Fig. 3.

(A) Genomic region surrounding EpCAM with exons labeled and CTE patient (P1, P2, P3, P4) mutations noted. Nucleotide and amino acid coordinates of mutations are shown assuming the A of the ATG codon is nucleotide 1. (B) Schematic representation of wild type and mutant RNA with variant splicing of Exon 4 as seen in patients P1 and P2 (C) EpCAM protein with Epidermal Growth Factor domains (EGF I and II) and transmembrane region (TM). Dashed box represents area coded by Exon 4. Predicted location of cysteine to tyrosine missense mutation at aa position 66 (C66Y) found in patient P4 (D) RT-PCR products of EpCAM from cDNA of control (WT) and affected patient (MUT) duodenal tissue demonstrating smaller product size of mutant (E) Fluorescent Immunohistochemistry of duodenal biopsies with 323/A3 EpCAM antibody (green) wild type (WT), mutant (MUT) and isotype control (IC). Demonstrating minimal non-specific staining of affected patient tissue (MUT) and epithelial predominance unaffected patient tissue (WT) (F) Patient Tissue Western Blot Analysis demonstrating decreased protein expression of EpCAM (mAB sc-25308 and 311-1k1) in CTE affected patient (P2) as compared with normal (N1, N4) and IBD patient (N6), equal levels of E-Cadherin (mAB EP700y) and Actin. 293 cell western blot analysis of 293 cells transfected with wild-type EpCAM (WT), mutant EpCAM (lacking exon 4) (MUT) and non-transfected cells (NT). Detectable bands corresponding to EpCAM using mAB sc-25308, but not 311-1k1 consistent with its epitope near the deleted exon 4.