Genes and Immunity (2013) 14, 310–316 & 2013 Macmillan Publishers Limited All rights reserved 1466-4879/13 www.nature.com/gene ORIGINAL ARTICLE Extended haplotype association study in Crohn’s disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-kB pathway gene, HEATR3 W Zhang1,14, KY Hui1,14, A Gusev2, N Warner3, SME Ng1, J Ferguson1, M Choi4, A Burberry3, C Abraham1, L Mayer5, RJ Desnick5, CJ Cardinale6, H Hakonarson6, M Waterman7, Y Chowers7, A Karban7, SR Brant8, MS Silverberg9, PK Gregersen10, S Katz11, RP Lifton4, H Zhao4,12, G Nun˜ez3, I Pe’er13, I Peter5 and JH Cho1,4 The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn’s disease (CD) compared with non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to CD etiology in this population, most notably at NOD2, in which three causal, uncommon and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes that showed significantly greater association to CD in the Ashkenazi Jewish population compared with a non-Jewish population (145 haplotypes and no haplotypes with P-value o10 À 3, respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established CD loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-kB signaling. The BRD7 signal showed conditional dependence with only the downstream rare CD-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association. Genes and Immunity (2013) 14, 310–316; doi:10.1038/gene.2013.19; published online 25 April 2013 Keywords: haplotype association; Ashkenazi Jewish; Crohn’s disease; NF-kB signaling; synthetic association INTRODUCTION Because genotyping platforms utilized thus far have focused on Crohn’s disease (CD) is a complex genetic disorder characterized common variants, it is possible that untested rare mutations may by chronic intestinal inflammation resulting from a dysregulated contribute significantly to complex disorders and account for some host immune response to intestinal microbiota.1 The NOD2 gene, portion of missing heritability. Furthermore, precise models of involved in innate immune responses to bacterial peptidoglycan, disease pathogenesis integrating multiple disease associations are is strongly associated with CD and was initially identified through largely lacking, reflecting in part the significant pathophysiological genetic linkage.2 Uncommon, loss-of-function coding mutations heterogeneity underlying the myriad associations reported thus far (Arg702Trp, Gly908Arg and Leu1007fsinsC) in NOD2 confer a 17.1- in CD. Approaches to reduce genetic complexity, such as through fold (95% confidence interval: 10.7–27.2) increased risk for focused studies in selected populations, may be of benefit. disease with homozygous or compound heterozygote risk allele The Ashkenazi Jewish population has a several-fold higher carriage.3 The advent of genome-wide association studies prevalence of CD compared with non-Jewish European ancestry (GWASs) has resulted in the identification of 140 loci with cohorts, with estimates of increased prevalence ranging between 5,6 genome-wide significance in CD, implicating numerous immune 4.3- to 7.7-fold. The Ashkenazim have a unique genetic history, mechanisms in disease pathogenesis. However, most identified characterized by population bottlenecks, expansions and 7 loci involve variants of modest effects, and the presently endogamy. However, the associated polymorphisms identified identified 140 genetic loci account for only 13.6% of the thus far through GWASs, as well as at NOD2, do not account for estimated heritability.4 the higher disease prevalence in the Ashkenazim. Although the 1Section of Digestive Diseases, Department of Medicine, Yale University School of Medicine, New Haven, CT, USA; 2Department of Epidemiology, Harvard University, Boston, MA, USA; 3Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; 4Department of Genetics, Yale University School of Medicine, New Haven, CT, USA; 5Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA; 6Center for Applied Genomics, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; 7Department of Gastroenterology, Rambam Health Care Campus, B. Rappaport, Institute for Research in the Medicine Science, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; 8Meyerhoff Inflammatory Bowel Disease Center, Department of Medicine, Johns Hopkins University School of Medicine, , Baltimore, MD, USA; 9IBD Group, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada; 10Robert S. Boas Center for Genomics and Human Genetics, Feinstein Institute for Medical Research, North Shore LIJ Health System, Manhasset, NY, USA; 11North Shore University Hospital-Long Island, Jewish Hospital Systems, St. Francis Hospital, Great Neck, NY, USA; 12Department of Epidemiology and Public Health, Yale School of Medicine, New Haven, CT, USA and 13Department of Computer Science, Columbia University, New York, NY, USA. Correspondence: Dr JH Cho, Departments of Medicine and Genetics, Yale University School of Medicine, 300 Cedar St, TAC S155A, New Haven, CT 06520, USA. E-mail: [email protected] 14These authors contributed equally to this work. Received 8 January 2013; revised 12 March 2013; accepted 21 March 2013; published online 25 April 2013 Extended haplotype association study in Crohn’s disease W Zhang et al 311 basis for the increased frequency of CD in the Ashkenazim is not analysis of a cohort comprising 397 Ashkenazi CD cases and 431 known, it is possible that unidentified, uncommon variants, controls, and 547 non-Ashkenazi CD cases and 549 controls.13,14 unique to or at a higher frequency within Ashkenazi Jews, Our analysis of associated extended haplotypes showed that there contribute to higher disease prevalence. An additional possibility, were markedly distinct patterns of haplotype association signals in given the greater linkage disequilibrium observed within the the Ashkenazim compared with the non-Jewish cohort (Figure 1). Ashkenazim, is that multiple functional polymorphisms inherited We observed 145 haplotypes demonstrating nominal evidence for in cis contribute to disease more commonly within Ashkenazi association with P-values o10 À 3, including three distinct regions Jewish compared with non-Jewish European ancestry populations. with P-values o10 À 4 (Supplementary Table 2). At this threshold, Such a configuration of mutations would be consistent with the no regions were found to be significant in the non-Jewish cohort. well-documented presence of longer haplotype blocks with Comparing the 145 haplotypes with 71 previously identified CD greater levels of linkage disequilibrium, relative to non-Jewish loci in a GWAS-based meta-analysis,15 we found that there were populations.8,9 As such, association testing to identify significant four haplotypes overlapping three established loci (Table 1); no extended regions may point the way to understanding the unique haplotypes overlapped the 69 additional CD-associated loci in a genetic architecture underlying CD in Ashkenazi Jews. larger immunochip-based study.4 Among all loci, the most Studying haplotype structure in the context of disease significant haplotype association signal was found at association also informs our conception of synthetic association, chromosome 16q12, in a region that includes the NOD2 gene. which has been hypothesized to be a major contributor to Three of these four haplotypes showed higher allele frequencies in genome-wide association signals. Under this framework, molecu- CD cases, corresponding to increased disease risk, whereas the lar evolution, which creates varying amounts of haplotype remaining haplotype on chromosome 2 appeared to confer a diversity, allows for significant disease association near clusters protective function. of rare causal variants.10 For example, in CD, NOD2 is distinguished by three uncommon functional mutations whose carriers are a Chromosome 16 haplotypes. Two of the significant haplotypes in subset of individuals who have a common background established CD loci (chr16_hap6721 and chr16_hap6830) were 3,11 haplotype. The multiple hierarchical levels in the tree-based located contiguously on chromosome 16, spanning 48.88–49.18 10 genealogy, originally proposed by Dickson et al. , are reflected in and 49.18–49.48 Mb, respectively (Table 1). Haplotype chr16_hap6830 various stringencies for the clustering parameters in an analysis of was carried by 25 CD Ashkenazi cases and 4 Ashkenazi controls, contemporary haplotype diversity; for analyses with strict and chr16_hap6721 was carried by 15 CD cases and no controls. parameters, causal variants may not lie within the associated The 15 CD cases that carried chr16_hap6721 were completely a haplotypes themselves. Using haplotypes may be a particularly subset of the 25 cases that carried chr16_hap6721. Importantly, of powerful approach