Daniel Wenning

The K+ Channel Opener 1-EBIO Potentiates Residual Function of Mutant CFTR in Rectal Biopsies from Cystic Fibrosis Patients

Background The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K+ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl− secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. Methods We studied the effects of 1-EBIO on CFTR-mediated Cl− secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl− secretion. Results Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl− secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl− secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca2+-activated and clotrimazole-sensitive KCNN4 K+ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl− secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl−conductance. Conclusions We conclude that 1-EBIO potentiates Cl−secretion in native CF tissues expressing CFTR mutants with residual Cl− channel function by activation of basolateral KCNN4 K+ channels that increase the driving force for luminal Cl− exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations.

The bile salt export pump BSEP mediates bile formation. Over 150 BSEP mutations are associated with progressive familial intrahepatic cholestasis type 2 (PFIC-2), with few characterised specifically. We examined liver tissues from two PFIC-2 patients compound heterozygous for the splice-site mutation c.150 + 3A > C and either c.2783_2787dup5 resulting in a frameshift with a premature termination codon (child 1) or p.R832C (child 2). Splicing was analysed with a minigene system and mRNA sequencing from patients’ livers. Protein expression was shown by immunofluorescence. Using the minigene, c.150 + 3A > C causes complete skipping of exon 3. In liver tissue of child 1, c.2783_2787dup5 was found on DNA but not on mRNA level, implying nonsense-mediated mRNA decay (NMD) when c.2783_2787dup5 is present. Still, BSEP protein as well as mRNA with and without exon 3 were detectable and can be assigned to the c.150 + 3A > C allele. Correctly spliced transcripts despite c.150 + 3A > C were also confirmed in liver of child 2. In conclusion, we provide evidence (1) for effective NMD due to a BSEP frameshift mutation and (2) partial exon-skipping due to c.150 + 3A > C. The results illustrate that the extent of exon-skipping depends on the genomic and cellular context and that regulation of splicing may have therapeutic potential.

High affinity anti‐BSEP antibodies after liver transplantation for PFIC‐2 – Successful treatment with immunoadsorption and B‐cell depletion

PFIC due to BSEP mutations (PFIC type 2) often necessitates OLT. It has recently been recognized that some PFIC‐2 patients develop phenotypic disease recurrence post‐OLT due to the appearance of anti‐BSEP antibodies. Here, we describe a boy who became cholestatic four yr after OLT during modification of immunosuppression. Canalicular antibody deposits were detected in biopsies of the transplant and antibodies specifically reacting with BSEP were identified at high titers in his serum. These antibodies bound extracellular epitopes of BSEP and inhibited BS transport and were assumed to cause disease recurrence. Consequently, anti‐BSEP antibody depletion was pursued by IA and B‐cell depletion by anti‐CD20 antibodies (rituximab) along with a switch of immunosuppression. This treatment resulted in prolonged relief of symptoms. Depletion of pathogenic anti‐BSEP antibodies causing AIBD after OLT in PFIC‐2 patients should be considered as a central therapeutic goal.

Antibiotic prophylaxis in the management of percutaneous endoscopic gastrostomy in infants and children

In randomized controlled trials in adult patients the use of prophylactic broad‐spectrum antibiotic reduces the number of insertion site and systemic infections, associated with placement of percutaneous endoscopic gastrostomy (PEG) tubes. For pediatric patients no such trials exist. The aim of this study was to assess the value of antibiotic prophylaxis in PEG placement in pediatric patients.

Phenotypic spectrum and diagnostic pitfalls of ABCB4 deficiency depending on age of onset

Genetic variants in the adenosine triphosphate‐binding cassette subfamily B member 4 (ABCB4) gene, which encodes hepatocanalicular phosphatidylcholine floppase, can lead to different phenotypes, such as progressive familial intrahepatic cholestasis (PFIC) type 3, low phospholipid‐associated cholelithiasis, and intrahepatic cholestasis of pregnancy. The aim of this multicenter project was to collect information on onset and progression of this entity in different age groups and to assess the relevance of this disease for the differential diagnosis of chronic liver disease. Clinical and laboratory data of 38 patients (17 males, 21 females, from 29 families) with homozygous or (compound) heterozygous ABCB4 mutations were retrospectively collected. For further analysis, patients were grouped according to the age at clinical diagnosis of ABCB4‐associated liver disease into younger age (<18 years) or adult age (≥18 years). All 26 patients diagnosed in childhood presented with pruritus (median age 1 year). Hepatomegaly and splenomegaly were present in 85% and 96% of these patients, respectively, followed by jaundice (62%) and portal hypertension (69%). Initial symptoms preceded diagnosis by 1 year, and 13 patients received a liver transplant (median age 6.9 years). Of note, 9 patients were misdiagnosed as biliary atresia, Alagille syndrome, or PFIC type 1. In the 12 patients with diagnosis in adulthood, the clinical phenotype was generally less severe, including intrahepatic cholestasis of pregnancy, low phospholipid‐associated cholelithiasis, or (non)cirrhotic PFIC3. Conclusion: ABCB4 deficiency with onset in younger patients caused a more severe PFIC type 3 phenotype with the need for liver transplantation in half the children. Patients with milder phenotypes are often not diagnosed before adulthood. One third of the children with PFIC type 3 were initially misdiagnosed, indicating the need for better diagnostic tools and medical education. (Hepatology Communications 2018;2:504‐514)

SCYL1 variants cause a syndrome with low γ-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (CALFAN)

AbstractPurposeBiallelic mutations in SCYL1 were recently identified as causing a syndromal disorder characterized by peripheral neuropathy, cerebellar atrophy, ataxia, and recurrent episodes of liver failure. The occurrence of SCYL1 deficiency among patients with previously undetermined infantile cholestasis or acute liver failure has not been studied; furthermore, little is known regarding the hepatic phenotype.MethodsWe aimed to identify patients with SCYL1 variants within an exome-sequencing study of individuals with infantile cholestasis or acute liver failure of unknown etiology. Deep clinical and biochemical phenotyping plus analysis of liver biopsies and functional studies on fibroblasts were performed.ResultsSeven patients from five families with biallelic SCYL1 variants were identified. The main clinical phenotype was recurrent low γ-glutamyl-transferase (GGT) cholestasis or acute liver failure with onset in infancy and a variable neurological phenotype of later onset (CALFAN syndrome). Liver crises were triggered by febrile infections and were transient, but fibrosis developed. Functional studies emphasize that SCYL1 deficiency is linked to impaired intracellular trafficking.ConclusionSCYL1 deficiency can cause recurrent low-GGT cholestatic liver dysfunction in conjunction with a variable neurological phenotype. Like NBAS deficiency, it is a member of the emerging group of congenital disorders of intracellular trafficking causing hepatopathy.

Die Lebensqualität positiv beeinflussen

Chronisch-entzündliche Darmerkrankungen (CED) schränken die Lebensqualität betroffener Kinder und vor allem Jugendlicher oft erheblich ein. Die Patienten sind häufig noch stärker betroffen als Kinder und Jugendliche mit anderen chronischen Erkrankungen.

An Assessment of Publication Status of Pediatric Liver Transplantation Studies.

Introduction Pediatric liver transplantation is a highly specialized, challenging field. Selective reporting may introduce bias into evidence based clinical decision making, but the precise extent of unpublished data in pediatric liver transplantation is unknown today. We therefore assessed the public availability of completed clinical trials in pediatric liver transplantation. Methods We determined the proportion of published and unpublished pre-registered, completed pediatric liver transplantation studies on ClinicalTrials.gov. The major trial and literature databases, i.e., clinicaltrials.gov, Pubmed, and Google Scholar were searched for publications. In addition, principal investigators or sponsors were contacted directly. STROBE criteria were applied for the descriptive analysis. Results Out of N = 33 studies focusing on pediatric liver transplantation registered as completed until March 2014 on clinicaltrials.gov, N = 19 (58%) studies were published until February 2015, whereas N = 14 (42%) studies remained unpublished. The unpublished trials contain data from N = 2105 (35%) patients out of a total population of N = 6044 study participants. Median time-to-publication, i.e., the period from completion of the trial until public availability of the data was 23 IQR 10 to 28 months. Most pertinent key questions in pediatric liver transplantation, i.e., surgical procedures, immunosuppression, concomitant infections, and graft rejection were addressed in 48% of studies (N = 16/33), half of which were published. Conclusion Half of the clinical trials in pediatric liver transplantation focused on key questions such as surgical procedures, immunosuppression, concomitant infections, and graft rejection. There is still a considerable amount of unpublished studies results in pediatric liver transplantation. Time from study completion to publication was almost twice as long as the 12 months mandatory FDAAA-timeline with a trend towards acceleration over time. The data should serve as a baseline for future progress in the field. More stringent publication of completed trials and focused multicenter research should be encouraged.