P076. A gene expression outlier strategy towards identifying molecular causes of Crohn's disease
G.W. Sewell1, P.J. Smith1, A.P. Levine1, C.M. McDonald1, S.L. Bloom2, A.P. Walker1, A.M. Smith1, A.W. Segal1
1UCL, Division of Medicine, United Kingdom; 2University College London Hospital, Department of Gastroenterology, United Kingdom
Background: Crohn's disease (CD) is a syndrome with multiple causes, the majority of which are unknown. Genome-wide association studies (GWAS) have accounted for only 23% of the heritability . We have identified a CD phenotype in which bacterial clearance is delayed as a consequence of impaired neutrophil accumulation. This results from reduced pro-inflammatory cytokine secretion from macrophages . Having demonstrated functional defects in CD macrophages, we examined transcription profiles of these cells to identify abnormal levels of mRNA as indicators of molecular pathology.
Methods: Macrophages were cultured from 61 quiescent CD patients (HarveyBradshaw score <3) and 43 healthy controls (HC), and RNA extracted. Gene expression was determined using Illumina WG6 v3 arrays. An outlier analysis was performed using proprietary software, in which expression of each probe in individual patients was compared to the expression in the HC cohort as a group. Expression of a probe was considered significantly different (an outlier) at p < 0.005 and a fold change of 1.5. Abnormal expression was confirmed by SYBR green real-time RT-PCR. Release of TNF was determined from primary macrophages and optineurin-depleted THP‑1 cells in response to Pam3CSK4 (a Toll-like receptor 2 (TLR2) agonist) stimulation using the L929 bioassay.
Results: The most frequently under-expressed gene expression outliers in CD macrophages included optineurin, a molecule implicated in vesicle trafficking and autophagy, and ADAM-like, decysin 1, a disintegrin and metalloproteinase. Furthermore, a number of patients demonstrated abnormal expression of genes previously implicated in CD susceptibility by GWAS. These included UTS2, CARD9 and CCL7. The abnormal expression of these genes was validated by quantitative PCR. Macrophages from optineurin outlier patients released significantly less TNF than HCs in response to TLR2 stimulation. Depletion of optineurin in THP‑1 cells was associated with reduced TNF release after TLR2 activation.
Conclusions: In conclusion, we have developed a successful strategy to identify abnormal expression of genes in individual CD patients relevant to diminished macrophage function. In a wider context, this approach may be equally applicable to delineating the molecular pathogenesis of other complex disorders.
1. A. Franke et al. (2010), Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci, Nat. Genet.
2. A. M. Smith et al. (2009), Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease, J. Exp. Med.