Independent and population-specific association of risk variants at the IRGM locus with Crohn's disease

Abstract

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohn's disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5' untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 x 10(-5) to 1.40 x 10(-9)), and that the CNV and the 5'-untranslated region variant -308(GTTT)(5) contribute independently to CD susceptibility (P = 2.6 x 10(-7) and P = 2 x 10(-5), respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10(-12)) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case-control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene-gene or gene-environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European-Asian split.

Figure 1.

Association of 35 SNPs at the IRGM-ZNF300 locus with CD (data from Ref. 16) indicated by –log of P-value. The physical location of the SNPs on chromosome 5 in Mbp is given on the x-axis with the relative position of the genes and CNV underneath.

Figure 2.

The structure of IRGM showing the location of the upstream CNV on chromosome 5. Horizontal arrows represent the relative positions of the three primers for the CNV PCR assay which uses the common left primer (black), and either the insertion right primer (grey) or deletion right (black). The position of the two risk SNPs are indicated by vertical arrows. The IRGM region consisting of the single coding exon 2 and upstream un-translated exon 1 containing the promoter (P) and U3/U5 repeats of the ERV9LTR is expanded above to indicate the relative positions of the two insertion/deletion polymorphisms (boxed). Potential TF binding sites adjacent to the −1644ins are underlined. The coding sequence (CDS) of IRGM is shaded in dark grey.

Figure 3.

Linkage disequilibrium of IRGM variants in UK CD cases and controls.

Figure 4.

Frequency of IRGM variants in five HapMap populations from Asia and Africa (YRI 120, MKK 145, LWK 145, JPT 88, CHB 88) and 2025 white population controls from the 1958 MRC British birth cohort (BC1958).

Figure 5.

Quantitative analysis of IRGM expression by real-time PCR in lymphocytes from CD patients of defined genotype for CNV (2, 1 and 0 copies of the CNV where 1 and 0 are the heterozygous and homozygous risk genotypes, respectively) −1644 (D = del allele, I = risk insertion allele) and −308 (where 2× GTTT repeats represent the non-risk allele and 5× GTTT repeats represent the risk allele. Relative expression is measured by, and inversely proportional to, mean ΔCt (see Materials and Methods).

Figure 6.

Relative expression of IRGM in HapMap populations from Asia (JPT & CHB), Africa (YRI, LWK & MKK) and Europe (CEU) by microarray analysis (26).