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Prevalence of the K469E polymorphism of intercellular adhesion molecule 1 gene in Italian patients with inflammatory bowel disease
Digestive and Liver Disease 36 (2004) 528–532
Alimentary Tract
Prevalence of the K469E polymorphism of intercellular adhesion molecule 1 gene in Italian patients with inflammatory bowel disease A. Papa a,∗ , R. Pola b , A. Flex b,c , S. Danese a , A. Armuzzi a , E. Gaetani b,c , L. Guidi a , I. De Vitis a , R. Urgesi a , A. Grillo a , M. Serricchio c , A.S. Proia b,c , G. Fedeli a , G. Gasbarrini a , P. Pola c , A. Gasbarrini c a
Department of Internal Medicine, Gastroenterology Unit, Catholic University of Rome, “A. Gemelli” University Hospital, Largo A. Gemelli 8, 00168 Rome, Italy b Laboratory of Vascular Biology and Genetics, Catholic University of Rome, Rome, Italy c Department of Internal Medicine and Angiology, Catholic University of Rome, Rome, Italy Received 7 October 2003; accepted 3 March 2004
Abstract Background. Intercellular adhesion molecule 1 plays an important role in the recruitment of leucocytes at sites of inflammation and is up-regulated in intestinal mucosa of inflammatory bowel disease. Intercellular adhesion molecule 1 gene lies on chromosome 19p13, implicated in determining susceptibility to inflammatory bowel disease. Recently, the polymorphism K469E of intercellular adhesion molecule 1 gene has been identified. Aim. To assess the potential association of this polymorphism with inflammatory bowel disease. Patients. A total of 165 inflammatory bowel disease patients, 75 with Crohn’s disease and 90 with ulcerative colitis, and 187 controls were studied. Methods. The K469E polymorphism was detected by polymerase chain reaction and restriction enzyme analysis. Statistical analysis was performed by χ2 -test. Results. In inflammatory bowel disease, the distribution of intercellular adhesion molecule 1 genotypes was 24.9% E/E, 44.2% E/K and 30.9% K/K. In controls, 11.8% showed E/E genotype, 55.6% E/K and 32.6% K/K. The frequency of the E/E genotype was significantly higher in inflammatory bowel disease (Crohn’s disease and ulcerative colitis) patients than in controls. Subgroup analysis showed that the frequency of the E469 allele was significantly increased only in Crohn’s disease patients with ileocolonic location of disease and penetrating behaviour compared with controls. Conclusions. We found an association of inflammatory bowel disease with the E/E genotype of intercellular adhesion molecule 1 gene, while allele E469 was associated with a subgroup of Crohn’s disease patients with more extensive location of disease and penetrating behaviour. However, further studies are needed to confirm our findings. © 2004 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Inflammatory bowel disease; Intercellular adhesion molecule 1
1. Introduction The aetiologies of Crohn’s disease (CD) and ulcerative colitis (UC), the two major forms of chronic inflammatory bowel disease (IBD), are unknown. However, genetic predisposition seems to play a crucial role as evidenced by the ∗ Corresponding author. Tel.: +39-06-3503726; fax: +39-06-35502775. E-mail address: [email protected] (A. Papa).
increased familial prevalence of IBD and increased concordance rates in siblings or twins [1]. In fact, in the last years, several association studies and linkage analyses have been performed to identify susceptibility genes in the whole genome [2–5]. Recently, Rioux et al. [6] have identified a linkage between IBD and chromosome 19p13, where intercellular adhesion molecule 1 (ICAM-1) gene is located. ICAM-1 is a surface glycoprotein that belongs to the immunoglobulin superfamily and plays an important role in the trafficking and activation of leucocytes [7]. Moreover,
1590-8658/$30 © 2004 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2004.03.015
A. Papa et al. / Digestive and Liver Disease 36 (2004) 528–532
several studies have shown that ICAM-1 is up-regulated in intestinal mucosa of patients with active IBD [8–10] and in experimental colitis [11]. Genetic variation in ICAM-1 gene structure may result in altered expression and/or function of the resulting adhesion molecule, thus potentially contributing to a genetic predisposition to inflammatory and immunomediated events. Recently, the K469E polymorphism of the ICAM-1 gene has been described and might be functionally important [12]. This polymorphism occurs in exon 6 of the ICAM-1 gene and results in a change from glutamic acid to lysine in Ig-like domain 5, which has been reported to affect interactions between ICAM-1 and the leucocyte function-associated antigen-1 (LFA-1) and to influence B-cell adhesion [13]. In the last years, some reports from both Western and Eastern countries have shown discordant data regarding the association of this polymorphism with IBD [14–16]. All these findings led us to investigate the prevalence of the K469E polymorphism in a population of Italian IBD patients.
2. Patients and methods 2.1. Patients and controls The study population included 165 consecutive patients with IBD (51 women and 114 men; mean age 38 ± 14.2 years, median age 35 years; range 17–80 years) referred to the Department of Internal Medicine and the Gastroenterology Unit of our university hospital. Of these patients, 90 had UC and 75 CD. All patients were of Italian ancestry. The diagnosis of IBD was based on established criteria of clinical, radiological, endoscopic and histological findings [17,18]. A detailed clinical history was also taken from each patient and their characteristics are described in Table 1. As controls, 187 subjects were considered, similar to the patients for Table 1 Epidemiological and clinical data of IBD patients and controls
Subjects (n) Gender (F/M) Mean age (±S.D.) Extent of CD Small bowel Colon Small bowel plus colon Clinical behaviour of CD Penetrating Stricturing Non-stricturing and non-penetrating Extent of UC Proctitis Left-sided colitis Entire colitis
Controls
CD patients
UC patients
187 58/129 40 ± 16.4
75 24/51 36 ± 12.8
90 27/63 40 ± 15.1
529
age and sex distribution (58 women and 129 men, mean age 40 ± 16.4 years, median age 38 years, range 19–82 years). In particular, the control group consisted of healthy subjects, without a history of neoplastic, metabolic or inflammatory disease. All controls came from the same geographical area as the patients. Patients and controls were enrolled in this study after giving their informed consent to the use of part of their blood samples for an experimental study. 2.2. Detection of ICAM-1 polymorphism Nucleic acid isolation from 200 l of peripheral blood was carried out by using a DNA extraction kit (NucleoSpin Blood QuickPure; Macherey–Nagel, Duren, Germany), as recommended by the supplier. Genomic DNAs were assayed with polymerase chain reaction for the detection of ICAM-1 gene, as previously described [19]. The amplified sequence was digested by BstUI restriction enzyme (New England Bio Labs; Beverly, MA, USA) at 60 ◦ C for 4 h. The digested products were electrophoresed in 2% agarose gel and visualised by ethidium bromide staining. The EE genotype corresponded to the contemporary presence of 136 and 87 base pair fragments. The EK genotype corresponded to the contemporary presence of 223, 136 and 87 base pair fragments. The KK genotype corresponded to a 223 base pair fragments. 2.3. Statistical analysis Allele and genotype frequencies between each of the patient groups and controls were compared by χ2 -test for 2×2 or 2 × 3 tables. To correct for multiple comparisons in the subgroups, corrected P values (Pc ) were obtained using Bonferroni’s correction factor by multiplying the P value by 2 (Pc = 2P) because the alleles were examined simultaneously. To analyse subgroups in IBD, Pc values were corrected using Fisher’s exact test when number of samples were small. After correction, Pc was considered significant when <0.05. The P value was two-tailed. Hardy–Weinberg equilibrium was tested in the control population. All analyses were carried out using Intercooled STATA 6.0 for Windows (Stata Corporation, College Station, TX, USA).
3. Results
22 11 42 23 16 36
19 48 23
The distribution of ICAM-1 genotypes and alleles in IBD patients and controls is shown in Table 2. The genotype frequencies follow Hardy–Weinberg distributions in controls. In particular, the prevalence of the homozygous E/E genotype was significantly higher in IBD patients than in controls. Also, considering CD and UC patients separately (Table 2), the E/E genotype resulted more frequently in controls. On the contrary, the allele frequency did not differ between groups of patients (CD and UC) and controls. Subdividing CD patients according to disease extension (small bowel,
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A. Papa et al. / Digestive and Liver Disease 36 (2004) 528–532
Table 2 ICAM-1 genotype and allele distribution in IBD patients overall, CD and UC patients separately and in controls Controls (N = 187)
IBD
CD
N = 165 Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
Allele K E
226 (60.4) 148 (39.6)
a
51 (30.9) 73 (44.2) 41 (24.9) 175 (53) 155 (47)
UC
P (Pc )
N = 75
P (Pc )
N = 90
P (Pc )
0.00023
19 (25.3) 38 (50.7) 18 (24.0)
0.00065
32 (35.6) 35 (38.9) 23 (25.5)
0.00002
0.0479 (0.0958)
76 (50.7) 74 (49.3)
0.041 (0.082)
99 (55) 81 (45)
0.224 (0.448)
Numbers in parentheses are percentage of total.
small bowel plus colon or colon) (Table 3) and clinical behaviour (penetrating, stricturing, and non-stricturing and non-penetrating) (Table 4) we found that the E/E genotype occurred more frequently in all subgroups of CD patients, independent of disease location and clinical behaviour, while the frequency of E469 allele was significantly higher only in patients with small bowel plus colon disease and penetrating behaviour. In UC patients, after stratification according to disease extension (proctitis, left-sided colitis or entire colitis) (Table 5), we found that only the frequency of the E/E genotype was increased in all subgroups of patients compared to controls, while no difference was observed in the frequency of the allele E469 between subgroups of UC patients and controls.
4. Discussion IBD are chronic inflammatory disorders of the intestine, characterised by immune dysregulation and leucocyte recruitment into gastrointestinal tissue [20]. Epidemiological data provide evidence that genetic predisposition to both CD and UC depends on the contribution of multiple genes instead of a single genetic factor, and genetic heterogeneity could explain different clinical features of the disease [1]. Accordingly, polymorphisms of genes coding for proteins involved in initiation and regulation of the immune response, such as cytokines and adhesion molecules, were investigated in order to show an association with IBD. ICAM-1 is a cell surface adhesion molecule that serves as a receptor
Table 3 ICAM-1 genotype and allele distribution according to CD location of disease Controls (N = 187)
Small bowel N = 22
Small bowel plus colon
Colon
P (Pc )
N = 42
N = 11
0.00072
0.0217 (0.0434)
P (Pc )
Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
4 (18.2) 13 (59.1) 5 (22.7)
0.00022
10 (23.8) 20 (47.6) 12 (28.6)
Allele K E
226 (60.4) 148 (39.6)
21 (47.7) 23 (52.3)
0.105 (0.21)
40 (47.6%) 44 (52.4%)
a
5 (45.5%) 5 (45.5%) 1 (9%) 15 (68.2%) 7 (31.8%)
P (Pc )
0.035
0.51 (NS)
Numbers in parentheses are percentage of total. NS, not significant compared to controls.
Table 4 ICAM-1 genotype and allele distribution according to CD clinical behaviour Controls (N = 187)
Penetrating N = 23
Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
2 (8.7) 15 (65.2) 6 (26.1)
Allele K E
226 (60.4) 148 (39.6)
19 (41.3) 27 (58.7)
a
Stricturing P (Pc )
<0.00001
0.013 (0.026)
N = 16 2 (12.5) 9 (56.3) 5 (31.2) 13 (40.6) 19 (59.4)
Non-stricturing and non-penetrating P (Pc )
N = 36
P (Pc )
<0.00001
15 (41.7) 14 (38.9) 7 (19.4)
0.0023
44 (61.1) 28 (38.9)
0.913 (NS)
0.0289 (0.0578)
Numbers in parentheses are percentage of total. NS, not significant compared to controls.
A. Papa et al. / Digestive and Liver Disease 36 (2004) 528–532
531
Table 5 ICAM-1 genotype and allele distribution according to UC location of disease Controls (N = 187)
Entire colitis N = 23
Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
Allele K E
226 (60.4) 148 (39.6)
a
Left-sided colitis
Proctitis
P (Pc )
N = 48
10 (43.5) 9 (39.1) 4 (17.4)
0.0031
18 (37.5) 17 (35.4) 13 (27.1)
0.00003
28 (63.6) 16 (36.4)
0.732 (NS)
53 (55.2) 43 (44.8)
0.353 (0.706)
P (Pc )
N = 19 4 (21) 9 (47.4) 6 (31.6) 17 (44.7) 21 (55.3)
P (Pc )
0.00043
0.061 (0.122)
Numbers in parentheses are percentage of total. NS, not significant compared to controls.
for the LFA-1 and the macrophage differentiation antigen-1 (Mac-1), and plays a central role in the regulation of leucocyte circulation and homing [21,22]. The up-regulation of ICAM-1 on endothelial cells by proinflammatory cytokines is a central event in regulating leucocyte localisation at inflammatory sites and can also play an important role in the regulation and amplification of the inflammatory response observed in IBD [7–10]. Thus, an anti-ICAM-1 therapeutic strategy with antisense oligonucleotide has been used in steroid dependent or refractory CD with conflicting efficacy data [23,24]. ICAM-1 gene lies on chromosome 19p13, in a region previously implicated in determining susceptibility to IBD [6]. Two polymorphisms in the ICAM-1 gene have been identified, one at position 241 of the coding region (exon 4) and the other at position 469 (exon 6), the first determining Gly/Arg substitution and the second a Lys/Glu variation [12]. The K469E polymorphism results in a non-conservative amino acid substitution in the fifth immunoglobulin-like domain of ICAM-1. This domain has been shown to play a role for adhesion of B cells and dendritic cells [25], and has also been demonstrated to be the ICAM-1 immunodominant epitope [26]. In the present study, we found an association of IBD with the E/E genotype of ICAM-1 gene. Other three previous studies assessed the prevalence of ICAM-1 K469E polymorphism in IBD patients, with conflicting results [14–16]. In particular, a recently published study by Matsuzawa et al. [16], reported an association between the K469 allele of ICAM-1 gene and IBD in a Japanese population. The author found that the allelic frequency of K469 was significantly higher in both CD and UC patients than in controls [16]. Braun et al. [15] found that the E/E genotype was significantly associated with both UC and CD patients with respect to controls, but no significant difference was observed in alleles frequencies between patients and controls, as also reported in the present study. The third study performed in United States did not show any significant association between the K469E polymorphism and IBD [14]. In the last two studies, the G241R polymorphism of ICAM-1 gene was also investigated, and IBD patients were stratified by antineutrophil cytoplasmic antibodies (ANCA) status [14,15]. In particular, Yang et al. [14] found a significantly increased fre-
quency of the G241R polymorphism both in ANCA-negative UC and in ANCA-positive CD patients, while Braun et al. [15] showed an association between the R241 allele and UC independent of the ANCA status. The G241R polymorphism is extremely rare in the general Italian population, since the frequency of the RR genotype and the R241 allele have been reported to be 0.4 and 3.1, respectively, in healthy controls [27]; thus, it was not analysed in the present study. A possible explanation for the discrepancy of results in the reported studies is probably the influence of the different geographic distribution of the genetic mutation. Indeed, Japanese patients have a genetic background that differs from Western patients as also recently shown for the NOD2/CARD15 gene polymorphisms [28,29], while the IBD population studied by Yang et al. [14] was in part constituted by patients of Jewish ethnicity. Finally, when patients were stratified according to disease extent and clinical features, we confirmed the correlation of the E/E genotype with all subgroups of patients, but interestingly, we observed an increased prevalence of E469 allele in CD patients with small bowel plus colon localisation and with penetrating behaviour. On the other hand, no association was found on comparing subgroups of UC patients with controls. However, this phenotype subgroup analysis has a potential limitation in the size of the studied population. In fact, the number of patients for each subgroup is small and our findings need to be confirmed for larger samples. In conclusion, this study reports the association of IBD with the E/E genotype of ICAM-1 gene and an higher frequency of E469 allele in CD patients with more extensive and aggressive disease. Conflict of interest statement None declared. List of abbreviations CD, Crohn’s disease; IBD, inflammatory bowel disease; ICAM-1, intercellular adhesion molecule 1; UC, ulcerative colitis.
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References [1] Annese V, Latiano A, Andriulli A. Genetics of inflammatory bowel disease the beginning of the end or the end of the beginning? Dig Liver Dis 2003;35:442–9. [2] Hugot JP, Laurent-Puig P, Gower-Rousseau C, Olson JM, Lee JC, Beaugerie L, et al. Mapping of a susceptibility locus for Crohn’s disease on chromosome 16. Nature 1996;379:821–3. [3] Satsangi J, Parkes M, Louis E, Hashimoto L, Kato N, Welsch K, et al. Two-stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosome 3, 7 and 12. Nat Genet 1996;14:199–202. [4] Cho JH, Nicolae DL, Gold LH, Fields CT, LaBuda MC, Rohal PM, et al. Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: evidence for epistasis between 1p and IBD1. Proc Natl Acad Sci USA 1998;95:7502–7. [5] Hampe J, Schreiber S, Shaw SH, Lau KF, Bridger S, Macpherson AJ, et al. A genome wide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort. Am J Hum Genet 1999;64:808–16. [6] Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, McLeod RS, Griffiths AM, et al. Genome wide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci. Am J Hum Genet 2000;66:1863–70. [7] Panés J, Granger DN. Leukocyte–endothelial cell interactions: molecular mechanisms and implications in gastrointestinal disease. Gastroenterology 1998;14:1066–90. [8] Sambataro A, Pera A, Gaia E, Valente G, Angeli A. Is adhesion molecule ICAM-1 expression correlated with Crohn’s clinical subgroups? Ital J Gastroenterol Hepatol 1998;30:663–5. [9] Vainer B, Nielsen OH, Horn T. Comparative studies of the colonic in situ expression of intercellular adhesion molecules (ICAM-1, -2, and -3), beta2 integrins (LFA-1, Mac-1, and p150,95), and PECAM-1 in ulcerative colitis and Crohn’s disease. Am J Surg Pathol 2000;24:1115–24. [10] Bernstein CN, Sargent M, Gallatin WM. Beta 2 integrin/ICAM expression in Crohn’s disease. Clin Immmunol Immunopathol 1998;86:147–60. [11] Sans M, Panes J, Ardite E, Elizalde JI, Arce Y, Elena M, et al. VCAM-1 and ICAM-1 mediate leukocyte–endothelial cell adhesion in rat experimental colitis. Gastroenterology 1999;116:874– 83. [12] Vora DK, Rosenbloom CL, Beaudet AL, Cottingham RW. Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster. Genomics 1994;21:473–7. [13] Staunton DE, Dustin ML, Erickson HP, Springer TA. The arrangements of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus. Cell 1990;61:243– 54. [14] Yang H, Vora DK, Targan SR, Toyoda H, Beaudet AL, Rotter JI. Intercellular adhesion molecule 1 gene associations with immunologic subsets of inflammatory bowel disease. Gastroenterology 1995;109:440–8.
[15] Braun C, Zahn R, Martin K, Albert E, Folwaczny C. Polymorphisms of the ICAM-1 gene are associated with inflammatory bowel disease, regardless of the p-ANCA status. Clin Immunol 2001;101:357–60. [16] Matsuzawa J, Sugimura K, Matsuda Y, Takazoe M, Ishizuka K, Mochizuki T, et al. Association between K469E allele of intercellular adhesion molecule 1 gene and inflammatory bowel disease in a Japanese population. Gut 2002;52:75–8. [17] Malchow H, Ewe K, Brandes JW, Goebell H, Ehms H, Sommer H, et al. European cooperative Crohn’s disease study (ECCDS): results of drug treatment. Gastroenterology 1984;86:249–66. [18] Rachmilewitz D. Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomized trial. Br Med J 1989;298:82–6. [19] Nejentsev S, Laine AP, Simmel O, Ilonen J. Intercellular adhesion molecule-1 (ICAM-1) K469E polymorphism: no association with type 1 diabetes among Finns. Tissue Antigens 2000;55:568–70. [20] Levine DS. Clinical features and complications of Crohn’s disease. In: Targan SR, Shanahan F, editors. Inflammatory bowel disease: from bench to bedside. Baltimore: Williams and Wilkins; 1993. p. 296–316. [21] Rothlein R, Dustin ML, Marlin SD, Springer TA. A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 1986;137:1270–4. [22] Smith CW, Marlin SD, Rothlein R, Toman C, Anderson DC. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J Clin Invest 1989;83:2008–17. [23] Schreiber S, Nikolaus S, Malchow H, Kruis W, Lochs H, Raedler A, et al. Absence of efficacy of subcutaneous antisense ICAM-1 treatment of chronic active Crohn’s disease. Gastroenterology 2001;120:1339–46. [24] Yacyshyn BR, Chey WY, Goff J, Salzberg B, Baerg R, Buchman AL, et al. Double blind, placebo controlled trial of the remission inducing and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn’s disease. Gut 2002;51:30–6. [25] Joling P, Boom S, Johnson J, Dekker ME, van den Tweel JG, Shuurman HJ, et al. Domain 5 of the intercellular adhesion molecule-1 (ICAM-1) is involved in adhesion of B-cells and follicular dendritic cells. Adv Exp Med Biol 1994;355:131–5. [26] Molgg M, Schwaeble W, Johnson JP, Dierich MP. Generation of recombinant, carbohydrate-free intercellular adhesion molecule-1 (ICAM-1) and ICAM-1 fragments in Escherichia coli and mapping of epitopes recognized by anti-ICAM-1 monoclonal antibodies. Immunol Lett 1991;28:237–43. [27] Boiardi L, Salvarani C, Casali B, Olivieri I, Ciancio G, Cantini F, et al. Intercellular adhesion molecule-1 gene polymorphisms in Behcet’s disease. J Rheumatol 2001;28:1283–7. [28] Yamazaki K, Takazoe M, Tanaka T, Kazumori T, Nakamura Y. Absence of mutation in the NOD2/CARD15 gene among 483 Japanese patients with Crohn’s disease. J Hum Genet 2002;47:469–72. [29] Inoue N, Tamura K, Kinouchi Y, Fukuda Y, Takahashi S, Ogura Y, et al. Lack of common NOD2 variants in Japanese patients with Crohn’s disease. Gastroenterology 2002;123:86–91.
Alimentary Tract
Prevalence of the K469E polymorphism of intercellular adhesion molecule 1 gene in Italian patients with inflammatory bowel disease A. Papa a,∗ , R. Pola b , A. Flex b,c , S. Danese a , A. Armuzzi a , E. Gaetani b,c , L. Guidi a , I. De Vitis a , R. Urgesi a , A. Grillo a , M. Serricchio c , A.S. Proia b,c , G. Fedeli a , G. Gasbarrini a , P. Pola c , A. Gasbarrini c a
Department of Internal Medicine, Gastroenterology Unit, Catholic University of Rome, “A. Gemelli” University Hospital, Largo A. Gemelli 8, 00168 Rome, Italy b Laboratory of Vascular Biology and Genetics, Catholic University of Rome, Rome, Italy c Department of Internal Medicine and Angiology, Catholic University of Rome, Rome, Italy Received 7 October 2003; accepted 3 March 2004
Abstract Background. Intercellular adhesion molecule 1 plays an important role in the recruitment of leucocytes at sites of inflammation and is up-regulated in intestinal mucosa of inflammatory bowel disease. Intercellular adhesion molecule 1 gene lies on chromosome 19p13, implicated in determining susceptibility to inflammatory bowel disease. Recently, the polymorphism K469E of intercellular adhesion molecule 1 gene has been identified. Aim. To assess the potential association of this polymorphism with inflammatory bowel disease. Patients. A total of 165 inflammatory bowel disease patients, 75 with Crohn’s disease and 90 with ulcerative colitis, and 187 controls were studied. Methods. The K469E polymorphism was detected by polymerase chain reaction and restriction enzyme analysis. Statistical analysis was performed by χ2 -test. Results. In inflammatory bowel disease, the distribution of intercellular adhesion molecule 1 genotypes was 24.9% E/E, 44.2% E/K and 30.9% K/K. In controls, 11.8% showed E/E genotype, 55.6% E/K and 32.6% K/K. The frequency of the E/E genotype was significantly higher in inflammatory bowel disease (Crohn’s disease and ulcerative colitis) patients than in controls. Subgroup analysis showed that the frequency of the E469 allele was significantly increased only in Crohn’s disease patients with ileocolonic location of disease and penetrating behaviour compared with controls. Conclusions. We found an association of inflammatory bowel disease with the E/E genotype of intercellular adhesion molecule 1 gene, while allele E469 was associated with a subgroup of Crohn’s disease patients with more extensive location of disease and penetrating behaviour. However, further studies are needed to confirm our findings. © 2004 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Inflammatory bowel disease; Intercellular adhesion molecule 1
1. Introduction The aetiologies of Crohn’s disease (CD) and ulcerative colitis (UC), the two major forms of chronic inflammatory bowel disease (IBD), are unknown. However, genetic predisposition seems to play a crucial role as evidenced by the ∗ Corresponding author. Tel.: +39-06-3503726; fax: +39-06-35502775. E-mail address: [email protected] (A. Papa).
increased familial prevalence of IBD and increased concordance rates in siblings or twins [1]. In fact, in the last years, several association studies and linkage analyses have been performed to identify susceptibility genes in the whole genome [2–5]. Recently, Rioux et al. [6] have identified a linkage between IBD and chromosome 19p13, where intercellular adhesion molecule 1 (ICAM-1) gene is located. ICAM-1 is a surface glycoprotein that belongs to the immunoglobulin superfamily and plays an important role in the trafficking and activation of leucocytes [7]. Moreover,
1590-8658/$30 © 2004 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2004.03.015
A. Papa et al. / Digestive and Liver Disease 36 (2004) 528–532
several studies have shown that ICAM-1 is up-regulated in intestinal mucosa of patients with active IBD [8–10] and in experimental colitis [11]. Genetic variation in ICAM-1 gene structure may result in altered expression and/or function of the resulting adhesion molecule, thus potentially contributing to a genetic predisposition to inflammatory and immunomediated events. Recently, the K469E polymorphism of the ICAM-1 gene has been described and might be functionally important [12]. This polymorphism occurs in exon 6 of the ICAM-1 gene and results in a change from glutamic acid to lysine in Ig-like domain 5, which has been reported to affect interactions between ICAM-1 and the leucocyte function-associated antigen-1 (LFA-1) and to influence B-cell adhesion [13]. In the last years, some reports from both Western and Eastern countries have shown discordant data regarding the association of this polymorphism with IBD [14–16]. All these findings led us to investigate the prevalence of the K469E polymorphism in a population of Italian IBD patients.
2. Patients and methods 2.1. Patients and controls The study population included 165 consecutive patients with IBD (51 women and 114 men; mean age 38 ± 14.2 years, median age 35 years; range 17–80 years) referred to the Department of Internal Medicine and the Gastroenterology Unit of our university hospital. Of these patients, 90 had UC and 75 CD. All patients were of Italian ancestry. The diagnosis of IBD was based on established criteria of clinical, radiological, endoscopic and histological findings [17,18]. A detailed clinical history was also taken from each patient and their characteristics are described in Table 1. As controls, 187 subjects were considered, similar to the patients for Table 1 Epidemiological and clinical data of IBD patients and controls
Subjects (n) Gender (F/M) Mean age (±S.D.) Extent of CD Small bowel Colon Small bowel plus colon Clinical behaviour of CD Penetrating Stricturing Non-stricturing and non-penetrating Extent of UC Proctitis Left-sided colitis Entire colitis
Controls
CD patients
UC patients
187 58/129 40 ± 16.4
75 24/51 36 ± 12.8
90 27/63 40 ± 15.1
529
age and sex distribution (58 women and 129 men, mean age 40 ± 16.4 years, median age 38 years, range 19–82 years). In particular, the control group consisted of healthy subjects, without a history of neoplastic, metabolic or inflammatory disease. All controls came from the same geographical area as the patients. Patients and controls were enrolled in this study after giving their informed consent to the use of part of their blood samples for an experimental study. 2.2. Detection of ICAM-1 polymorphism Nucleic acid isolation from 200 l of peripheral blood was carried out by using a DNA extraction kit (NucleoSpin Blood QuickPure; Macherey–Nagel, Duren, Germany), as recommended by the supplier. Genomic DNAs were assayed with polymerase chain reaction for the detection of ICAM-1 gene, as previously described [19]. The amplified sequence was digested by BstUI restriction enzyme (New England Bio Labs; Beverly, MA, USA) at 60 ◦ C for 4 h. The digested products were electrophoresed in 2% agarose gel and visualised by ethidium bromide staining. The EE genotype corresponded to the contemporary presence of 136 and 87 base pair fragments. The EK genotype corresponded to the contemporary presence of 223, 136 and 87 base pair fragments. The KK genotype corresponded to a 223 base pair fragments. 2.3. Statistical analysis Allele and genotype frequencies between each of the patient groups and controls were compared by χ2 -test for 2×2 or 2 × 3 tables. To correct for multiple comparisons in the subgroups, corrected P values (Pc ) were obtained using Bonferroni’s correction factor by multiplying the P value by 2 (Pc = 2P) because the alleles were examined simultaneously. To analyse subgroups in IBD, Pc values were corrected using Fisher’s exact test when number of samples were small. After correction, Pc was considered significant when <0.05. The P value was two-tailed. Hardy–Weinberg equilibrium was tested in the control population. All analyses were carried out using Intercooled STATA 6.0 for Windows (Stata Corporation, College Station, TX, USA).
3. Results
22 11 42 23 16 36
19 48 23
The distribution of ICAM-1 genotypes and alleles in IBD patients and controls is shown in Table 2. The genotype frequencies follow Hardy–Weinberg distributions in controls. In particular, the prevalence of the homozygous E/E genotype was significantly higher in IBD patients than in controls. Also, considering CD and UC patients separately (Table 2), the E/E genotype resulted more frequently in controls. On the contrary, the allele frequency did not differ between groups of patients (CD and UC) and controls. Subdividing CD patients according to disease extension (small bowel,
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Table 2 ICAM-1 genotype and allele distribution in IBD patients overall, CD and UC patients separately and in controls Controls (N = 187)
IBD
CD
N = 165 Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
Allele K E
226 (60.4) 148 (39.6)
a
51 (30.9) 73 (44.2) 41 (24.9) 175 (53) 155 (47)
UC
P (Pc )
N = 75
P (Pc )
N = 90
P (Pc )
0.00023
19 (25.3) 38 (50.7) 18 (24.0)
0.00065
32 (35.6) 35 (38.9) 23 (25.5)
0.00002
0.0479 (0.0958)
76 (50.7) 74 (49.3)
0.041 (0.082)
99 (55) 81 (45)
0.224 (0.448)
Numbers in parentheses are percentage of total.
small bowel plus colon or colon) (Table 3) and clinical behaviour (penetrating, stricturing, and non-stricturing and non-penetrating) (Table 4) we found that the E/E genotype occurred more frequently in all subgroups of CD patients, independent of disease location and clinical behaviour, while the frequency of E469 allele was significantly higher only in patients with small bowel plus colon disease and penetrating behaviour. In UC patients, after stratification according to disease extension (proctitis, left-sided colitis or entire colitis) (Table 5), we found that only the frequency of the E/E genotype was increased in all subgroups of patients compared to controls, while no difference was observed in the frequency of the allele E469 between subgroups of UC patients and controls.
4. Discussion IBD are chronic inflammatory disorders of the intestine, characterised by immune dysregulation and leucocyte recruitment into gastrointestinal tissue [20]. Epidemiological data provide evidence that genetic predisposition to both CD and UC depends on the contribution of multiple genes instead of a single genetic factor, and genetic heterogeneity could explain different clinical features of the disease [1]. Accordingly, polymorphisms of genes coding for proteins involved in initiation and regulation of the immune response, such as cytokines and adhesion molecules, were investigated in order to show an association with IBD. ICAM-1 is a cell surface adhesion molecule that serves as a receptor
Table 3 ICAM-1 genotype and allele distribution according to CD location of disease Controls (N = 187)
Small bowel N = 22
Small bowel plus colon
Colon
P (Pc )
N = 42
N = 11
0.00072
0.0217 (0.0434)
P (Pc )
Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
4 (18.2) 13 (59.1) 5 (22.7)
0.00022
10 (23.8) 20 (47.6) 12 (28.6)
Allele K E
226 (60.4) 148 (39.6)
21 (47.7) 23 (52.3)
0.105 (0.21)
40 (47.6%) 44 (52.4%)
a
5 (45.5%) 5 (45.5%) 1 (9%) 15 (68.2%) 7 (31.8%)
P (Pc )
0.035
0.51 (NS)
Numbers in parentheses are percentage of total. NS, not significant compared to controls.
Table 4 ICAM-1 genotype and allele distribution according to CD clinical behaviour Controls (N = 187)
Penetrating N = 23
Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
2 (8.7) 15 (65.2) 6 (26.1)
Allele K E
226 (60.4) 148 (39.6)
19 (41.3) 27 (58.7)
a
Stricturing P (Pc )
<0.00001
0.013 (0.026)
N = 16 2 (12.5) 9 (56.3) 5 (31.2) 13 (40.6) 19 (59.4)
Non-stricturing and non-penetrating P (Pc )
N = 36
P (Pc )
<0.00001
15 (41.7) 14 (38.9) 7 (19.4)
0.0023
44 (61.1) 28 (38.9)
0.913 (NS)
0.0289 (0.0578)
Numbers in parentheses are percentage of total. NS, not significant compared to controls.
A. Papa et al. / Digestive and Liver Disease 36 (2004) 528–532
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Table 5 ICAM-1 genotype and allele distribution according to UC location of disease Controls (N = 187)
Entire colitis N = 23
Genotype K/K E/K E/E
61 (32.6)a 104 (55.6) 22 (11.8)
Allele K E
226 (60.4) 148 (39.6)
a
Left-sided colitis
Proctitis
P (Pc )
N = 48
10 (43.5) 9 (39.1) 4 (17.4)
0.0031
18 (37.5) 17 (35.4) 13 (27.1)
0.00003
28 (63.6) 16 (36.4)
0.732 (NS)
53 (55.2) 43 (44.8)
0.353 (0.706)
P (Pc )
N = 19 4 (21) 9 (47.4) 6 (31.6) 17 (44.7) 21 (55.3)
P (Pc )
0.00043
0.061 (0.122)
Numbers in parentheses are percentage of total. NS, not significant compared to controls.
for the LFA-1 and the macrophage differentiation antigen-1 (Mac-1), and plays a central role in the regulation of leucocyte circulation and homing [21,22]. The up-regulation of ICAM-1 on endothelial cells by proinflammatory cytokines is a central event in regulating leucocyte localisation at inflammatory sites and can also play an important role in the regulation and amplification of the inflammatory response observed in IBD [7–10]. Thus, an anti-ICAM-1 therapeutic strategy with antisense oligonucleotide has been used in steroid dependent or refractory CD with conflicting efficacy data [23,24]. ICAM-1 gene lies on chromosome 19p13, in a region previously implicated in determining susceptibility to IBD [6]. Two polymorphisms in the ICAM-1 gene have been identified, one at position 241 of the coding region (exon 4) and the other at position 469 (exon 6), the first determining Gly/Arg substitution and the second a Lys/Glu variation [12]. The K469E polymorphism results in a non-conservative amino acid substitution in the fifth immunoglobulin-like domain of ICAM-1. This domain has been shown to play a role for adhesion of B cells and dendritic cells [25], and has also been demonstrated to be the ICAM-1 immunodominant epitope [26]. In the present study, we found an association of IBD with the E/E genotype of ICAM-1 gene. Other three previous studies assessed the prevalence of ICAM-1 K469E polymorphism in IBD patients, with conflicting results [14–16]. In particular, a recently published study by Matsuzawa et al. [16], reported an association between the K469 allele of ICAM-1 gene and IBD in a Japanese population. The author found that the allelic frequency of K469 was significantly higher in both CD and UC patients than in controls [16]. Braun et al. [15] found that the E/E genotype was significantly associated with both UC and CD patients with respect to controls, but no significant difference was observed in alleles frequencies between patients and controls, as also reported in the present study. The third study performed in United States did not show any significant association between the K469E polymorphism and IBD [14]. In the last two studies, the G241R polymorphism of ICAM-1 gene was also investigated, and IBD patients were stratified by antineutrophil cytoplasmic antibodies (ANCA) status [14,15]. In particular, Yang et al. [14] found a significantly increased fre-
quency of the G241R polymorphism both in ANCA-negative UC and in ANCA-positive CD patients, while Braun et al. [15] showed an association between the R241 allele and UC independent of the ANCA status. The G241R polymorphism is extremely rare in the general Italian population, since the frequency of the RR genotype and the R241 allele have been reported to be 0.4 and 3.1, respectively, in healthy controls [27]; thus, it was not analysed in the present study. A possible explanation for the discrepancy of results in the reported studies is probably the influence of the different geographic distribution of the genetic mutation. Indeed, Japanese patients have a genetic background that differs from Western patients as also recently shown for the NOD2/CARD15 gene polymorphisms [28,29], while the IBD population studied by Yang et al. [14] was in part constituted by patients of Jewish ethnicity. Finally, when patients were stratified according to disease extent and clinical features, we confirmed the correlation of the E/E genotype with all subgroups of patients, but interestingly, we observed an increased prevalence of E469 allele in CD patients with small bowel plus colon localisation and with penetrating behaviour. On the other hand, no association was found on comparing subgroups of UC patients with controls. However, this phenotype subgroup analysis has a potential limitation in the size of the studied population. In fact, the number of patients for each subgroup is small and our findings need to be confirmed for larger samples. In conclusion, this study reports the association of IBD with the E/E genotype of ICAM-1 gene and an higher frequency of E469 allele in CD patients with more extensive and aggressive disease. Conflict of interest statement None declared. List of abbreviations CD, Crohn’s disease; IBD, inflammatory bowel disease; ICAM-1, intercellular adhesion molecule 1; UC, ulcerative colitis.
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