculation and particular haplotype we evidenced here was not ob- leukocyte emigration: the multistep paradigm. Cell 1994; 76: served probably because of the small sample size.19 301–314. The ICAM-1 molecule consists of five immunoglobu- 8 Pryce G, Male D, Campbell I, Greenwood J. Factors controlling lin-like (Ig) domains (Ig-1 to Ig-5), each of them encoded T- across rat cerebral endothelium in vitro. by its own exon. Exon 4 polymorphism (G/R241) J Neuroimmunol 1997; 75: 84–94. changes an arginine into a lysine in domain Ig-3. Exon 9 Male D, Rahman J, Pryce G, Tamatani T, Miyasaka M. 6 polymorphism (K/E469) substitutes a glutamic acid to Lymphocyte migration into the CNS modelled in vitro: a lysine in Ig-like domain 5. 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Binding efficacy of the interaction several fold.36 The second of the Mac-1 (CD11b/CD18) to the third immunoglo- hypothesis relates to the role of ICAM-1 as a viral bulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell 1991; 65: 961–971. receptor. 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The major human 1 molecule may influence susceptibility to viral infection, rhinovirus receptor is ICAM-1. Cell 1989; 56: 839–847. an often suspected environmental factor in MS patho- 16 Olson NH, Kolatkar PR, Oliveira MA et al. Structure of a physiology. human rhinovirus complexed with its receptor molecule. Proc In conclusion, in a large population of MS case–parent Natl Acad Sci 1993; 90: 507–511. trios, we found that a particular haplotype of ICAM-1 17 Mycko MP, Kwinkowski M, Tronczynska E, Szymanska B, may have a protective role in MS. Although this Selmaj KW. Multiple sclerosis: the increased frequency of the haplotype is a rare haplotype, the association is strong ICAM-1 exon 6 gene point mutation genetic type K469. Ann enough to be detected in the French population. Neurol 1998; 44: 70–75. 18 Luomala M, Elovaara I, Koivula T, Lehtimaki T. Intercellular adhesion molecule-1 K/E 469 polymorphism and multiple sclerosis. 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