Sharon F. Taylor

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.

Digested formula but not digested fresh human milk causes death of intestinal cells in vitro: implications for necrotizing enterocolitis

Background:Premature infants fed formula are more likely to develop necrotizing enterocolitis (NEC) than those who are breastfed, but the mechanisms of intestinal necrosis in NEC and protection by breast milk are unknown. We hypothesized that after lipase digestion, formula, but not fresh breast milk, contains levels of unbound free fatty acids (FFAs) that are cytotoxic to intestinal cells.Methods:We digested multiple term and preterm infant formulas or human milk with pancreatic lipase, proteases (trypsin and chymotrypsin), lipase + proteases, or luminal fluid from a rat small intestine and tested FFA levels and cytotoxicity in vitro on intestinal epithelial cells, endothelial cells, and neutrophils.Results:Lipase digestion of formula, but not milk, caused significant death of neutrophils (ranging from 47 to 99% with formulas vs. 6% with milk) with similar results in endothelial and epithelial cells. FFAs were significantly elevated in digested formula vs. milk and death from formula was significantly decreased with lipase inhibitor pretreatment, or treatments to bind FFAs. Protease digestion significantly increased FFA binding capacity of formula and milk but only enough to decrease cytotoxicity from milk.Conclusion:FFA-induced cytotoxicity may contribute to the pathogenesis of NEC.

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

Effect of Digestion and Storage of Human Milk on Free Fatty Acid Concentration and Cytotoxicity

Objectives: Fat is digested in the intestine into free fatty acids (FFAs), which are detergents and therefore toxic to cells at micromolar concentration. The mucosal barrier protects cells in the adult intestine, but this barrier may not be fully developed in premature infants. Lipase-digested infant formula, but not fresh human milk, has elevated FFAs and is cytotoxic to intestinal cells, and therefore could contribute to intestinal injury in necrotizing enterocolitis (NEC), but even infants exclusively fed breast milk may develop NEC. Our objective was to determine whether stored milk and milk from donor milk (DM) banks could also become cytotoxic, especially after digestion. Methods: We exposed cultured rat intestinal epithelial cells or human neutrophils to DM and milk collected fresh and stored at 4°C or −20°C for up to 12 weeks and then treated for 2 hours (37°C) with 0.1 or 1 mg/mL pancreatic lipase and/or trypsin and chymotrypsin. Results: DM and milk stored 3 days (at 4°C or −20°C) and then digested were cytotoxic. Storage at −20°C for 8 and 12 weeks resulted in an additional increase in cytotoxicity. Protease digestion decreased, but did not eliminate cell death. Conclusions: Present storage practices may allow milk to become cytotoxic and contribute to intestinal damage in NEC.

Breast Milk Protects Against Gastrointestinal Symptoms in Infants at High Risk for Autism During Early Development.

Objectives: Parents of children with autism spectrum disorders (ASDs) often report gastrointestinal (GI) dysfunction in their children. The objectives of the present study were to determine whether infants at high risk for developing ASD (ie, siblings of children diagnosed as having ASD) show greater prevalence of GI problems and whether this prevalence is associated with diet and age at weaning from breast milk. Methods: Using questionnaires, diet history and GI problems were tracked prospectively and retrospectively in 57 high-risk infants and for comparison in 114 low-risk infants (infants from families without ASD history). Results: In low-risk infants, prevalence of GI symptoms, in aggregate, did not vary with diet or age of weaning. By contrast, high-risk infants with GI symptoms were weaned earlier than those without symptoms (P < 0.04), and high-risk infants showed greater prevalence of GI symptoms, in aggregate, on a no breast milk diet than on an exclusive breast milk diet (P < 0.017). Constipation, in particular, was more prevalent in high-risk infants compared with low-risk infants (P = 0.01), especially on a no breast milk diet (P = 0.002). High-risk infants who completed weaning earlier than 6 months showed greater prevalence of constipation (P = 0.001) and abdominal distress (P = 0.004) than those fully weaned after 6 months. Conclusions: The greater prevalence of GI symptoms in high-risk infants suggests that GI dysfunction during early infant development may be a part of the ASD endophenotype. Late weaning and exclusive breast milk were associated with protection against GI symptoms in high-risk infants.

Editor’s Focus

The risk factors for diastolic dysfunction in hypertrophic cardiomyopathy (HCM) are poorly understood. Alkon and colleagues investigated the association of variants in hypoxia-response genes with phenotype severity in pediatric HCM. They found that hypoxiainducible factor upregulation and/ or vascular endothelial growth factor downregulation genotypes were associated with more severe septal hypertrophy and diastolic dysfunction and may provide genetic markers to improve risk prediction in HCM. See page 583