Isabelle Scheers

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype–phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca2+o) homeostasis. To elucidate the role of AP2σ2 in Ca2+o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Diagnostic and Therapeutic Role of Endoscopic Ultrasound in Pediatric Pancreaticobiliary Disorders.

Objectives: The diagnostic role of endoscopic ultrasound (EUS) in children has only recently been demonstrated, and that also to a lesser extent than in adults. Data on the techniques therapeutic indications remain scarce. We therefore sought to evaluate diagnostic and interventional EUS indications, safety, and impact in children with pancreaticobiliary disorders. Methods: We retrospectively reviewed our single pediatric center records, covering a 14-year period. Results: From January 2000 to January 2014, 52 EUS procedures were performed in 48 children (mean age: 12 years; range: 2–17 years) with pancreaticobiliary disorders for the following indications: suspected biliary obstruction (n = 20/52), acute/chronic pancreatitis (n = 20), pancreatic mass (n = 3), pancreatic trauma (n = 7), and ampullary adenoma (n = 2). EUS was found to have a positive impact in 51 of 52 procedures, enabling us to avoid endoscopic retrograde cholangiopancreatography (ERCP) (n = 13 biliary; n = 6 pancreatic), focusing instead on endotherapy (n = 7 biliary; n = 14 pancreatic) or reorienting therapy toward surgery (n = 7). EUS-guided fine-needle aspiration was carried out on 12 patients for pancreatic tumor (n = 4), pancreatic cyst fluid analysis (n = 4), autoimmune pancreatitis (n = 2), and suspicion of biliary tumor (n = 2). A total of 13 therapeutic EUS procedures (11 children) were conducted, including 9 combined EUS–ERCP procedures (7 children, mean age: 8 years, range: 4–11 years), 3 EUS-guided pseudocyst drainage (2 children), and 1 EUS-guided transgastric biliary drainage. Conclusions: Our study reports on a large pediatric EUS series for diagnostic and therapeutic pancreaticobiliary disorders, demonstrating the impact of diagnostic EUS and affording insights into novel EUS and combined EUS–ERCP therapeutic applications. We suggest considering EUS as a diagnostic and therapeutic tool in the management of pediatric pancreaticobiliary diseases.

Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection

The role of cell differentiation state on hepatitis B virus (HBV) replication has been well demonstrated, whereas how it determines cell susceptibility to HBV entry is far less understood. We previously showed that umbilical cord matrix stem cells (UCMSC) can be differentiated towards hepatocyte‐like cells in vitro. In this study we infected undifferentiated (UD‐) and differentiated (D‐) UCMSCs with HBV and studied the infection kinetics, comparing them to primary human hepatocytes (PHHs). UD‐UCMSCs, although permissive to viral binding, had a very limited uptake capacity, whereas D‐UCMSCs showed binding and uptake capabilities similar to PHHs. Likewise, asialoglycoprotein receptor (ASGPR) was up‐regulated in UCMSCs upon differentiation. In D‐UCMSCs, a dose‐dependent inhibition of HBV binding and uptake was observed when ASGPR was saturated with known specific ligands. Subsequent viral replication was shown in D‐UCMSCs but not in UD‐UCMSCs. Susceptibility of UCMSCs to viral replication correlated with the degree of differentiation. Replication efficiency was low compared to PHHs, but was confirmed by (1) a dose‐dependent inhibition by specific antiviral treatment using tenofovir; (2) the increase of viral RNAs along time; (3) de novo synthesis of viral proteins; and (4) secretion of infectious viral progeny. Conclusion: UCMSCs become supportive of the entire HBV life cycle upon in vitro hepatic differentiation. Despite low replication efficiency, D‐UCMSCs proved to be fully capable of HBV uptake. Overall, UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection that could prove useful to study early infection events and the role of the cell differentiation state on such events. (HEPATOLOGY 2013)

Retargeting of bile salt export pump and favorable outcome in children with progressive familial intrahepatic cholestasis type 2

We investigated predictors of clinical evolution in progressive familial intrahepatic cholestasis type 2 patients and how they relate to bile salt export pump (BSEP) expression and its (re)targeting. Our retrospective study included 22 children with progressive familial intrahepatic cholestasis type 2. Clinical, biochemical, and histological characteristics were reviewed on admittance and following treatment with either ursodeoxycholic acid alone (10 mg/kg thrice daily, n = 19) or partial biliary diversion (n = 3). Immunostaining of BSEP was performed in 20 patients. Response to treatment was defined as normalization of pruritus, disappearance of jaundice, and alanine aminotransferase (ALT) levels <1.5 times the upper limit of normal. Ten of 22 patients were responders, and paired biopsies were available in six. De novo or retargeted canalicular expression of BSEP occurred in four of these six, two of whom exhibited baseline intracellular expression. Twelve of 22 were nonresponders and exhibited earlier onset of jaundice (<9 months), neonatal cholestasis, and higher ALT levels. An ALT >165 IU/L produced 72% sensitivity and 55% specificity in predicting nonresponse. Seven patients were still responding at last follow‐up (median = 20 months, range 5‐67 months). Three responders relapsed after 56, 72, and 82 months, respectively. Of nine surviving responders, median relapse‐free survival time was 72 months (95% confidence interval 48‐96 months) and 5‐year relapse‐free survival was 75% (95% confidence interval 33‐100%). Intracellular BSEP at baseline was seen in six, of whom five were responders. Genetic analysis was performed in 17 of 22, confirming diagnosis in 13 (76%) and in four (24%) in whom only heterozygous mutation was identified. Conclusion: De novo or retargeted canalicular expression of BSEP occurs in treatment responders; children with late‐onset presentation, lower ALT, and intracellular BSEP expression are likely to respond, at least transiently, to nontransplant treatment. (Hepatology 2015;62:198‐206)

Autoimmune Pancreatitis in Children: Characteristic Features, Diagnosis, and Management

Objectives:Autoimmune pancreatitis (AIP) is an increasingly recognized disease entity, but data in children are limited. AIP presentation and outcome in children might differ from the adult experience. We aim to determine the characteristic features of AIP in children.Methods:Data about clinical symptoms, imaging, histology, and treatment were collected using two sources: (i) a systematic literature search and (ii) the INSPPIRE database, the largest international multicenter study of pancreatitis in children and the Cliniques Universitaires St-Luc (CUSL) registry.Results:We identified 48 AIP cases: 30 from literature review, 14 from INSPPIRE, and 4 from CUSL. The median age at diagnosis was 13 years (range 2–17 years). Abdominal pain (43/47, 91%) and/or obstructive jaundice (20/47, 42%) were the most common symptoms at diagnosis. Elevated serum IgG4 levels were only observed in 9/40 (22%) children. Cross-sectional imaging studies were abnormal in all children including hypointense global or focal gland enlargement (39/47, 83%), main pancreatic duct irregularity (30/47, 64%), and common bile duct stricture (26/47, 55%). A combination of lymphoplasmacytic inflammation, pancreatic fibrosis, and ductal granulocyte infiltration were the main histological findings (18/25, 72%). Children with AIP had a prompt clinical response to steroids. Complications of AIP included failure of exocrine (4/25, 16%) and endocrine (3/27, 11%) pancreas function.Conclusions:Pediatric AIP has a distinct presentation with features similar to type 2 AIP in adults. This comprehensive report on the largest group of children with AIP to date is expected to help with the diagnosis and management of this disease and pave the way for future research studies.

Hepato-biliary profile of potential candidate liver progenitor cells from healthy rat liver

AIM To evaluate the presence of progenitor cells in healthy adult rat liver displaying the equivalent advanced hepatogenic profile as that obtained in human. METHODS Rat fibroblastic-like liver derived cells (rFLDC) were obtained from collagenase-isolated liver cell suspensions and characterized and their phenotype profile determined using flow cytometry, immunocytochemistry, reverse transcription polymerase chain reaction and functional assays. RESULTS rFLDC exhibit fibroblastoid morphology, express mesenchymal (CD73, CD90, vimentin, α-smooth muscle actin), hepatocyte (UGT1A1, CK8) and biliary (CK19) markers. Moreover, these cells are able to store glycogen, and have glucose 6 phosphatase activity, but not UGT1A1 activity. Under the hepatogenic differentiation protocol, rFLDC display an up-regulation of hepatocyte markers expression (albumin, tryptophan 2,3-dioxygenase, G6Pase) correlated to a down-regulation of the expression of the biliary marker CK19. CONCLUSION Advanced hepatic features observed in human liver progenitor cells could not be demonstrated in rFLDC. However, we demonstrated the presence of an original rodent hepato-biliary cell type.

Sofosbuvir/ledipasvir and ribavirin tolerability and efficacy in pediatric liver transplant recipients

The longterm results of pediatric liver transplantation (LT) have become exceptionally good, with >95% longterm survival, but hepatitis C virus (HCV) recurrence for a liver graft was an important cause of morbidity, making prevention of disease relapse after transplantation a major objective. Treating HCV in children remains challenging, but the development of oral direct-acting antivirals (DAAs) has revolutionized the modern approach for HCV treatment in adults. We describe 2 cases of pediatric HCV patients treated with sofosbuvir (SOF)/ledipasvir (LDV) and ribavirin: one treated before LT (a 1-year-old boy transplanted for biliary atresia) and the other after LT (a 16-year-old girl transplanted for Budd-Chiari syndrome and cirrhosis). The protocol used to treat these patients was approved by the local ethical committee, and informed consent was obtained for both patients. Both patients were HCV genotype 1b: the first was diagnosed during a pretransplant check-up, and the second was a recurrence 5 weeks after LT. Both received a 12-week course of SOF/LDV (Harvoni) and ribavirin, which is in accordance with recent recommendations for adults in the LT context. The first patient received 5 weeks of treatment before LT, and the remaining course was continued after LT (dosage was SOF 100mg/LDV 22.5mg once daily and ribavirin 7.5mg/kg twice daily); the second child received the full treatment course after LT. Interestingly, HCV polymerase chain reaction (PCR) on the explanted liver correlated well with the blood PCR after 5 weeks of treatment (both< 30 IU/mL) for the youngest child. Treatment response with nondetectable HCV on PCR was observed at the end of the 12 weeks of treatment, leading to a sustained virological response (SVR) 24 weeks after the end of treatment for both children. None of the commonly reported side effects were observed. For the 1-year-old, singledose and multiple-dose pharmacokinetic (PK) parameters of SOF, its predominant circulating metabolite GS-331007, and LDV were estimated using noncompartmental methods and are summarized in Table 1. Following the first-dose administration, SOF was detectable in 2 samples only; as such, the single-dose PK parameters for SOF could not be estimated reliably and are not included in the summary. The PK was repeated at day 7; mean steady-state exposure parameters for SOF were higher, whereas GS-331007 and LDV exposures were lower than the mean exposures observed in LDV/SOF adult phase 2/3 population PK analyses. Notably, all estimates remained within the ranges (minimum to maximum) of exposures in the phase 2/3 population. In conclusion, a 12-week treatment regimen with SOF/LDV/ribavirin for HCV was well tolerated and led to a SVR in 2 HCV (type 1b)– infected pediatric patients. The PK data in the 1-yearold child was within the ranges reported for adults, using a quarter of the adult DAA dosages. These first 2 successfully treated HCV patients provide us with preliminary evidence that these drugs are safe and Abbreviations: %CV, % coefficient of variation; AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; DAA, direct-acting antiviral; HCV, hepatitis C virus; LDV, ledipasvir; LT, liver transplantation; PCR, polymerase chain reaction; PK, pharmacokinetic; SCT, stem cell transplantation; SOF, sofosbuvir; SVR, sustained virological response.

Influence de la kinésithérapie respiratoire sur le reflux gastro-œsophagien chez l’enfant

INTRODUCTION Chest physiotherapy is regularly prescribed for children, particularly in cystic fibrosis. Gastro-oesophageal reflux is common in this disease and is associated with certain chest physiotherapy manoeuvres. AIM OF THE STUDY To evaluate the influence of two chest physiotherapy techniques on gastro-oesophageal reflux in children. MATERIAL AND METHOD Twenty-nine children were investigated by routine pHmetry. During the examination, they performed two chest physiotherapy manoeuvres in a seated position for 10 minutes each with a 5 minutes rest between them. The two manoeuvres used were a slow expiration technique (ELPr) and positive expiratory pressure (PEP). It was a prospective study and the order of manoeuvres was randomised. The pH traces were analysed blindly when all the studies had been completed. RESULTS In the sample, 21% of children had gastro-oesophageal reflux during the physiotherapy session. No relationship was found between reflux during physiotherapy and pathological reflux (P=0.411) nor the physiotherapy technique used (P=0.219). CONCLUSION The use of these two chest physiotherapy techniques in children in a seated position can produce gastro-oesophageal reflux.

Living-donor liver transplantation for mild Zellweger spectrum disorder: Up to 17 years follow-up.

Mild Zellweger spectrum disorder, also described as Infantile Refsum disease, is attributable to mutations in PEX genes. Its clinical course is characterized by progressive hearing and vision loss, and neurodevelopmental regression. Supportive management is currently considered the standard of care, as no treatment has shown clinical benefits. LT was shown to correct levels of circulating toxic metabolites, partly responsible for chronic neurological impairment. Of three patients having undergone LT for mild ZSD, one died after LT, while the other two displayed significant neurodevelopmental improvement on both the long‐term (17 years post‐LT) and short‐term (9 months post‐LT) follow‐up. We documented a sustained improvement of biochemical functions, with a complete normalization of plasma phytanic, pristanic, and pipecolic acid levels. This was associated with stabilization of hearing and visual functions, and improved neurodevelopmental status, which has enabled the older patient to lead a relatively autonomous lifestyle on the long term. The psychomotor acquisitions have been markedly improved as compared to their affected siblings, who did not undergo LT and exhibited a poor neurological outcome with severe disabilities. We speculate that LT performed before the onset of severe sensorineural defects in mild ZSD enables partial metabolic remission and improved long‐term clinical outcomes.

Evaluation of the Explorer Endoscopy Mask(©) for esogastroduodenoscopy in children: a retrospective study of 173 cases.

The aim of this study was to evaluate the usability and safety of the Explorer Endoscopy Mask® (EM) as an alternative to endotracheal intubation in children undergoing elective esogastroduodenoscopy (EGD) under general anesthesia (GA).