Noncoding deletions reveal a gene that is critical for intestinal function

Abstract

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants^1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

Extended Data Figure 1.. Family pedigrees.

Filled black symbols represent affected individuals, and deletion genotypes are indicated in red. Exome sequencing was done for individuals 1.1, 2.1, 3.1, 4.1, 4.2; whole genome sequencing was done for individual 2.1. Transcriptome analysis done for 2.1, 2.4. Patient 1.1 (*) was found to have uniparental disomy (UPD).

Extended Data Figure 2.. Genetic analysis in IDIS.

a, Analysis of SNP genotyping performed on six of the patients in families 1-5 and their 22 relatives detected a single significant telomeric linkage interval on chr16 with a maximum LOD score of 4.26. Haplotype reconstruction confirmed this interval with flanking marker rs207435 (chr16: 2,984,868) and showed two distinct disease haplotypes in an either homozygous setting in affected individuals for disease allele 1 (i.e. ΔL) in families 2, 3, 5, or a compound heterozygous setting for disease alleles 1 and 2 (i.e. ΔS) in family 4. All affected individuals carrying disease allele 1 showed an identical disease haplotype from rs533184 (chr16: 1,155,025) to rs397435 (chr16: 2,010,138). b, Schematic of reads covering exons in the C16orf91 gene, for the five exome- sequenced patients and for three controls sequenced under identical conditions. The first three patients with a ΔL/ΔL genotype had zero-coverage in the three upstream exons (right). The last two patients with a ΔL/ΔS genotype had non-zero-coverage in these exons, but coverage was significantly lower than in controls. The downstream exons (left) had high coverage in all subjects. Numbers indicate scale in sequencing reads per base.

Extended Data Figure 3.. Targeted deletion of the ICR non-coding sequence in mice.

a, Overview of targeting approach. See Methods for details. b, Genotyping results obtained from genomic DNA (n=554) isolated from the tails of homozygous and heterozygous ICR deletion mice, compared to a wild type control. See Methods for primers and details. c, Percc1 expression derived from mouse RNA-Seq from control littermates (left) and knockout mice (right). Tissues and timepoints are indicated to the left of each plot.

Extended Data Figure 4.. Gastrointestinal and microbiome analysis in chr17^ΔICR/ΔICR mice.

a, Modified intestinal content in wild type (left) compared to chr17^ΔICR/ΔICR mice at postnatal day 10 (n=45, right). b,c,d, ICR deletion causes changes in intestinal and fecal microbiome composition. Microbial communities in different intestinal compartments and feces were profiled by 16S rRNA-based sequence profiling. b, Family-level relative abundance profiles of the top fifteen most abundant prokaryotic families for wild type (n=22) and chr17^ΔICR/ΔICR (n=21) intestinal and fecal samples, organized by sample type. The most pronounced changes were observed in colon and fecal samples. c, Heatmap of log-transformed read counts for those genera exhibiting the greatest variance (top 60%) across all fecal samples. The abundance profiles exhibit perfect clustering of the fecal samples (rows) into wild type (n=6) and chr17^ΔICR/ΔICR (n=7) groups. d, Bar plots of Shannon’s diversity for all fecal samples from panel b grouped into wild type and chr17^ΔICR/ΔICR sample types.

Extended Data Figure 5.. Gastrointestinal X-gal (ß-gal) staining of ICR-reporter transgenic embryos compared to Percc1 mRNA in situ hybridization.

a,b E14.5 sections from a beta-galactosidase ICR-driven transgene. c,d Percc1 mRNA in situ hybridization analysis on E14.5 wild-type sections. For X-gal staining and in situ hybridization experiments, two embryos for each experiment and each condition were collected at E14.5 and a minimum of three sections from each embryo were examined. Representative sections are shown.

Extended Data Figure 6.. Body weight analysis in Percc1 knockout and transgenic mice.

a, Percc1 knockout mice have reduced body weight. Percc1 knockout mice (n=38) weight comparison to littermate controls (n=25) (red Percc1 knockouts, blue littermate controls). Lines represent the mean. Shaded areas represent +/− 1SD. Percc1 knockout mice were generated in an FVB/N genetic background. b, Percc1 transgenic rescue of the body weight phenotype found in chr17^ΔICR/ΔICR mice. An 8.5kb Percc1 mini gene was constructed (Supp. Table 10) and used to generate a Percc1 mouse line over-expressing PERCC1. Through introduction of this trangene into the chr17^ΔICR/ΔICR mouse genetic background, we observed rescue of all the phenotypes found in chr17^ΔICR/ΔICR mice including severe body weight reduction. Chr17^ΔICR/ΔICR mice were generated in a mixed 129/C57Bl6 background. Center point of lines represent the mean. Shaded areas represent +/− 1SD. P values were determined using a 2-tailed T-test. n.s.= p value 0.8-1.0.

Extended Data Figure 7.. Characterization of PERCC1 in mice and patients.

a, Western blot analysis of PERCC1:mCherry fusion protein. Two stable transgenic lines (B3269 and B3309) were established through standard pronuclear microinjection of fertilized mouse eggs. Protein extracts from juvenile mice (P13/14) were separated by SDS-PAGE and transferred for western hybridization. Lanes: 1, Molecular weight marker. 2/3, Line B3269. 4/5, Line B3309. 6, wild type control. 7, mCherry positive control protein. 8, Molecular weight marker. mCherry is predicted to be 28.8kD and the PERCC1:mCherry fusion protein 59kD with both proteins running ~5kD larger. Line B3309 does not express the fusion protein in contrast to Line B3269 (likely due to a position effect). These experiments were performed four times. b,c,d,e, Identification of cells with PERCC1+ identity and impact of PERCC1 ablation in gastrointestinal tissues. b, A subpopulation of PERCC1+ cells (red) in the corpus epithelium (mucosa) expresses Synaptophysin (Syp, green) at postnatal day (p) 8. Arrowheads mark double positive cells. Arrows mark a minor fraction of PERCC1+ cells detected in longitudinal smooth muscle cells (lSM). DAPI-stained nuclei are shown blue. c, Dispersed PERCC1+ cells (red) are observed in the villi of the duodenum at p8. Upper panel: Cross-section through villi illustrates absence of endocrine identity (green) in these cells. Lower panel: Sagittal section showing distribution of PERCC1+ cells in the epithelium of villi (Cdh1, green). d, Upper left: Schematic depicting the anatomical compartments of the distal stomach and location of sections used for cell counting. Upper right: Reduction of the fraction of G-cells observed predominantly in the pyloric antrum of Percc1-deficient (ΔICR/ΔICR) mice at p8. Box plots indicate median, interquartile values, range and individual biological replicates. Outliers are shown as circled data points. p, unpaired, two-tailed t-test. Lower panels: Comparative immunofluorescence analysis illustrating reduced numbers of Gastrin-expressing cells (red) in the absence of Percc1 (ΔICR/ΔICR) in the pyloric antrum at p8. Syp-expressing endocrine cells are colored green and nuclei gray. e, Immunofluorescence from Human intestinal organoids (HIOs) derived from control (+/+) and patient (ΔL/ΔL) iPSC lines. Detection of FOXA2 (blue) and anti-CDH1 (red) was used to visualize the HIO epithelium, and enteroendocrine cells were localized at 21 and 42 days based on Synaptophysin (SYP - green) expression and counted. The average number of detected SYP positive (SYP+) cells per 1000 epithelial (Epi) cells from cell counts in n=2 technical replicates from independent HIO preparations is indicated (p= 1.75E-18 for reduced number of SYP+ cells in ΔL/ΔL HIOs, Fisher’s Exact Test). n represents independent biological replicates with similar results. Scale bars, 50μm.

Extended Data Figure 8.. Percc1 analysis in mouse intestinal single cell transcriptomes.

a, Bar chart on the left shows the fraction of the total cells profiled in Haber et al. 2017 (n=11,665) assigned to each one of the major cell types identified. Bar chart on the right shows the same information but limited to those cells expressing Percc1 (n=8). b, Same as (a) but limited to Enteroendocrine Cells. (a-b) p-values were calculated using a Chi-squared test, using data from the corresponding left panel as reference. c, Box plots showing the distributions of the normalized expression values for known enteroendocrine-cell-associated transcription factors and hormones in the eight Percc1-positive cells from (a). In boxplots, the middle line denotes median; box denotes interquartile range (IQR); and whiskers denote 1.5 x IQR.

Extended Data Figure 9.. Validation of human RNA-Seq data by quantitative RT-PCR in duodenum tissue from 2 different patients and control tissue.

Six peptide hormones relative expression levels are presented compared with normal duodenum tissue (control), which is represented as 1. Relative expression levels for patients represent the average between two patients (patient 1.1 and 5.1). Gene symbols are provided beneath each pairwise comparison. NTS, neurotensin. GCG, glucagon. CCK, choleocystokinin. GAST, gastrin. SST, somatostatin. GIP, gastric inhibitory polypeptide.

Extended Data Figure 10.. Characterization of human intestinal organoids (HOIs) and iPSC lines.

a, HIOs generated from an affected patient, carrier, and unaffected sibling all showing normal morphology. Differential to HIOs was performed in duplicated with qPCR and histological analyses yielding similar results. b, iPSC lines from an affected patient, carrier and unaffected sibling display a normal karyotype. This was a single experiment for each sample as a quality control assessment.

Figure 1.. Overview of human and mouse locus and key findings.

a/b, Selected family pedigrees and genotyping results for patients compound heterozygous for the two deletion alleles (a) and homozygous for one of the deletion alleles (b). c/d, Genomic map of the deletion alleles in human (c; genome build GRCh37) and mouse (d), indicating the location of ΔL and ΔS, as well as their minimal overlapping region ICR. Exome sequencing data is capped at up to 5 overlapping tags for visualization; vertebrate conservation is 100-vertebrate PhyloP; only selected transcription factor binding sites and DNase hypersensitivity clusters with signal in >20/125 ENCODE cell types shown. e, General appearance of wildtype (n=50) and chr17^ΔICR/ΔICR (n=46) mice at 21 days after birth, showing overall significantly reduced size (see Fig 2d). g, Abnormal appearance of fecal pellets from chr17^ΔICR/ΔICR mice (n=46).

Figure 2.. Enhancer activity of the ICR and mouse deletion phenotypes.

a-b, Enhancer reporter activity in transgenic mouse embryos. a, Mouse embryo cross-sections showing X-gal staining for β-galactosidase activity in E13.5 stomach, pancreas and duodenum as marked. b, E14.5 cross-section showing immunofluorescence with anti-β-galactosidase (ICR activity, red), anti-endomucin (endothelial cells, green), and DAPI (DNA, blue). a/b, Two embryos for each experiment and each condition were collected and a minimum of three sections from each embryo were examined. Representative sections are shown. c, Chr17^ΔICR/ΔICR mice (n=46) are viable but show a reduction in size and weight compared to wild-type littermates (n=50). d/e, Reduction in body weight among surviving offspring (d) and increased mortality (e) of chr17^ΔICR/ΔICR compared to wild-type. Body weight of female mice shown in (d); male wildtype and chr17^ΔICR/ΔICR mice had similar genotype-dependent weight differences.

Figure 3.. Discovery of a novel gene, Percc1, flanking the ICR.

a, Gene expression levels of Percc1 in gastrointestinal tissues from wildtype (wt) and chr17^ΔICR/ΔICR mice. Highest levels of expression were detected in stomach at postnatal day 10 (P10). b, Mouse genome view of Percc1 gene localization and structure. Stranded RNA-seq data indicating expression loss of Percc1 in knockout mice compared to controls (bottom panel). c, Detailed view of the Percc1 gene and evolutionary conservation. d, Percc1 codon position analysis illustrating the relaxation of constraint in the third codon position of the predicted Percc1 protein (n = 274). dN/dS ratio and corresponding P-value calculated using Phylogenetic Analysis using Maximum Likelihood (Chi-square distribution).

Figure 4:. PERCC1 is abundant in G-cells and its genetic disruption impairs gastrointestinal peptide hormone expression and enteroendocrine cell development.

a, Top: Generation of a reporter fusion transgene to track PERCC1 localization in murine gastrointestinal tissues. The genomic sequence spanning the ICR and Percc1 ORF were fused to mCherry. Lower left panels: PERCC1+ cells (red) in the pyloric antrum at postnatal day 8 (P8) show endocrine identity marked by Synaptophysin (Syp, green) and extensive overlap with the endocrine subset of Gastrin-expressing G-cells (blue). Arrowheads indicate triple-positive cells. Nuclei are shown in gray. Lower right panels: PERCC1+ cells in the pyloric canal carry either endocrine G-cell identity (arrowheads) or appear frequently clustered at the gland base (arrows). n represents independent biological replicates with similar results. Scale bars, 50μm. b, RNA-seq from mouse stomach samples across different timepoints (n = 1 biological replicates) reveals reduced transcript levels of different gastric peptide hormones in ΔICR/ΔICR mice. Sst, Somatostatin; Gast, Gastrin; Grhl, Ghrelin; Pyy, Peptide YY; Gcg, Glucagon; Fndc5, Fibronectin type III domain-containing protein 5; Adipoq, Adiponectin precursor. c, Quantitative RT-PCR analysis of induced pluripotent stem cell (iPS)-derived human intestinal organoids (HIOs) from patients with disrupted PERCC1 (ΔL/ΔL) vs. control siblings. In contrast to Chromogranin A (CHGA), Synaptophysin (SYP) is significantly downregulated in patient-derived HIOs (p<0.05) (two-tailed, unpaired t-test). iPS, induced pluripotent stem cells. Box plot indicates median, interquartile values, range, outliers (circled dots) and individual technical replicates (from independent organoid preparations).