Genes 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 gene 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 cytokines 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 locus.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.