Identification of EpCAM as the gene for congenital tufting enteropathy

Abstract

Background & aims: Congenital tufting enteropathy (CTE) is a rare autosomal recessive diarrheal disorder presenting in the neonatal period. CTE is characterized by intestinal epithelial cell dysplasia leading to severe malabsorption and significant morbidity and mortality. The pathogenesis and genetics of this disorder are not well understood. The objective of this study was to identify the gene responsible for CTE.

Methods: A family with 2 children affected with CTE was identified. The affected children are double second cousins providing significant statistical power for linkage. Using Affymetrix 50K single nucleotide polymorphism (SNP) chips, genotyping was performed on only 2 patients and 1 unaffected sibling. Direct DNA sequencing of candidate genes, reverse-transcription polymerase chain reaction, immunohistochemistry, and Western blotting were performed on specimens from patients and controls.

Results: SNP homozygosity mapping identified a unique 6.5-Mbp haplotype of homozygous SNPs on chromosome 2p21 where approximately 40 genes are located. Direct sequencing of genes in this region revealed homozygous G>A substitution at the donor splice site of exon 4 in epithelial cell adhesion molecule (EpCAM) of affected patients. Reverse-transcription polymerase chain reaction of duodenal tissue demonstrated a novel alternative splice form with deletion of exon 4 in affected patients. Immunohistochemistry and Western blot of patient intestinal tissue revealed decreased expression of EpCAM. Direct sequencing of EpCAM from 2 additional unrelated patients revealed novel mutations in the gene.

Conclusions: Mutations in the gene for EpCAM are responsible for CTE. This information will be used to gain further insight into the molecular mechanisms of this disease.

Fig. 1

Schematic of duodenal mucosa showing histology of (A) Normal intestinal villus and (B) Congenital tufting enteropathy villus with crowded epithelial cells forming tufts, villus atrophy. (C) H&E stained duodenal tissue (20X magnification) from affected patients (P1,P2) exhibiting tufting and crowding of epithelial cells.

Fig. 2

(A) Pedigree 1: family with congenital tufting enteropathy including affected patients (P1, P2) and unaffected siblings (U1, U2). Generations are indicated by Roman numerals on the left. Affecteds (P1, P2) are double second cousins. (B) Homozygosity Mapping: Genome-wide homozygosity intervals are plotted for individuals P1 and P2. The y-axis indicates the length of the intervals and the x-axis shows relative chromosomal position with chromosomes indicated across the top of each panel. The homozygous blocks shared between P1 and P2 are shown in the bottom bar graph (P1 & P2). The interval identified as homozygous in both individuals is highlighted with a gray box bar across all three panels (C) Haplotype Structure of the Pedigree. Homozygous blocks on chromosome 2p21 that are shared by the two affected descendants (P1, P2) are shown as black bars with genotypes at the flanking ends shown with SNP IDs from dbSNP. 220 SNPs in the middle of each block are not shown for simplicity as all 220 SNPs are homozygous and common in both affected individuals. (D) EpCAM exon 4 donor splice site mutation in Pedigree 1: Nucleotides across x-axis with mutation in bold. Coding nucleotides in capital letters and intronic nucleotides in lower case. Homozygous mutant affected patients (P1, P2), Heterozygous unaffected sibling (U2), Normal Control (C1)

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.