Characterizing IBD risk alleles on chromosome IP36

A708 AGA ABSTRACTS

GASTROENTEROLOGY Vol. 118, No.4

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THE INTERNATIONAL GENETICS CONSORTIUM CONFIRMS LINKAGE OF CROHN'S DISEASE TO A LOCUS ON CHROMOSOME 16 nann, The Ibd Genetics Consortium, Juleen A. Cavanaugh, The Canberra Hosp, Canberra, Australia. Background: Inflammatory bowel disease (IBD), consisting of Crohn's disease (CD) and ulcerative colitis (UC), is genetically complex. Two putative localizations on chromosomes 16 (mOl) and 12 (IBD2) have been identified [1,2], with varying degrees of support for linkage of CD andlor UC to these regions from subsequent studies. Power to detect linkage, particularly in a complex disease, is directly related to the number of families studied. Aims: To assemble a large sample of sibling pairs affected with IBD and their parents from eleven centers around the world to evaluate the significance of these putative localisations. Methods: We have genotyped 581 families (382CD:9IUC:108MIXED) with 6 microsatellite markers on each of chromosomes 12 and 16, spanning the previously reported IBDI and IBD2 loci. Non-parametric analyses were performed using the Aspex, Genehunter and Analyse programs, yielding concordant estimates. Results: We demonstrate very significant evidence for linkage between a locus on chromosome 16 and CD (Maximum Lod Score[MLS]=5.2) but not UC (MLS=O.I). We do not observe any differences between Jewish and non-Jewish families at this locus. We estimate that this locus is responsible for approximately 10 -IS % of the inheritance of CD. We show minimal support for a localisation on chromosome 12 where the MLS for IBD, UC and CD are 1.8, 1.2 and 1.1 respectively. Conclusions: While the IBDI gene has yet to be identified, this is the first complex disease where a novel locus has been identified by linkage analysis of genome scan data rather than by the candidate gene approach. Our results also demonstrate the importance of the systematic analysis of a large number of families to detect weak gene effects by linkage analysis for a complex disease. References: I Hugot, J.-P. et (1996) Nature 379: 821-8232 Satsangi, 1. et al (1996) Nature Genetics 14:199-202 3863 A GENOME-WIDE SCAN IN A CANADIAN INFLAMMATORY BOWEL DISEASE POPULATION REVEALS TWO NOVEL SUSCEPTIBILITY LOCI. John D. Rioux, Mark S. Silverberg, Mark J. Daly, A. Hillary Steinhart, Robin S. McLeod, Anne M. Griffiths, Shelley B. Bull, Gordon R. Greenberg, Zane Cohen, Eric S. Lander, Thomas 1. Hudson, Katherine A. Siminovitch, Whitehead InstitutelMIT Ctr for Genome Research, Cambridge, MA; Mount Sinai Hosp, Toronto, ON, Canada; Hosp for Sick Children, Univ of Toronto, Toronto, ON, Canada. Aims: Genetic factors play an important role in the etiology of Crohn's disease (CD) and ulcerative colitis (UC). As is consistent with the genetic heterogeneity of IBD, genome-wide scans of iridependent IBD populations have now identified multiple loci representing potential sites of IBD susceptibility genes. To further elucidate chromosomal loci relevant to IBD susceptibility, a genome-wide search has been undertaken in a Canadian IBD population. Methods: 181 IBD-affected sibling pairs (122 CD; 25 DC; 34 mixed) and their parents were recruited from the Greater Toronto Area and disease documented using standardized criteria at the MSH Inflammatory Bowel Disease Centre. Genotyping was performed using 312 microsatellite markers with an average spacing of 12 centiMorgans (cM). Nonparametric multipoint linkage analysis was performed using Genehunter 2.0. Results: Lirikage analysis of the genome scan data identified a locus on chromosome 19 of genome-wide significance (LaD 4.6; p=0.OOOOO3). This represents a novel IBD locus that contributes to genetic susceptibility in both CD and UC patients of Jewish and non-Jewish origin. Three other regions surpassed the threshold for suggestive linkage on chromosomes 5 (LaD 3.0;p=0.OOO2), 3 (LaD 2.4;p=0.OOO9) and 6 (LaD 2.3;p =0.OOII). Higher density mapping performed in the chromosome 5 region has identified a significant locus (LaD 3.9) important in the susceptibility to CD of both Jewish and non-Jewish individuals. The other two suggestive loci are within 20 cM of previously reported loci and higher density mapping is still required. Analysis of the data failed to show suggestive linkage to the previously reported IBDI and IBD2 loci. Exclusion mapping of the data demonstrated that loci of modest effect (As> 2) could be excluded for chromosome 12, however, on chromosome 16, only loci conferring a As > 4 could be excluded, suggesting IBD] may be a relevant susceptibility locus in this population. Conclusions: The search for IBD susceptibility genes has resulted in the identification of putative loci that only account for a fraction of the total heritability of IBD. This genome-wide analysis in a Canadian IBD population has identified two novel loci. Specifically, a CD susceptibility locus on chromosome 5 was identified as well as an IBD susceptibility locus on chromosome 19. Regions on chromosomes 3 and 6 also demonstrated evidence for suggestive linkage to IBD further supporting the hypothesis that substantial genetic heterogeneity exists in IBD.

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A GENOME SCAN AT 751 MICROSATELLITE LOCI REVEALS LINKAGE BETWEEN CROHN'S DISEASE AND CHROMOSOME 14Ql1-12, THE IBD4 LOCUS. Richard H. Duerr, M. Michael Barmada, Leilei Zhang, Roland Pfiitzer, Daniel E. Weeks, Univ of Pittsburgh, Pittsburgh, PA. Epidemiological studies have shown that genetic factors contribute to the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC). Recent genome scans, extension and replication studies have led to the identification of replicated linkage between CD and a locus on chromosome 16 (the IBDI locus), replicated linkage between IBD (especially UC) and a locus on chromosome 12q (the IBD2 locus), and significant but not replicated evidence for linkage between IBD and a locus on chromosome Ip (the putative IBD310cus). Since the estimated locus-specific relative risk to siblings of affected individuals (A,) for the two regions of replicated linkage do not account for the overall As in CD, and since the published genome scans in IBD show at least nominal evidence for linkage to regions on all but two chromosomes, we performed an independent genome scan at 751 microsatellite loci in 127 CD-affected relative pairs from 62 families. Single-point nonparametric linkage analysis using the GENEHUNTER-PLUS program shows evidence for lirikage to the adjacent DI4S261 and 014S283 loci on chromsome l4qIl-12 (LaD = 3.00 and 1.70, respectively), and the maximal multipoint LaD score is observed at DI4S261 (LOD = 3.60). In the multipoint analysis, nominal evidence for linkage (p-values < 0.05) is observed near D2S117 (LaD = 1.25), near O3S3045 (LaD = 1.31), between D7S40 and D7S648 (LaD = 0.91), and near DI8S61 (LaD = 1.15). Our finding of significant linkage to D14S261 and the finding of suggestive lirikage (multipoint LaD = 2.8) between CD and the same locus in an independent study by other investigators (Ma et al. Inflammatory Bowel Diseases 1999; 5:271-278) satisfies criteria for confirmed lirikage, so we propose that the region of interest on chromosome 14q11-12 should be designated as the IBD4 locus. 3865 CROHN'S DISEASE DIAGNOSIS BEFORE AGE 22 AND WITH GREATER SEVERITY OF DISEASE IDENTIFIES MULTIPLEX PEDIGREES AT GREATER RISK FOR LOCUS lBDl. Steven R. Brant, Carolien Panhuysen, Joan Bailey-Wilson, Sinda Lee, Jasdeep Mann, Patrick M. Rohal, Michael F. Picco, Barbara S. Kirschner, Stephen B. Hanauer, Judy H. Cho, Theodore M. Bayless, Johns Hopkins Univ, Baltimore, MD; Boston Univ, Boston, MA; NIH, Baltimore, MD; Univ of Chicago, Chicago, IL; Mayo Clin, Jacksonville, FL. Introduction: Linkage studies have identified a Crohn's disease (CD) locus that maps to the pericentromeric region of chromosome 16, IBDI. Of nine linkage investigations, six studies showed strong evidence for IBDI, but three showed no evidence. Familial CD occurs at a younger age of onset (median 22 versus 26) and with greater severity than sporadic CD. The purpose of this study was to determine if younger age of onset and more severe disease could identify pedigrees at greater risk for IBD] and possibly explain study differences. Methods: Age at diagnosis, surgical bowel resection and immunosuppresant use were determined in 226 affected relatives from 90 pure CD multiplex pedigrees, 37% Ashkenazi Jewish. Linkage analysis was performed using genotyping data from 14 chromosome 16 markers with Genehunter Plus. Sub-analyses were performed to determine the effect of age of diagnosis and disease severity on linkage evidence. Results: In 57 pedigrees (group I) with at least one relative having severe disease and disease diagnosed before age 22, there was genome- wide linkage evidence for locus IBDI. Peak non-parametric multipoint lod score (Mlod)was 3.8, p=2.9 x 10-5 at 39 cM, within the IBDI candidate region. In 33 pedigrees (group 2) with no severe disease or disease diagnosed >22 in all relatives Mlod was near zero from 10 to 80 eM. For all pure CD pedigrees Mlod = 2.12, p=0.OOI7. Group I pedigrees were more likely than group 2 pedigrees to have earlier diagnosis, even in non-proband affecteds, be of Ashkenazi Jewish ancestry and have more than 2 affected relatives. Conclusions: The combination of more severe disease with onset before age 22 identifies pedigrees at greatest risk for IBDI, or alternately excludes those pedigrees less likely to have a genetic source for familial disease aggregation. Such classifications may be useful to further refine the IBD] locus. 3866 CHARACTERIZING ran RISK ALLELES ON CHROMOSOME IP36. Denise K. Bonen, Richard Ramos, Sanggyu Lee, Sarah Corradino, Heidi M. Britton, Rafael Espinosa, Michelle M. LeBeau, Barbara S. Kirschner, Steven R. Brant, Jason Bodzin, Stephen B. Hanauer, Judy H. Cho, Univ of Chicago, Chicago, IL; Johns Hopkins Univ Sch of Medicine, Baltimore, MD; William Beaumont Hosp, Royal Oak, MI. Background: Linkage has been observed on chromosome Ip36 in pure CD and pure UC families but not "mixed" families (both CD and UC). Furthermore, linkage is observed in non-Ashkenazim Caucasian, but not Ashkenazim families. Epistasis was observed between chrlp36 and the CD locus, IBDi, on chromosome 16cen. Aim: To refine localization and characterize risk haplotypesfor IBD on chrl p36 in isolatedand outbred populations. Methods: PI plasmid contigs were developed using sequence-taggedsites in the region. Fluorescent-in-situ hybridization and radiation hybrid mapping

April 2000

were performed to determine map order. Novel, short tandem repeat markers (STRs) and single nucleotide polymorphisms (SNPs) were developed. Homozygosity mapping was performed in four multiply-affected American Iraqi Chaldean families, a genetic isolate with a high prevalence of IBD. Linkage and transmission-disequilibrium testing (TOn was performed in 135outbred families. Results: A shared haplotype (DlS507 to DIS1628) was observed over 27 cM between two unrelated Chaldean families, with homozygous sharing in at least one affected member in all families, narrowing the risk regionto 130kb nearDlS2697 and DIS3669. Confirmation of homozygosity was established with 34 SNPs in the region. Markers not in linkagedisequilibrium were typed in the outhred population. TOT using a single, randomly selected affected from each multiplex familywas performed. A risk haplotype was defined by two STRs and four SNPs (SNPS9,10, 31and24) spanning the regionestablished in the Chaldeans, characterized by 17 transmissions and 2 non-transmissions (chi-square 11.8, p = 0.0006, uncorrected). This haplotype Was uniquefrom the Chaldeans. Of the 17 transmitting families, 10 are pure CD, 5 mixed, and 2 UC; II are non-Ashkenazim Caucasians, 6 Ashkenazim. Families withthe at-riskhaplotype do not completely account for theobserved evidence for linkage, indicating thatthis haplotype represents onlya fraction of the families possessing a risk allele here. Conclusions: The outbred IBD risk allele is common to CD and UC, and non-Ashkenazim and Ashkenazim families. The absence of linkage in mixed and Ashkenazim families likely reflects this locus' smallercontribution to overalldiseaserisk in these groups. We hypothesize the risk allele at chrlp36 induces a generic susceptibility to IBD, with concomitant inheritance of the IBD risk alleleon chrl6cen,IBDI, producing a CD phenotype.

3867 UNDERSTANDING GENE-GENE INTERACTIONS IN IBD: DISEASE GENES ON CHROMOSOMES I AND 16 INTERACT TO INDUCE DISEASE SUSCEPTmILITY, JUdy H. Cho, Dan Nicolae, Steven R. Brant, Paul Pavli, M. Gorska, M. E. Bryce, P. M. Stanford, Barbara S. Kirschner, E. Quak, Theodore M. Bayless, Stephen B. Hanauer, Juleen A. Cavanaugh, Univ of Chicago, Chicago, IL; Johns Hopkins Univ Sch of Medicine, Baltimore, MD; The Canberra Hosp, Garren, ACf, Australia; The Canberra Hosp, Garran, ACf, Australia; The Univ of Chicago, Chicago, IL; Thr Univ of Chicago, Chicago, IL. Background: The nature of risk alleles for multigenic, complex genetic disorders such as inflammatory bowel disease (lBD) will likely be fundamentally different than that observed for monogenic disorders. In IBD, a single disease gene may contribute only a modest portion of overall disease risk, and by itself, be insufficient to produce disease. It is postulated that, for complex disorders, simultaneous inheritance of risk alleles on multiple genes may be required for full disease expression. It is well established that a disease gene for Crohn's disease (CD) resides in the pericentromeric region of chromosome 16, with highly significantlinkage scores previously reported in the Australian population (Lod = 6.3). Suggestive evidence for linkage was previously observed in an American cohort of multiply affected families with both CD and ulcerative colitis (UC) on chromosome Ip36. Subset analysis suggested a positive correlation between the chromosomes Ip36 and 16cen loci. Aim: To extend evidence for linkage on chromosome Ip36 in Australian families with IBD and more precisely characterize the interaction between chromosomes Ip36 and 16cen. Methods: 169 American and 70 Australian multiplex families were genotyped and analyzed throughout chromosomes I and 16 using Genehunter plus. A likelihood test of interaction was developed to analyze datasets with incomplete information on meiotic events. This interactive lod score corresponds to the Pearson correlation coefficient between linkage scores for each family at the two tested loci if complete informationwere available in all families. Results: Genome-wide levels of significance were observed at chromosome Ip36 (multipoint lod score, MLS= 3.95, p = 1.02 x 10'5) in the combined cohort. Most of the evidence for linkage originated from CD families (MLS = 3.22, P = 5.91 x 10"), compared to mixed (MLS = 0.26, P = 0.14) and UC families ( MLS = 1.50, P = 0.0043). The interactive lod score comparing only the peak regions of linkage (chrIp: 37 cM; chrl6cen: 48.06 cM) was highly significant (lod = 2.33, P = 0.00053). Conclusions: Chromosome Ip36 contains an IBD disease gene common to CD and UC. The highly significant interactive lod score between chromosomes Ip36 and 16cen provides additional support for the presence of disease genes at these two loci. Those families demonstrating positive evidence for linkage at both loci represent a more homogenous subset which can be utilized to more precisely refine localization of both disease genes.

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3868 HYDROGEN PEROXIDE (H202) DECREASES CAT LOWER ESOPHAGEAL SPHINCTER (LES) TONE: POSSIBLE ROLE IN ACUTE EXPERIMENTAL ESOPHAGITIS (AE). Weibiao Cao, Karen M. Hamett, Jose Behar, Piero Biancani, Michael T. Kirber, RI Hosp and Brown Univ, Providence, RI. We have previously shown that experimental esophagitis damages intracellular Caz+ stores in cat LES, and changes the intracellular contractile pathways activated by ACh from Caz.. release- calmodulin- dependent in normal cells to Caz+ influx - PKC de~ndent in esophagitis. Thapsigargininduced depletion of intracellular Ca + stores in normal cells reproduces the changes in signal transduction pathways induced by AE, suggesting that the switch in the transduction pathway may be due to AE-induced reduction in intracellular Caz + stores. To explain these changes we propose that esophageal inflammation is associated with infiltration of neutrophils and macrophagesand production of HzOz. HzOz may induce production of prostanoids and generate free radicals such as 0z- and -OH, causing depletion of intracellular calcium stores. To test this hypothesis. normal LES strips were incubated in HzO z for at least 30 min. HzOz dosedependently reduced LES tone and induced formation of prostaglandin Ez, which relaxes LES muscle. The reduction in tone was prevented by the HzOz scavengercatalase and by the o; scavenger superoxidedisrnutase but not by the -OH scavenger deferri-oxarnine. To further examine the mechanism of HzOz-induced inhibition of tone, we used LES muscle cells isolated by enzymatic digestion. Thapsigargin inhibits Ca2+ uptake and enhances Caz+ release from intracellular stores, causing an initial contraction of LES cells, and subsequent depletion of Caz+ stores. Thapsigargininduced contraction was antagonized by pre-incubation in HzOz and. conversely, "zOz caused contraction of LES cells, which was antagonized by preincubation in thapsigar;rn. These data support the hypothesis that HzOz causes depletion of Ca from stores. Finally, HzOz-induced CaH release was directly documented by fura 2 imaging of LES cells. In fura 2-loaded cells, 5mM HzOz significantly increased cytosolic CaH levels. The release of intracellular Caz+ induced by caffeine or by ACh was decreased by pretreatment with 100 ILM HzOz, further confirming that HzOz can deplete the intracellular calcium stores. We conclude that hydrogen peroxide reduces cat LES tone by production of PGEz and by depletion of intracellular Caz+ stores. Supported by NIH ROI-DK-28614 and NIH R29 DK-47223.

3869 INTERLEUKIN IB-INDUCED REDUCfION OF LES TONE. Weibiao Cao, Ling Cheng, Karen M. Hamett, Claudio Fiocchi, Jose Behar, Piero Biancani, RI Hosp and Brown Univ, Providence, RI; Rhode Island Hosp, Providence, RI; Case Western Reserve Univ, Cleveland, OR. In a cat model of acute experimental esophagitis, the amplitude of esophageal contraction in response to direct stimulation by the endogenous neurotransmitterACh is not affected, but the in vivo response to swallowing and the in vitro response to electrical stimulation are significantly reduced. These data suggest that esophageal circular smooth muscle is not damaged by acid perfusion, but that the neural mechanisms responsible for release of excitatory neurotransmitters may be affected. We have previously shown that interleukin (IL)-I (3, but not other cytokines, mimic these esophagitis-induced changes in the body of the esophagus, supporting a role of IL-l{3 as a possible inflammatory mediator in esophagitis. In the present study we examined whether IL-l{3may also reproduce esophagitisinduced changes in cat lower esophageal sphincter (LES) tone. IL-113 (1-1000 U/ml) dose-dependentlydecreased tone of in vitro LES strips. The reduction was partially reversed by catalase (87 Ulml) and by superoxide dismutase (300 Vlml), suggesting that IL-113 reduces tone by producing hydrogen peroxide (" zOz) and superoxide anion, which have been shown to cause Caz", release and depletion of Ca2+ stores. In addition. the reduction in tone induced by IL- J(3 was partially blocked by the cytosolic phospholipase Az(cPLAz) inhibitor AACOCF3 (10 ILM ), indicating that arachidonic acid metabolites may also be involved in the reduction in LES tone. Because prostaglandin Ez (PGEz) relaxes LES circular muscle, we measured PGEz production in response to IL-I{3. After 2h incubation in IL-I{3 (200 U/ml), PGEz production was significantly increased, and the increase in PGEz was significantly reduced by AACOCF3 (10 ILM ), supporting a role of cPLA2 in IL-I {3-induced PGEz production. In addition, IL-I{3 (200 Ulml) significantly increased the phosphorylation ofp38 MAP kinase, and the IL-I{3-induced PGEzincrease was reduced by the p38 MAP kinase inhibitor SB203580 (10 ILM ), supporting a role of p38 MAP kinase in PGEz production. These data suggest that IL-I f3 reduces LES tone by producing reactive oxygen species and by increasing PGEz synthesis, which was mediated by a p38 MAP kinase dependent pathway. Supported by NIH ROI-DK-28614