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Somatic inflammatory gene mutations in human ulcerative colitis epithelium

Abstract

With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1,2,3,4,5,6,7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling—including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8,9,10,11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.

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Fig. 1: Somatic mutations in UC epithelium.
Fig. 2: Targeted sequencing of bulk UC organoids.
Fig. 3: Phenotypic characterization of UC-related mutations.
Fig. 4: iNOS mediates IL-17A-induced apoptosis.

Data availability

Gene expression datasets are available from the Gene Expression Omnibus (GEO) under the accession code GSE127757. Whole-exome sequencing data have been deposited to the Japanese Genotype-phenotype Archive under the accession number JGAS00000000199. Source data for Figs. 1, 3 and 4 and Extended Data Figs. 4, 6, 8 and 9 are provided with the paper. All relevant data that are included with this study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by AMED (grant numbers JP19cm0106206 and JP19gm5010002), AMED-CREST (grant number JP19gm1210001) and the JSPS KAKENHI (grant numbers JP17H06176 and JP26115007). K.T., Y.O. and M.F. were supported by the Japan Society for the Promotion of Science Research Fellowships for Young Scientists. We also thank the Collaborative Research Resources at the School of Medicine, Keio University for providing technical assistance.

Author information

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Authors

Contributions

K.N., M.F. and T.S. designed the study. T.H., S.I. and T.K. provided specimens. Y.O. and S.T. performed histochemical analysis. K.N., M.F., M.M., S.S., K.K. and K.I. performed sample preparation. K.N., M.F., M.M., S.N., A.T., S.S., K.I., K.Y. and N.S. performed organoid experiments. M.F., M.S., S.D., Y.N. and K.T. performed sequencing and bioinformatics analysis. R.I. supervised statistical analyses. K.N., M.F. and T.S. wrote the manuscript.

Corresponding author

Correspondence to Toshiro Sato.

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Competing interests

T.S. is an inventor on several patents related to organoid culture.

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Peer review information Nature thanks Judy Cho, Ramnik Xavier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Study design.

a, Nomenclature of organoids. b, Patient demographics and the breakdown of the organoids used for each analysis.

Extended Data Fig. 2 Mutational signatures of HC, UC and CAN organoids.

a, Relative contribution of the nucleotide transition patterns in each sample group in healthy (n = 28), UCuninf (n = 15), UCinf without CAN (n = 15) and UCinf with CAN (n = 17) organoids. Data are shown as the mean (±s.d.) relative contribution of base substitution types in each sample type. b, Cosine similarity of the mutational signature of each organoid clone to the COSMIC signatures (1–30). UCinf organoids with an MLH1 mutation (UC08T_IS1, UC08T_IS2) are highlighted in red. One organoid line with a contribution of APOBEC signatures (signatures 2 and 13) is highlighted in green characters (UC10T_IS). c, Number of truncating mutations in the indicated genes in healthy, UCuninf (n = 43) and UCinf (n = 31) organoids. Genes with two or more organoid clones that have truncating mutations are shown. MLH1-mutated organoids (UC08T_IS1, 2) were excluded from this analysis. *P = 0.004; q < 0.1 (two-sided Fisher's exact test with Benjamini–Hochberg adjustment). d, dN/dS ratios of NFKBIZ and PIGR (genes with pglobal < 0.05 and qglobal < 0.1), calculated using the whole-exome sequencing data.

Extended Data Fig. 3 Copy-number analysis of UC organoids.

a, Heat map showing the average minor allele frequencies (MAFs) of SNPs that were heterozygous in the normal counterpart (bin size 25 Mb). Genomic regions with LOH are indicated with red boxes. Genomic positions of PIGR, NFKBIZ and TRAF3IP2 are shown at the top. Organoid lines that were subjected to CGH–SNP array analysis are highlighted in red. Organoid lines with MLH1 mutations (UC08) are not shown. b, DNA copy-number analysis using CGH–SNP microarray analysis. For each organoid line, the genome-wide chromosome state is demonstrated by the log2-transformed signal ratio (top) and the allelic difference (bottom) values. Genomic regions with CN-LOH are shown in red. Deletions in common fragile sites are shown in green. Genetic lesions associated with UC-related mutations are shown in black. c, Loss of PIGR protein in UC12T_IA organoids with CN-LOH at the PIGR locus. Intact PIGR protein expression in UC12T_IS organoids is also shown. β-actin was used as a loading control. Data are representative of n = 2 technical replicates with similar results. d, Focal deletion of fragile sites in UCinf organoids, detected by CGH analysis. Chromosome states are visualized using the log2-transformed signal ratio. Deletions in chromosomal fragile sites at positions 3p14 and 16p13.2 were uniquely detected in the UCinf epithelium.

Extended Data Fig. 4 Characterization of UC-related mutations.

a, Increased expression of NFKBIZ mRNA in purified UC epithelium (E-MTAB-5464). NFKBIZ expression was compared between healthy control individuals (n = 11) and patients with UC (n = 11) using the exact negative binomial test in edgeR (P = 5.0 × 10−6). Bars represent the median. b, Gene set enrichment analysis using public datasets (GSE38713, left; GSE59071, right) and in-house IL-17 target genes (Supplementary Table 3). NES; normalized enrichment score. Left, n = 15 (active colitis), n = 13 (healthy control); right, n = 82 (active colitis), n = 11 (healthy control). ch, IL-17A-induced expression of NFKBIZ (IκBζ) and PIGR in colon organoids. Organoids were stimulated with 100 ng ml−1 IL-17A for 3 h (c, f) or 24 h (d, e, g, h). The expression of NFKBIZ (IκBζ) and PIGR in control, NFKBIZ-mutant and TRAF3IP2-mutant organoids was analysed by quantitative PCR with reverse transcription (c, f) and immunoassay (d, e, g, h). Matched UCuninf organoids that were derived from the same patients were used as controls. mRNA expression levels are shown relative to the expression of ACTB. Expression levels of PIGR protein relative to β-actin are shown at the bottom of the pseudo-blot images. Results are shown from two (ce) and one (fh) biologically independent experiments, and two technical replicates (g, h). Data are shown as mean + s.e.m. (c, f). i, Genetic mutations detected by whole-exome sequencing in CAN organoids. Patterns of mutations are specified using the same symbols as Fig. 2a. Source data

Extended Data Fig. 5 Targeted sequencing of UC-related genes.

a, Consistency of VAFs between fresh epithelium samples and organoids (n = 11 pairs). Red dots and lines show truncating and non-synonymous mutations in UC-mutated genes (NFKBIZ, PIGR, IL17RA, TRAF3IP2 and ZC3H12A) (n = 12 mutations). Black dots and lines show synonymous mutations in genes that are mutated in UC and any mutations in the other genes (n = 19 mutations). VAFs did not significantly differ between the sample pairs (P = 1, two-sided Wilcoxon signed-rank test). b, dN/dS ratios of the indicated genes in the targeted sequencing data. Genes with with q-values <0.1 (that is, statistically significant) are shown. c, Genetic mutations in UCuninf and control organoids, detected by targeted sequencing. Top, for each gene, the number of different variants is shown by density. Bottom, the VAF in each analysed sample. Each dot represents the fraction of each altered allele per total reads mapped to the gene. Genes are shown in different colours as specified on the left. d, PIGR (top), secretory IgA (middle) and cytokeratin20 (CK20) (bottom) immunostaining of UCinf epithelium (UC09T_IS2 and UC17T_IA) with focal loss of PIGR and secretory IgA expression. Magnified areas show boundaries between regions in which PIGR expression is intact and regions in which PIGR expression is lost. Images are representative images of four regions in which PIGR expression is lost, in eight patients with UC. Scale bars, 500 μm (low magnification); 100 μm (high magnification).

Extended Data Fig. 6 Targeted sequencing of UC-related genes (continued).

a, b, Reduced response to treatment with IL-17A in patient-derived cloned ZC3H12AD437Y UCinf organoids (UC23N_IS) in comparison to wild-type counterpart organoids (UC23N_UA). Results are representative of n = 2 technical replicates with similar results. c, Design of CRISPR–Cas9-mediated ZC3H12AD437Y knock-in. The donor vector contains two silent mutations at the protospacer adjacent motifs flanking the CRISPR targets, in addition to the D437Y mutation. Each allele of the knocked-in clone used for analysis is shown on the right. The clone is heterozygous for a knocked-in D437Y mutation and a 42-bp in-frame deletion. d, e, Reduced response to treatment with IL-17A in genetically engineered ZC3H12AD437Y-KI organoids compared to parental wild-type organoids. Results are representative of n = 2 technical replicates with similar results. f, Per-patient data of the targeted sequencing result. For each patient, mutations in organoids that were derived from the most-distal sample are shown. Top, for each gene, the number of different variants is shown by density. Bottom, dots represent the VAF of each variant. Genes are shown in different colours as specified on the left. Data are shown as mean + s.e.m. (a, d). Source data

Extended Data Fig. 7 Targeted sequencing of cytokine and PAMP receptor genes and cancer driver genes.

ac, Mutations in cytokine and PAMP receptor genes (a) and sporadic colon cancer driver genes (b, c) identified in UCinf, UCuninf and control organoids by targeted sequencing. Number of different variants per gene are shown by density (top). Top, for each gene, the number of different variants is shown by density. Bottom, dots represent the VAF of each variant. Genes are shown in different colours as specified on the left. d, Sporadic colon cancer gene mutations identified in CAN organoids.

Extended Data Fig. 8 IL-17A-induced cytotoxic response in human colon organoids.

a, Fold-change values of individual sgRNAs in two experiments that used different lines of organoids. sgRNAs are coloured according to their targeting genes, as specified at the top. b, Response of wild-type organoids to treatment with IL-17A in differing growth factor conditions. The organoids showed a cytotoxic response to treatment with IL-17A only in the absence of Noggin. The WNRAIF condition is the medium condition including WNT3A, Noggin, R-spondin-1 (Rspo), A83-01, IGF-1 and FGF-2, but lacking EGF. Data are representative of n = 2 biologically independent experiments with similar results. c, sgRNA targets and confirmation of CRISPR–Cas9-mediated knockout of IL17RA, TRAF3IP2 and NFKBIZ. For knockout of NFKBIZ, three NFKBIZKO lines (#1–3) were generated from different parental organoids. Successful knockout was confirmed by Sanger sequencing in all lines. Loss of IκBζ protein was also confirmed by an immunoassay using an organoid stimulated with IL-17A for 24 h. Result is from a single experiment. d, IL17RAKO and TRAF3IP2KO organoids are resistant to treatment with IL-17A in a Noggin-deficient condition. Right, dots show the relative area of organoids in each well. Results are representative of n = 3 technical replicates. e, NFKBIZKO organoid line 1 tolerates treatment with IL-17A. The other two knockout lines showed a similar response. f, Patient-derived NFKBIZ-mutant organoids (UC02N_IS) are resistant to treatment with IL-17A. Wild-type counterpart organoids (UC02N_UA) were used as a control. Data are representative of n = 2 biologically independent experiments with similar results. g, Patient-derived ZC3H12AD437Y-mutant organoids (UC23N_IS) are tolerant of treatment with IL-17A compared to their wild-type counterparts (UC23N_UA). Data are representative of n = 2 technical replicates with similar results. h, Knockout of TP53 mitigates IL-17A-induced cytotoxicity. Data are representative of n = 2 biologically independent experiments with similar results. Mean + s.e.m (dh). P values determined by two-sided Welch’s t-test. Scale bars, 1 mm (b, dh). Source data

Extended Data Fig. 9 iNOS-dependent apoptosis in human colon organoids treated with IL-17A.

a, Detection of cleaved caspase-3 by immunoassay. Treatment with IL-17A increases the expression of cleaved caspase-3 in wild-type, but not NFKBIZKO, organoids in the absence of Noggin. b, Analysis of NOS2 transcript expression by quantitative PCR. IL-17A induces expression of NOS2 transcript in wild-type, but not in NFKBIZKO, TRAF3IP2KO and IL17RAKO, organoids in the absence of Noggin. Source data

Extended Data Table 1 Clinical information on the participants in the study

Supplementary information

Supplementary Figure

Supplementary Figure 1: Source images of capillary based immunodetection.

Reporting Summary

Supplementary Table

Supplementary Table 1: Somatic mutations identified by whole exome sequencing

Supplementary Table

Supplementary Table 2: Somatic mutations identified by targeted sequencing.

Supplementary Table

Supplementary Table 3: List of IL-17 target genes, and sequences of qPCR primers, sgRNA targets and double strand DNAs.

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Nanki, K., Fujii, M., Shimokawa, M. et al. Somatic inflammatory gene mutations in human ulcerative colitis epithelium. Nature 577, 254–259 (2020). https://doi.org/10.1038/s41586-019-1844-5

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