An Integrated Taxonomy for Monogenic Inflammatory Bowel Disease

Abstract

Background & aims: Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis.

Methods: To build a taxonomy model, we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by (1) overlapping syndromic features, (2) response to hematopoietic stem cell transplantation, (3) bulk RNA-sequencing of 32 tissues, (4) single-cell RNA-sequencing of >50 cell subsets from the intestine of healthy individuals and patients with IBD (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4).

Results: Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8⁺ T cells, neutrophils, epithelial subsets, and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mammalian target of rapamycin activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD.

Conclusion: Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.

Keywords: Genomics; Immunodeficiency; Next-Generation Sequencing; RNA-seq.

Graphic abstract

Figure 1. Approach to investigating syndromes and Mendelian disorders associated with monogenic inflammatory bowel disease (IBD)

Monogenic gene defects were classified according to whether they had (b) high-penetrance, (c) moderate-penetrance or (d) insufficient evidence of intestinal inflammation, 90% confidence intervals are shown. For each gene, the total number of patients with IBD-like intestinal inflammation is presented above as an additional marker of clinical confidence.

Figure 2. Phenotypic characteristics of people with monogenic IBD gene defects indicate a spectrum of onset age and syndromic associations

(a) Onset age (circles) of individuals’ intestinal inflammation of monogenic IBD (n=338). Dashed line denotes the median. The age of onset in polygenic IBD is shown for comparison (grey, right, n=1608). (b) Median age of onset for high- and moderate-penetrance IBD genes (P= 0.0065, Mann Whitney test, one-tailed) with individual gene defects represented (circle). (c) Syndromic gene-phenotype associations and outcomes of intestinal inflammation following hematopoietic stem cell transplant (HSCT). SCID= severe combined immunodeficiency

Figure 3. scRNA-seq expression of monogenic IBD genes from healthy participants shows specificity to cellular compartments

(a-d) Monogenic IBD genes are enriched in specific cell subsets from healthy adult colon (n=12) (a), healthy pediatric ileum samples (n=8) (c) and their corresponding 1^st and 2^nd principal components ((b) and (d) respectively). Scaled mean expression of monogenic IBD genes (columns) across healthy cell subsets (rows) from different cell lineages (color legend), black outlines: q<0.05. (e) Unsupervised clustering of mean protein copy numbers (encoded by monogenic IBD genes) from 3-4 donors of peripheral hematopoietic cells, by quantitative proteomics of cells sorted by fluorescence-activated cell sorting (Rieckmann et al 2017). There was no data available for n=14 monogenic IBD genes, isoforms are specified. (f) Mean protein copy numbers of 3-4 donors in neutrophils vs classical monocytes in healthy participants. ILCs= innate lymphoid cells; T_regs= regulatory T cells; NKs= natural killer cells; CD4+LP= CD4+ lamina propria T cells; GC=germinal centre cells, DC= dendritic cells; M cell= ‘Microfold’ cells; TA= transit amplifying; WNT…= fibroblast subsets; MAIT cell= mucosal-associated invariant T cells

Figure 4. Cell-type specific monogenic IBD gene expression in IBD

(a-d) Monogenic IBD genes are enriched in specific cell subsets from inflamed adult colon (n=18) (a), inflamed paediatric ileum (n=7) (c) and their corresponding 1^st and 2^nd principal components ((b) and (d) respectively). Scaled mean expression of monogenic IBD genes (columns) across inflamed cell subsets (rows) from different cell lineages (color legend), black outlines: q<0.05. (e) Differentially-expressed (DE) genes (columns) in inflamed vs. healthy samples across cell subsets (rows) annotated by cell lineage (color legend) from colonic samples. Dot size: fraction of expressing cells from inflamed samples; dot color: DE model coefficients (q < 0.05; discrete model coefficient from MAST). (f) Cytokine/receptor cascades involving monogenic IBD genes based on scRNA-seq of non-inflamed colon. Monogenic IBD genes are indicated with an asterisk.

Figure 5. Overlap in genes, cell types, cell modules and transcriptional regulatory networks between polygenic and monogenic IBD

(a) There is a significant overlap of 13 genes between 81 monogenic IBD genes and 278 polygenic IBD candidate loci (representation factor: 6.5; p < 3.034e-05). (b) Monogenic and polygenic IBD genes are enriched in overlapping cell types. Mean expression of polygenic IBD genes (y axis) vs. monogenic IBD genes (x axis) for each cell type from the healthy human colon, with select cell types annotated and colored by lineage (legend). (c) Monogenic and polygenic IBD genes are co-expressed in gene modules. For gene modules of 250 co-expressed genes in healthy and inflamed cells from the colon (top) and ileum (bottom), shown is the top enriched KEGG term (y axis), the number of monogenic and polygenic IBD genes (left), the cell type distribution colored by cell lineage (middle), and annotated monogenic genes (right, black) and polygenic genes (right, grey) in the module. (d) For monogenic IBD genes (dark grey) or a background set of genes with the same expression statistics (light grey), the probability that genes from the gene set are co-expressed with polygenic IBD genes in the same cell type (left) or module (right) for colon and ileum cells (x axis). Error bars: SEM, P-values, *** P < 0.001. (e-f) 40 monogenic IBD genes (the ‘seed set’) were found within a Bayesian Gene Regulatory Network of gut biopsy transcriptomes and paediatric CD genotyping from the RISK cohort (Peters et al 2017) cohort, which integrated 7568 nodes (genes) and 14389 edges. Shown are the networks formed by adding (e) 1 or (f) 2 additional network layers to the seed set of these 40 monogenic IBD genes. 39 of the 40 genes were connected to each other in (f), along with a many adult GWAS polygenic genes, with significant enrichment found, suggesting a common transcriptional landscape.