Abstract
Neutrophil dysfunction and GM-CSF auto-antibodies are observed in pediatric and adult patients with Crohn’s disease (CD). We associated damaging coding variants with low GM-CSF induced STAT5 stimulation index (GMSI) in pediatric CD patients and implicated variation of neutrophil GM-CSF signaling in cell function and disease complications. Because many CD patients with low GMSI do not carry damaging coding mutations, we sought to test the hypothesis that non-coding variants contribute to this phenotype. We enrolled, performed whole genome sequencing, and measured the GMSI in 77 CD and ulcerative colitis (UC) patients (24 low and 53 normal GMSI). We identified 4 non-coding variants (rs3808851, rs10974787, rs10974788 and rs10974789) in RCL1 significantly associated with variation of GMSI level (p < 0.011). They were validated in two independent cohorts with: RNAseq data (n = 50) and blood eQTL dataset (n = 31,684). These variants are in LD and affect expression of JAK2 (p 0.005 to 0.013), RCL1 (p 8.17E-13 to 2.98E-11) and AK3 (p 2.00E-68 to 3.03E-55) genes. Additionally, they influence proteins involved in differentiation of gut epithelium, inflammation, and immune system regulation. In summary, our study outlines the contribution of non-coding variants in neutrophil GM-CSF signaling and the potential importance of RCL1 and AK3 in CD pathogenesis.
Highlights
Inflammatory bowel disease (IBD) is comprised of Crohn’s Disease (CD) and ulcerative colitis (UC)
Using whole exome sequencing (WES) in pediatric Crohn’s disease (CD), we previously identified potentially damaging rare missense coding mutations in Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) signaling genes[6] and we reported that the protein variant p.Ala17Gly (MAF = 9%) of GM-CSF Receptor Alpha Chain (CSF2RA) was associated with low GM-CSF induced STAT5 stimulation index (GMSI) in CD patients[6]
To identify non-coding regulatory variants for gene expression in neutrophils that affect GM-CSF signaling and to replicate previous findings for association between GM-CSF:STAT5 signaling pathway in neutrophils, and coding variants, we recruited 77 patients with known GMSI level comprised of 39 CD, 36 UC, 1 inflammatory bowel disease-undetermined (IBD-U) and 1 control (Supplementary Fig. 1 and Supplementary Table 1) and we performed whole genome sequencing (WGS) on them
Summary
Inflammatory bowel disease (IBD) is comprised of Crohn’s Disease (CD) and ulcerative colitis (UC). Using peripheral blood samples of 543 pediatric IBD patients and whole exome sequencing (WES), we tested for association between coding variants and the GM-CSF:STAT5 signaling pathway in neutrophils, and disease complications[6]. We noted that damaging missense mutations are not found in most of the CD patients who have low GMSI, which suggests the hypothesis that the low GMSI trait could be influenced by non-coding variants that regulate GM-CSF signaling. Many genotypes associated with diseases are located in regulatory regions and are more likely to be expression quantitative trait loci (eQTLs)[8,9] These non-coding and regulatory variants are best ascertained through comprehensive genomic dissection via whole genome sequencing (WGS). We asked whether CD patients selected on the basis of their low neutrophil GMSI level would carry non-coding regulatory variants for gene expression in neutrophils, that affect GM-CSF signaling. We attempted to replicate variants previously associated with neutrophil GMSI signaling in pediatric CD6
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