Exome Analysis of Rare and Common Variants within the NOD Signaling Pathway

Abstract

Pediatric inflammatory bowel disease (pIBD) is a chronic heterogeneous disorder. This study looks at the burden of common and rare coding mutations within 41 genes comprising the NOD signaling pathway in pIBD patients. 136 pIBD and 106 control samples underwent whole-exome sequencing. We compared the burden of common, rare and private mutation between these two groups using the SKAT-O test. An independent replication cohort of 33 cases and 111 controls was used to validate significant findings. We observed variation in 40 of 41 genes comprising the NOD signaling pathway. Four genes were significantly associated with disease in the discovery cohort (BIRC2 p = 0.004, NFKB1 p = 0.005, NOD2 p = 0.029 and SUGT1 p = 0.047). Statistical significance was replicated for BIRC2 (p = 0.041) and NOD2 (p = 0.045) in an independent validation cohort. A gene based test on the combined discovery and replication cohort confirmed association for BIRC2 (p = 0.030). We successfully applied burden of mutation testing that jointly assesses common and rare variants, identifying two previously implicated genes (NFKB1 and NOD2) and confirmed a possible role in disease risk in a previously unreported gene (BIRC2). The identification of this novel gene provides a wider role for the inhibitor of apoptosis gene family in IBD pathogenesis.

Figure 1. Proteins acting within the NOD signaling pathway.

The recognistion of NOD1 and NOD2 of the bacterial peptidoglycan (PGN) promotes the formation of the multi-protein complex named inflammasome. The complex recruits the kinase receptor interacting protein 2 (RIP2), which is ubiquitinated by the ubiquitin ligases XIAP, BIRC2 and BIRC3 proteins. The polyubiquitinated RIP2 recruits the kinase TAK1 and TAK binding protein 1 (TAB1), TAB2 and TAB3 which leads to the activation of the MAPK kinases, p38 and c-Jun N-terminal kinase (JNK) through the activation of mitogen-activated protein kinase kinase (MKK). RIP2 polyubiquitinated also interacts with the IKK complex (IKKα, IKKβ and NEMO). The IKK complex mediates the phosphorylation of the IKKβ subunit of IKK by TAK1 and results in the phosphorylation and degradation of the NF-κB inhibitor (IκBα) which results in the cytoplasmic release and translocation of NF-κB dimers p65 and p50 in the nucleus to activate of the expression of the NF-κB proinflammatory genes.

Figure 2. NOD2 gene and protein.

NOD2 is a gene composed of 12 exons (black rectangles). The NOD2 protein consists of two N-terminal caspase activation recruitment (CARD) domains, a central nucleotide-binding oligomerization (NBD) domain and a terminal sequence rich in leucine (LRR). The CARD domains interact with RIP2 protein to activate the immune response in the gut and the leucine-rich domain recognizes the bacterial peptidoglycan. Mutations within the NBD have been shown to increase the inflammatory cascade. The 31 mutations observed by interrogating exome data from 136 pIBD and 106 controls are depicted using arrows and the corresponding protein change noted. Known IBD biomarkers are in red.

Figure 3. BIRC2 gene and protein.

BIRC2 is a gene composed of 9 exons (black rectangles). BIRC2 encodes an inhibitor of apoptosis protein, which contributes to innate immune responses by acting as inhibitor of cell death. All the members of the inhibitor of apoptosis (IAP) gene family share three tandem specific motifs: BIR belonging to the zinc-finger domain that mediates protein-protein interaction, a CARD domain is involved in CARD-CARD mediated interaction; and a C-terminal RING domain conferring an E3-ubiquitin ligase activity. The RING domain of BIRC2, BIRC3, and XIAP is required for the ubiquitin activity of the IAPs. Studies have reported that the CARD domain in BIRC2 and BIRC3 act as an inhibitor of the ubiquitin ligase activity. Mutations within the BIR1 domain in BIRC2 alters molecular interaction with TNF receptor associated factor 1 (TRAF1) and TRAF2. The six mutations found by interrogating exome data from 136 pIBD and 106 controls are depicted using arrows and the corresponding protein change shown.