Systematic association mapping identifies NELL1 as a novel IBD disease gene

Abstract

Crohn disease (CD), a sub-entity of inflammatory bowel disease (IBD), is a complex polygenic disorder. Although recent studies have successfully identified CD-associated genetic variants, these susceptibility loci explain only a fraction of the heritability of the disease. Here, we report on a multi-stage genome-wide scan of 393 German CD cases and 399 controls. Among the 116,161 single-nucleotide polymorphisms tested, an association with the known CD susceptibility gene NOD2, the 5q31 haplotype, and the recently reported CD locus at 5p13.1 was confirmed. In addition, SNP rs1793004 in the gene encoding nel-like 1 precursor (NELL1, chromosome 11p15.1) showed a consistent disease-association in independent German population- and family-based samples (942 cases, 1082 controls, 375 trios). Subsequent fine mapping and replication in an independent sample of 454 French/Canadian CD trios supported the authenticity of the NELL1 association. Further confirmation in a large German ulcerative colitis (UC) sample indicated that NELL1 is a ubiquitous IBD susceptibility locus (combined p<10(-6); OR = 1.66, 95% CI: 1.30-2.11). The novel 5p13.1 locus was also replicated in the French/Canadian sample and in an independent UK CD patient panel (453 cases, 521 controls, combined p<10(-6) for SNP rs1992660). Several associations were replicated in at least one independent sample, point to an involvement of ITGB6 (upstream), GRM8 (downstream), OR5V1 (downstream), PPP3R2 (downstream), NM_152575 (upstream) and HNF4G (intron).

Figure 1. Overview of the CD association findings for the NELL1 gene.

(A) Plot of the negative natural logarithm of the p-values obtained in the different stages of the study across a 1 Mb region. The red line shows the significance threshold (i.e. p = 0.05). Results for SNP rs1793004 are highlighted in pink. The main replication signal localizes to the 5′ region of the NELL1 gene. (B) Plot of the recombination rate (in cM/Mb), showing that the peak replication signal is delineated by two regions of increased recombination. (C, D) Linkage-disequilibrium (LD) plots from HapMap, using metrics D′ (section C) and r² (section D). Genotypes of trios with Northern- and Western-European ancestry were retrieved from HapMap for 1261 SNPs (CR≥90%, MAF≥1%, p_HWE≥0.01, Mendel errors≤3). Positions are from NCBI build 35.

Figure 2. Expression and localization of NELL1.

(A) Transcript levels of NELL1 in a set of different tissues were quantified by RT-PCR. Parallel amplification of β-actin (ACTB) is shown. Expression and localization of the NELL1 protein in healthy colonic tissue is demonstrated in sections B (20×) and C (40×; bar = 10 µm) by immunohistochemistry. Immunoreactivity is confined to mononuclear/lymphocytic cells in the lamina propria (brown DAB reaction product, arrows). A control without the primary antibody is shown in section D. No major expression differences between colonic specimen from normal controls (N) and Crohn disease (CD) were detected in the Western blot (section E) with the same antibody as applied in sections B and C. The single detected band (90 kDa) corresponds to the predicted molecular weight of the isoform encoded by GenBank AK127805 (UniProt accession number Q92832).

Figure 3. In silico protein analysis.

Domain architectures of NELL1/NELL2 and TSP1. The positions of variant amino acids are annotated. Abbreviations are as follows: SP: signal peptide; TSPN: thrombospondin N-terminal domain; CC: coiled-coil region; VWC: von Willebrand factor, type C domain; EGF: EGF domain; TSP-3: thrombospondin-3 repeat; TSP_C: thrombospondin C-terminal domain.

Figure 4. In silico protein analysis.

Computationally derived 3D structure model of the N-terminal domain of the NELL1 protein. The model was created using the TSPN (PDB code 1z78, chain A) as a structure template for NELL1. The locations of variant amino acids as well as of two cysteines forming a disulfide bridge are annotated.