Crohn's disease IRGM risk alleles are associated with altered gene expression in human tissues

Abstract

Crohn's disease (CD) is a chronic inflammatory gastrointestinal disorder. Genetic association studies have implicated dysregulated autophagy in CD. Among risk loci identified are a promoter single nucleotide polymorphism (SNP)( rs13361189 ) and two intragenic SNPs ( rs9637876 , rs10065172 ) in immunity-related GTPase family M ( IRGM) a gene that encodes a protein of the autophagy initiation complex. All three SNPs have been proposed to modify IRGM expression, but reports have been divergent and largely derived from cell lines. Here, analyzing RNA-Sequencing data of human tissues from the Genotype-Tissue Expression Project, we found that rs13361189 minor allele carriers had reduced IRGM expression in whole blood and terminal ileum, and upregulation in ileum of ZNF300P1, a locus adjacent to IRGM on chromosome 5q33.1 that encodes a long noncoding RNA. Whole blood and ileum from minor allele carriers had altered expression of multiple additional genes that have previously been linked to colitis and/or autophagy. Notable among these was an increase in ileum of LTF (lactoferrin), an established fecal inflammatory biomarker of CD, and in whole blood of TNF, a key cytokine in CD pathogenesis. Last, we confirmed that risk alleles at all three loci associated with increased risk for CD but not ulcerative colitis in a case-control study. Taken together, our findings suggest that genetically encoded IRGM deficiency may predispose to CD through dysregulation of inflammatory gene networks. Gene expression profiling of disease target tissues in genetically susceptible populations is a promising strategy for revealing new leads for the study of molecular pathogenesis and, potentially, for precision medicine. NEW & NOTEWORTHY Single nucleotide polymorphisms in immunity-related GTPase family M ( IRGM), a gene that encodes an autophagy initiation protein, have been linked epidemiologically to increased risk for Crohn's disease (CD). Here, we show for the first time that subjects with risk alleles at two such loci, rs13361189 and rs10065172 , have reduced IRGM expression in whole blood and terminal ileum, as well as dysregulated expression of a wide array of additional genes that regulate inflammation and autophagy.

Keywords: autophagy; inflammatory bowel disease; ulcerative colitis.

Fig. 1.

Relationship of immunity-related GTPase family M (IRGM) single nucleotide polymorphism to IRGM expression in human terminal ileum and whole blood. RNA-Seq data in the Genotype-Tissue Expression Project portal (https://gtexportal.org/home/) was analyzed to define the relationship of genotypes at rs13361189 (A), rs10065172 (B), and rs9637876 (C) to IRGM mRNA levels in human terminal ileum and whole blood. Box plots depict median and interquartile ranges of transcripts per million (TPM) for mRNA expression of IRGM defined by RefSeq gene models. Numbers below IRGM genotypes indicate the number of independent human specimens sampled. Tables at right depict P values for intergenotype comparisons.

Fig. 2.

Relationship of immunity-related GTPase family M (IRGM) single nucleotide polymorphism TNF expression in human terminal ileum and whole blood. RNA-Seq data in the Genotype-Tissue Expression Project portal (gtexportal.org) was analyzed to define the relationship of genotypes at rs13361189 (A), rs10065172 (B), and rs9637876 (C) to TNF mRNA levels in human terminal ileum and whole blood. Box plots depict median and interquartile ranges of transcripts per million (TPM) for TNF mRNA expression. Numbers below IRGM genotypes indicate the number of independent human specimens sampled. Tables at right depict P values for intergenotype comparisons.

Fig. 3.

immunity-related GTPase family M (IRGM) disruption augments TNF expression by human cells. A: TNF mRNA was quantified by RT-qPCR in parental HeLa cells and two HeLa IRGM disruption CRISPR clones after exposure for 4 h to buffer (control), LPS, or TNF. Data are mean ± SE and are representative of 3 independent experiments. *P = 0.03; ψP = 0.01; ***P < 0.001 (comparison to parental cells under same treatment). B: HeLa cells and human monocyte-derived macrophages (Mϕ) were exposed to LPS as shown and then immunoblotted for IRGM and β-actin (loading control).