Peter Heinz-Erian

MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.

Following homozygosity mapping in a single kindred, we identified nonsense and missense mutations in MYO5B, encoding type Vb myosin motor protein, in individuals with microvillus inclusion disease (MVID). MVID is characterized by lack of microvilli on the surface of enterocytes and occurrence of intracellular vacuolar structures containing microvilli. In addition, mislocalization of transferrin receptor in MVID enterocytes suggests that MYO5B deficiency causes defective trafficking of apical and basolateral proteins in MVID.

Loss-of-function of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model†

Autosomal recessive microvillus inclusion disease (MVID) is characterized by an intractable diarrhea starting within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid‐Schiff (PAS)‐positive vesicles in enterocytes. Recently, we identified mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first cohort of nine MVID patients. In this study, we identified 15 novel nonsense and missense mutations in MYO5B in 11 unrelated MVID patients. Fluorescence microscopy, Western blotting, and electron microscopy were applied to analyze the effects of MYO5B siRNA knock‐down in polarized, brush border possessing CaCo‐2 cells. Loss of surface microvilli, increased formation of microvillus inclusions, and subapical enrichment of PAS‐positive endomembrane compartments were induced in polarized, filter‐grown CaCo‐2 cells, following MYO5B knock‐down. Our data indicate that MYO5B mutations are a major cause of microvillus inclusion disease and that MYO5B knock‐down recapitulates most of the cellular phenotype in vitro, thus independently showing loss of MYO5B function as the cause of microvillus inclusion disease. Hum Mutat 31:1–8, 2010.

Mutations in SPINT2 Cause a Syndromic Form of Congenital Sodium Diarrhea

Autosomal-recessive congenital sodium diarrhea (CSD) is characterized by perinatal onset of a persistent watery diarrhea with nonproportionally high fecal sodium excretion. Defective jejunal brush-border Na(+)/H(+) exchange has been reported in three sporadic patients, but the molecular basis of the disease has not been elucidated. We reviewed data from a large cohort of CSD patients (n = 24) and distinguished CSD associated with choanal or anal atresia, hypertelorism, and corneal erosions--i.e., a syndromic form of CSD--occurring in ten families from an isolated form--i.e., classic CSD--presenting in seven families. Patients from both groups have a high risk of mortality due to immediate electrolyte imbalances and complications from long-term parenteral nutrition in the first years of life, but survivors can eventually adapt to partial or complete enteral nutrition. A genome-wide SNP scan was applied and identified a homozygous c.593-1G-->A splicing mutation in SPINT2, encoding a Kunitz-type serine-protease inhibitor, in one extended kindred with syndromic CSD. The same mutation and four distinct, homozygous or compound heterozygous mutations (p.Y163C, c.1A-->T, c.337+2T-->C, c.553+2T-->A) were identified in all syndromic patients. No SPINT2 mutations were found in classic-CSD patients. SPINT2 mutations were associated with loss of protein synthesis or failure to inhibit the serine protease trypsin in vitro. We delineate syndromic CSD as a distinct disease entity caused by SPINT2 loss-of-function mutations. SPINT2 mutations might lead to an excess of yet unknown serine protease activity in affected tissues.

Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea

Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

Amino Acid Cerebrospinal Fluid/Plasma Ratios in Children: Influence of Age, Gender, and Antiepileptic Medication

OBJECTIVE. The purpose of this work was to investigate the influence of age, gender, and antiepileptic therapy on amino acid cerebrospinal fluid/plasma ratios in children. PATIENTS AND METHODS. Concentrations of 17 amino acids measured by ion-exchange chromatography with ninhydrin detection in plasma and cerebrospinal fluid from 68 patients with neurologic diseases were used to calculate their cerebrospinal fluid/plasma ratios (70 measurements; 28 female patients [29 punctures] and 40 male patients [41 punctures]). Age dependence and the effects of gender and antiepileptic medication on amino acid cerebrospinal fluid/plasma ratios were investigated by linear multiple regression analysis, and nonstandardized predicted mean values for 2 age groups were calculated (cutoff: 3 years old). RESULTS. The cerebrospinal fluid/plasma ratios ranged between 0.02 for glycine and 0.93 for glutamine. Age had a significant influence on cerebrospinal fluid/plasma ratios for valine, isoleucine, leucine, and tyrosine, with higher ratios in younger children. Gender had a significant influence only on the glutamine cerebrospinal fluid/plasma ratio (female patients had lower ratios). Cerebrospinal fluid/plasma ratios of glutamine and tyrosine were significantly elevated by valproate therapy and those of serine, asparagine, glutamine, valine, methionine, and phenylalanine by phenobarbital therapy. No significant influence of age, gender, and antiepileptic drugs was detectable on cerebrospinal fluid/plasma ratios of threonine, proline, glycine, alanine, histidine, ornithine, lysine, and arginine. CONCLUSIONS. Cerebrospinal fluid/plasma ratios, especially for essential neutral amino acids and for serine, asparagine, and glutamine were influenced to different degrees by age, gender, and antiepileptic therapy.

Gastric lactobezoar - a rare disorder?

Gastric lactobezoar, a pathological conglomeration of milk and mucus in the stomach of milk-fed infants often causing gastric outlet obstruction, is a rarely reported disorder (96 cases since its first description in 1959). While most patients were described 1975-1985 only 26 children have been published since 1986. Clinically, gastric lactobezoars frequently manifest as acute abdomen with abdominal distension (61.0% of 96 patients), vomiting (54.2%), diarrhea (21.9%), and/or a palpable abdominal mass (19.8%). Respiratory (23.0%) and cardiocirculatory (16.7%) symptoms are not uncommon. The pathogenesis of lactobezoar formation is multifactorial: exogenous influences such as high casein content (54.2%), medium chain triglycerides (54.2%) or enhanced caloric density (65.6%) of infant milk as well as endogenous factors including immature gastrointestinal functions (66.0%), dehydration (27.5%) and many other mechanisms have been suggested. Diagnosis is easy if the potential presence of a gastric lactobezoar is thought of, and is based on a history of inappropriate milk feeding, signs of acute abdomen and characteristic features of diagnostic imaging. Previously, plain and/or air-, clear fluid- or opaque contrast medium radiography techniques were used to demonstrate a mass free-floating in the lumen of the stomach. This feature differentiates a gastric lactobezoar from intussusception or an abdominal neoplasm. Currently, abdominal ultrasound, showing highly echogenic intrabezoaric air trapping, is the diagnostic method of choice. However, identifying a gastric lactobezoar requires an investigator experienced in gastrointestinal problems of infancy as can be appreciated from the results of our review which show that in not even a single patient gastric lactobezoar was initially considered as a possible differential diagnosis. Furthermore, in over 30% of plain radiographs reported, diagnosis was initially missed although a lactobezoar was clearly demonstrable on repeat evaluation of the same X-ray films. Enhanced diagnostic sensitivity would be most rewarding since management consisting of cessation of oral feedings combined with administration of intravenous fluids and gastric lavage is easy and resolves over 85% of gastric lactobezoars. In conclusion, gastric lactobezoar is a disorder of unknown prevalence and is nowadays very rarely published, possibly because of inadequate diagnostic sensitivity and/or not yet identified but beneficial modifications of patient management.

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C

Objective Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. Design We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. Results We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. Conclusions Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD.

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

Universal Mass Vaccination Against Rotavirus: Indirect Effects on Rotavirus Infections in Neonates and Unvaccinated Young Infants Not Eligible for Vaccination

BACKGROUND Rotavirus (RV)-associated infections account for high numbers of hospitalizations in neonates and young infants. Universal mass vaccination (UMV) has been shown to prevent the burden of disease in vaccinated children. METHODS The present study investigated the long-term effects of UMV on RV-associated hospitalizations in children with particular focus on neonates and young infants (≤42 days old) not eligible for vaccination. Ten years of Austrian surveillance data were compared, including 10 960 laboratory-confirmed RV cases before (prevaccination period [PreVP]) and after (postvaccination period [PostVP]) introduction of UMV. RESULTS A postvaccination decrease in hospitalized community-acquired RV infections by 89.3% was seen in all age groups, including unvaccinated neonates and young infants. Of the latter, 27.6% had a nosocomial RV infection in PreVP, and 19.3% in PostVP. Overall, the proportion of nosocomial RV infections increased from 5.5% in PreVP to 13.0% in PostVP. Breakthrough infections, usually after incomplete RV vaccination, could be identified in 6.2% of patients. CONCLUSIONS Unvaccinated neonates and infants ≤42 days old may indirectly benefit from UMV by reduction of RV infections. Breakthrough infections underline the importance of early and complete protection by the vaccine. In older patients, heightened awareness of nosocomial RV infections is warranted. Identification of RV reservoirs is also needed.

Disintegration of large gastric lactobezoars by N-acetylcysteine.

108 oar is a freely floating A gastric lactobez mass in the lumen of the stomach composed of undigested milk and mucous secretions. In infants and small toddlers it is the most common type of bezoar as compared with other bezoar types such as tricho-, phyto-, mixed food, or medication bezoars (1). The etiopathogenetic factors implicated are inadequate milk composition, and disturbed gastric emptying, which impedes food fragmentation as well as abnormal secretion of gastric acid, pepsin, and mucus. Bezoar formation has been reported with every type of milk, including human breast milk (2,3), cow’s milk, and cow’s milkor soy-based commercial infant formulas (4,5). Gastric lactobezoars may grow to considerable sizes. With a high index of suspicion, diagnosis can be easily made by plain or contrast radiography and, provided an experienced examiner, also by ultrasonography. Successful treatment measures include discontinuation of oral feeding, intravenous fluids, and gastric lavage (4,6). However, despite these therapies, in a few published cases lactobezoars did not disintegrate as expected and caused serious complications such as persistent gastric outlet obstruction or gastric perforation (7–12). We here describe 3 patients with large gastric lactobezoars that did not readily dissolve with the standard treatment of nil per mouth and intravenous fluids. Having in mind serious complications reported in the literature (8–10), we looked for options other than endoscopy or surgery to quickly disintegrate lactobezoars resistant to the established standard therapy. N-Acetylcysteine (ACC) has been known as an effective bronchial mucolytic agent for a long time and was successfully used for the treatment of a mixed food bezoar in an adult (13). We report for the first time the use of intragastric ACC for the rapid dissolution of large gastric lactobezoars in 3 female toddlers.