Figure 1.
Endoscopic phenotype of four representative sessile serrated polyps/adenomas (SSA/Ps) located in the ascending colon of patients with the serrated polyposis syndrome.
Panel A. Large 15 mm diameter SSA/P with a mucus cap. Panel B. 20 mm diameter SSA/P. Panel C. 10 mm diameter SSA/P. Panel D. Small 4 mm diameter SSA/P. The size of polyps was estimated using biopsy forceps as a reference. Histopathology analyses were consistent with SSA/Ps (M. Bronner).
Table 1.
Demographics of Serrated Polyposis Syndrome Patients.
Figure 2.
Differentially expressed genes in sessile serrated adenoma/polyps (SSA/Ps) by RNA sequencing (RNA-seq) and microarray analyses.
Panel A. RNA-seq analysis identified 1294 genes (875 increased, 419 decreased) that were significantly differentially expressed (fold change ≥1.5, FDR<0.05) in SSA/Ps as compared to control colon biopsies. Differentially expressed genes in SSA/Ps that were found by RNA-seq analysis (red) and those found in a microarray study (green; 101 total, 59 increased, 42 decreased) are shown in the Venn diagram (23). Panel B. Hierarchical clustering of the differentially expressed genes in Panel A. Note: only 782 genes could be compared in the hierarchical clustering analysis because fewer genes were interrogated in the microarray analysis. Panel C. Hierarchical clustering of differentially expressed genes in SSA/Ps identified by RNA-seq analysis and in adenomatous polyps (APs) identified by microarray analysis (24). 136 genes (75 increased, 61 decreased) with a fold change ≥10 and FDR of <0.05 from both datasets were compared. Four distinct clusters are shown, cluster 1 represents genes increased in only SSA/Ps, cluster 2 represents genes increased in both SSA/Ps and APs, cluster 3 represents genes decreased only in APs, and cluster 4 represents genes decreased in both SSA/Ps and APs. Note: the full range of fold change is not reflected in color bar scale, the maximum fold change in RNA-seq analysis was 582-fold (MUC5AC) in SSA/Ps and 208-fold (GCG) in APs by microarray analysis.
Table 2.
Top 50 gene transcripts increased by RNA sequencing in sessile serrated polyps (SSA/P) in serrated polyposis patients compared to controls.
Figure 3.
Expression of mucin 17 (MUC17), V-set and immunoglobulin domain containing 1 (VSIG1), gap junction protein, beta 5 (GJB5) and regenerating islet-derived family member 4 (REG4) in SSA/Ps, adenomatous polyps (APs) and controls as measured by RNA-seq analysis.
Panel A1. MUC17 RNA-seq results. The y-axis represents the number of uniquely mapped sequencing reads per kilobase of transcript length per million total reads (RPKM) mapped to the MUC17 locus. The x-axis represents the chromosome (Chr) 7 coordinates and gene structure of the MUC17 transcript. Analysis showed an 82-fold increase in MUC17 mRNA in SSA/Ps (red, n = 7 polyps) compared to uninvolved colon (patient matched uninvolved, blue, n = 6) and control colon (screening colon without polyps; green, n = 2). The sequencing read length was 50 base pairs. Panel A2. MUC17 expression measured by qPCR analysis in SSA/Ps, adenomatous polyps and controls in additional patients. Relative mRNA levels of MUC17 in large (>1 cm) and small (<1 cm) SSA/Ps (n = 21), adenomatous polyps (n = 10), uninvolved colon and normal control colon biopsies (n = 10 each) are shown. In small and large SSA/Ps, MUC17 expression was increased by 38 and 71-fold, respectively, compared to controls. qPCR results were normalized to β-actin. The average MUC17 expression level in uninvolved colon tissue was chosen as the baseline. P-values were calculated using the Mann-Whitney U-test. Panel B1. VSIG1 (Chr X) RNA-seq results. A 106-fold increase in expression of VSIG1 was found in SSA/Ps as compared to controls. Panel B2. VSIG1 qPCR results. In small and large SSA/Ps, VSIG1 expression was increased 969 and 1393-fold, respectively. Panel C1. GJB5 (Chr 1) RNA-seq results. A 27-fold increase in GJB5 mRNA was found in SSA/Ps. Panel C2. GJB5 qPCR results. In small and large SSA/Ps, GJB5 expression was increased 446 and 523-fold, respectively. Panel D1. REG4 (Chr 1) RNA-seq results. An 87-fold increase in REG4 mRNA was found in SSA/Ps. Panel D2. REG4 qPCR results. In small and large SSA/Ps, REG4 mRNA was increased 68 and 116-fold, respectively.
Figure 4.
Immunostaining for VSIG1, MUC17, CTSE and TFF2 in control colon, SSA/Ps, hyperplastic and adenomatous polyps.
Representative images of immunoperoxidase staining with affinity purified polyclonal antibodies and formalin-fixed, paraffin-embedded biopsies of patient matched and normal control colon (Panel A, n = 15, see Methods), syndromic SSA/Ps (Panel B, n = 10), sporadic SSA/Ps (Panel C, n = 15), hyperplastic polyps (Panel D, n = 10) and adenomatous polyps (Panel E, n = 10) are shown. Representative immunohistochemical stains for REG4 in control and polyp specimens are provided in Figure S2 in File S1.
Table 3.
Immunohistochemical analysis of different serrated and adenomatous polyp types for proteins encoded by genes found to be highly differentially expressed in SSA/Ps.