Cell-based BSEP trans-inhibition: A novel, non-invasive test for diagnosis of antibody-induced BSEP deficiency.

Background & Aims: Antibody-induced bile salt export pump deficiency (AIBD) is an acquired form of intrahepatic cholestasis, which may develop following orthotopic liver transplantation (OLT) for progressive familial intrahepatic cholestasis type 2 (PFIC-2). Approximately 8-33% of patients with PFIC-2 who underwent a transplant develop bile salt export pump (BSEP) antibodies, which trans-inhibit this bile salt transporter from the extracellular, biliary side. AIBD is diagnosed by demonstration of BSEP-reactive and BSEP-inhibitory antibodies in patient serum. We developed a cell-based test directly measuring BSEP trans-inhibition by antibodies in serum samples to confirm AIBD diagnosis. Methods: Sera from healthy controls and cholestatic non-AIBD or AIBD cases were tested (1) for anticanalicular reactivity by immunofluorescence staining of human liver cryosections, (2) for anti-BSEP reactivity by immunofluorescence staining of human embryonic kidney 293 (HEK293) cells expressing BSEP-enhanced yellow fluorescent protein (EYFP) and immunodetection of BSEP-EYFP on Western blot, and (3) for BSEP trans-inhibition using HEK293 cells stably expressing Na+/taurocholate cotransporting polypeptide (NTCP)-mCherry and BSEP-EYFP. The trans-inhibition test uses [3H]-taurocholate as substrate and is divided into an uptake phase dominated by NTCP followed by BSEP-mediated export. For functional analysis, sera were bile salt depleted. Results: We found BSEP trans-inhibition by seven sera containing anti-BSEP antibodies, but not by five cholestatic or nine control sera, all lacking BSEP reactivity. Prospective screening of a patient with PFIC-2 post OLT showed seroconversion to AIBD, and the novel test method allowed monitoring of treatment response. Notably, we identified a patient with PFIC-2 post OLT with anti-BSEP antibodies yet without BSEP trans-inhibition activity, in line with asymptomatic presentation at serum sampling. Conclusions: Our cell-based assay is the first direct functional test for AIBD and allows confirmation of diagnosis as well as monitoring under therapy. We propose an updated workflow for AIBD diagnosis including this functional assay. Impact and Implications: Antibody-induced BSEP deficiency (AIBD) is a potentially serious complication that may affect patients with PFIC-2 after liver transplantation. To improve its early diagnosis and thus immediate treatment, we developed a novel functional assay to confirm AIBD diagnosis using a patient's serum and propose an updated diagnostic algorithm for AIBD.

Functional characterization of novel or yet uncharacterized ATP7B missense variants detected in patients with clinical Wilson's disease.

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.

Native liver survival in bile salt export pump deficiency: results of a retrospective cohort study.

BACKGROUND: Bile salt export pump (ABCB11) deficiency [Progressive familial intrahepatic cholestasis (PFIC2)] is the most common genetic cause of PFIC and is associated with pruritus and progressive liver disease. Surgical biliary diversion or pharmacological [ileal bile acid transporter inhibitor (IBATi)] approaches can be used to block the recirculation of bile acids to the liver. There is a paucity of detailed data on the natural history and, in particular, the longitudinal evolution of bile acid levels to predict treatment response. Cross-sectional data from large international consortia suggested a maximum cutoff value of bile acids after the intervention to predict a successful outcome. METHODS: This retrospective, single-center, cohort study included all patients with confirmed biallelic pathogenic ABCB11 genotype PFIC2 treated at our institution with ≥2 years follow-up. The outcomes of interventions and predictors of long-term health were analyzed. RESULTS: Forty-eight cases were identified with PFIC2. Eighteen received partial external biliary diversion (PEBD) surgery, and 22 patients underwent liver transplantation. Two patients developed HCC and 2 died. Improved survival with native liver was closely associated with genotype, complete normalization of serum bile acids following PEBD, and alleviation of pruritus. Persistence of mild-to-moderate elevation of bile acids or a secondary rise following normalization was associated with liver disease progression and led to transplantation, suggesting that any prolonged elevation of bile acids worsens the chance of native liver survival. Higher-grade fibrosis at the time of PEBD was not associated with reduced long-term native liver survival. Patients with PFIC2 benefit from PEBD even at a stage of advanced fibrosis. CONCLUSION: Serum bile acid levels are an early predictor of treatment response and might serve as the gold standard in the evaluation of novel therapies including IBATi.

Long-Term Outcome Following Liver Transplantation for Primary Hepatic Tumors-A Single Centre Observational Study over 40 Years.

The incidence of pediatric liver tumors in general has been rising over the last years and so is the number of children undergoing liver transplantation for this indication. To contribute to the ongoing improvement of pre- and post-transplant care, we aim to describe outcome and risk factors in our patient cohort. We have compared characteristics and outcome for patients transplanted for hepatoblastoma to other liver malignancies in our center between 1983 and 2022 and analysed influential factors on tumor recurrence and mortality using nominal logistic regression analysis. Of 39 children (16 f) who had transplants for liver malignancy, 31 were diagnosed with hepatoblastoma. The proportion of malignant tumors in the transplant cohort rose from 1.9% (1983-1992) to 9.1% in the current decade (p < 0.0001). Hepatoblastoma patients were transplanted at a younger age and were more likely to have tumor extent beyond the liver. Post-transplant bile flow impairment requiring intervention was significantly higher compared to our total cohort (48 vs. 24%, p > 0.0001). Hearing loss was a common side effect of ototoxic chemotherapy in hepatoblastoma patients (48%). The most common maintenance immunosuppression were mTor-inhibitors. Risk factors for tumor recurrence in patients with hepatoblastoma were higher AFP before transplant (AFPpre-LTX), a low ratio of AFPmax to AFPpre-LTX and salvage transplantation. Liver malignancies represent a rising number of indications for liver transplantation in childhood. Primary tumor resection can spare a liver transplant with all its long-term complications, but in case of tumor recurrence, transplantation might have inferior outcome. The rate of acute biopsy-proven rejections and biliary complications in comparison to our total transplant cohort needs further investigations.

Acute severe non-A-E-hepatitis of unknown origin in children - A 30-year retrospective observational study from north-west Germany.

BACKGROUND & AIMS: The etiology of the current acute severe non-A-E hepatitis epidemic in children remains unclear. We aimed to describe the occurrence and outcomes of acute severe hepatitis in pediatric patients in North-West Germany over a period of more than 30 years and in the context of the current epidemic. METHODS: We analyzed all cases of acute severe hepatitis in childhood, as defined by the World Health Organization, at Hannover Medical School from 1990 and at the University Hospital of Essen from 2009 to 16 May 2022. We separated cases into a historic cohort (1990-2018) and a COVID-19 era cohort (2019-2022). RESULTS: After application of exclusion criteria, 107 patients with acute severe hepatitis were identified (2.32 cases/center/year). Annual incidence per center rose significantly from 2.2 (historic cohort until 2018) to 4.25/center/year (from 2019, p = 0.002). Of all cases, 75.7% presented with jaundice, while 53.3% had clinical signs of infection. Two cases of adenovirus were proven (2004/2016), other pathogens detected were HHV-6 (4), CMV, HSV, EBV(3). Sixty-nine patients (64.5%) met the criteria of pediatric acute liver failure, with 44 requiring liver transplantation. In the current cohort, patients with infection, gastrointestinal symptoms and higher alanine aminotransferase had a better chance of transplant-free survival, whereas hepatic encephalopathy, higher international normalized ratio and bilirubin predicted a poor outcome. Twenty-five patients developed hepatitis-associated aplastic anemia and 19 patients (17.8%) died. CONCLUSIONS: Acute non-A-E-hepatitis in children is a rare but severe entity, often leading to acute liver failure. Clinical presentation in our current cohort resembles 2022 NAEH cases, with improved outcomes compared to historic controls. The rising incidence of NAEH in our centers since 2019, in the absence of adenoviral infection, indicates other potential triggers of similar NAEH cases. IMPACT AND IMPLICATIONS: As the current epidemic of severe acute non-A-E-hepatitis cases in children highlights our limited understanding in the field, we aim to describe current cases, characterizing the presentation over time, and defining similarities and discrepancies before and during the COVID-19 pandemic. Our data show a rising incidence of non-A-E-hepatitis cases since the beginning of the COVID-19 pandemic. These cases were not associated with adenoviral infections, suggesting that the recently described accumulation of adenovirus infections in relationship to hepatitis is a new trigger for a known phenomenon, rather than a new disease entity. Therefore, the role of protective isolation and subsequent lack of contact with trivial infections in children during the pandemic should be the subject of further examinations. We expect our data to contribute to a better understanding of severe acute hepatitis in childhood, increased vigilance for this potentially lethal disease beyond the current epidemic, and ultimately improved clinical diagnosis and care.

Accumulation of Postoperative Unexpected Events Assessed by the Comprehensive Complication Index® as Prognostic Outcome Parameters for Kasai Procedure.

Introduction The Kasai procedure in children with biliary atresia (BA) is associated with several complications in the short-term. The Comprehensive Complication Index (CCI®) is a validated metric in adult surgery for the analysis of complications and morbidity in surgical patients. We aimed to analyze the CCI® for the first time in BA infants and to correlate its association with outcomes. Material and Methods We conducted a retrospective review of medical records of infants with type III BA undergoing the Kasai procedure between January 2011 and December 2021 at our institution. All unexpected events were ranked according to the Clavien−Dindo classification, and the CCI® per patient was subsequently calculated. Clavien−Dindo grades, individual events, CCI®, and total event numbers per patient were correlated with one- and two-year outcomes post-surgery. Results A total of 131 events were identified in 101 patients (ranging 0−11 per patient). Forty-four Grade I (33.6%), 67 Grade II (51.1%), 18 Grade III (13.7%), and two sentinel events [>Grade IV] (1.5%) were documented according to Clavien−Dindo, including one death in a cardiac-associated BA patient. None of the complications significantly correlated with a poor outcome. Sixty-three (62.4%) CCI® scores were calculated (range 0−100). The mean CCI® score during the in-patient treatment post-surgery was significantly higher in patients with a poorer outcome than patients with native liver survival at one- and two-year follow-up (22.7 ± 21.7 vs. 13.2 ± 18.1; p = 0.02). Conclusion Not the severity of complications, but the accumulation of numerous events related to Kasai procedure were associated with a poorer outcome. Therefore, the CCI® is an excellent instrument for the postoperative morbidity assessment of BA patients.

Cold Ischemia Time and Graft Fibrosis Are Associated with Autoantibodies after Pediatric Liver Transplantation: A Retrospective Cohort Study of the European Reference Network TransplantChild.

The reported prevalence of autoantibodies (AAB) (ANA, SMA, LKM, SLA) after pediatric liver transplantation (pLTX) varies considerably from 26-75%, but their clinical impact on outcome is uncertain. We aimed to study the prevalence of AAB after pLTX, their association with donor-, transplant-, and recipient-characteristics, and their relation to outcome. In our multicenter retrospective study, we aimed to clarify conflicting results from earlier studies. Six ERN TransplantChild centers reported data on 242 patients, of whom 61% were AAB positive. Prevalence varied across these centers. Independent of the interval between pLTX and AAB analysis, a one-hour increase in CIT resulted in an odds ratio (OR) of 1.37 (95% CI 1.11-1.69) for SMA positivity and an OR of 1.42 (95%CI 1.18-1.72) for ANA positivity. Steroid-free immunosuppression (IS) versus steroid-including IS (OR 5.28; 95% CI 1.45-19.28) was a risk factor for SMA positivity. Liver enzymes were not associated with ANA or SMA positivity. We did not observe an association of rejection activity index with ANA or SMA. However, the liver fibrosis score in follow-up biopsies was associated with ANA titer and donor age. In conclusion, this first multicenter study on AAB after pLTX showed high AAB prevalence and varied widely between centers. Longer CIT and prednisolone-free-IS were associated with AAB positivity, whereas AAB were not indicative of rejection, but instead were associated with graft fibrosis. The detection of AAB may be a marker of liver fibrosis and may be taken into consideration when indications for liver biopsy and immunosuppressive regimes, or reduction of immunosuppression in long-term follow-up, are being discussed. Prospective immunological profiling of pLTX patients, including AAB, is important to further improve our understanding of transplant immunology and silent graft fibrosis.

Extrahepatic manifestations of progressive familial intrahepatic cholestasis syndromes: Presentation of a case series and literature review.

BACKGROUND AND AIMS: Progressive familial intrahepatic cholestasis (PFIC) is a collective term for a heterogenous group of rare, inherited cholestasis syndromes. The number of genes underlying the clinical PFIC phenotype is still increasing. While progressive liver disease and its sequelae such as portal hypertension, pruritus and hepatocellular carcinoma determine transplant-free survival, extrahepatic manifestations may cause relevant morbidity. METHODS: We performed a literature search for extrahepatic manifestations of PFIC associated with pathogenic gene variants in ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 and MYO5B. To illustrate the extrahepatic symptoms described in the literature, PFIC cases from our centres were revisited. RESULTS: Extrahepatic symptoms are common in PFIC subtypes, where the affected gene is expressed at high levels in other tissues. While most liver-associated complications resolve after successful orthotopic liver transplantation (OLT), some extrahepatic symptoms show no response or even worsen after OLT. CONCLUSION: The spectrum of extrahepatic manifestations in PFIC highlights essential, non-redundant roles of the affected genes in other organs. Extrahepatic features contribute towards low health-related quality of life (HRQOL) and morbidity in PFIC. While OLT is often the only remaining, curative treatment, potential extrahepatic manifestations need to be carefully monitored and addressed.

[Solid Pseudopapillary Neoplasm of the Pancreas in Children and Adolescents - Tailored Diagnostic and Operative Concepts in Four Patients]. / Solide pseudopapilläre Pankreasneoplasie bei Kindern und Jugendlichen.

BACKGROUND: Pancreatic tumors in children and adolescents are rare entities that can stay asymptomatic for long periods. They often become apparent as incidental findings or due to clinical symptoms, like abdominal pain. Solid pseudopapillary neoplasms of the pancreas (SPN) are rare representatives of this group, being low-grade malignant processes and requiring radical surgical treatment. We present four cases of SPN with different diagnostic and therapeutic approaches. METHODS: A retrospective analysis of four cases of SPN treated between 2015 and 2020 was performed. RESULTS: Four female patients (11-17 years) were found to have SPN during diagnostic procedures. Three of them were located in the pancreatic head. Histological confirmation was obtained with endosonographic-, CT-guided and open biopsy, respectively. R0 resection was achieved by a pylorus preserving, partial duodenopancreatectomy according to Traverso-Longmire. In one patient the tumor was located in the pancreatic tail with tumor adherence to the splenic vessels. A CT guided biopsy confirmed an SPN. A distal pancreatectomy and splenectomy was performed. Follow-up (6 months - 6 years) revealed no evidence of tumor recurrence, metastasis, or pancreoprive diabetes. CONCLUSION: For the treatment plan preoperative histological confirmation of SPN is necessary. Based on the tailored diagnostic and operative concepts, treatment at a center with a specialized pediatric surgery, pediatric oncology, pediatric gastroenterology, pediatric radiology and pathology is mandatory.

Kasai Procedure in Patients Older Than 90 Days: Worth a Cut.

INTRODUCTION: Age at Kasai portoenterostomy (KPE) has been identified as a predictive factor for native-liver survival in patients with biliary atresia (BA). Outcomes of pediatric liver transplantation (LT) have improved over recent years. It has been proposed to consider primary LT as a treatment option for late-presenting BA infants instead of attempting KPE. We present our experience with patients older than 90 days undergoing KPE. MATERIALS AND METHODS: A retrospective chart review of patients with BA undergoing KPE at our institution between January 2010 and December 2020 was performed. Patients 90 days and older at the time of surgery were included. Patients' characteristics, perioperative data, and follow-up results were collected. Eleven patients matched the inclusion criteria. Mean age at KPE was 108 days (range: 90-133 days). RESULTS: Postoperative jaundice clearance (bilirubin < 2 mg/dL) at 2-year follow-up was achieved in three patients (27%). Eight patients (73%) received a liver transplant at a mean of 626 days (range: 57-2,109 days) after KPE. Four patients (36%) were transplanted within 12 months post-KPE. Two patients died 237 and 139 days after KPE due to disease-related complications. One patient is still alive with his native liver, currently 10 years old. CONCLUSION: Even when performed at an advanced age, KPE can help prolong native-liver survival in BA patients and offers an important bridge to transplant. In our opinion, it continues to represent a viable primary treatment option for late-presenting infants with BA.

Centralization of Biliary Atresia: Has Germany Learned Its Lessons?

INTRODUCTION: The majority of pediatric surgeons and hepatologists recommend the centralization of biliary atresia (BA) treatment within experienced liver units. We aimed to investigate whether voluntary self-restriction and acceptance of the need for this change in practice changed the BA referral policy in Germany during the last decade. MATERIALS AND METHODS: In cooperation with pediatric surgeons, gastroenterologists or hepatologists, and pediatric liver transplant units, the 2-year follow-up data of infants with BA born in Germany between 2010 and 2014 were collected using www.bard-online.com or pseudonymized data transfer. Results were compared with our previous analysis of the outcome data of infants with BA born between 2001 and 2005 in Germany. RESULT: Overall, 173 infants with BA were identified, of whom 160 underwent Kasai portoenterostomy (KPE; 92.5%) and 13 (7.5%) underwent primary liver transplantation at 21 German centers. At 2-year follow-up, overall survival was 87.7% (vs. 81.9% in 2001-2005 [p = 0.19]), survival with native liver post-KPE was 29.2% (vs. 22.8% in 2001-2005 [p = 0.24]), and jaundice-free survival with native liver post-KPE was 24.0% (vs. 20.1% in 2001-2005 [p = 0.5]). Compared with the 2001-2005 analysis, all criteria showed improvement but the differences are statistically not significant. CONCLUSION: Our observation shows that KPE management requires improvement in Germany. Centralization of BA patients to German reference liver units is not yet mandatory. However, European and national efforts with regard to the centralization of rare diseases support our common endeavor in this direction.

Recipient-Specific Risk Factors Impairing Patient and Graft Outcome after Pediatric Liver Transplantation-Analysis of 858 Transplantations in 38 Years.

(1) Background and Aim: Despite excellent long-term results in pediatric liver transplantation (pLTx), mortality and graft loss still are to be diminished. We aim to describe time-dependent changes and long-term outcome of a large single-center pLTx cohort and to identify independent recipient-related risk factors impairing patient and graft survival. (2) Methods: This is a retrospective single-center study analyzing all pediatric liver transplants from 1983-2020. Risk factors for mortality and graft loss were identified by univariable and multi-linear regression analysis. (3) Results: We analyzed 858 liver transplantations in 705 pediatric patients. Five-year patient/graft survival increased from 60.9%/48.0% (1983-1992) to 97.5%/86.5% (OR = 12.5; p < 0.0001/OR = 6.5; p < 0.0001) (2014-2020). Indications changed significantly over time, with a higher proportion of patients being transplanted for malignancies and metabolic disease and indications of PFIC and α1AT-deficiency declining. The era of transplantation (log7.378/9.657; p < 0.0001) and indication of acute liver failure (log = 1.944/2.667; HR = 2.015/1.772; p = 0.0114/0.002) impairs patient/graft survival significantly in the multivariate analysis. Furthermore, patient survival is worsened by re-transplantation (log = 1.755; HR = 1.744; p = 0.0176) and prolonged waiting times in high-urgency status (log = 2.588; HR = 1.073; p = 0.0026), whereas the indication of biliary atresia improved outcome (log = 1.502; HR = 0.575; p = 0.0315). Graft survival was additionally impaired by pre-existing portal vein thrombosis (log = 1.482; HR = 2.016; p = 0.0330). (4) Conclusions: Despite more complex indications, patient and graft survival after pLTx continue to improve.. Acute liver failure remains the indication with poorest outcome, and listing for high urgency liver transplantation should be considered carefully and early to keep waiting time on HU list short. Furthermore, pre-transplant portal vein thrombosis should be prevented whenever possible to improve graft survival.

Survival Benefits Following Liver Transplantation: A Matched-pair Analysis in Pediatric Patients With Cystic Fibrosis.

OBJECTIVES: Cystic fibrosis-related liver disease (CFLD) with consecutive cirrhosis is the third most common cause of death in CF patients. The aim of this study was to identify the potential long-term benefits of liver transplantation (LTx) in a match-control comparison. METHODS: Retrospective single-center data analysis of all pediatric LTx for CFLD between 1998 and 2014. A control group was selected from the local CF patient registry. Data were collected from case report forms and included clinical and laboratory data, lung function tests, the indication for LTx, and details of surgical procedures. RESULTS: At our institution, 23 patients with severe CFLD median age 13.8 years (range 8.7-17.4; 16 boys) underwent LTx between 1998 and 2014. In all patients, normalization of hepatic CF manifestations were achieved after LTx. But obviously there was no significant positive influence on nutritional status. Signs of posttransplant liver steatosis were documented by ultrasound in 17 patients. Liver biopsies after LTx were performed in 19 patients, in 42% (n = 8) of these biopsies a fatty degeneration was observed. Five patients died after LTx, none because of primary hepatic dysfunction (1 because of posttransplant proliferative disorder, 4 because of infection). Analysis of matched control pairs revealed that liver function, anthropometry, pulmonary function, and life expectancy of CFLD patients with LTx are comparable with matched CF peers without CFLD. CONCLUSIONS: Isolated LTx normalizes the hepatic manifestation of CF disease. LTx enables children and adolescents with severe CFLD to have a comparable prognosis in terms of growth, life expectancy, and lung function as CF patients without advanced liver involvement. Our data clarifies the long-term perspectives of affected patients.

Hepatitis-associated Aplastic Anaemia in Children.

BACKGROUND: Children with idiopathic acute liver failure (IALF) are at a high risk of developing life-threatening bone marrow failure (BMF). The aim of the study was to describe the development, therapy and prognosis of this hepatitis-associated aplastic anaemia (HAAA) in comparison to isolated acquired aplastic anaemia. RESULTS: We retrospectively found 18 patients (9 female) of HAAA between 1984 and 2017 with an age of 1.4-16.4 years. Fifteen of them fulfilled the SAA criteria, 3 had a bone marrow hypoplasia. Eleven of these children received liver transplantation (LTx) (these were 11 of 42 (26%) children receiving LTx for IALF), 6 patients recovered without LTx. The first signs of BMF, thrombocytopaenia and leucocytopaenia, occurred before LTx in all cases. During the follow-up period 8 patients reached haematological remission, 6 received haematopoietic stem cell transplantation (HSCT). Seven children died in a median of 304 days after the first symptoms mostly because of bleedings and infections. To date, extensive investigations failed to detect a genetically, viral or immunological aetiology. No AA was diagnosed in the 41 patients receiving liver transplants during the same period for ALF of known aetiology. As a comparison group, we collected the data of patients with isolated SAA. 73% achieved a remission after Immunosuppressive therapy (IST) without HSCT, and none of them died during the follow-up period. CONCLUSION: Blood counts should be examined early and regularly (0-22 days after onset) in patients with IALF. Aggressive treatment with LTx, IST and HSCT appears to improve the prognosis.

MTF1 binds to metal-responsive element e within the ATP7B promoter and is a strong candidate in regulating the ATP7B expression.

Wilson's disease is an autosomal recessive disorder resulting from copper excess. Some patients with clinical Wilson's disease symptoms exhibit no or only heterozygous pathogenic variants in the coding region of the disease-causing ATP7B gene. Therefore, the ATP7B promoter region is of special interest. Metal-responsive elements (MREs) located in the ATP7B promoter are promising motifs in modulating the ATP7B expression. We studied protein interaction of MREe, MREc, and MREd by electrophoretic mobility shift assays and revealed specific interactions for all MREs. We further narrowed down the specific binding site. Proteins potentially binding to the three MREs were identified by MatInspector analyses. Metal regulatory transcription factor 1 (MTF1) could be validated to bind to MREe by electrophoretic mobility shift assays. ATP7B promoter-driven reporter gene expression was significantly increased because of this interaction. MTF1 is a strong candidate in regulating the ATP7B expression through MREe binding.

Homozygous frame shift variant in ATP7B exon 1 leads to bypass of nonsense-mediated mRNA decay and to a protein capable of copper export.

Wilson disease (WD) is an autosomal recessive disease of copper excess due to pathogenic variants in the ATP7B gene coding for a copper-transporting ATPase. We present a 5-year-old girl with the homozygous frame shift variant NM_000053.3: c.19_20del in exon 1 of ATP7B (consecutive exon numbering with c.1 as first nucleotide of exon 1), detected by whole-exome sequencing as a secondary finding. The variant leads to a premature termination codon in exon 2. The girl exhibited no WD symptoms and no abnormalities in liver biopsy. ATP7B liver mRNA expression was comparable to healthy controls suggesting that nonsense-mediated mRNA decay (NMD) could be bypassed by the mechanism of translation reinitiation. To verify this hypothesis, a CMV-driven ATP7B minigene (pcDNA3) was equipped with the authentic ATP7B 5' untranslated region  and a truncated intron 2. We introduced c.19_20del by site-directed mutagenesis and overexpressed the constructs in HEK293T cells. We analyzed ATP7B expression by qRT-PCR, northern and western blot, and examined protein function by copper export capacity assays. Northern blot, qRT-PCR, and western blot revealed that c.19_20del ATP7B mRNA and protein is expressed in size and amount comparable to wild-type. Copper export capacity was also comparable to wild-type. Our results indicate that c.19_20del in ATP7B is able to bypass NMD by translation reinitiation, demonstrating that the classification of truncating variants as pathogenic without additional investigations should be done carefully.

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).

Immune monitoring after pediatric liver transplantation - the prospective ChilSFree cohort study.

BACKGROUND: Although trough levels of immunosuppressive drugs are largely used to monitor immunosuppressive therapy after solid organ transplantation, there is still no established tool that allows for a validated assessment of functional degree of immunosuppression or the identification of clinically relevant over- or under-immunosuppression, depending on graft homeostasis. Reliable non-invasive markers to predict biopsy proven acute rejection (BPAR) do not exist. Literature data suggest that longitudinal measurements of immune markers might be predictive of BPAR, but data in children are scarce. We therefore propose an observational prospective cohort study focusing on immune monitoring in children after liver transplantation. We aim to describe immune function in a cohort of children before and during the first year after liver transplantation and plan to investigate how the immune function profile is associated with clinical and laboratory findings. METHODS: In an international multicenter prospective approach, children with end-stage liver disease who undergo liver transplantation are enrolled to the study and receive extensive immune monitoring before and at 1, 2, 3, 4 weeks and 3, 6, 12 months after transplantation, and whenever a clinically indicated liver biopsy is scheduled. Blood samples are analyzed for immune cell numbers and circulating levels of cytokines, chemokines and factors of angiogenesis reflecting immune cell activation. Statistical analysis will focus on the identification of trajectorial patterns of immune reactivity predictive for systemic non-inflammatory states, infectious complications or BPAR using joint modelling approaches. DISCUSSION: The ChilSFree study will help to understand the immune response after pLTx in different states of infection or rejection. It may provide insight into response mechanisms eventually facilitating immune tolerance towards the graft. Our analysis may yield an applicable immune panel for non-invasive early detection of acute cellular rejection, with the prospect of individually tailoring immunosuppressive therapy. The international collaborative set-up of this study allows for an appropriate sample size which is otherwise difficult to achieve in the field of pediatric liver transplantation.

Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants.

BACKGROUND & AIMS: The bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4) and the ATPase familial intrahepatic cholestasis 1 (FIC1, ATP8B1) mediate bile formation. This study aimed to determine the contribution of mutations and common variants in the FIC1, BSEP and MDR3 genes to cholestatic disorders of differing disease onset and severity. METHODS: Coding exons with flanking intron regions of ATP8B1, ABCB11, and ABCB4 were sequenced in cholestatic patients with assumed genetic cause. The effects of new variants were evaluated by bioinformatic tools and 3D protein modeling. RESULTS: In 427 patients with suspected inherited cholestasis, 149 patients carried at least one disease-causing mutation in FIC1, BSEP or MDR3, respectively. Overall, 154 different mutations were identified, of which 25 were novel. All 13 novel missense mutations were disease-causing according to bioinformatics analyses and homology modeling. Eighty-two percent of patients with at least one disease-causing mutation in either of the three genes were children. One or more common polymorphism(s) were found in FIC1 in 35.3%, BSEP in 64.3% and MDR3 in 72.6% of patients without disease-causing mutations in the respective gene. Minor allele frequencies of common polymorphisms in BSEP and MDR3 varied in our cohort compared to the general population, as described by gnomAD. However, differences in ethnic background may contribute to this effect. CONCLUSIONS: In a large cohort of patients, 154 different variants were detected in FIC1, BSEP, and MDR3, 25 of which were novel. In our cohort, frequencies for risk alleles of BSEP (p.V444A) and MDR3 (p.I237I) polymorphisms were significantly overrepresented in patients without disease-causing mutation in the respective gene, indicating that these common variants can contribute to a cholestatic phenotype. LAY SUMMARY: FIC1, BSEP, and MDR3 represent hepatobiliary transport proteins essential for bile formation. Genetic variants in these transporters underlie a broad spectrum of cholestatic liver diseases. To confirm a genetic contribution to the patients' phenotypes, gene sequencing of these three major cholestasis-related genes was performed in 427 patients and revealed 154 different variants of which 25 have not been previously reported in a database. In patients without a disease-causing mutation, common genetic variants were detected in a high number of cases, indicating that these common variants may contribute to cholestasis development.