1. Genetics and Genomics
  2. Immunology and Inflammation
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SCGN deficiency results in colitis susceptibility

  1. Luis F Sifuentes-Dominguez
  2. Haiying Li
  3. Ernesto Llano
  4. Zhe Liu
  5. Amika Singla
  6. Ashish S Patel
  7. Mahesh Kathania
  8. Areen Khoury
  9. Nicholas Norris
  10. Jonathan J Rios
  11. Petro Starokadomskyy
  12. Jason Y Park
  13. Purva Gopal
  14. Qi Liu
  15. Shuai Tan
  16. Lillienne Chan
  17. Theodora Ross
  18. Steven Harrison
  19. K Venuprasad
  20. Linda A Baker
  21. Da Jia
  22. Ezra Burstein  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Sichuan University, China
  3. Texas Scottish Rite Hospital for Children, United States
Research Article
Cite this article as: eLife 2019;8:e49910 doi: 10.7554/eLife.49910
7 figures, 1 table, 1 data set and 6 additional files

Figures

Figure 1 with 2 supplements
A mutation in SCGN is linked to early-onset ulcerative colitis.

(a) Pedigree of index family. Probands (P1, P2, and P3) and their siblings (S1 and S2) are indicated. SCGN genotypes are noted (+ = WT allele, - = R77H allele) (b) Representative endoscopic images of the rectum from affected individuals. (c) Multispecies alignment of SCGN protein sequences is shown; the residue affected in the rare coding variant found in affected patients (R77) is indicated. (d) Frequencies of allele variants of SCGN found in ExAC are plotted along the SCGN protein sequence, with the location of the six EF-hands also indicated. R77 is noted by an arrow.

https://doi.org/10.7554/eLife.49910.002
Figure 1—figure supplement 1
Clinical course of affected probands.

Critical clinical events are noted. Full line represents active follow-up and dashed line represents no active follow-up. Red bars along each proband’s timeline represent periods of time of reported colitic symptoms (bloody diarrhea and abdominal pain). Time of diagnosis, infliximab therapy initiation and time of colectomy are also noted.

https://doi.org/10.7554/eLife.49910.003
Figure 1—figure supplement 2
Areas of shared LOH in P1 and P2 as analyzed by WGS.

The average heterozygosity across 100 kb genomic windows are shown for both affected siblings. This analysis identified single contiguous regions of homozygosity on 6 p and 12q in both probands.

https://doi.org/10.7554/eLife.49910.004
Figure 2 with 3 supplements
SCGN is expressed neuroendocrine cells.

(a) Immunohistochemistry (IHC) staining for SCGN in rectal biopsies obtained from a healthy individual. 40x magnification. (b) IHC staining for SCGN in rectal biopsies obtained from P2, one of the probands in the index family. 40x magnification. (c) SCGN and chromogranin A (CGA) immunofluorescence staining of murine colonic epithelium (scale bar 200 µm – inset 20 µm). (d) Morphometric quantification of SCGN and CGA staining patterns in epithelial cells of three sites of the murine gut. Cell population percentages are shown. SB: Small bowel (e) SCGN and synaptophysin (SYP) immunofluorescence staining in subepithelial cells of the murine large intestine (scale bar 200 µm – inset 20 µm). (f) SCGN and CGA immunofluorescence staining in murine pancreatic islets (scale bar 200 µm - inset 100 µm).

https://doi.org/10.7554/eLife.49910.007
Figure 2—figure supplement 1
Immunohistochemistry (IHC) staining for SCGN in colonic biopsies obtained from probands P1, P2, and P3. 40x magnification.
https://doi.org/10.7554/eLife.49910.008
Figure 2—figure supplement 2
Immunofluorescence staining for markers of EEC lineage in healthy human colon (scale bar 200µ - inset 40 µm).

Arrows indicate cells that are exclusively positive for SCGN. 5-HT, 5-hydroxytryptophan (serotonin); CGB, chromogranin B; GCG, glucagon precursor peptide.

https://doi.org/10.7554/eLife.49910.009
Figure 2—figure supplement 3
Immunofluorescence staining for neuronal markers in murine colon of C57BL/6 mice (scale bar 200 µm - inset 50 µm).

TUJ1, neuron-specific class III β-tubulin; SNAP25, synaptosomal nerve-associated protein 25.

https://doi.org/10.7554/eLife.49910.010
Figure 3 with 3 supplements
SCGN p.R77H is a hypomorphic allele.

(a) Basal and DHA fatty acid induced GLP-1 release from parental and Scgn deleted (KO) clones. GLP-1 values are normalized to total protein content. (b) Basal and DHA stimulated GLP-1 secretion from rescue clones expressing human SCGNWT or SCGNR77H. (c) Immunofluorescence images showing subcellular localization of SNAP25 and SCGN in parental STC-1 cells and the indicated SCGN KO and rescue cell lines. Scale bar 15 µm. (d) For the experiment depicted in (c), SNAP25 staining intensity ratio between the membranous compartment and the total cellular signal was plotted in the indicated cell lines. Dots indicate individual cells, horizontal bars correspond to the mean within each group. (e) Midbrain size of zebrafish after scgn targeting with morpholinos or rescue with human SCGNWT or SCGNR77H. Dots indicate individual embryos, horizontal bars correspond to the mean within each group. The zebrafish experiments were performed in triplicate. *p<0.05, **p<0.01, ****p<0.0001 unpaired student t test in (a), (b) and (d). ****p<0.0001 multiple comparison ANOVA in (e). Error bars in (a) and (b) represent the S.E.M.

https://doi.org/10.7554/eLife.49910.012
Figure 3—figure supplement 1
Scgn deficient clones of STC-1 cells were generated by CRISPR/Cas9 technology.

Protein knockout (KO) was confirmed by immunoblotting. Expression of SCGNWT or SCGNR77H in rescue clones is also shown.

https://doi.org/10.7554/eLife.49910.013
Figure 3—figure supplement 2
SNAP25 co-precipitation from SCGN rescue cell lines.

Re-expressed SCGN was HA tagged.

https://doi.org/10.7554/eLife.49910.014
Figure 3—figure supplement 3
Bright-field images of zebrafish after morpholino injections (MO).

In-situ hybridization of HuC (elavl3) was performed in embryos at 48 hr post fertilization to highlight the developing brain. Control: control MO injection; MO: scgn MO injection; MO+WT: SCGN MO and human SCGN WT mRNA co-injection; MO+R77H: SCGN MO and human SCGN R77H mRNA co-injection. All injections are performed at one-cell stage of the development.

https://doi.org/10.7554/eLife.49910.015
Figure 4 with 1 supplement
Scgn deficient mice display intact mucosal architecture and microbiota at baseline.

(a) Representative colon histologic images from Secret1 and WT animals under untreated conditions. HE is shown on the left, alcian blue staining on the right. Scale bar 100 µm. (b) Baseline expression of prototypical intestinal epithelial cell lineage-specific markers from small bowel in Secret1 and WT mice was determined by qRT-PCR (n = 3 in each group). (c) Microbiome alpha diversity as measured by observed OTU mean counts of fecal 16 s rRNA sequencing. (d) Beta diversity by unweighted UNIFRAC principal coordinate analysis (PCoA) of fecal 16 s rRNA sequencing. (e) Class level taxonomic composition for stool 16 s sequencing. WT n = 16 Secret1 n = 20. S.E.M. was used for error bars in (b).

https://doi.org/10.7554/eLife.49910.020
Figure 4—figure supplement 1
Engineering Scgn deficient animals.

(a) Diagram of CRISPR/Cas9 targeted exon 3 of Scgn. 20 nucleotide guide RNA sequence (blue), PAM (red). Reference sequence (top) and sequences from founder animals (middle and bottom) with varying length deletions are depicted. (b) Genotyping of mice carrying CRISPR/Cas9 induced deletions. (c) RT-PCR for Scgn using RNA extracted from colonocytes from WT and Secret1 animals. (d) Sequence alignment of WT and Secret1 Sanger sequencing results of RT-PCR product. (e) Fluorescent immunostaining of SCGN and CGA in pancreatic islets of Scgn sufficient (Scgn+/+) and Scgn deficient (Secret1 and Secret2) mouse lines. Scale bar 50 µm.

https://doi.org/10.7554/eLife.49910.021
Figure 5 with 2 supplements
SCGN loss leads to increased sensitivity to DSS colitis.

(a–c) Body weight, disease activity index (DAI), and survival of male WT (n = 13) and Secret1 (n = 13) mice treated for 6 days with 2% DSS in their drinking water. (d) Histologic score of male and female WT (n = 11) and Secret1 (n = 13) animals treated for 6 days with 2% DSS. (e) Representative microphotographs (20x) of colonic epithelium of DSS-treated mice. (f) Inflammatory gene expression measured by qRT-PCR from ceca of DSS-treated mice. Data in (a), (b) and (c) are representative of 2 experiments. *p<0.05. **p<0.01, two tailed unpaired t test in (a), (b) and (f). *p<0.05. one tailed unpaired t test in (d). S.E.M. was used for error bars in (a), (b), (d), (f). Log rank test was used in (c).

https://doi.org/10.7554/eLife.49910.025
Figure 5—figure supplement 1
Increased sensitivity to DSS in Scgn deficient animals.

(a) Body weight of male and female Secret1 (n = 27) and WT (n = 21) mice treated with 2% DSS for 6 days. This is the aggregate data of 2 separate experiments. (b) Body weight of male Secret2 and WT mice during 6 day treatment with 1% DSS. (c) Inflammatory gene expression from ceca of experimental mice as measured by qRT-PCR. WT n = 9 Secret2 n = 12. **p<0.01 two tailed unpaired t test in (b) *p<0.05 one tailed unpaired t test in (c). Error bars in (a), (b), (c). S.E.M.

https://doi.org/10.7554/eLife.49910.026
Figure 5—figure supplement 2
Immunophenotying of WT and Secret1 mice.

Lamina propria leukocytes were extracted from colon and small intestine (SI) of WT and Secret1 mice and stained for flow cytometry. The flow cytometry images show pooled analysis of (a) CD45+CD4+ cells T cells, (b) CD45+CD19+ B cells, (c) CD45+NK1.1+ NKT cells, (d) CD45+CD4+CD25+ Tregs, (e) CD45+LinNKp46IL23R+ ILC3, (f) CD45+CD11b+Ly6G+ neutrophils, (g) CD45+CD11b+ macrophages, and (h) CD45+CD11b+CD11c+ dendritic cells. (i) Representative images showing gross appearance of the spleen and MLN from WT and Secret1 mice (n = 5 mice in each group).

https://doi.org/10.7554/eLife.49910.030
Loss of colonic EECs does not confer DSS susceptibility.

(a) Diagram depicting the mating strategy used to generate colonic EEC (Neurog3ΔCol) deficient mice. (b) CGA immunofluorescent staining of colon and small bowel from wild-type (Neurog3f/f) and colonic EEC deficient (Neurog3ΔCol) mice (c) qRT-PCR of epithelial lineage makers from colonic epithelium from Neurog3f/f and Neurog3ΔCol mice (n = 3 in each group). (d) Heat map presentation of top differentially expressed genes from RNA-seq of colonic epithelia of Neurog3f/f and Neurog3ΔCol mice. (e) Ex-vivo basal and DHA-stimulated GLP-1 and GLP-2 secretion from colonic explants of Secret1 and EEC deficient mice. (GLP-1: Neurog3f/f n = 7, Neurog3ΔCol n = 6, WT n = 6, Secret1 n = 6) (GLP2: Neurog3f/f n = 5, Neurog3ΔCol n = 5, WT n = 6, Secret1 n = 4). Bars represent the mean and error bars the S.E.M. (f–g) Body weight and DAI of conventionally raised Neurog3f/f and Neurog3ΔCol mice treated for 6 days with 3% DSS (n = 15 in each group). Scale bar in (b) 50 µm. *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001 two tailed unpaired t test in (c) and (e). S.E.M. was used for error bars in (c), (e), (f) and (g).

https://doi.org/10.7554/eLife.49910.037
Author response image 1
HEK293T cells, which do not express SNAP25, were transfected with HA-tagged SCGN (WT or R77H).

These proteins were subsequently purified through an HA binding resin. The purified proteins, immobilized on the resin were then incubated with cellular lysates of STC-1 cells (0.75mg of lysate offered), and SNAP25 binding was determined by immunoblotting. Three technical replicates of the IP were performed (top three panels on the left). Input from HEK293 cells are shown on the right. This experiment is representative of 3 independent iterations.

Tables

Table 1
Areas of shared loss-of-heterozygosity (LOH) among affected probands as defined by SNP array.
https://doi.org/10.7554/eLife.49910.006
StartEnd
Chr 1271,016,15772,070,710
72,320,25173,669,855
73,671,27675,891,939
77,249,40079,204,389
79,302,41982,139,004
82,147,48783,157,896
83,541,96688,441,286
88,594,15989,910,070
89,915,48491,765,486
91,766,72093,152,115
93,251,75095,410,845
Chr 616,893,01118,222,277
18,262,60719,682,599
19,686,12319,803,768
19,804,18821,778,105
23,584,37526,148,311

Data availability

Sequencing data have been deposited in GEO under accession code GSE134202. Data generated during this study is included in the manuscript.

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. LF Sifuentes-Dominguez
    2. E Burstein
    (2019)
    ID GSE134202. Transcriptome-wide gene-expression analysis of colonic epithelium from enteroendocrine cell-deficient mice.

Additional files

Supplementary file 1

Copy number variation and loss of heterozygosity (LOH) analysis by SNP array.

https://doi.org/10.7554/eLife.49910.043
Supplementary file 2

Scoring system for inflammation-associated histological changes in the colon (DSS).

https://doi.org/10.7554/eLife.49910.044
Supplementary file 3

Disease activity index for colitis model.

https://doi.org/10.7554/eLife.49910.045
Supplementary file 4

Primer Sequences.

https://doi.org/10.7554/eLife.49910.046
Supplementary file 5

Key resource table.

https://doi.org/10.7554/eLife.49910.047
Transparent reporting form
https://doi.org/10.7554/eLife.49910.048

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