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sand wild type allele carrying patients (n=15), simultaneously. Expression of CSF2RB wasdetermined by flowcytometry and confirmed by qPCR. ROS production upon stimulationof cells with fMLP with or without prior priming with GMCSF or GCSF was measured byflowcytometry. Uptake and killing of E. Coli were measured using fluorescence spectopho-tometry. CSF2RB signaling was studied after stimulation with GMCSF and IL5. Results:Membrane expression of CSF2RB is similar between patients bearing either T or C allele(n=10; 323±40 and 323±54 MFI). GMCSF-primed ROS production was greatly diminishedin C allele patients compared to T allele patients (n=10, 221±41 MFI vs 464±52 MFI, p=0.002), wereas GCSF-primed ROS production was unaffected (210±30 vs 225±20 MFI, p=0.79). indicating a specific GMCSF receptor signaling defect. E.Coli uptake was not different,whereas a trend towards diminished killing was observed in C allele patients (171±28 vs280±86 CFU). In C-allele patients, GMCSF stimulation induced less activation of STAT3(n=5, 1.8±0.8 vs 3.5±1.1 vs); AKT (11±5 vs 23±13) and ERK (30±20 vs 40±10) in C-allelecarriers. IL5 activated lower STAT5 (n=4, 0.2± 0.1 vs 0.5±0.2) and AKT signaling (0.03±0.06vs 0.7±0.04). Conclusion: We show a specific defect in CSFR2B signaling in patients carryingthe NCF4 risk allele. The CSFR2B receptor is used by both GMCSF and IL5, and bothcytokines induce reduced signaling in C-allele patients. In contrast, priming of PMN byGCSF, which signals through a different receptor, was normal. Thus, the NCF4 risk alleleconfers a defect in CSFR2B signaling, and may identify a subset of CD patients whoseinflammation is influenced by impaired innate immunity induced by neutrophils. GM-CSFtreatment, which is beneficial for some patients, may be limited by NCF4 genetic backgroundof patients.

975

Novel Associations of Uncommon Crohn's Disease Risk Alleles With HigherFrequencies in the Ashkenazi Jewish PopulationKen Hui, Wei Zhang, Beatrice M. Bowen, Talin Haritunians, Mark S. Silverberg, John D.Rioux, Seymour Katz, Adam S. Cheifetz, Steven R. Brant, Dermot P. McGovern, HongyuZhao, Richard H. Duerr, Inga Peter, Judy H. Cho

Crohn's disease (CD) is 4.3-7.7 times more prevalent in the Ashkenazi Jewish (AJ) populationcompared to non-Jewish (NJ) European-ancestry populations. Additionally, AJ individualscarry an increased lifetime risk of inflammatory bowel disease for first-degree relatives of aCD proband (7.8% AJ vs. 5.2% NJ). The unique demographic history of the AJ population,characterized by an extreme bottleneck, subsequent explosive population growth, and endo-gamy, has resulted in decreased genetic variation and increased sharing of long haplotypes.Therefore, surveying variation within a moderately sized sample of individuals should yielda more complete representation of the population's genetic variants. We showed previouslyin a genome-wide association study of CD in the Ashkenazim that common polymorphismsin established CD loci did not account for the population's increased population diseaseprevalence. In this current study, we hypothesized that uncommon variation - particularlywithin in protein-coding exons and poorly assayed by GWAS - contributes uniquely to CDin the AJ population. We performed exome sequencing of 50 AJ individuals with CD andidentified 4,277 novel coding polymorphisms of interest, which we combined with markerson the Illumina HumanExome beadchip. Using this custom assay, we conducted genotypingin 1,463 CD unrelated cases and 2,770 independent healthy controls, which were geneticallyvalidated as having complete AJ ancestry. A vast majority of variants were low-frequency;72.7% of polymorphic markers had MAF , 1% in the AJ control samples, compared to74.1% in 3,973 NJ healthy controls, which were genotyped in parallel. We detected anovel AJ-enriched association signal in leucine-rich repeat kinase 2 ( LRRK2) with maximumsignificance at Y2189C, R50H, and G116R (P = 2.03x10-12, MAF = 9%), distinct from thealleles most associated to Parkinson's disease. The minor allele at this position, which confersincreased disease risk (OR = 1.87), is over 3 times more common in the AJ cohort than inthe NJ samples. In our prior report, the LRRK2 region demonstrated a CD LOD score of2.08, driven more by AJ (137 affected CD relative pairs) than NJ (386 pairs) families, withLOD scores of 1.26 and 0.74, respectively. In follow-up to the exomic analysis, we havealso completed whole-genome sequencing of 144 AJ healthy controls using the CompleteGenomics platform and are using these samples as a reference panel for imputation of non-exomic polymorphisms in our full cohort; we will present additional association results fromthis analysis. We expect that these data, in conjunction with our analytical results to date,will further demonstrate the critical role that uncommon risk alleles play in CD heritability,as well as the complex relationships between them that can be uncovered by performing afocused study in the Ashkenazim.

976

Early Onset Crohn's Disease Exhibit Distinct Allele Architecture Differencesand Reveal New IBD LociSubra Kugathasan, Marla Dubinsky, Stephen L. Guthery, David T. Okou, Promita Bose,Michael E. Zwick, David J. Cutler, Jon P. Waters, Jeffrey S. Hyams, Robert Baldassano,Michael C. Stephens, Melvin B. Heyman, Anne M. Griffiths, Wallace Crandall, JamesMarkowitz, Jonah B. Essers, Scott B. Snapper, Thomas D. Walters, Bruce J. Aronow, LeeDenson

Genome Wide Association Studies (GWAS) have not specifically focused on early onset ( ≤10yrs) Crohn disease (EO CD). A pediatric IBD GWAS with a mean age of onset of 12 years(85% ≥11 years old) reported association/IBD loci similar to those seen in adult IBD (NatGenet. 2009;41:133). However, the role of genetic factors in EO CD is hypothesized to begreater due to age of onset, increased family history, greater disease severity. EO CD wasnot represented well in pediatric GWAS studies and lacked power to reveal the allelicarchitecture of common variants. We aim to detremine if the allelic architecture/ frequencyof common variants in EO CD is different than that of older children and adult CD. DNAwas obtained from the RISK study, a prospective, longitudinal study where subjects ≤16years with a new diagnosis of IBD are enrolled. We genotyped ~1570 subjects from RISK(including 1132 CD using immunochip. Healthy non-RISK subjects were used as control(n=3000, age and gender matched) and the CD group (only Caucasians)was also stratifiedby age with EO CD (n=375) and older ( .10 yrs) (n=725). ~125,000 SNPs passed ourstringent QC (less than 5% missing data, no evidence of genotyping error in duplicates,

S-178AGA Abstracts

both alleles observed, no departure from HW at p , 10-3 in controls). Analysis showedfew well known loci with genome-wide significance (NOD2, ATG16L1 and IL23R) betweencases and controls, and no novel associations were found to be genome-wide significance.The allele frequencies of known 163 IBD loci among older IBD subjects were similar tothose recently published (Nature 2012;491:119). when we used ≤10 years CD as ‘cases'and over 10 years CD as ‘controls', there were 14 SNPs with p values between 10-4 and10-7 of which seven (50%) are in the IL19 locus. Population sub-structure analysis amongCD yielded a genomic inflation of 1.05. The SNP with lowest p value within IL19 loci hadan OR of 2 with a MAF of 16% in ‘cases' compared with 8.4 % in ‘controls'. There were16 SNPs that were further identified in IL19 with p,10-3. Additionally, there were hundredsof nominally significant SNPs that showed differences in allele frequencies between and theyounger and older CD cohort and many of those alleles belong to common loci ascertainedby GWAS and previously reported to be associated with adult CD (CARD9, IL23, PTPN2STAT4, ERAP2 andmore). Conclusion: Differences exist in allelic frequencies and architecturebetween EO CD and older children. IL-19 which is known to suppress bacteria inducedmacrophage pro-inflammatory cytokine production. IL19 belongs to IL-10 superfamily. IL10is an established genetic risk for IBD, and it is not clear if IL19 signal is exhibited via IL10.IL10 and IL19 genes are linked together through genetic similarity and intron-exon genestructure. Further investigation in a larger cohort is underway for replication.

977

Gene-Environment Interactions: IBD Risk Loci and Hygene-Related ChildhoodEnvironmental Factors in the Puerto Rico IBD PopulationFrancisco E. Garcia Gonzalez, Esther A. Torres, Roberto Vendrell, Nelly Rabell, Julio A.Peguero, Jonathan Rodriguez, Xiuqing Guo, Stephan R. Targan, Dermot P. McGovern,Jerome I. Rotter, Kent D. Taylor

Introduction: A recent genome-wide association study has identified 163 IBD risk loci.However, these loci explain only a minority of the variance in disease risk. Gene-environmentinteractions are among the factors believed to explain the missing heritability. Supportingthis concept is the fact that murine models of IBD have demonstrated that the ATG16L1gene interacts with norovirus in the environment to produce intestinal inflammation. InPuerto Rico, where a dramatic increase in incidence has paralleled an abrupt modernization,we have observed an increased IBD risk with hygiene-related childhood environmentalexposures. Aim: To test whether susceptibility to IBD conferred by known IBD risk loci ismodified by hygiene-related childhood environmental exposures in the Puerto Rico popula-tion. Methods: Of the subjects recruited for the NIDDK IBD Genetics Research Consortium,536 Puerto Ricans with IBD and 375 controls with environmental questionnaire data andImmunoChip data were available for this study. Since cases, controls and their families wererecruited, association was tested using genome-wide association analyses with family data.Covariates included were global continental ancestry from Africa and Ancient America, andone of the environmental factors (EF) from the questionnaire. Continental ancestry wasestimated using Admixture 1.22. The environmental factors tested were childhood exposureto a rural v. urban environment (rural), presence of running water in the home (water),presence of toilet inside the home (toilet), or connection of home to public sewer (sewer).Results: Fourteen of the 163 previously identified IBD loci showed a SNP by EF effect witha p-value less than 0.001 as shown in table 1. Conclusion: The particular environmentalfactor(s) leading to increased IBD susceptibility is/are currently unknown, but we proposethat the childhood exposure studied here is marking an important determinant of IBDsusceptibility in Puerto Rico. Using such a marker, we report evidence of gene-environmentinteractions between hygiene-related childhood environmental exposures in Puerto Rico andmultiple, functionally diverse, IBD risk genes. This finding supports the current notion thatIBD risk genes share pathways in IBD pathogenesis which are modified by environmentalfactors. Because two of the regions represented, 5p13.1 and the Major HistocompatibilityComplex (MHC), have been intractable to further genetic analysis up to this time, ourfindings suggest that gene by environment studies may be the way forward for these loci.Further studies on the mechanisms by which these exposures modify IBD gene pathways,including their effect on the host-microbiota relationship, are warranted in the search forthe missing heritability in IBD.Table 1

Chr = Chromosome Mb = Mega base pairs SNP = Single nucleotide polymorphism EF =Environmental Factor