UvA-DARE (Digital Academic Repository) On genes and inflammatory bowel disease Stokkers, P.C.F. Link to publication

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1 UvA-DARE (Digital Academic Repository) On genes and inflammatory bowel disease Stokkers, P.C.F. Link to publication Citation for published version (APA): Stokkers, P. C. F. (1999). On genes and inflammatory bowel disease. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 14 Apr 2019

2 Chapter 7 Genetic Analysis of Inflammatory Bowel Disease in a Large European Cohort Supports Linkage to Chromosomes 12 and 16 ME. Curran ', K.F. Lau ', C. Mathew 2, J. Hampe, S. Bridger, A.J.S. Macpherson 2, L.R. Cardon 2, H. Sakul ', T.J.R. Harris ', P.CF. Stokkers 5, S.J.H. van Deventer 5, M. Mirza 2, A. Raedler 6, W. Kruis 7, U. Meckler \ D. Theuer 9, T. Herrmann \ P. Gionchetti 10, J. Lee ", S. Schreiber \ J. Lennard-Jones. Gastroenterology, 1998; 115 (5): AxyS Pharmaceuticals Inc., La Jolla, C A/US A '. Guy's Hospital, London 1st Medical Dept., Cristian-Albrechts-University, Kiel/Germany. King's College School of Medicine, London 4. AMC, Amsterdam/Netherlands. Tabea 1BD Center, Hamburg/Germany 6. Kalk Hospital Cologne/Germany SchloBbergklinik, Gedern/Germany Gastroenterol 8. Clinic Heilbronn/Germany. Univ. Bologna/Italy '. St. Mark's Hospital, Harrow/UK ".

3 Chapter 7 ABSTRACT Inflammatory bowel disease (IBD) is a complex disorder of unknown etiology. Epidemiological investigations suggest a genetic basis for IBD. Recent genetic studies have identified several IBD linkages. Significance of these linkages will be determined by studies in large patient collections. The aim of this study was to replicate IBD linkages on chromsomes 12 and 16 in a large European cohort. 359 affected siblingpairs from 274 kindreds were genotyped using microsatellite markers spanning chromosomes 12 and 16. Affection status of the sibling pairs was defined as Crohn's disease or ulcerative colitis. Non-parametric statistical analyses demonstrated linkage for both chromosomes. Two-point results for chromosome 12 peaked at D12S303 (LOD 2.15, p=0.003) for Crohn's disease and D12S75 (LOD 0.92, p=0.03) for ulcerative colitis. Multipoint analyses produced a peak LOD of 1.8 for Crohn's disease. Chromosome 16 demonstrated linkage for Crohn's disease at marker D16S415 (LOD 1.52, p=0.007). Multipoint support peaked above markers D16S409 and D16S411 (LOD= 1.7). These data are consistent with linkage of IBD to chromosomes 12 and 16. The replication of genetic risk loci in a large independent family collection indicates important and common susceptibility genes in these regions and will facilitate identification of genes involved in IBD. 130

4 Linkage analysis chromosomes 12 & 16 INTRODUCTION Inflammatory bowel disease (IBD) is a disorder of unknown etiology characterized by chronic relapsing inflammation of the gastrointestinal tract. On the basis of clinical and histopathological features, IBD is categorized into two subtypes, Crohn's disease (CD) and ulcerative colitis (UC). The phenotypic overlap between CD and UC, combined with a lack of clear biochemical markers, complicates differential diagnosis and clinical management of IBD. Elucidation of genetic factors involved in predisposition to IBD will facilitate our understanding of these disorders and provide for improved diagnosis and treatment Epidemiologic studies have consistently shown a familial clustering of IBD ( 1-3) as well as an increased concordance among monozygotic twins (4, 5). The underlying genetic basis of IBD is likely to be complex, suggesting that multiple molecular variants will combine to interact with multiple environmental factors, ultimately producing a spectrum of disease severity. Functional hypotheses for IBD have centered predominantly on the mucosal immune system and molecules that mediate cell-cell interactions and activation of lymphocytes. Many studies have attempted to implicate genes by showing association with IBD phenotypes. The human leukocyte antigens (HLA) have been studied extensively and have produced conflicting results. Several studies have demonstrated association with HLA class 1 (6-9) and II (10-12) genes while others have failed to detect a significant influence of the HLA genotype on IBD (13-17). Other factors implicated by association include: ICAM-1 (18), IL-1B and ILl-ra (19-20), TNT-A (21), natural resistance-associated macrophage protein gene (22), and intestinal mucin-2 (23). Thus far the analysis of functional candidate genes has not provided conclusive evidence for an etiologic role in IBD. Therefore, a systematic genomewide genetic approach was indicated. Genome-wide linkage analyses are beginning to provide insight into the genetic basis of IBD and are an important step towards isolating the molecular determinants of this disorder. Many putative linkages (24-25) have been reported suggesting significant heterogeneity of IBD. Hugot and colleagues (24) defined the first IBD linkage and localized a susceptibility locus to chromosome 16. This finding is supported by a study of North American families for CD, but not for UC (26). This observation is complicated, however, by results from analyses of European families which support chromosome 16 linkage to the UC phenotype (27). A second genomewide scan consisting of European sibling-pair families identified strong linkage to 131

5 Chapter 7 several IBD loci on chromosomes 3, 7 and 12, but provided little support for the chromosome 16 locus. Genetic analyses of complex diseases have indicated the need to replicate primary linkage findings by analysis of large family collections (28, 29). In this study we conduct a replication study of IBD linkages to both chromosomes 12 and 16 in a large cohort of families ascertained from Northern Europe. MATERIALS AND METHODS Patient Recruitment and Phenotyping Individual kindreds consisting of at least two siblings diagnosed with either Crohn's disease or ulcerative colitis were identified and collected in collaboration with IBD programs located at the following European institutions: King's College School of Medicine, Guy's Hospital and St. Mark's Hospital (London, UK), Charité University Hospital (Berlin, DE), AMC (Amsterdam), Tabea IBD Center (Hamburg, DE), Kalk Hospital (Cologne, DE) and other central European centers. Informed, written consent was obtained from all study participants. Collection protocols were subject to approval by institutional review committees at each participating center. Diagnosis of IBD and definition of Crohn's disease and ulcerative colitis phenotypes were determined by standard diagnostic criteria (30). The structure of the investigated family cohort is presented in table 1, with the number of affected sibling pairs (ASP) totaling 359. Table 1: Structure of the family cohort: affected sibling pairs are ordered according to sibship size and disease type (Mixed refers to UC-CD sibships) within the family. The number of sibships and affected sib pairs (ASP in brackets) is given in each category. Sibship size CD UC Mixed All 2 119(119) 78 (78) 39(39) 236 (2.36) 3 14(42) 12(36) 9(27) 35(105) 4 1(6) 0 (0) 2(12) 3(18) Total 134(167) 90(114) 50 (78) 274 (359) 132

6 Linkage analysis chromosomes 12 & 16 Genotyping Blood samples were obtained from all siblings and - when possible - from one or both parents. Genomic DNA was prepared using the Puregene system (Gentra Systems, Minneapolis, MN). A total of 30 highly polymorphic microsatellite markers, 20 spanning chromosome 12 and 10 spanning chromosome 16 were genotyped using fluorescent methods as described (31). Briefly, individual DNA samples were arrayed in 96 well microtiter plates and subjected to PCR with individual marker amplicons. Marker sets were pooled post-pcr, denatured and electrophoresed on denaturing Polyacrylamide gels. Data collection utilized ABl 377 automated DNA sequencers and data analyses were performed with the Genescan 672 and Genotyper software programs. Allele analyses and individual allele calling were performed as described (31,32). Stalistical Analyses Two point and multipoint analyses were performed using the Mapmaker/SIBS program. (29) Probability values for all maximum LOD scores (MLS) were determined by simulation of the genotypes in the families, in which all allele frequencies, familial relationships, phenotypic data, and missing genotype patterns were preserved. Simulations were run 5,000 times for each MLS score. An additional program, TWOLOC (33), was used to conduct tests of interactions between the loci on chromosomes 12 and 16. RESULTS Maximum LOD score (MLS) curves for CD, UC and all IBD pairs for chromosome 12 are shown in Figure 1A. Although the magnitude of MLS varies among disease categories, the linkage region is defined by the same markers for Crohn's and allpairs, D12S85 and D12S346, in a 47cM region. MLS curves for both Crohn's and allpairs had a similar pattern. The UC disease category had a somewhat similar shape, but was associated with smaller LOD scores. The highest multipoint MLS was 1.82 for Crohn's (D12S303-DI2S326) and 1.60 (D12S379-D12S88) for all-pairs. Twopoint analyses confirmed the presence of these peaks (Table 2). Two-point MLS was 2.15 (p=.003) at D12S303 for Crohn's and 1.10 (p=.02) at D12S88 for all-pairs, with average allele sharing of 0.60 and 0.55, respectively. The apparent shoulder proximal to the linkage peak seemed to replicate between Crohn's and all-pairs, and was included in the 47cM region. 133

7 Chapter 7 Figure 1: Multipoint MLS curves for chromosomes 12 (Panel A) and 16 (Panel B). Multipoint analyses were carried out using the MAPMAK.ER/SIBS program. Results for Crohn's disease (CD. thick solid line), ulcerative colitis (UC. thin solid line) and all-pairs (dashed line) are shown. Genetic distances between markers, estimated from the data set, arc shown in Table 2. Marker allele frequencies were calculated from the full sample. Average heterozygosity was 0.77 for chromosome 12 (range: ), and 0.76 for chromosome 16 (range: ). r :.6 iv f / 1 / A y / ^^^^r i \ / \,- s/ V ê / >* ê \\ / Q Q 134

8 Linkage analysis chromosomes 12 & 16 Table 2 Maximum LOD Scores (MLS) and empirical p-values (in brackets) from analyses of ulcerative colitis sib pairs. Crohn's disease, and all-pairs combined on chromosomes 12 and 16. UC CD All Marker Distance Average MLS (P)" Average MLS (P) 1 ' Average MLS (P) b (Alf sharing sharing sharing D12S (.54) (.58) (.57) D12S (.28) (.56) ,00 (.40) D12S (.56) (25) (.54) D12S (.07) (.45) (.39) D12S (.14) (.56) (.56) D12S (.25) (.02) (.11) D (.04) (.12) (.17) D12S (.35) (.05) ( 10) D12S (.04) (.10) (.03) D12S (.03) (.003) (.02) D12S (.30) (007) (.10) D12S (.33) (035) (.09) D12S (.55) (.08) (.02) D12S (.20) (.08) (.13) D12S (.53) (.11) (.37) D12S (.54) (.53) (.37) D12S (.11) (.58) (.51) D12S (.53) (29) (.31) D12S (.30) (.41) (.11) D12S (25) (.44) (.I7) D16S (.08) (.45) (.11) D16S (.26) (.41) (.24) D16S4Ü (.42) ,40 (.006) (.042) D16S (.32) (.01) (.01) D16S (.07) (.01) (.003) D16S4Ü8) (.55) ( (.15) D16S (.17) (.055) (03) D16S (.13) (.43) (.57) D16S (.04) (.018) (.02) D16S (.16) (.58) (.37) Ä Marker distances were calculated from the families analyzed using the program CRIMAP (35) B Probability values for all maximum LOD scores (MLS) were determined by Monte Carlo simulation of the genotypes in the families under analysis, in which all allele frequencies, familial relationships, phenotypic data, and missing genotype patterns were preserved. Probability values shown thus represent the proportion of times that MLS values greater than or equal to the observed statistic were observed in the simulations. The simulations were conducted times for each MLS score Complete genotype data for the markers on chromosome 12 were available for and 359 affected sibling pairs tor l.'c. Crohns disease, and all families combined, respectively. Complete genotype data for the markers on chromosome 16 were available tor and 358 affected sibling pairs for UC. Crohn's disease, and all families combined, respectively. 135

9 Chapter 7 MLS curves for Crohn's and all-pairs were similar for chromosome 16, with two apparent peaks at D16S409-DI6S411 and at D16S503 (figure IB). The UC category again showed a different, and mostly nonsignificant, LOD curve pattern. The highest multipoint MLS was observed for Crohn's (1.71) over DI6S409 and D16S41 1. The two-point LOD scores were 1.52 (p=007) and 1.4 (p=.006) for D16S411 and D16S409, respectively. Average allele sharing was 0.58 and 0.59, respectively. The second peak carried a multipoint MLS of approximately 1.4 and 1.3 for all-pairs and Crohn's, respectively. Two-point scores were marginally significant for either disease category (table 2). Two loci, D12S303 and D16S411, closest to the peak on each chromosome, were chosen to investigate possible epistatic interactions using the TWOLOC program. (33) Two-point MLS results from TWOLOC were similar to the results obtained from single point MAPMAKER/SIBS (2.29 for D12S303 and 1.59 for D16S41 1). Two different models were then tested: 1) a general model in which both additive effects and epistatic interaction were allowed and 2) an additive model with additive effects but no epistatic interactions. MLS values for the two models were 3.93 and 3.90, respectively. This indicates no significant evidence for epistatic interaction between the two loci suggesting that their effects may be additive. DISCUSSION Genome-wide linkage analyses have implicated multiple candidate regions for IBD. (24, 25) The strongest evidence for linkage in the initial studies was provided for the susceptibility regions on chromosomes 12 and 16. To evaluate the importance of these linkages in IBD, it is important to replicate these findings in a large independent sample. In this study, we demonstrate support for IBD susceptibility regions on both chromosome 12 and 16. Interestingly, these data show strongest support for both loci when analyzed for the CD phenotype and for all-pairs. This observation is consistent with conclusions from prior studies of chromosome 16. (26, 34) To investigate the genetic relationship of these susceptibility loci, we tested for epistatic interaction using the TWOLOC program. (33) Results from these analyses suggest that the loci on chromosome 12 and 16 influence the phenotype additively rather than epistatically. The relevance of different levels of statistical significance with regard to potential genetic differences between the CD and UC phenotypes is unclear. Interpretation of these data is confounded by the difficult clinical overlap between CD and UC (30), and the small number of UC pairs as compared to CD pairs available for 136

10 Linkage analysis chromosomes 12 & 16 analysis. Intriguingly, while the two-point results for UC pairs alone are not significant, the underlying multipoint curves are similar in shape to the CD and allpairs curves. It is possible, therefore, that chromosomes 12 and 16 harbor genetic variants that are important for both CD and UC. This hypothesis is supported by a recent study of extended UC families which demonstrated support for a locus on chromosome 16 (27). This issue will not be resolved until the actual predisposing mutations are identified. In conclusion, these data support linkage to both chromosomes 12 and 16 in a large European family collection, suggest that these loci act in additive fashion, and provide important replication of linkage upon which efforts to identify IBD genes can build. ACKNOWLEDGMENTS The authors thank the physicians, IBD patients and their families for participating in this study. The cooperation of the German Crohn's and colits foundation (DCCV e. V), Dr. Wewalka /Linz, Dr. Knofloch / Wels, both in Austria, Prof. Lochs, Dr. Wedel, Dr. Ntimberg / Berlin, Dr. Herchenbach / Recklinghausen, Prof. Scheurlen / Wiirzburg, Dr. Demharter / Augsburg, Dr. Simon / Munich, Dr. Purrmann / Moers, Dr. Jessen / Kiel, Dr. Zehnter / Dortmund, Dr. Ltibke, Dr. Weismiiller / Koblenz, Dr. Eiche / Denkendorf, Dr. Schdnfelder / Aachen, Prof. Fleig, Halle all in Germany and Prof. Campieri/Bologna - Italy is gratefully acknowledged. We thank Sarah Shaw for assistance with the genetic analysis and acknowledge the expert technical assistance of Jonalyn Matusalem, Larenia Pedriguez and Hye Jin Yang. Grant support. This work was supported by AxyS Pharmaceuticals Inc, the National Association for Colitis & Crohn's disease (UK), Crohn's in Childhood Research Association (UK), the Sir Halley Stewart Trust, the Deutsche Forschungsgemeinschaft (Sehr 512/1-2) and MFG. REFERENCES Orholm M. Munkholm P. Langholz E, Nielsen OH. Sorensen IA, Binder V. Familial occurrence of inflammatory bowel disease. New England Journal of Medicine 1991;324(2):84-8.

11 Chapter 7 2. Colombel JF. Grandbasticn B. Gower-Rousseau C, et al. Clinical characteristics of Crohn's disease in 72 families. Gastroenterology 1996:111(3): Satsangi J. Grootscholten C. Holt H. Jewell DP. Clinical patterns of familial inflammatory bowel disease. Gut 1996;38(5): Tysk C. Lindberg E. Jarnerot G. Flodenis-Mvrhed B. Ulcerative colitis and Crohn's disease in an unselected population of monozygotic and dizygotic twins. A study of hcritability and the influence of smoking. Gut 1988;29(7): Thompson NP, Driscoll R. Pounder RE. Wakefield AJ. Genetics versus environment in inflammatory bowel disease: results of a British twin study. BMJ 1996;312(7023): Glecson MH. Walker JS. Wentzel J. Chapman JA. Harris R. Human leucocyte antigens in Crohn's disease and ulcerative colitis. Gut 1972;13(6): van den Berg-Loonen EM. Dekker-Sacys BJ. Meuwissen SG. Nijenhuis LE. Engclfrict CP. Histocompatibility antigens and other genetic markers in ankylosing spondylitis and inflammatory bowel diseases. J Immunogcnet 1977:4(3): Biemond I. Burnham WR. D'Amaro J. Langman MJ. HLA-A and -B antigens in inflammatory bowel disease. Gut 1986;27(8): Purrmann J. Korsten S. Bertrams J. ct al. HLA-Haplotypsludie bei familiärem Morbus Crohn. Zeitschrift fur Gastroenterologie 1985;23(8): Fujita K. Naito S. Okabc N. Yao T. Immunological studies in Crohn's disease. 1. Association with HLA systems in the Japanese. J Clin Lab Immunol 1984;14(2): Toyoda H, Wang SJ, Yang HY. et al. Distinct associations of HLA class II genes with inflammatory bowel disease. Gastroenterology 1993;104(3): Nakajima A. Matsuhashi N, Kodama T, Yazaki Y. Takazoe M, Kimura A. HLA-linked susceptibility and resistance genes in Crohn's disease. Gastroenterology 1995; 109(5): Russell AS, Percy JS, Schlaut J, ct al. Transplantation antigens in Crohn's disease: Linkage of associated ankylosing spondylitis with HL-Aw27. Am J Dig Dis 1975;20(4): Delpre G, Kadish U. Gazit E, Joshua H, Zamir R. HLA antigens in ulcerative colitis and Crohn's disease in Israel. Gastroenterology 1980:78(6): Smolen JS. Gangl A, Polteraucr P. Menzel EJ. Mayr WR. HLA antigens in inflammatory bowel disease. Gastroenterology 1982:82(1 ): Satsangi J. Welsh KL Bunce M. et al. Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotypc in inflammatory' bowel disease. Lancet 1996:347(9010): Naom I, Lee J, Ford D, et al. Analysis of the contribution of HLA genes to genetic predisposition in inflammatory bowel disease. Am J Hum Genet 1996;59(l): Yang H. Vora DK. Targan SR. Toyoda H. Beaudet AL. Rotter Jl. Intercellular adhesion molecule 1 gene associations with immunologic subsets of inflammatory bowel disease. Gastroenterology 1995;109(2): Mansfield JC. Holden H. Tarlow JK, et al. Novel genetic association between ulcerative colitis and the anti-inflammatory cytokine intcrleukin-1 receptor antagonist. Gastroenterology 1994;106(3):

12 Linkage analysis chromosomes 12 & Bioque G, Crusius JB. Koutroubakis I. ct al. Allelic polymorphism in IL-1 beta and IL-1 receptor antagonist (IL-IRa) genes in inflammatory bowel disease. Clinical & Experimental Immunology 1995;102(2): Plevy SE, Targan SR, Yang H, Fernandez D. Rotter Jl, Toyoda H. Tumor necrosis factor microsatellites define a Crohn's disease-associated haplotype on chromosome 6. Gastroenterology 1996:110(4): Hofmeister A, Neibergs HL, Pokorny RM, Galandiuk S. The natural resistance-associated macrophage protein gene is associated with Crohn's disease. Surgery 1997; 122(2): 173-8; discussion Parkes M, Satsangi J, Simmons J. Bell JI. Lathrop GM. Jewell DP. Preliminary evidence links the MUC2 gene to inflammatroy bowel disease susceptibility. Gastroenterology 1997;112:A Hugot JP, Laurent-Puig P. Gower-Rousseau C. et al. Mapping of a susceptibility locus for Crohn's disease on chromosome 16. Nature 1996;379(6568): Satsangi J, Parkes M, Louis E, ct al. Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3. 7 and 12. Nature Genetics 1996;14(2): Ohmen JD, Yang HY, Yamamoto KK. et al. Susceptibility locus for inflammatory bowel disease on chromosome 16 has a role in Crohn's disease, but not in ulcerative colitis. Human Molecular Genetics 1996:5(10): Mirza MM, Lee J, Teare D. et al. Evidence of linkage of the inflammatory bowel disease susceptibility locus on chromosome 16 (IBD1) to ulcerative colitis. J Med Genet 1998;35(3): Suarez BK. Hampe CL. Van Eerdewegh PD. Problems in replicating linkage claims in psychiatry. In: Gershon ES, Cloninger CR. eds. Genetic Approaches to Mental Disorders. Washington: American Psychiatric Press, 1994: Lander E. Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nature Genetics 1995;ll(3): Podolsky DK. Inflammatory bowel disease (1). New England Journal of Medicine 1991;325(13): Hall J. Nanthakumar E. Automated fluorescent gcnotyping. In: Boyle AL. ed. Current protocols in human genetics. John Wiley & Sons, 1997: Idury RM. Cardon LR. A simple method for automated allele binning in microsatellite markers. Genome Res 1997:7(11): Farrall M. Affected sibpair linkage tests for multiple linked susceptibility genes. Genet Epidemiol 1997; 14(2): 1Ü Parkes M, Satsangi J, Lathrop GM, Bell JI, Jewell DP. Susceptibility loci in inflammatory bowel disease. Lancet 1996:348(9041): Lander ES. Green P. Construction of multilocus genetic linkage maps in humans. Proc Natl Acad Sei U S A 1987;84(8):

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