Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis

Abstract

We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10(-10)) with an intergenic region on 10q21.1 and a coding variant in ATG16L1, the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B, NCF4 and a predicted gene on 16q24.1 (FAM92B). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.

Figure 1. Genome-wide association results for 946 ileal CD patient and 977 control samples

Single marker association results for the combined Non-Jewish and Jewish cohorts using the Cochran-Mantel-Haenszel (CMH) test. Each chromosome is depicted as a different color and the blue and red lines indicate the suggestive and significant thresholds, respectively, based on a Bonferroni correction for multiple testing. A modest correction factor (lambda 1.06-1.07) is necessary to apply in order to correct for stratification between cases and controls, but does not modify the current results.

Figure 2. Expression of autophagy components in human cell-lines and primary immune cells

Quantitative real-time PCR was used to determine the expression patterns of ATG5, 7 and 16L1 in a variety of human immune and epithelial cell lines. Panel A - Expression pattern of ATG5 in human cell lines. ATG5 seems broadly expressed in the cell lines studied, with between 1.5-and 4-fold variations in expression level across the cell lines. Panel B - Expression pattern of ATG7 in human cell lines. ATG7 expression levels appeared broadly comparable to those of ATG5, with the exception of THP1 cells, which exhibited a 35-fold higher level than SW480 cells. Panel C - Expression pattern of ATG16L1 in human cell lines. ATG16L1 expression was also broadly similar to those of ATG5 and 7 (with the exception of ATG7 expression in THP1 cells) with moderate variations in expression levels across the cell lines tested (1.5- to 3.5-fold). Panel D - Expression pattern of ATG16L1 in human primary immune cells. In a resting human immune cell RNA panel (Clontech, CA) ATG16L1 showed a clear peak of expression in the T cell compartment, with both CD4+ and CD8+ cells expressing high levels of transcript (15- to 20-fold more than control samples). Modest levels were detected in CD19+ cells, and low levels in CD14+ monocytes and unfractionated mononuclear cells. In all cases, real-time quantitative RTPCR reactions were performed in duplicate and the means plotted, error bars represent 1 standard deviation. Reactions containing no template RNA were performed to control for reaction contamination (water controls). Expression levels were normalized by comparison to GAPDH controls and arbitrary relative expression units plotted where SW480 or placental RNA is equal to 1 (for cell lines, or primary cells respectively). All RNAs were isolated from resting cells in the absence of stimulation or activation.

Figure 3. ATG16L1 is required for autophagy of Salmonella Typhimurium

Panel A. Specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 is achieved in HEK293 cells within 48 hours of transfection with oligo duplexes. Protein levels of Flag-ATG16L1 were undetectable by Western blotting following treatment with specific siRNA constructs, but expression was maintained with control duplexes. Panel B. Endogenous ATG16L1 mRNA is efficiently knocked-down by siRNA 2 in HeLa cells within 48 hours of transfection. HeLa cells were transfected with siRNA duplexes and allowed to grow for 48 hours, RNA was then isolated and real-time quantitative RT-PCR performed. Data was normalized to GAPDH controls and siRNA control mRNA levels were set at 1.0. Compared to control duplex, siRNA 2 yielded an 89% reduction in mRNA transcript. RT-PCR reactions were performed in triplicate and means are plotted with error bars representing 1 standard deviation, results are representative of two independent experiments. Panel C. Knockdown of ATG16L1 prevents effective autophagy of S. Typhimurium in HeLa cells. 48 hours following co-transfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 hour with Salmonella Typhimurium SL1344, fixed and microscopically examined. The mean % of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) and standard errors are shown. Bacterial counts were pooled from two separate experiments, each counting a minimum of 100 infected cells. Significance was assessed using a two-tailed Students T-test, assuming unequal variances. Panel D. Representative images of intracellular S. Typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells, but loss of autophagic targeting when ATG16L1 knockdown is affected. HeLa cells were transfected with LC3-GFP plasmid and siRNA and subsequently infected as decribed above. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. Typhiumurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of the areas marked in rows 1 and 3 respectively. Images were obtained using confocal microscopy and are flat projection of Z-stacks taken throughout the plane of the infected cell. Scale bars represent 5 μm.