Scandinavian Journal of Gastroenterology. 2014; 49: 1201–1206
ORIGINAL ARTICLE
Association of IL23R and ATG16L1 with susceptibility of Crohn’s disease in Chinese population
JIE ZHANG1,2, JIEBIN CHEN2, JIANJUN GU2, HUIMIN GUO3 & WEICHANG CHEN1 1
Department of Gastroenterology, the First Affiliated Hospital of Soochow University, Suzhou, China, 2Department of Gastroenterology, Jiangsu Taizhou People’s Hospital, Taizhou, China, and 3Department of Gastroenterology, The Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
Abstract Objectives. To investigate whether gene polymorphisms of ATG16L1 and IL23R are associated with the susceptibility of Crohn’s disease (CD) in Chinese population. Methods. A total of 420 patients with CD and 450 age- and sex-matched healthy volunteers from Chinese Han population were included in this study. Single nucleotide polymorphisms (SNPs) rs2241880 of ATG16L1 and rs11209026, rs1004819, and rs1495965 of IL23R were genotyped. The differences of genotype and allele distributions between CD patients and healthy controls were assessed using the Chi-squared test. Besides, subgroup analysis of disease groups was performed using the Chi-squared test. Results. For ATG16L1, patients were found to have significantly higher proportion of genotype GG (18.3%), when compared with the normal controls (12.4%). Allele G was found to be the risk allele for the disease (34.3% vs. 29.0%, p = 0.016) with an odds ratio of 1.18. For IL23R, all three SNPs were found not to be associated with the development of CD. None of these four SNPs was found to be associated with the clinical features of the patients, including age at diagnosis, disease location, and behavior. Conclusion. The original genome-wide association studies finding on ATG16L1 gene should be robust and this gene does play a role in the pathogenesis of CD in the Chinese population. However, the role of IL23R gene in the occurrence of CD remains obscure.
Key Words: ATG16L1, Chinese population, Crohn’s disease, gene polymorphism, IL23R
Introduction As one common form of chronic inflammatory bowel disease (IBD), Crohn’s disease (CD) is an immunerelated disorder that may affect any part of the gut with discontinuous penetrating lesions [1–3]. It is well documented that CD has a complex etiology possibly involving interplay between multiple genes that interact with unknown environmental factors leading to predisposition to the disease [4–7]. Previous familial clustering and twin studies implied that CD could be a polygenic disorder. The following linkage or association analysis identified many predisposition genes as the susceptibility loci, with convincing causative variants such as NOD2, HSP70, IBD5, and DLG5 validated in different populations [8–12]. In
recent years, tremendous insights into the genetic architecture of CD have been yielded through recent genome-wide association studies (GWAS). Hampe et al. [13] performed a GWAS of CD in 735 patients and 368 controls using 19,779 nonsynonymous single nucleotide polymorphisms (SNPs), revealing that ATG16L1 gene could carry the risk allele of CD. This finding was subsequently confirmed by another independent GWAS of CD presented by Rioux et al. [14]. In a recent GWAS in Korean population, Yang et al. [15] reported for the first time that ATG16L2 gene could be a novel susceptibility gene for CD and rs11235604 was a potential causal variant of the association. As suggested by the differences in genetic susceptibility of CD between different populations, the
Correspondence: Dr. Weichang Chen, Department of Gastroenterology, the First Affiliated Hospital of Soochow University, Shizi Street No. 256, Suzhou 215006, China. E-mail: [email protected]
(Received 14 April 2014; revised 10 June 2014; accepted 15 June 2014) ISSN 0036-5521 print/ISSN 1502-7708 online 2014 Informa Healthcare DOI: 10.3109/00365521.2014.936031
1202
J. Zhang et al.
heritability of CD has not been fully explained. As well-established CD susceptibility genes in Caucasian population, ATG16L1 and IL23R failed to be replicated in Asians [16–24]. Yamazaki et al. [20] selected 29 SNPs out of regions of the IL23R and ATG16L1. After comparing the distributions of these markers between 484 CD patients and 439 controls from the Japanese population, the authors concluded that there is a significant ethnical divergence between Japanese and Caucasian populations regarding the association of IL23R and ATG16L1 with pathogenesis of CD. In an independent study performed in the Korean population, the association of the IL23R gene with CD was supported, although ATG16L1 was still reported to have no association with CD [21]. These inconclusive results can be partially due to ethnic difference, inherent genetic heterogeneity, and the substantially small effect of this locus on the development of CD. Considering that there exist remarkable ethnic differences concerning the heritability of CD, replication studies would be helpful in finding remaining risk loci and in expanding our understanding of the genetic architecture of CD. To our knowledge, there is still a lack of knowledge concerning the association of ATG16L1 and IL23R gene with CD in the Chinese population. The primary purpose of this study was to investigate whether ATG16L1 and IL23R gene can function as susceptibility loci for CD in Chinese population and to further narrate their roles in the etiology of CD.
classified into four types including terminal ileum, colon, ileocolon, and isolated upper disease. Disease behavior was grouped as follows: B1, non-stricturing and non-penetrating; B2, stricturing; B3, penetrating; p, perianal disease modifier defined as the presence of a perianal abscess, fistulas, and/or ulceration. Disease severity was determined by the presence of prior treatment, such as surgical intervention and corticosteroids, immunosuppressant agents, or infliximab treatment.
Genotyping protocols Blood samples were collected from the subjects for the extraction of DNA, with informed consent obtained from the subjects. Genomic DNA was extracted from blood leukocytes using standard protocols (Qiagen K. K., Tokyo, Japan). SNPs rs2241880 of ATG16L1 and rs11209026, rs1004819, and rs1495965 of IL23R were genotyped using TaqMan SNP Genotyping Assay according to the manufacturer’s instructions, which was read with an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). Besides, three SNPs of CARD15 (rs2066844, rs2066845, and rs2066847) were genotyped to detect the potential interaction between CARD15 and ATG16L1, as previously reported [13]. About 20% of the samples were selected randomly to validate the reproducibility of the genotyping results.
Methods Subjects Under the approval of the Local Ethical Committee, the current multicenter study retrospectively included 420 patients with CD from Chinese Han population who had received treatment since 2007. CD was diagnosed according to the standard clinical, radiological, endoscopic, and histological criteria [3]. A total of 450 age- and ethnic-matched healthy volunteers were recruited as the control group after ruling out those with known IBD, family history of IBD, or other autoimmune disease. Clinical characteristics of the patients Data on the clinical characteristics of the patients were obtained according to Montreal classification, including age at diagnosis, disease location, disease behavior, and previous treatment [25]. Briefly, age at diagnosis was classified as follows: £16 years, between 17 and 40 years, and >40 years. Disease location was
Statistical analysis Clinical characteristics such as age, height, and weight between patients and controls were compared by means of an independent t-test. The Hardy-Weinberg equilibrium (HWE) test was used for each SNP between the patient and control groups. The differences of genotype and allele distributions between cases and controls were analyzed with the Chisquared test, which was also applied to subgroup analysis of disease groups. Odds ratio (OR) of the significant associated SNP was calculated using the ancestral allele as reference. Logistic regression analysis was used to test the potential statistical interaction between ATG16L1 and CARD15. Modeled as a 3-level factor, CARD15 statuses were grouped as wild-type, heterozygous carriers of one of the three common CARD15 mutations, or homozygous mutant carriers [13]. The SPSS software (version 13.0, Chicago, IL, USA) was used for statistical analyses. A p-Value of 40 years Location† Terminal ileum Colon Ileocolon Upper GI disease Behavior¶ Non-stricturing and non-penetrating Stricturing Penetrating Perianal disease
Number
Percentage (%)
30 280 110
7.1 66.7 26.2
82 58 248 32
19.5 13.8 59.1 7.6
165 91 102 62
39.3 21.7 24.3 14.7
† Patients can be added to the other three groups when having concomitant upper GI disease. ¶ Patients can be added to the other three groups when there is concomitant perianal disease. Abbreviation: GI = Gastrointestinal.
1203
Distributions of genotype and allele frequencies of ATG16L1 and IL23R in the cases and the controls are shown in Table III. There were significant differences between the two groups in terms of genotype and allele frequencies of ATG16L1. Patients were found to have significantly higher proportion of genotype GG (18.3%), when compared with the normal controls (12.4%). Allele G was found to be the risk allele for the disease (34.3% vs. 29.0%, p = 0.016) with an OR of 1.18. For IL23R and CARD15, all six SNPs were found not to be associated with the development of CD. The logistic regression analysis indicated that ATG16L1 contributed independently to the risk of the disease (p = 0.028), which however had no significant association with CARD15 (p = 0.72). Comparisons of subgroup genotypes according to the Montreal Classification Comparisons of genotypes of CD among the subgroups classified according to the Montreal Classification are shown in Table IV. None of the four SNPs was found to be associated with the clinical features of the CD, including age at diagnosis, disease location, and behavior. Discussion CD is a complex disease with strong genetic factors that can lead to aberrant mucosal inflammation of the intestinal tract when combined with environmental or microbial triggers. During the past decade, there has been an increasing interest in the exploration of genetic variants associated with CD [4,26–29]. To date, over 100 independent genetic loci were reported to potentially play a role in the pathogenesis of CD, thus yielding an extensive framework that unraveled the genetic basis and clinical predictors of CD. As a critical tool for the identification of susceptibility genes for complex diseases, GWAS can produce more robust results based on its biological, technical, and statistical foundations. Among those reported CD-associated genetic loci, the IL23R gene and the ATG16L1 gene were both identified by GWAS and were subsequently validated in large independent Caucasian samples. Dusatkova et al. [17] confirmed the role of IL23R and ATG16L1 in the CD susceptibility in the Czech population and found a weak protective effect of IL23R p.381Gln against upper gastrointestinal tract involvement. Lakatos et al. [22] confirmed that both ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Moreover, Latiano et al. [23] reported that polymorphisms of IL23R and ATG16L1 were associated
1204
J. Zhang et al.
Table III. Associations between ATG16L1, IL23R, and CARD15 gene polymorphisms and disease susceptibility of CD. Genotypea SNPs rs2241880 (A/G) rs11209026 (G/A) rs1004819 (T/C) rs1495965 (G/A) rs2066844 (T/C) rs2066845 (C/G) rs2066847 (insC)
Allele type
p-Value
Patient (n = 420)
Control (n = 500)
Patient
Control
Genotype
Allele
Odds ratio (95%CI)b
209/134/77 403/12/5 151/209/60 95/212/113 1/1/418 1/0/419 0/0/420
272/166/62 473/18/9 170/237/93 131/225/144 1/0/499 0/0/500 1/1/498
552/288 818/22 511/329 402/438 3/837 2/838 0/840
710/290 964/36 577/423 487/513 2/998 0/1000 3/997
0.04 0.61 0.21 0.23 0.54 0.52 0.43
0.016 0.28 0.18 0.74 0.66 0.21 0.25
1.18 0.72 0.93 1.02 0.998 1.003 0.998
(1.03–1.35) (0.43–1.22) (0.83–1.03) (0.93–1.11) (0.994–1.003) (1.001–1.006) (0.994–1.001)
The three values in the ‘genotype’ column indicate the numbers of homozygotes with respect to the ancestral allele, heterozygotes, and homozygotes with respect to the wild-type allele, respectively, that is, for rs2241880, the three values indicate numbers of genotype AA, AG, and GG, respectively. b Calculated for the alleles. a
Table IV. Comparisons of subgroup genotypes according to the Montreal Classification.
Age at diagnosis £16 years (n = 30) 17–40 years (n = 280) >40 years (n = 110) p-Value Location Terminal ileum (n = 82) Colon (n = 58) Ileocolon (n = 248) Upper GI disease (n = 32) p-Value Behavior Non-stricturing and non-penetrating (n = 165) Stricturing (n = 91) Penetrating (n = 102) Perianal disease (n = 62) p-Value
a
rs11209026
rs1004819
rs1495965
16/10/4 137/90/53 56/34/20 0.95
29/1/0 268/8/4 106/3/1 0.96
11/15/4 99/141/40 41/53/16 0.3
6/16/8 65/140/75 24/56/30 0.99
42/27/13 27/22/9 124/74/50 16/11/5 0.89
79/2/1 55/2/1 239/7/2 30/1/1 0.95
28/42/12 22/28/8 88/124/36 13/15/4 0.99
18/42/22 12/28/18 58/125/65 7/17/8 0.99
86/49/30 42/33/16 52/30/20 29/22/11 0.92
159/4/2 86/4/1 98/3/1 60/1/1 0.96
55/86/24 37/41/13 37/48/17 22/34/6 0.77
35/83/43 21/48/22 23/50/29 12/31/19 0.98
rs2241880
Abbreviation: GI = Gastrointestinal. The indications of three values for genotypes were the same with those in Table III.
a
with CD in both adult- and pediatric-onset subsets from Italy population. Collectively, these findings supported the theory that IL23R and ATG16L1 were common susceptible genes to CD in Caucasian populations. To date, there is still a lack of common susceptible variants of CD between Asian and Caucasian populations, possibly due to great ethnical diversity of susceptible genes to CD between Asian and European ancestries. Although IL23R and ATG16L1 were reported as well-established predisposition genes of CD in Caucasian population, these candidate genes were not common variants to CD among the Japanese and Caucasian populations [20]. Differently, the association of IL23R with Korean CD patients was confirmed, partly supporting the previous findings in Caucasian populations [21]. To better clarify the role of these two genes in the pathogenesis of CD in Asian
population, we examined the association of IL23R and ATG16L1 with CD in a large cohort of Chinese patients. In our study, a coding SNP in the ATG16L1 gene (rs2241880) was demonstrated to be associated with the susceptibility of CD in Chinese population. Moreover, G allele was confirmed to be the risk allele of the disease which can increase the incidence of CD by 1.18-folds. The contributable risk of the ATG16L1 G allele reported in our study (OR = 1.18) was similar to the published studies performed in UK (OR = 1.30) and Hungary (OR = 1.39), and a meta-analysis performed in German, Dutch, and Hungarian cohorts (OR = 1.32) [22,30,31]. Regarding polymorphism of the IL23R gene, our study failed to confirm the recently described associations between variants in the IL23R gene in Caucasian or Korean CD patients. Recently Yamazaki et al. [20] also failed to find any positive association of the
Association of IL23R and ATG16L1 with Crohn’s disease IL23R gene polymorphism with CD in the Japanese population. We believe that this discrepancy can be attributed to the distinct ethnic difference of genetic backgrounds of CD that has been reported previously for other genes between Asian and Caucasian populations. In the current study, the possible interaction between ATG16L1 and CARD15 in contributing to the risk of CD was also investigated. In line with the finding of Leong et al. [32], we found that CARD15 was not significantly associated with the occurrence of CD in Chinese population. Moreover, through logistic regression analysis, we confirmed that ATG16L1 is a CD susceptibility gene and found no evidence of potential interaction with CARD15. These findings were mostly consistent with previous studies which failed to confirm the association of CARD15 with CD in Asian populations [33,34]. Since genotypes remain unchanged during the lifetime, the identification of genetic variants that may predict disease behavior and progress are of great interest, usually superior to other predictive factors such as serological or clinical parameters. With the ever-growing number of susceptibility loci in CD, there are also a few variants reported to be significantly associated with clinical outcome and prognosis. One example was the identification of NOD2/ CARD15, which was found to be associated with a more aggressive clinical course involving higher risk of intestinal strictures and need for early surgical treatment [8,27]. Other genetic variants that can affect the disease behavior include SNP rs4958847 of IRGM, which was significantly associated with frequency of surgery in patients with ileocecal CD [35]. Besides, Prager et al. [9] demonstrated that patients with the C risk allele of rs10758669 in JAK2 could have increased epithelial permeability, indicating that the influence on disease behavior may result in alterations in overall intestinal permeability. In the current study, we also investigated the relationship between genotypes and phenotypes including the age at diagnosis, disease location, behavior, and the disease severity. Although remarkably associated with the occurrence of CD, ATG16L1 gene seems to have no significant relationship with the phenotypes mentioned above. Further, in agreement with previous studies, we did not observe any association of the polymorphism of the IL23R gene with the disease phenotype. In conclusion, it is unlikely that these two genes are implicated in the progression of CD. To our knowledge, this is the first replication study investigating the association of ATG16L1 and IL23R gene with CD in the Chinese population, which provides novel insights into the role of these two genes in the pathogenesis of CD. One limitation of our study lies in that hierarchical clustering was not
1205
applied to the statistical analysis, which can potentially provide some useful information on the association between the genetic variants and the risk of the disease. In summary, our results indicate that the original GWAS finding on ATG16L1 gene should be robust and that this gene does play a role in the pathogenesis of CD in the Chinese population. However, the role of IL23R gene in the development of CD remains obscure, and further investigations are warranted to clearly illustrate its association with this disease. Collectively, these genetic data are helpful information that will possibly lead to the ultimate discovery of etiologies for CD.
Acknowledgment The authors sincerely thank Dr Leilei Xu from the Department of Spine Surgery of Drum Tower Hospital who made the primary contribution to the proofreading of the manuscript. Declaration of interest: The authors involved in this study do not have anything to disclose regarding funding or any conflicts of interest with respect to this manuscript.
References [1] Gibson J, Collins A, Morton N. Individual disease risk and multimetric analysis of Crohn disease. Proc Natl Acad Sci USA 2008;105:15843–7. [2] Mazal J. Crohn disease: pathophysiology, diagnosis, and treatment. Radiol Technol 2014;85:297–316. [3] Vermeire S, Van Assche G, Rutgeerts P. Classification of inflammatory bowel disease: the old and the new. Curr Opin Gastroenterol 2012;28:321–6. [4] Ellinghaus D, Ellinghaus E, Nair RP, Stuart PE, Esko T, Metspalu A, et al. Combined analysis of genome-wide association studies for Crohn disease and psoriasis identifies seven shared susceptibility loci. Am J Hum Genet 2012; 90:636–47. [5] Esaki M, Furuse M, Matsumoto T, Aoyagi K, Jo Y, Yamagata H, et al. Polymorphism of heat-shock protein gene HSP70-2 in Crohn disease: possible genetic marker for two forms of Crohn disease. Scand J Gastroenterol 1999; 34:703–7. [6] Meucci G, Vecchi M, Torgano G, Arrigoni M, Prada A, Rocca F, et al. Familial aggregation of inflammatory bowel disease in northern Italy: a multicenter study. The Gruppo di Studio per le Malattie Infiammatorie Intestinali (IBD Study Group). Gastroenterology 1992;103:514–19. [7] Russel MG, Pastoor CJ, Janssen KM, van Deursen CT, Muris JW, van Wijlick EH, et al. Familial aggregation of inflammatory bowel disease: a population-based study in South Limburg, The Netherlands. The South Limburg IBD Study Group. Scand J Gastroenterol Suppl 1997;223:88–91. [8] Alvarez-Lobos M, Arostegui JI, Sans M, Tassies D, Plaza S, Delgado S, et al. Crohn’s disease patients carrying Nod2/
1206
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
J. Zhang et al.
CARD15 gene variants have an increased and early need for first surgery due to stricturing disease and higher rate of surgical recurrence. Ann Surg 2005;242:693–700. Prager M, Buttner J, Haas V, Baumgart DC, Sturm A, Zeitz M, et al. The JAK2 variant rs10758669 in Crohn’s disease: altering the intestinal barrier as one mechanism of action. Int J Colorectal Dis 2012;27:565–73. Sugimura K, Taylor KD, Lin YC, Hang T, Wang D, Tang YM, et al. A novel NOD2/CARD15 haplotype conferring risk for Crohn disease in Ashkenazi Jews. Am J Hum Genet 2003;72:509–18. Tomer G, Wetzler G, Keddache M, Denson LA. Polymorphisms in the IBD5 locus are associated with Crohn disease in pediatric Ashkenazi Jewish patients. J Pediatr Gastroenterol Nutr 2009;48:531–7. Yamazaki K, Takazoe M, Tanaka T, Ichimori T, Saito S, Iida A, et al. Association analysis of SLC22A4, SLC22A5 and DLG5 in Japanese patients with Crohn disease. J Hum Genet 2004;49:664–8. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 2007;39:207–11. Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet 2007;39:596–604. Yang SK, Hong M, Zhao W, Jung Y, Baek J, Tayebi N, et al. Genome-wide association study of Crohn’s disease in Koreans revealed three new susceptibility loci and common attributes of genetic susceptibility across ethnic populations. Gut 2014;63:80–7. Cadwell K, Patel KK, Komatsu M, Virgin HT, Stappenbeck TS. A common role for Atg16L1, Atg5 and Atg7 in small intestinal Paneth cells and Crohn disease. Autophagy 2009;5:250–2. Dusatkova P, Hradsky O, Lenicek M, Bronsky J, Nevoral J, Kotalova R, et al. Association of IL23R p.381Gln and ATG16L1 p.197Ala with Crohn disease in the Czech population. J Pediatr Gastroenterol Nutr 2009;49:405–10. Fujita N, Saitoh T, Kageyama S, Akira S, Noda T, Yoshimori T. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts. J Biol Chem 2009; 284:32602–9. Raju D, Hussey S, Jones NL. Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans. Autophagy 2012;8:1387–8. Yamazaki K, Onouchi Y, Takazoe M, Kubo M, Nakamura Y, Hata A. Association analysis of genetic variants in IL23R, ATG16L1 and 5p13.1 loci with Crohn’s disease in Japanese patients. J Hum Genet 2007;52:575–83. Yang SK, Park M, Lim J, Park SH, Ye BD, Lee I, et al. Contribution of IL23R but not ATG16L1 to Crohn’s disease susceptibility in Koreans. Inflamm Bowel Dis 2009;15: 1385–90. Lakatos PL, Szamosi T, Szilvasi A, Molnar E, Lakatos L, Kovacs A, et al. ATG16L1 and IL23 receptor (IL23R) genes
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
are associated with disease susceptibility in Hungarian CD patients. Dig Liver Dis 2008;40:867–73. Latiano A, Palmieri O, Valvano MR, D’Inca R, Cucchiara S, Riegler G, et al. Replication of interleukin 23 receptor and autophagy-related 16-like 1 association in adult- and pediatric-onset inflammatory bowel disease in Italy. World J Gastroenterol 2008;14:4643–51. Amre DK, Mack DR, Morgan K, Krupoves A, Costea I, Lambrette P, et al. Autophagy gene ATG16L1 but not IRGM is associated with Crohn’s disease in Canadian children. Inflamm Bowel Dis 2009;15:501–7. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, Brant SR, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005;19:5A–36A. Chapman JM, Onnie CM, Prescott NJ, Fisher SA, Mansfield JC, Mathew CG, et al. Searching for genotypephenotype structure: using hierarchical log-linear models in Crohn disease. Am J Hum Genet 2009;84:178–87. Cleynen I, Gonzalez JR, Figueroa C, Franke A, McGovern D, Bortlik M, et al. Genetic factors conferring an increased susceptibility to develop Crohn’s disease also influence disease phenotype: results from the IBDchip European Project. Gut 2013;62:1556–65. Elding H, Lau W, Swallow DM, Maniatis N. Dissecting the genetics of complex inheritance: linkage disequilibrium mapping provides insight into Crohn disease. Am J Hum Genet 2011;89:798–805. Elding H, Lau W, Swallow DM, Maniatis N. Refinement in localization and identification of gene regions associated with Crohn disease. Am J Hum Genet 2013;92:107–13. Prescott NJ, Fisher SA, Franke A, Hampe J, Onnie CM, Soars D, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn’s disease and is independent of CARD15 and IBD5. Gastroenterology 2007;132:1665–71. Buning C, Durmus T, Molnar T, de Jong DJ, Drenth JP, Fiedler T, et al. A study in three European IBD cohorts confirms that the ATG16L1 c.898A>G (p.Thr300Ala) variant is a susceptibility factor for Crohn’s disease. J Crohns Colitis 2007;1:70–6. Leong RW, Armuzzi A, Ahmad T, Wong ML, Tse P, Jewell DP, et al. NOD2/CARD15 gene polymorphisms and Crohn’s disease in the Chinese population. Aliment Pharmacol Ther 2003;17:1465–70. 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. Croucher PJ, Mascheretti S, Hampe J, Huse K, Frenzel H, Stoll M, et al. Haplotype structure and association to Crohn’s disease of CARD15 mutations in two ethnically divergent populations. Eur J Hum Genet 2003;11:6–16. Sehgal R, Berg A, Polinski JI, Hegarty JP, Lin Z, McKenna KJ, et al. Mutations in IRGM are associated with more frequent need for surgery in patients with ileocolonic Crohn’s disease. Dis Colon Rectum 2012;55:115–21.
Copyright of Scandinavian Journal of Gastroenterology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
ORIGINAL ARTICLE
Association of IL23R and ATG16L1 with susceptibility of Crohn’s disease in Chinese population
JIE ZHANG1,2, JIEBIN CHEN2, JIANJUN GU2, HUIMIN GUO3 & WEICHANG CHEN1 1
Department of Gastroenterology, the First Affiliated Hospital of Soochow University, Suzhou, China, 2Department of Gastroenterology, Jiangsu Taizhou People’s Hospital, Taizhou, China, and 3Department of Gastroenterology, The Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
Abstract Objectives. To investigate whether gene polymorphisms of ATG16L1 and IL23R are associated with the susceptibility of Crohn’s disease (CD) in Chinese population. Methods. A total of 420 patients with CD and 450 age- and sex-matched healthy volunteers from Chinese Han population were included in this study. Single nucleotide polymorphisms (SNPs) rs2241880 of ATG16L1 and rs11209026, rs1004819, and rs1495965 of IL23R were genotyped. The differences of genotype and allele distributions between CD patients and healthy controls were assessed using the Chi-squared test. Besides, subgroup analysis of disease groups was performed using the Chi-squared test. Results. For ATG16L1, patients were found to have significantly higher proportion of genotype GG (18.3%), when compared with the normal controls (12.4%). Allele G was found to be the risk allele for the disease (34.3% vs. 29.0%, p = 0.016) with an odds ratio of 1.18. For IL23R, all three SNPs were found not to be associated with the development of CD. None of these four SNPs was found to be associated with the clinical features of the patients, including age at diagnosis, disease location, and behavior. Conclusion. The original genome-wide association studies finding on ATG16L1 gene should be robust and this gene does play a role in the pathogenesis of CD in the Chinese population. However, the role of IL23R gene in the occurrence of CD remains obscure.
Key Words: ATG16L1, Chinese population, Crohn’s disease, gene polymorphism, IL23R
Introduction As one common form of chronic inflammatory bowel disease (IBD), Crohn’s disease (CD) is an immunerelated disorder that may affect any part of the gut with discontinuous penetrating lesions [1–3]. It is well documented that CD has a complex etiology possibly involving interplay between multiple genes that interact with unknown environmental factors leading to predisposition to the disease [4–7]. Previous familial clustering and twin studies implied that CD could be a polygenic disorder. The following linkage or association analysis identified many predisposition genes as the susceptibility loci, with convincing causative variants such as NOD2, HSP70, IBD5, and DLG5 validated in different populations [8–12]. In
recent years, tremendous insights into the genetic architecture of CD have been yielded through recent genome-wide association studies (GWAS). Hampe et al. [13] performed a GWAS of CD in 735 patients and 368 controls using 19,779 nonsynonymous single nucleotide polymorphisms (SNPs), revealing that ATG16L1 gene could carry the risk allele of CD. This finding was subsequently confirmed by another independent GWAS of CD presented by Rioux et al. [14]. In a recent GWAS in Korean population, Yang et al. [15] reported for the first time that ATG16L2 gene could be a novel susceptibility gene for CD and rs11235604 was a potential causal variant of the association. As suggested by the differences in genetic susceptibility of CD between different populations, the
Correspondence: Dr. Weichang Chen, Department of Gastroenterology, the First Affiliated Hospital of Soochow University, Shizi Street No. 256, Suzhou 215006, China. E-mail: [email protected]
(Received 14 April 2014; revised 10 June 2014; accepted 15 June 2014) ISSN 0036-5521 print/ISSN 1502-7708 online 2014 Informa Healthcare DOI: 10.3109/00365521.2014.936031
1202
J. Zhang et al.
heritability of CD has not been fully explained. As well-established CD susceptibility genes in Caucasian population, ATG16L1 and IL23R failed to be replicated in Asians [16–24]. Yamazaki et al. [20] selected 29 SNPs out of regions of the IL23R and ATG16L1. After comparing the distributions of these markers between 484 CD patients and 439 controls from the Japanese population, the authors concluded that there is a significant ethnical divergence between Japanese and Caucasian populations regarding the association of IL23R and ATG16L1 with pathogenesis of CD. In an independent study performed in the Korean population, the association of the IL23R gene with CD was supported, although ATG16L1 was still reported to have no association with CD [21]. These inconclusive results can be partially due to ethnic difference, inherent genetic heterogeneity, and the substantially small effect of this locus on the development of CD. Considering that there exist remarkable ethnic differences concerning the heritability of CD, replication studies would be helpful in finding remaining risk loci and in expanding our understanding of the genetic architecture of CD. To our knowledge, there is still a lack of knowledge concerning the association of ATG16L1 and IL23R gene with CD in the Chinese population. The primary purpose of this study was to investigate whether ATG16L1 and IL23R gene can function as susceptibility loci for CD in Chinese population and to further narrate their roles in the etiology of CD.
classified into four types including terminal ileum, colon, ileocolon, and isolated upper disease. Disease behavior was grouped as follows: B1, non-stricturing and non-penetrating; B2, stricturing; B3, penetrating; p, perianal disease modifier defined as the presence of a perianal abscess, fistulas, and/or ulceration. Disease severity was determined by the presence of prior treatment, such as surgical intervention and corticosteroids, immunosuppressant agents, or infliximab treatment.
Genotyping protocols Blood samples were collected from the subjects for the extraction of DNA, with informed consent obtained from the subjects. Genomic DNA was extracted from blood leukocytes using standard protocols (Qiagen K. K., Tokyo, Japan). SNPs rs2241880 of ATG16L1 and rs11209026, rs1004819, and rs1495965 of IL23R were genotyped using TaqMan SNP Genotyping Assay according to the manufacturer’s instructions, which was read with an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). Besides, three SNPs of CARD15 (rs2066844, rs2066845, and rs2066847) were genotyped to detect the potential interaction between CARD15 and ATG16L1, as previously reported [13]. About 20% of the samples were selected randomly to validate the reproducibility of the genotyping results.
Methods Subjects Under the approval of the Local Ethical Committee, the current multicenter study retrospectively included 420 patients with CD from Chinese Han population who had received treatment since 2007. CD was diagnosed according to the standard clinical, radiological, endoscopic, and histological criteria [3]. A total of 450 age- and ethnic-matched healthy volunteers were recruited as the control group after ruling out those with known IBD, family history of IBD, or other autoimmune disease. Clinical characteristics of the patients Data on the clinical characteristics of the patients were obtained according to Montreal classification, including age at diagnosis, disease location, disease behavior, and previous treatment [25]. Briefly, age at diagnosis was classified as follows: £16 years, between 17 and 40 years, and >40 years. Disease location was
Statistical analysis Clinical characteristics such as age, height, and weight between patients and controls were compared by means of an independent t-test. The Hardy-Weinberg equilibrium (HWE) test was used for each SNP between the patient and control groups. The differences of genotype and allele distributions between cases and controls were analyzed with the Chisquared test, which was also applied to subgroup analysis of disease groups. Odds ratio (OR) of the significant associated SNP was calculated using the ancestral allele as reference. Logistic regression analysis was used to test the potential statistical interaction between ATG16L1 and CARD15. Modeled as a 3-level factor, CARD15 statuses were grouped as wild-type, heterozygous carriers of one of the three common CARD15 mutations, or homozygous mutant carriers [13]. The SPSS software (version 13.0, Chicago, IL, USA) was used for statistical analyses. A p-Value of 40 years Location† Terminal ileum Colon Ileocolon Upper GI disease Behavior¶ Non-stricturing and non-penetrating Stricturing Penetrating Perianal disease
Number
Percentage (%)
30 280 110
7.1 66.7 26.2
82 58 248 32
19.5 13.8 59.1 7.6
165 91 102 62
39.3 21.7 24.3 14.7
† Patients can be added to the other three groups when having concomitant upper GI disease. ¶ Patients can be added to the other three groups when there is concomitant perianal disease. Abbreviation: GI = Gastrointestinal.
1203
Distributions of genotype and allele frequencies of ATG16L1 and IL23R in the cases and the controls are shown in Table III. There were significant differences between the two groups in terms of genotype and allele frequencies of ATG16L1. Patients were found to have significantly higher proportion of genotype GG (18.3%), when compared with the normal controls (12.4%). Allele G was found to be the risk allele for the disease (34.3% vs. 29.0%, p = 0.016) with an OR of 1.18. For IL23R and CARD15, all six SNPs were found not to be associated with the development of CD. The logistic regression analysis indicated that ATG16L1 contributed independently to the risk of the disease (p = 0.028), which however had no significant association with CARD15 (p = 0.72). Comparisons of subgroup genotypes according to the Montreal Classification Comparisons of genotypes of CD among the subgroups classified according to the Montreal Classification are shown in Table IV. None of the four SNPs was found to be associated with the clinical features of the CD, including age at diagnosis, disease location, and behavior. Discussion CD is a complex disease with strong genetic factors that can lead to aberrant mucosal inflammation of the intestinal tract when combined with environmental or microbial triggers. During the past decade, there has been an increasing interest in the exploration of genetic variants associated with CD [4,26–29]. To date, over 100 independent genetic loci were reported to potentially play a role in the pathogenesis of CD, thus yielding an extensive framework that unraveled the genetic basis and clinical predictors of CD. As a critical tool for the identification of susceptibility genes for complex diseases, GWAS can produce more robust results based on its biological, technical, and statistical foundations. Among those reported CD-associated genetic loci, the IL23R gene and the ATG16L1 gene were both identified by GWAS and were subsequently validated in large independent Caucasian samples. Dusatkova et al. [17] confirmed the role of IL23R and ATG16L1 in the CD susceptibility in the Czech population and found a weak protective effect of IL23R p.381Gln against upper gastrointestinal tract involvement. Lakatos et al. [22] confirmed that both ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Moreover, Latiano et al. [23] reported that polymorphisms of IL23R and ATG16L1 were associated
1204
J. Zhang et al.
Table III. Associations between ATG16L1, IL23R, and CARD15 gene polymorphisms and disease susceptibility of CD. Genotypea SNPs rs2241880 (A/G) rs11209026 (G/A) rs1004819 (T/C) rs1495965 (G/A) rs2066844 (T/C) rs2066845 (C/G) rs2066847 (insC)
Allele type
p-Value
Patient (n = 420)
Control (n = 500)
Patient
Control
Genotype
Allele
Odds ratio (95%CI)b
209/134/77 403/12/5 151/209/60 95/212/113 1/1/418 1/0/419 0/0/420
272/166/62 473/18/9 170/237/93 131/225/144 1/0/499 0/0/500 1/1/498
552/288 818/22 511/329 402/438 3/837 2/838 0/840
710/290 964/36 577/423 487/513 2/998 0/1000 3/997
0.04 0.61 0.21 0.23 0.54 0.52 0.43
0.016 0.28 0.18 0.74 0.66 0.21 0.25
1.18 0.72 0.93 1.02 0.998 1.003 0.998
(1.03–1.35) (0.43–1.22) (0.83–1.03) (0.93–1.11) (0.994–1.003) (1.001–1.006) (0.994–1.001)
The three values in the ‘genotype’ column indicate the numbers of homozygotes with respect to the ancestral allele, heterozygotes, and homozygotes with respect to the wild-type allele, respectively, that is, for rs2241880, the three values indicate numbers of genotype AA, AG, and GG, respectively. b Calculated for the alleles. a
Table IV. Comparisons of subgroup genotypes according to the Montreal Classification.
Age at diagnosis £16 years (n = 30) 17–40 years (n = 280) >40 years (n = 110) p-Value Location Terminal ileum (n = 82) Colon (n = 58) Ileocolon (n = 248) Upper GI disease (n = 32) p-Value Behavior Non-stricturing and non-penetrating (n = 165) Stricturing (n = 91) Penetrating (n = 102) Perianal disease (n = 62) p-Value
a
rs11209026
rs1004819
rs1495965
16/10/4 137/90/53 56/34/20 0.95
29/1/0 268/8/4 106/3/1 0.96
11/15/4 99/141/40 41/53/16 0.3
6/16/8 65/140/75 24/56/30 0.99
42/27/13 27/22/9 124/74/50 16/11/5 0.89
79/2/1 55/2/1 239/7/2 30/1/1 0.95
28/42/12 22/28/8 88/124/36 13/15/4 0.99
18/42/22 12/28/18 58/125/65 7/17/8 0.99
86/49/30 42/33/16 52/30/20 29/22/11 0.92
159/4/2 86/4/1 98/3/1 60/1/1 0.96
55/86/24 37/41/13 37/48/17 22/34/6 0.77
35/83/43 21/48/22 23/50/29 12/31/19 0.98
rs2241880
Abbreviation: GI = Gastrointestinal. The indications of three values for genotypes were the same with those in Table III.
a
with CD in both adult- and pediatric-onset subsets from Italy population. Collectively, these findings supported the theory that IL23R and ATG16L1 were common susceptible genes to CD in Caucasian populations. To date, there is still a lack of common susceptible variants of CD between Asian and Caucasian populations, possibly due to great ethnical diversity of susceptible genes to CD between Asian and European ancestries. Although IL23R and ATG16L1 were reported as well-established predisposition genes of CD in Caucasian population, these candidate genes were not common variants to CD among the Japanese and Caucasian populations [20]. Differently, the association of IL23R with Korean CD patients was confirmed, partly supporting the previous findings in Caucasian populations [21]. To better clarify the role of these two genes in the pathogenesis of CD in Asian
population, we examined the association of IL23R and ATG16L1 with CD in a large cohort of Chinese patients. In our study, a coding SNP in the ATG16L1 gene (rs2241880) was demonstrated to be associated with the susceptibility of CD in Chinese population. Moreover, G allele was confirmed to be the risk allele of the disease which can increase the incidence of CD by 1.18-folds. The contributable risk of the ATG16L1 G allele reported in our study (OR = 1.18) was similar to the published studies performed in UK (OR = 1.30) and Hungary (OR = 1.39), and a meta-analysis performed in German, Dutch, and Hungarian cohorts (OR = 1.32) [22,30,31]. Regarding polymorphism of the IL23R gene, our study failed to confirm the recently described associations between variants in the IL23R gene in Caucasian or Korean CD patients. Recently Yamazaki et al. [20] also failed to find any positive association of the
Association of IL23R and ATG16L1 with Crohn’s disease IL23R gene polymorphism with CD in the Japanese population. We believe that this discrepancy can be attributed to the distinct ethnic difference of genetic backgrounds of CD that has been reported previously for other genes between Asian and Caucasian populations. In the current study, the possible interaction between ATG16L1 and CARD15 in contributing to the risk of CD was also investigated. In line with the finding of Leong et al. [32], we found that CARD15 was not significantly associated with the occurrence of CD in Chinese population. Moreover, through logistic regression analysis, we confirmed that ATG16L1 is a CD susceptibility gene and found no evidence of potential interaction with CARD15. These findings were mostly consistent with previous studies which failed to confirm the association of CARD15 with CD in Asian populations [33,34]. Since genotypes remain unchanged during the lifetime, the identification of genetic variants that may predict disease behavior and progress are of great interest, usually superior to other predictive factors such as serological or clinical parameters. With the ever-growing number of susceptibility loci in CD, there are also a few variants reported to be significantly associated with clinical outcome and prognosis. One example was the identification of NOD2/ CARD15, which was found to be associated with a more aggressive clinical course involving higher risk of intestinal strictures and need for early surgical treatment [8,27]. Other genetic variants that can affect the disease behavior include SNP rs4958847 of IRGM, which was significantly associated with frequency of surgery in patients with ileocecal CD [35]. Besides, Prager et al. [9] demonstrated that patients with the C risk allele of rs10758669 in JAK2 could have increased epithelial permeability, indicating that the influence on disease behavior may result in alterations in overall intestinal permeability. In the current study, we also investigated the relationship between genotypes and phenotypes including the age at diagnosis, disease location, behavior, and the disease severity. Although remarkably associated with the occurrence of CD, ATG16L1 gene seems to have no significant relationship with the phenotypes mentioned above. Further, in agreement with previous studies, we did not observe any association of the polymorphism of the IL23R gene with the disease phenotype. In conclusion, it is unlikely that these two genes are implicated in the progression of CD. To our knowledge, this is the first replication study investigating the association of ATG16L1 and IL23R gene with CD in the Chinese population, which provides novel insights into the role of these two genes in the pathogenesis of CD. One limitation of our study lies in that hierarchical clustering was not
1205
applied to the statistical analysis, which can potentially provide some useful information on the association between the genetic variants and the risk of the disease. In summary, our results indicate that the original GWAS finding on ATG16L1 gene should be robust and that this gene does play a role in the pathogenesis of CD in the Chinese population. However, the role of IL23R gene in the development of CD remains obscure, and further investigations are warranted to clearly illustrate its association with this disease. Collectively, these genetic data are helpful information that will possibly lead to the ultimate discovery of etiologies for CD.
Acknowledgment The authors sincerely thank Dr Leilei Xu from the Department of Spine Surgery of Drum Tower Hospital who made the primary contribution to the proofreading of the manuscript. Declaration of interest: The authors involved in this study do not have anything to disclose regarding funding or any conflicts of interest with respect to this manuscript.
References [1] Gibson J, Collins A, Morton N. Individual disease risk and multimetric analysis of Crohn disease. Proc Natl Acad Sci USA 2008;105:15843–7. [2] Mazal J. Crohn disease: pathophysiology, diagnosis, and treatment. Radiol Technol 2014;85:297–316. [3] Vermeire S, Van Assche G, Rutgeerts P. Classification of inflammatory bowel disease: the old and the new. Curr Opin Gastroenterol 2012;28:321–6. [4] Ellinghaus D, Ellinghaus E, Nair RP, Stuart PE, Esko T, Metspalu A, et al. Combined analysis of genome-wide association studies for Crohn disease and psoriasis identifies seven shared susceptibility loci. Am J Hum Genet 2012; 90:636–47. [5] Esaki M, Furuse M, Matsumoto T, Aoyagi K, Jo Y, Yamagata H, et al. Polymorphism of heat-shock protein gene HSP70-2 in Crohn disease: possible genetic marker for two forms of Crohn disease. Scand J Gastroenterol 1999; 34:703–7. [6] Meucci G, Vecchi M, Torgano G, Arrigoni M, Prada A, Rocca F, et al. Familial aggregation of inflammatory bowel disease in northern Italy: a multicenter study. The Gruppo di Studio per le Malattie Infiammatorie Intestinali (IBD Study Group). Gastroenterology 1992;103:514–19. [7] Russel MG, Pastoor CJ, Janssen KM, van Deursen CT, Muris JW, van Wijlick EH, et al. Familial aggregation of inflammatory bowel disease: a population-based study in South Limburg, The Netherlands. The South Limburg IBD Study Group. Scand J Gastroenterol Suppl 1997;223:88–91. [8] Alvarez-Lobos M, Arostegui JI, Sans M, Tassies D, Plaza S, Delgado S, et al. Crohn’s disease patients carrying Nod2/
1206
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
J. Zhang et al.
CARD15 gene variants have an increased and early need for first surgery due to stricturing disease and higher rate of surgical recurrence. Ann Surg 2005;242:693–700. Prager M, Buttner J, Haas V, Baumgart DC, Sturm A, Zeitz M, et al. The JAK2 variant rs10758669 in Crohn’s disease: altering the intestinal barrier as one mechanism of action. Int J Colorectal Dis 2012;27:565–73. Sugimura K, Taylor KD, Lin YC, Hang T, Wang D, Tang YM, et al. A novel NOD2/CARD15 haplotype conferring risk for Crohn disease in Ashkenazi Jews. Am J Hum Genet 2003;72:509–18. Tomer G, Wetzler G, Keddache M, Denson LA. Polymorphisms in the IBD5 locus are associated with Crohn disease in pediatric Ashkenazi Jewish patients. J Pediatr Gastroenterol Nutr 2009;48:531–7. Yamazaki K, Takazoe M, Tanaka T, Ichimori T, Saito S, Iida A, et al. Association analysis of SLC22A4, SLC22A5 and DLG5 in Japanese patients with Crohn disease. J Hum Genet 2004;49:664–8. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 2007;39:207–11. Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet 2007;39:596–604. Yang SK, Hong M, Zhao W, Jung Y, Baek J, Tayebi N, et al. Genome-wide association study of Crohn’s disease in Koreans revealed three new susceptibility loci and common attributes of genetic susceptibility across ethnic populations. Gut 2014;63:80–7. Cadwell K, Patel KK, Komatsu M, Virgin HT, Stappenbeck TS. A common role for Atg16L1, Atg5 and Atg7 in small intestinal Paneth cells and Crohn disease. Autophagy 2009;5:250–2. Dusatkova P, Hradsky O, Lenicek M, Bronsky J, Nevoral J, Kotalova R, et al. Association of IL23R p.381Gln and ATG16L1 p.197Ala with Crohn disease in the Czech population. J Pediatr Gastroenterol Nutr 2009;49:405–10. Fujita N, Saitoh T, Kageyama S, Akira S, Noda T, Yoshimori T. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts. J Biol Chem 2009; 284:32602–9. Raju D, Hussey S, Jones NL. Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans. Autophagy 2012;8:1387–8. Yamazaki K, Onouchi Y, Takazoe M, Kubo M, Nakamura Y, Hata A. Association analysis of genetic variants in IL23R, ATG16L1 and 5p13.1 loci with Crohn’s disease in Japanese patients. J Hum Genet 2007;52:575–83. Yang SK, Park M, Lim J, Park SH, Ye BD, Lee I, et al. Contribution of IL23R but not ATG16L1 to Crohn’s disease susceptibility in Koreans. Inflamm Bowel Dis 2009;15: 1385–90. Lakatos PL, Szamosi T, Szilvasi A, Molnar E, Lakatos L, Kovacs A, et al. ATG16L1 and IL23 receptor (IL23R) genes
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
are associated with disease susceptibility in Hungarian CD patients. Dig Liver Dis 2008;40:867–73. Latiano A, Palmieri O, Valvano MR, D’Inca R, Cucchiara S, Riegler G, et al. Replication of interleukin 23 receptor and autophagy-related 16-like 1 association in adult- and pediatric-onset inflammatory bowel disease in Italy. World J Gastroenterol 2008;14:4643–51. Amre DK, Mack DR, Morgan K, Krupoves A, Costea I, Lambrette P, et al. Autophagy gene ATG16L1 but not IRGM is associated with Crohn’s disease in Canadian children. Inflamm Bowel Dis 2009;15:501–7. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, Brant SR, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005;19:5A–36A. Chapman JM, Onnie CM, Prescott NJ, Fisher SA, Mansfield JC, Mathew CG, et al. Searching for genotypephenotype structure: using hierarchical log-linear models in Crohn disease. Am J Hum Genet 2009;84:178–87. Cleynen I, Gonzalez JR, Figueroa C, Franke A, McGovern D, Bortlik M, et al. Genetic factors conferring an increased susceptibility to develop Crohn’s disease also influence disease phenotype: results from the IBDchip European Project. Gut 2013;62:1556–65. Elding H, Lau W, Swallow DM, Maniatis N. Dissecting the genetics of complex inheritance: linkage disequilibrium mapping provides insight into Crohn disease. Am J Hum Genet 2011;89:798–805. Elding H, Lau W, Swallow DM, Maniatis N. Refinement in localization and identification of gene regions associated with Crohn disease. Am J Hum Genet 2013;92:107–13. Prescott NJ, Fisher SA, Franke A, Hampe J, Onnie CM, Soars D, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn’s disease and is independent of CARD15 and IBD5. Gastroenterology 2007;132:1665–71. Buning C, Durmus T, Molnar T, de Jong DJ, Drenth JP, Fiedler T, et al. A study in three European IBD cohorts confirms that the ATG16L1 c.898A>G (p.Thr300Ala) variant is a susceptibility factor for Crohn’s disease. J Crohns Colitis 2007;1:70–6. Leong RW, Armuzzi A, Ahmad T, Wong ML, Tse P, Jewell DP, et al. NOD2/CARD15 gene polymorphisms and Crohn’s disease in the Chinese population. Aliment Pharmacol Ther 2003;17:1465–70. 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. Croucher PJ, Mascheretti S, Hampe J, Huse K, Frenzel H, Stoll M, et al. Haplotype structure and association to Crohn’s disease of CARD15 mutations in two ethnically divergent populations. Eur J Hum Genet 2003;11:6–16. Sehgal R, Berg A, Polinski JI, Hegarty JP, Lin Z, McKenna KJ, et al. Mutations in IRGM are associated with more frequent need for surgery in patients with ileocolonic Crohn’s disease. Dis Colon Rectum 2012;55:115–21.
Copyright of Scandinavian Journal of Gastroenterology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
