Congenital Tufting Enteropathy is a rare autosomal recessive diarrheal disorder presenting in the neonatal period with significant morbidity and mortality. Using a family with 2 children affected with CTE, Single Nucleotide Polymorphism genotyping was performed revealing a unique 6.5 MB haplotype of homozygous SNPs on chromosome 2p21. 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 of affected patients. RT-PCR 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 two additional unrelated patients revealed 2 additional mutations in the gene. The identification of EpCAM as the gene responsible for CTE will not only improve the diagnosis of this congenital diarrhea, but it is an important step in the understanding of the underlying pathophysiology and mechanisms involved in normal and abnormal intestinal morphogenesis and differentiation.
This study highlights the power of modern genetic technology to identify disease genes associated with rare diseases using a small number of affected patients. SNP genotyping allows for dense whole genome analysis and identification of linkage that was previously impossible. Recently, several large genome wide association studies using this methodology have provided clues to the genetic basis of common diseases, such as diabetes mellitus, breast cancer and Crohn’s disease, using large cohorts of patients and controls18, 19
. However, this technology has not been widely applied to the study of rare disease genes. Traditional microsatellite marker mapping has been used in the mapping of rare disease genes, even in relatively small inbred families20
. Here, we have applied SNP technology for the identification of a gene responsible for CTE by analysis of only two distantly related affected patients with a unique relationship. The use of SNPs allows for a high likelihood for the identification of linkage.
Like most cell adhesion molecules, the primary function of EpCAM appears to be cell-cell interaction. This is supported by studies with L929 fibroblasts which are normally incapable of cellular adhesion, but form multicellular aggregates of cells when expressing EpCAM, suggesting involvement in homotypic cell-cell interactions21
. EpCAM is known to recruit intracellular α-actinin to the sites of homophilic contacts22
. EpCAM also co-localizes with E-cadherin in the areas of cell-cell junctions and directly associates with claudin-7, a tight junction protein23
Using RT-PCR we demonstrate a 66 bp in-frame deletion in EpCAM
small intestinal mRNA in the affected patients only. This variation does not result in a frameshift; therefore translation of the C terminal portion of the EpCAM protein is not likely to be affected. Lack of immunofluorescent staining and lower levels EpCAM on Western Blot in affected patient tissue suggests the splice site mutation affects protein expression in the intestinal tissue. The function of the domain coded for by exon 4 is not known (), but its deletion could affect protein stability, localization, or function. This may occur by altering post-translational modifications such as proteolytic cleavage, homophilic adhesion (mediated by the EGF domains), or transmembrane domain anchoring of EpCAM to intestinal epithelial cell membranes. Mutations in EpCAM
may disrupt its association with α-actinin, claudin-7 or E-Cadherin leading to mucosal integrity breakage and intestinal failure seen in CTE. Interestingly, deletion of a small portion of the extracellular domain of EpCAM completely abolishes the interaction of the intracellular domain with α-actinin24
. It is possible that deletion of exon 4 has a similar consequence.
Cell adhesion molecules have also received much attention for their morphoregulatory roles in development and influence in specifying cell fate in numerous tissues25
. In fact, EpCAM is primarily known for its potential role in tumorigenesis resulting from increased expression on the cell surface of human carcinoma cells including tumors of the gastrointestinal system, breast, thyroid, and kidney22
and therefore it is being studied as a target for cancer therapy26
We speculate that EpCAM plays a role in normal intestinal development, as described in the pancreas27
, resulting in the pathologic findings observed in CTE. EpCAM function may be important for the development of the crypt villus axis, where epithelial cells originate from stem cells in the crypt and migrate distally to the tip of the villus prior to shedding. Mechanisms that lead to apoptosis of intestinal epithelial cells are still not completely clear, but it is plausible that dysregulation of apoptosis in intestinal epithelial cells may play a pathogenic role in diseases such as intestinal dysplasia and carcinomas28, 29
Many families reported with CTE are consanguineous or follow a pattern consistent with autosomal recessive inheritance2, 9
, as was demonstrated in our initial family. However, analysis of patient P4 revealed a heterozygous missense mutation in an exon coding for an important extracellular functional domain (EGF2), and no additional coding mutations were identified. In this patient, it is possible that CTE is transmitted in an autosomal dominant fashion. Alternatively, compound heterozygosity with a second mutation in an unsequenced noncoding region is also possible.
Residual gut function and longevity vary among CTE patients. In addition, some patients have associated malformations including punctated keratitis, choanal atresia, and esophageal atresia10, 30
. The clinical phenotype spectrum in CTE may be explained by different mutations in the same gene or mutations in other genes. An EpCAM
mutation was not found in one of our patients suggesting genetic heterogeneity. Interestingly, this patient (P5) is currently 20 years old and his survival suggests less severe disease. It is also possible that sequence variants within the promoter or intronic regions of the EpCAM
gene could contribute to disease development. Genes that code for proteins with significant homology, similar function, or that form complexes with EpCAM
are also candidate genes for further study.
The identification of this CTE mutation will improve our understanding of this disorder, and offer new research directions in this field. Furthermore, our findings should elucidate the essential role of adhesion molecules in the development of the gastrointestinal system. This approach illustrates the utility of using unique kindreds and powerful new genetic technology to better characterize difficult to study rare diseases.