Mamata Sivagnanam

Identification of EpCAM as the Gene for Congenital Tufting Enteropathy

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.

Absence of cell-surface EpCAM in congenital tufting enteropathy

Mutations in the epithelial cell adhesion molecule (EpCAM; CD326) gene are causal for congenital tufting enteropathy (CTE), a disease characterized by intestinal abnormalities resulting in lethal diarrhea in newborns. Why the different mutations all lead to the same disease is not clear. Here, we report that most mutations, including a novel intronic variant, will result in lack of EpCAMs transmembrane domain, whereas two mutations allow transmembrane localization. We find that these mutants are not routed to the plasma membrane, and that truncated mutants are secreted or degraded. Thus, all epcam mutations lead to loss of cell-surface EpCAM, resulting in CTE.

Further evidence for EpCAM as the gene for congenital tufting enteropathy

Further Evidence for EpCAM as the Gene for Congenital Tufting Enteropathy Mamata Sivagnanam, Tiffany Schaible, Reka Szigeti, Robert H. Byrd, Milton J. Finegold, Sarangarajan Ranganathan, G.S. Gopalakrishna, Nina Tatevian, and Richard Kellermayer* Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of California San Diego, La Jolla, California Rady Children’s Hospital, San Diego, California Section of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, Baylor College of Medicine, Houston, Texas Department of Pathology, Baylor College of Medicine, Houston, Texas Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania Department of Pathology, University of Texas, Houston, Texas

Acute hepatotoxicity after ingestion of Morinda citrifolia (Noni Berry) juice in a 14-year-old boy.

We present a case of a 14-year-old previously healthy boy with acute hepatotoxicity after noni berry juice consumption. As the popularity of noni berry consumption continues to increase, heightened awareness of the relation between noni berry consumption and acute hepatotoxicity is important.

A new syndrome of tufting enteropathy and choanal atresia, with ophthalmologic, hematologic and hair abnormalities.

Three siblings are reported with a syndrome of intractable diarrhea of infancy (owing to tufting enteropathy) and choanal atresia/stenosis. Additional components of this condition are a mild shortness of stature, a prominent and broad nasal bridge, micrognathia, single palmar creases, chronic corneal inflammation, episodic cytopenia, and abnormal hair texture. Intelligence is normal, and there is no immunodeficiency distinguishing this syndrome from that reported by Girault et al. (1994). Additional features that might occur in this syndrome include bifid uvula, preauricular pits, and 2/3 toe syndactyly. We compare this syndrome with previously reported intractable diarrhea syndromes and speculate on the developmental mechanisms that could account for many of the features demonstrated by this sibship.

Case of syndromic tufting enteropathy harbors SPINT2 mutation seen in congenital sodium diarrhea.

Mamata Sivagnanam, Andreas R. Janecke, Thomas Müller, Peter Heinz-Erian, Sharon Taylor and Lynne M. Bird, Divisions of Pediatric Gastroenterology, Hepatology and Nutrition, Dysmorphology and Genetics, Department of Pediatrics, University of California, Rady Children’s Hospital, San Diego, California, USA, Division of Clinical Genetics and Department of Pediatrics II, Innsbruck Medical University, Innsbruck, Austria

Functional consequences of EpCam mutation in mice and men

Congenital tufting enteropathy (CTE) is a severe diarrheal disease of infancy characterized by villous changes and epithelial tufts. We previously identified mutations in epithelial cell adhesion molecule (EpCAM) as the cause of CTE. We developed an in vivo mouse model of CTE based on EpCAM mutations found in patients with the aim to further elucidate the in vivo role of EpCAM and allow for a direct comparison to human CTE. Using Cre-LoxP recombination technology, we generated a construct lacking exon 4 in Epcam. Epcam(Δ4/Δ4) mice and CTE patient intestinal tissue integrity was analyzed by histology using both light immunohistochemistry and electron microscopy. Epcam(Δ4/Δ4) mice demonstrate neonatal lethality and growth retardation with pathological features, including epithelial tufts, enterocyte crowding, altered desmosomes, and intercellular gaps, similar to human CTE patients. Mutant EpCAM protein is present at low levels and is mislocalized in the intestine of Epcam(Δ4/Δ4) mice and CTE patients. Deletion of exon 4 was found to decrease expression of both EpCAM and claudin-7 causing a loss of colocalization, functionally disrupting the EpCAM/claudin-7 complex, a finding for the first time confirmed in CTE patients. Furthermore, compared with unaffected mice, mutation of Epcam leads to enhanced permeability and intestinal cell migration, uncovering underlying disease mechanisms.

Transcriptional Read-Through Induction Treatment Trial in Intestinal Failure Induced by an EpCAM Nonsense Mutation

Congenital tufting enteropathy (CTE) is a rare autosomal recessive diarrheal disorder where epithelial tufts can be present from the duodenum to the large intestine. CTE has been linked to mutations in the epithelial cell adhesion molecule gene (EpCAM) Sivagnanam et al. (2008). We recently reported the first case with a nonsense mutation in EpCAM Sivagnanam et al. (2010). Here, we explored the clinical and molecular effects of enterally administered gentamicin in this CTE patient. Altogether, our findings indicate that the therapy employed was insufficient to produce notable read-through induction of the EpCAM premature termination codon. This report highlights the utility of genetic testing not only in respect of diagnostics, prognostics, and family planning, but potential mutation-specific therapeutic considerations as well.

Biallelic Mismatch Repair Deficiency in an Adolescent Female

Constitutional (Biallelic) Mismatch Repair Deficiency is a rare autosomal recessive disorder characterized by numerous cancers presenting as early as the first decade of life. Biallelic germline variants in one of four mismatch repair genes (MLH1, MSH2, MSH6, or PMS2) cause this devastating disease. Given the rarity of the syndrome, often-asymptomatic tumors, and overlap with neurofibromatosis-1, diagnosis is frequently unrecognized or delayed. We present a unique case of a 14-year-old female with minimal gastrointestinal symptoms diagnosed with invasive adenocarcinoma secondary to biallelic PMS2 variants.

Fever and Jaundice in a Previously Healthy Teenager

A previously healthy 14-year-old girl was transferred to our facility from an outside hospital with a 10-day history of nausea, malaise, intermittent fever (peak 39.5°C), and fatigue. In the 72 hours prior to admission, she had developed progressive yellowing of the skin and scleral icterus. She had not traveled recently and her immunizations were up-to-date. She denied being sexually active or using illicit substances. She was a well-developed Caucasian adolescent who on initial presentation appeared fatigued and jaundiced. Vital signs on admission were the following: temperature, 38.8°C; heart rate, 96 beats per minute; respiratory rate, 18 breaths per minute; and blood pressure, 112/58 mm Hg. Her physical examination revealed scleral icterus, pharyngeal erythema with tonsillar exudates, anterior cervical lymphadenopathy, and right-sided abdominal tenderness. The liver edge was palpable at 1 cm below the right costal margin. Laboratory evaluation from the outside hospital revealed transaminase elevation and elevated serum bilirubin levels (Table 1). Complete blood count did not demonstrate evidence of anemia, thrombocytopenia, leukocytosis, or bandemia. Lipase level was normal at 32 U/L (23-300 U/L), though a C-reactive protein was mildly elevated at 1.20 mg/dL (<0.99 mg/ dL). Acetaminophen level was negative at <10.0 μg/ mL (10-25 μg/mL). Coagulation studies were within the reference range. An initial ultrasound of the abdomen showed diffuse thickening of the gallbladder wall, measuring up to 8.5 mm in thickness, with no evidence of biliary duct dilatation or the presence of stones (Figure 1). Splenomegaly was also seen. The patient did report right upper quadrant tenderness during the ultrasound, exhibiting the positive sonographic Murphy’s sign. These findings were consistent with a working diagnosis of acute acalculous cholecystitis (AAC), and empiric broad-spectrum antibiotic therapy with piperacillin-tazobactam was initiated. All oral feeds were held and aggressive intravenous hydration was initiated. Hospital Course