Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4

Abstract

To identify novel susceptibility loci for Crohn disease (CD), we undertook a genome-wide association study with more than 300,000 SNPs characterized in 547 patients and 928 controls. We found three chromosome regions that provided evidence of disease association with p-values between 10(-6) and 10(-9). Two of these (IL23R on Chromosome 1 and CARD15 on Chromosome 16) correspond to genes previously reported to be associated with CD. In addition, a 250-kb region of Chromosome 5p13.1 was found to contain multiple markers with strongly suggestive evidence of disease association (including four markers with p < 10(-7)). We replicated the results for 5p13.1 by studying 1,266 additional CD patients, 559 additional controls, and 428 trios. Significant evidence of association (p < 4 x 10(-4)) was found in case/control comparisons with the replication data, while associated alleles were over-transmitted to affected offspring (p < 0.05), thus confirming that the 5p13.1 locus contributes to CD susceptibility. The CD-associated 250-kb region was saturated with 111 SNP markers. Haplotype analysis supports a complex locus architecture with multiple variants contributing to disease susceptibility. The novel 5p13.1 CD locus is contained within a 1.25-Mb gene desert. We present evidence that disease-associated alleles correlate with quantitative expression levels of the prostaglandin receptor EP4, PTGER4, the gene that resides closest to the associated region. Our results identify a major new susceptibility locus for CD, and suggest that genetic variants associated with disease risk at this locus could modulate cis-acting regulatory elements of PTGER4.

Figure 1. Results of the GWA for CD

p-values (−log[p]) for the 10,000 best SNPs out of 302,451 are shown (gray circles). The position of previously described susceptibility loci are marked by red arrows. The p-values obtained in our cohorts with the reportedly associated SNPs/mutations are shown by the red dots, and the corresponding odds ratios are indicated. The p-values obtained with SNPs included in the Illumina panel ≤50 kb from these SNPs/mutations are marked by black circles. SNPs genotyped in the confirmation cohort are shown as green dots.

Figure 2. LD, Association and Haplotype Analyses of the 5p13.1. Susceptibility Locus

(A) Pair-wise LD analysis between the 111 SNPs in the 250-kb window. r ² (lower left) and D′ (upper right) values were computed using standard procedures from the genotypes phased with PHASE [11]. Values >0.93 are marked in red, values ≤0.93 in blue. The five LD blocks are easily identified and marked by corresponding boxes I to V. (B) Red dots: results of single-marker association analyses for CD using 111 SNPs located in a 250-kb window spanning the positions of the most significant 5p13.1 markers in the GWA. The results are expressed as log(1/p), where p corresponds to the p-value of the association determined by chi-squared analysis. The positions of the 111 markers are indicated by the small triangles. The limits between the LD blocks (I–V) are indicated. Blue diamonds: log(1/p) values of the effect of marker genotype on PTGER4 expression levels for the 26 HumanHap300 Genotyping Beadchip SNPs mapping to the 250-kb window. Values are only shown when exceeding 2. (C) Haplotype analysis of LD blocks II, III, and IV. Haplotypes accounting jointly for >93% of studied chromosomes are shown. The ancestral allele is in grey when known. Within each block, similar haplotypes are grouped in “clades” (e.g., IIA, IIB, and IIC) marked by different colors (red, blue or green). For blocks II and III, supposedly recombinant haplotypes are represented under the major clades and colored accordingly. The frequency of the corresponding haplotypes (black and white) and clades (colored) in CD patients and controls are given. p-values (chi-squared test) of the clade-based association tests for CD are given underneath for intervals bounded by recombination events. The approximate positions of within-block recombinations are marked by vertical lines between p-values. The two haplotypes forming the IIIBa sub-clade are indicated. CD, CD patients; CTR, controls.