Intercellular adhesion molecule-1 K469E polymorphism: study of association with multiple sclerosis

Intercellular Adhesion Molecule-1 K469E Polymorphism: Study of Association With Multiple Sclerosis Sergey Nejentsev, Mikko Laaksonen, Pentti J. Tienari, Oscar Fernandez, Heather Cordell, Juhani Ruutiainen, Juhani Wikstro¨m, Tomi Pastinen, Satu Kuokkanen, Jan Hillert, and Jorma Ilonen ABSTRACT: Intercellular adhesion molecule-1 (ICAM-1) is involved in the pathogenesis of multiple sclerosis (MS), whereas sequence variations in the ICAM-1 gene could potentially be responsible for the genetic susceptibility to MS. We studied an association of MS with the 13,848A⬎G (K469E) polymorphism of the ICAM-1 gene in Finnish and Spanish cases and controls and affected families. An increased risk for the AA (Lys469/ Lys469) genotype was found in both populations. The effect observed was found to be strongest among the ABBREVIATIONS 95% CI 95% confidence interval HLA human leukocyte antigens ICAM-1 intercellular adhesion molecule-1 GRR genotype relative risk

INTRODUCTION Multiple sclerosis (MS) is a putative autoimmune disease of the central nervous system characterized by lymphocyte-mediated demyelination. Lymphocyte infiltration of the brain tissue is regulated by adhesion molecules.

From the Departments of Virology (S.N., M.L., J.I.) and Neurology (M.L.), University of Turku, Turku, Finland; Department of Neurology (P.J.T., J.W.), University of Helsinki, Helsinki, Finland; Department of Neurology (O.F.), Regional Hospital Carlos Haya, Malaga, Spain; JDRF/WT Diabetes and Inflammation Laboratory (H.C.), Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; Masku Neurological Rehabilitation Centre (J.R.), Masku, Finland; National Public Health Institute (T.P., S.K.), Helsinki, Finland, Department of Neurology (J.H.), Karolinska Institute, Stockholm, Sweden. Address reprint requests to: Dr. Sergey Nejentsev, JDRF/WT Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke’s Hospital, Cambridge CB2 2XY, UK; Tel: (⫹44)0 1223 762106; Fax: (⫹44)0 1223 762102; E-mail: [email protected]. Received July 24, 2002; revised December 4, 2002; accepted December 9, 2002. Human Immunology 64, 345–349 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Science Inc.

HLA-DQB1*0602–positive subjects, which implies genetic heterogeneity of MS. Meta-analysis of all published datasets supports increased risk of MS for the ICAM-1 Lys469 homozygotes (relative risk ⫽ 1.3, p ⫽ 0.002). Human Immunology 64, 345–349 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Science Inc. KEYWORDS: gene, ICAM-1, SNP, interaction, heterogeneity

MS OR TDT

multiple sclerosis odds ratio transmission disequilibrium test

Intercellular adhesion molecule-1 (ICAM-1), which is known to be involved in the process of lymphocyte migration [1] and activation [2], is expressed at increased levels in the brain microvessels of MS patients [3]. Its soluble form is also present at higher concentrations in the cerebrospinal fluid [4] and serum [5, 6] of MS patients. Thus ICAM-1 is involved in the MS pathogenesis, whereas sequence variations in the ICAM-1 gene could potentially be responsible for the genetic susceptibility to MS. ICAM1 is located on chromosome 19p13 and two nonsynonymous single nucleotide polymorphisms (SNPs) are known to be common in European populations: 12,959G⬎A, encoding Gly241/Arg241 (G241R) and 13,848A⬎G, encoding Lys469/Glu469 (K469E) [7]. Several studies examined association of the K469E polymorphism with MS [8 –11]. Association of the ICAM1 AA genotype (Lys469/Lys469) was found in the Polish population [8], but was not confirmed in the 0198-8859/03/$–see front matter doi:10.1016/S0198-8859(02)00825-X

346

Finnish [9], Dutch [10], or Sardinian [11] studies, although the number of samples tested was small. In this article, we report an association study of the ICAM-1 K469E polymorphism and meta-analysis of all published data. We have studied MS patients and ethnically matched controls from Finland and Spain. We also analyzed allele transmission in the MS families. The Finnish sample collection used in this study does not include samples examined by Luomala et al. [9] and is the largest analyzed to date. MATERIALS AND METHODS The Finnish MS patient cohort included 235 unrelated cases with both parents available for the study, 16 multiplex families with two affected individuals (the eldest affected sibling was included in the association analysis), and 26 unrelated cases with no family members available—a total of 277 unrelated cases collected in all parts of Finland. Information on the MS clinical type was available for 223 patients: 20 had primary progressive MS, 50 had secondary progressive MS, and 153 had relapsing-remitting MS. Two hundred fifteen newborn infants consecutively enrolled in the population-based Diabetes Prediction and Prevention Trial [12] in Turku, Finland, and 358 blood donors from all over Finland—a total of 573 individuals—represented background population. The Spanish MS patient cohort included 130 unrelated cases with no family members available for the study from Malaga, 4 unrelated cases with both parents available, and 6 multiplex families with two or more affected individuals. Of these, 3 patients had primary progressive MS, 43 had secondary progressive MS, and 72 had relapsing-remitting MS. The control group included 113 healthy blood donors from Malaga. In both cohorts, MS was diagnosed according to Poser’s criteria. The study was approved by the Ethical Committees of the participating Hospitals. An informed consent was obtained from the participating subjects and parents. The A/G polymorphism in the exon 6 of ICAM1 was studied using restriction fragment length polymorphism as described elsewhere [13]. To reveal possible interaction between human leukocyte antigen (HLA) and ICAM-1, HLA-DQB1 genotypes were studied in MS patients [14]. A ␹2 test was used for genotype frequency comparisons between cases and controls, either comparing all three genotypes or comparing AA homozygotes against AG heterozygotes and GG homozygotes combined. Analysis of allele transmission in families was done by means of the transmission disequilibrium test (TDT) [15] using TDTPHASE program written by F. Dudbridge (http://www-gene.cimr.cam.ac.uk/todd/). We performed an analysis of full genotype transmission by means of the appropriate score statistics using a

S. Nejentsev et al.

recessive genetic model (GREC) [16] with Stata 7.0 (http://www.stata.com). This method is based on reconstructing genotypes of a case and three matched pseudocontrols from the alleles that could have been transmitted within each family, and thus is resistant to population stratification. It has more power than TDT when recessive effects are studied [16]. Bonferroni correction was used to account for multiple testing. Significance level was set at p ⬍ 0.05. Standard odds ratio (OR) calculations were performed for case-control comparisons. For the family studies, genotype relative risk (GRR) was calculated assuming a recessive genetic model (because only AA homozygotes have increased MS risk) using Stata 7.0. Because no genotype data were available for the Sardinian family study [11], genotype relative risk was calculated from the observed allele transmission, assuming a recessive model [17]. Meta-analysis was carried out on the estimated log relative risk (RR) and its variance using a standard fixed-effects approach [18]. Statistical power to show association at significance level p ⫽ 0.05 was calculated assuming a gene effect with OR ⫽ 1.3, allele frequency of 0.5, and a recessive genetic model. RESULTS In the control Finnish population, the allele frequency was 0.52 for A and 0.48 for G and was 0.5 for each allele among Spanish controls. Genotypes in both control populations were in Hardy-Weinberg equilibrium, and no heterogeneity was observed between the subgroups of the Finnish controls. An association of the ICAM1 AA genotype with MS was found among Finns. AA genotype was found in 33.2% of the patients versus 25.5% of the controls (␹2 ⫽ 5.5 [1 degree of freedom (df)], p ⫽ 0.019, OR ⫽ 1.5, 95% confidence interval [CI] 1.1—2.0). In the Spanish population the AA genotype frequency was also increased in patients (35.0%) versus controls (29.2%), with the OR ⫽ 1.3. However, owing to small sample size, this effect was not significant. In the combined sample sets the AA genotype association reached ␹2 ⫽ 7.5 (1 df), p ⫽ 0.0062, with OR ⫽ 1.4, 95% CI 1.1—1.9 (Table 1). The p values obtained were not subject to a multiple testing correction because the hypothesis that MS is associated with the ICAM1 AA genotype was specifically tested in this study. Association of the AA genotype with MS was found to be strongest among the HLA-DQB1*0602–positive patients (see Table 1). Only in this group of patients was the association observed and was significant after correction, whereas DQB1*0602–negative patients did not differ from control population. We observed this effect separately for the Finnish and Spanish population and in the combined dataset (see Table 1). Stratification of the

ICAM-1 in MS

347

TABLE 1 ICAM1 A/G (K469E) alleles and genotypes in MS patients and controls

Finnish controls Finnish patients DQB1*0602–positive DQB1*0602–negative Spanish controls Spanish patients DQB1*0602–positive DQB1*0602–negative Combined controls Combined patients DQB1*0602–positive DQB1*0602–negative

n

AA,%

AG,%

GG,%

573 277 162 110 113 140 63 77 686 417 225 187

25.5 33.2 40.1 23.6 29.2 35.0 41.3 29.9 26.1 33.8 40.4 26.2

52.7 43.0 37.7 50.9 41.6 39.3 31.7 45.5 50.9 41.7 36.0 48.1

21.8 23.8 22.2 25.5 29.2 25.7 27.0 24.7 23.0 24.5 23.6 25.1

p (pc) 0.02 (NA) 0.0005 (0.01) NS NS NS NS 0.006 (NA) 0.00005 (0.001) NS

AA,%

GG⫹AG,%

25.5 33.2 40.1 23.6 29.2 35.0 41.3 29.9 26.1 33.8 40.4 26.7

74.5 66.8 59.9 76.4 70.8 65.0 58.7 70.1 73.9 66.2 59.6 73.3

p (pc)

OR (95% CI)

0.02 (NA) 0.0003 (0.007) NS

1.5 (1.1–2.0) 2.0 (1.4–2.8) 0.9 (0.6–1.5)

NS 0.1 (NS) NS

1.3 (0.8–2.2) 1.7 (0.9–3.2) 1.0 (0.5–1.9)

0.006 (NA) 0.00004 (0.001) NS

1.4 (1.1–1.9) 1.9 (1.4–2.6) 1.0 (0.7–1.5)

p values of 0.1 or less are shown. Abbreviations: MS ⫽ multiple sclerosis; pc ⫽ p ⫻ 24; OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval; NA ⫽ not applied; NS ⫽ not significant.

patients according to their clinical type (primary progressive, secondary progressive, or relapsing-remitting) or sex did not reveal any further differences between groups studied (data not shown). Allele transmission was then studied in 276 MS families. Owing to the small number of Spanish families it was not feasible to analyze them separately. Using TDT, allele transmission to the MS patients was found to be random—50% for each allele (Table 2). Stratification of families according to the sex or clinical type of the proband did not reveal any significant deviation from the random allele transmission (data not shown). Transmission of the A allele to the DQB1*0602– positive patients, however, was increased, although not significantly (55.6%, TDT ␹2 ⫽ 1.6 [1 df], p ⫽ 0.2, not significant). However, GREC revealed significant association in the group of DQB1*0602–positive patients, p ⫽ 0.005, p ⫽ 0.04 (Table 2). This finding is in line with the association observed in our case-control study. We also detected a preferential transmission of allele G (62.8%, p ⫽ 0.02) to the DQB1*0602–negative patients, whereas it was no longer significant after correction for multiple testing and no significant effect was found using GREC. Because no evidence for the disease association of G-positive subjects was found in casecontrol comparisons, including a group of the

DQB1*0602–negative patients, we conclude that this is a random statistical fluctuation. DISCUSSION Several studies that examined the role of the K469E polymorphism in the ICAM-1 gene have been published. Significant association of the AA genotype (Lys469/ Lys469) with MS was observed only in the first study of the Polish population [8]. These data were not confirmed in the consequent reports, and may be artefactual, because genotypes in the Polish control group deviated from the Hardy-Weinberg equilibrium [9]. However, in the present study we found association of the AA genotype with MS. This inconsistency can be explained by comparison of the statistical power to detect association in the studies published (Table 3). Indeed, statistical power to show association was less than 35% for each case-control study, including this one. It is no surprise that a significant difference between MS patients and controls was observed only twice, whereas increased relative risks for the AA genotype were observed in all studies, except that of the Dutch (Table 3). We used a meta-analysis approach to combine all published data sets. Combining all case-control data (Table 3), the association was significant, p ⫽ 0.009, with RR ⫽ 1.3.

TABLE 2 Transmission of ICAM1 A/G (K469E) alleles in MS families Transmission to offspring All affected DQB1*0602–positive DQB1*0602–negative

Number of probands (Finnish ⫹ Spanish) 276 (266⫹10) 146 (141⫹ 5) 95 (93 ⫹ 2)

Allele A (%T)

Allele G (%T)

TDT p (pc)

GREC p (pc)

115 (50) 69 (55.6) 29 (37.2)

115 (50) 55 (44.4) 49 (62.8)

NS NS 0.02 (NS)

NS 0.005 (0.04) NS

Abbreviations: MS ⫽ multiple sclerosis; TDT ⫽ transmission disequilibrium test; pc ⫽ p ⫻ 8; GREC ⫽ recessive genetic model.

348

S. Nejentsev et al.

TABLE 3 Statistical power to detect association of the ICAM1 AA (Lys469/Lys469) genotype with MS Case control studies Polish [8] Finnish [9] Dutch [10] Spanish (this study) Finnish (this study) All case-control

Family-based studies Sardinian [11] Finnish (this study) All family-based studies

Cases vs controls

Statistical power, %

OR (95% CI) for the AA genotype

p

11 14 15 15 35 67

2.2 (1.1–4.3) 1.5 (0.8–2.7) 0.7 (0.4–1.1) 1.3 (0.8–2.2) 1.5 (1.1–2.0) 1.3 (1.1–1.6)

0.02 0.17 0.14 0.16 0.009 0.009

Families

Statistical power, %

GRR (95% CI)

p

157 276 433

14 22 31

1.3 (0.7–2.5) 1.3 (0.9–1.9) 1.3 (1.0–1.8)

0.45 0.15 0.1

1.3 (1.1–1.6)

0.002

79 104 145 140 277 745

vs vs vs vs vs vs

68 111 106 113 573 971

All studies

Abbreviations: MS ⫽ multiple sclerosis; OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval; GRR ⫽ genotype relative risk.

We could not confirm this association analyzing allele transmission in the MS families, although GRR was estimated at 1.3. Similar results were observed in the Sardinian MS families [11]. However, statistical power to detect association was less than 22% in each of these studies and 31% in the combined data set. Combining all published case-control and family studies using a meta-analysis approach, ICAM1 AA genotype was associated with MS, p ⫽ 0.002, although this effect is weak, RR ⫽ 1.3 (95% CI 1.1—1.6). We found that association in both Finnish and Spanish case-control comparisons was seen only among the DQB1*0602–positive MS patients and no association was found among the DQB1*0602–negative patients. A similar significant result was observed in our family study using score statistics for recessive model (GREC). Unfortunately, no data on the combined ICAM-1–HLA analysis were published in the previous reports. If confirmed in future studies, this association may reflect genetic heterogeneity of MS. Locus heterogeneity at HLA was previously reported for MS [19, 20]. These findings imply differences in the pathologic process leading to MS, in which only some pathways involve ICAM-1 and HLA molecules. We can conclude that ICAM1 AA genotype (Lys469/ Lys469) may be associated with increased risk for MS, although this increase is modest and large studies are required to demonstrate it. K469E polymorphism results in an amino acid substitution and may have a significant functional effect. Alternatively, it may merely reflect linkage disequilibrium with another causal variant. Studies of additional polymorphisms in the ICAM-1 gene and functional studies are required to clarify this.

ACKNOWLEDGMENTS

We thank T. Laure´ n, R. Suominen, and M. Karlsson for their skillful technical assistance and Prof. John Todd for his valuable comments. The study was supported by grants from European Commission (BMH4-97-2422), Academy of Finland, Emil Aaltonen Foundation, Turku University Central Hospital, Helsinki University Central Hospital, and the Sigrid Juse´ lius Foundation.

REFERENCES 1. McMurray RW: Adhesion molecules in autoimmune disease. Semin Arthritis Rheum 25:215, 1996. 2. Van Seventer GA, Shimizu Y, Horgan KJ, Shaw S: The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor-mediated activation of resting T cells. J Immunol 144:4579, 1990. 3. Washington R, Burton J, Todd 3rd RF, Newman W, Dragovic L, Dore Duffy P: Expression of immunologically relevant endothelial cell activation antigens on isolated central nervous system microvessels from patients with multiple sclerosis. Ann Neurol 35:89, 1994. 4. Tsukada N, Matsuda M, Miyagi K, Yanagisawa N: Increased levels of intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor receptor in the cerebrospinal fluid of patients with multiple sclerosis. Neurology 43:2679, 1993. 5. Hartung HP, Michels M, Reiners K, Seeldrayers P, Archelos JJ, Toyka KV: Soluble ICAM-1 serum levels in multiple sclerosis and viral encephalitis. Neurology 43: 2331, 1993. 6. Jander S, Heidenreich F, Stoll G: Serum and CSF levels of soluble intercellular adhesion molecule-1 (ICAM-1) in

ICAM-1 in MS

7.

8.

9.

10.

11.

12.

13.

inflammatory neurologic diseases. Neurology 43:1809, 1993. Vora DK, Rosenbloom CL, Beaudet AL, Cottingham RW: Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster. Genomics 21:473, 1994. Mycko MP, Kwinkowski M, Tronczynska E, Szymanska B, Selmaj KW: Multiple sclerosis: the increased frequency of the ICAM-1 exon 6 gene point mutation genetic type K469. Ann Neurol 44:70, 1998. Luomala M, Elovaara I, Koivula T, Lehtimaki T: Intercellular adhesion molecule-1 K/E 469 polymorphism and multiple sclerosis. Ann Neurol 45:546, 1999. Killestein J, Schrijver HM, Crusius JB, Perez C, Uitdehaag BM, Pena AS, Polman CH: Intracellular adhesion molecule-1 polymorphisms and genetic susceptibility to multiple sclerosis: additional data and meta-analysis. Ann Neurol 47:277, 2000. Marrosu MG, Schirru L, Fadda E, Mancosu C, Lai M, Cocco E, Cucca F: ICAM-1 gene is not associated with multiple sclerosis in Sardinian patients. J Neurol 247: 677, 2000. Ilonen J, Reijonen H, Knip M, Simell O: Populationbased genetic screening for IDDM susceptibility as a source of HLA-genotyped control subjects. Diabetologia 39:123, 1996. Nejentsev S, Laine AP, Simell O, Ilonen J: Intercellular adhesion molecule-1 (ICAM-1) K469E polymorphism: no

349

14.

15.

16.

17.

18.

19.

20.

association with type 1 diabetes among Finns. Tissue Antigens 55:568, 2000. Sjo¨ roos M, Iitia¨ A, Ilonen J, Reijonen H, Lo¨ vgren T: Triple-label hybridization assay for type-1 diabetes-related HLA alleles. Biotechniques 18:870, 1995. Spielman RS, McGinnis RE, Ewens WJ: Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 52:506, 1993. Schaid DJ: General score tests for associations of genetic markers with disease using cases and their parents. Genet Epidemiol 13:423, 1996. Camp NJ: Genomewide transmission/disequilibrium testing— consideration of the genotypic relative risks at disease loci. Am J Hum Genet 61:1424, 1997. Whitehead A, Whitehead J: A general parametric approach to the meta-analysis of randomized clinical trials. Stat Med 10:1665, 1991. Haines JL, Terwedow HA, Burgess K, Pericak Vance MA, Rimmler JB, Martin ER, Oksenberg JR, Lincoln R, Zhang DY, Banatao DR, Gatto N, Goodkin DE, Hauser SL: Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group. Hum Mol Genet 7:1229, 1998. Barcellos LF, Oksenberg JR, Green AJ, Bucher P, Rimmler JB, Schmidt S, Garcia ME, Lincoln RR, Pericak Vance MA, Haines JL, Hauser SL: Genetic basis for clinical expression in multiple sclerosis. Brain 125:150, 2002.