Molecular and Clinical Links between Drug-Induced Cholestasis and Familial Intrahepatic Cholestasis.

Idiosyncratic Drug-Induced Liver Injury (iDILI) represents an actual health challenge, accounting for more than 40% of hepatitis cases in adults over 50 years and more than 50% of acute fulminant hepatic failure cases. In addition, approximately 30% of iDILI are cholestatic (drug-induced cholestasis (DIC)). The liver's metabolism and clearance of lipophilic drugs depend on their emission into the bile. Therefore, many medications cause cholestasis through their interaction with hepatic transporters. The main canalicular efflux transport proteins include: 1. the bile salt export pump (BSEP) protein (ABCB11); 2. the multidrug resistance protein-2 (MRP2, ABCC2) regulating the bile salts' independent flow by excretion of glutathione; 3. the multidrug resistance-1 protein (MDR1, ABCB1) that transports organic cations; 4. the multidrug resistance-3 protein (MDR3, ABCB4). Two of the most known proteins involved in bile acids' (BAs) metabolism and transport are BSEP and MDR3. BSEP inhibition by drugs leads to reduced BAs' secretion and their retention within hepatocytes, exiting in cholestasis, while mutations in the ABCB4 gene expose the biliary epithelium to the injurious detergent actions of BAs, thus increasing susceptibility to DIC. Herein, we review the leading molecular pathways behind the DIC, the links with the other clinical forms of familial intrahepatic cholestasis, and, finally, the main cholestasis-inducing drugs.

The molecular landscape of progressive familial intrahepatic cholestasis in Turkey: Defining the molecular profiles and expanding the variant spectrum.

Progressive familial intrahepatic cholestasis (PFIC) is a rare genetically heterogeneous group of autosomal recessive liver disorders that manifests as intrahepatic cholestasis during the neonatal period. ATP8B1, ABCB11, and ABCB4 genes are responsible for PFIC type 1, PFIC type 2, and PFIC type 3, respectively. To determine the underlying molecular etiology of PFIC, 80 patients from 77 families were investigated. The molecular genetic diagnosis was applied by using next-generation sequencing (NGS) and revealed 29 different variants from 32 patients. In this study, we evaluated these variants according to mechanisms, clinical sub-groups, and genotype-phenotype correlation.

Disordered farnesoid X receptor signaling is associated with liver carcinogenesis in Abcb11-deficient mice.

ABCB11 encodes the bile salt export pump (BSEP), a key regulator in maintaining bile acid (BA) homeostasis. Although inherited ABCB11 mutations have previously been linked to primary liver cancer, whether ABCB11 deficiency leads to liver cancer remains unknown. Here, we analyzed ABCB11 mRNA expression levels in liver tumor specimens [29 with hepatocellular carcinoma (HCC), one with intrahepatic cholangiocarcinoma (ICC), and one with mixed HCC/ICC] with adjacent normal specimens and published human datasets. Liver tissues obtained from Abcb11-deficient (Abcb11-/- ) mice and wild-type mice at different ages were compared by histologic, RNA-sequencing, and BA analyses. ABCB11 was significantly downregulated in human liver tumors compared with normal controls. Abcb11-/- mice demonstrated progressive intrahepatic cholestasis and liver fibrosis, and spontaneously developed HCC and ICC over 12 months of age. Abcb11 deficiency increased BAs in the liver and serum in mice, most of which are farnesoid X receptor (FXR) antagonists/non-agonists. Accordingly, the hepatic expression and transcriptional activity of FXR were downregulated in Abcb11-/- mouse livers. Administration of the FXR agonist obeticholic acid reduced liver injury and tumor incidence in Abcb11-/- mice. In conclusion, ABCB11 is aberrantly downregulated and plays a vital role in liver carcinogenesis. The cholestatic liver injury and liver tumors developed in Abcb11-/- mice are associated with increased FXR antagonist BAs and thereby decreased activation of FXR. FXR activation might be a therapeutic strategy in ABCB11 deficiency diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Heterozygous mutations of ATP8B1, ABCB11 and ABCB4 cause mild forms of Progressive Familial Intrahepatic Cholestasis in a pediatric cohort.

INTRODUCTION: Heterozygous defects in genes implicated in Progressive Familial Intrahepatic Cholestasis have been described in milder forms of cholestatic diseases. Our aim is to describe clinical, laboratory and imaging characteristics as well as treatment and outcome of a cohort of pediatric patients with heterozygous mutations in ATP8B1, ABCB11 or ABCB4. PATIENTS AND METHODS: We present a retrospective descriptive study including pediatric patients with at least one heterozygosis defect in ATP8B1, ABCB11 or ABCB4 diagnosed after a cholestatic episode. Clinical, diagnostic and outcome data were collected including gene analysis (panel of PFIC NextGeneDx®). RESULTS: 7 patients showed a heterozygous mutation: 3 patients in ABCB4, 1 in ABCB11, 2 in ABCB4 and ABCB11 and 1 in ATP8B1. The median onset age was 5.5 years with a median time of follow-up of 6 years. The initial presentation was pruritus followed by asymptomatic hypertransaminasemia and persistent cholestasis. Two patients had family history of gallbladder stones and mild hepatitis. All showed elevated transaminases and bile acids, high gamma glutamyl-transferase (GGT) in 3 and conjugated bilirubin in 2 patients. Liver biopsy showed inflammatory infiltrate or mild fibrosis with normal immunohistochemistry. All patients were treated with ursodeoxycholic acid, two patients requiring the addition of resincholestyramine. During follow-up, 3 patients suffered limited relapses of pruritus. No disease progression was observed. CONCLUSION: Heterozygous mutations in genes coding proteins of the hepatocellular transport system can cause cholestatic diseases with great phenotypic variability. The presence of repeated episodes of hypertransaminasemia or cholestasis after a trigger should force us to rule out the presence of these heterozygous mutations in genes involved in CIFP.

The Bile Salt Export Pump: Molecular Structure, Study Models and Small-Molecule Drugs for the Treatment of Inherited BSEP Deficiencies.

The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.

Molecular Regulation of Canalicular ABC Transporters.

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

Genotype correlates with the natural history of severe bile salt export pump deficiency.

BACKGROUND & AIMS: Mutations in ABCB11 can cause deficiency of the bile salt export pump (BSEP), leading to cholestasis and end-stage liver disease. Owing to the rarity of the disease, the associations between genotype and natural history, or outcomes following surgical biliary diversion (SBD), remain elusive. We aimed to determine these associations by assembling the largest genetically defined cohort of patients with severe BSEP deficiency to date. METHODS: This multicentre, retrospective cohort study included 264 patients with homozygous or compound heterozygous pathological ABCB11 mutations. Patients were categorized according to genotypic severity (BSEP1, BSEP2, BSEP3). The predicted residual BSEP transport function decreased with each category. RESULTS: Genotype severity was strongly associated with native liver survival (NLS, BSEP1 median 20.4 years; BSEP2, 7.0 years; BSEP3, 3.5 years; p <0.001). At 15 years of age, the proportion of patients with hepatocellular carcinoma was 4% in BSEP1, 7% in BSEP2 and 34% in BSEP3 (p = 0.001). SBD was associated with significantly increased NLS (hazard ratio 0.50; 95% CI 0.27-0.94: p = 0.03) in BSEP1 and BSEP2. A serum bile acid concentration below 102 µmol/L or a decrease of at least 75%, each shortly after SBD, reliably predicted NLS of ≥15 years following SBD (each p <0.001). CONCLUSIONS: The genotype of severe BSEP deficiency strongly predicts long-term NLS, the risk of developing hepatocellular carcinoma, and the chance that SBD will increase NLS. Serum bile acid parameters shortly after SBD can predict long-term NLS. LAY SUMMARY: This study presents data from the largest genetically defined cohort of patients with severe bile salt export pump deficiency to date. The genotype of patients with severe bile salt export pump deficiency is associated with clinical outcomes and the success of therapeutic interventions. Therefore, genotypic data should be used to guide personalized clinical care throughout childhood and adulthood in patients with this disease.

Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells.

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects, we first established a liver organoid using human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids, resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after in vivo organoid transplantation. To assess the paracrine contributions, we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form, their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs, resulting in the expression of bile salt export pump. In conclusion, the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids, but organoid self-organization requires cell-to-cell surface contact. Our in vitro model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes.

ABCB11 deficiency presenting as transient neonatal cholestasis: Correlation with genotypes and BSEP expression.

BACKGROUND & AIMS: ABCB11 deficiency presenting in infancy is believed generally to manifest as persistent/progressive cholestasis. We describe a group of patients with biallelic ABCB11 variants whose disorder manifested as transient neonatal cholestasis (TNC). METHODS: Neonatal intrahepatic cholestasis in 68 children (31 males) with biallelic predictedly pathogenic variants (PPV) in ABCB11 was classified as transient (TNC group, n = 23, 11 males), intermittent (benign recurrent intrahepatic cholestasis [BRIC] group, n = 3, 1 male) or persistent/ progressive (progressive familial intrahepatic cholestasis [PFIC] group, n = 42, 19 males). Clinical, genetic and bile salt export pump (BSEP) expression information was correlated with outcomes. RESULTS: The median onset age of jaundice was 3 days (birth to 2 months) for the TNC group and 10.5 days (birth to 3 months) for the PFIC group (P = .034). The median length of follow-up of TNC patients was 44 months (12 months-168 months). At presentation, hepatobiliary-injury biomarker values were similar between the groups (P > .05). TNC patients (17/23) more often than PFIC patients (20/42, P = .041) harboured biallelic non-null variants (predicted not to terminate translation prematurely). TNC patient livers (7/7) more often than PFIC patient livers (5/16, P = .005) expressed immunohistochemically detectable BSEP. Kaplan-Meier analysis showed better prognosis for patients with BSEP expression (P = .009). Too few BRIC patients were available for statistical study. CONCLUSIONS: Neonatal cholestasis associated with biallelic PPV in ABCB11 can resolve temporarily or persistently in one third of cases. Resolution is more likely in patients with biallelic non-null PPV or with liver BSEP expression.

The effect of ursodeoxycholic acid on the relative expression of the lipid metabolism genes in mouse cholesterol gallstone models.

BACKGROUND: Many studies indicate that gallstone formation has genetic components. The abnormal expression of lipid-related genes could be the basis for particular forms of cholesterol gallstone disease. The aim of this study was to obtain insight into lipid metabolism disorder during cholesterol gallstone formation and to evaluate the effect of ursodeoxycholic acid (UDCA) on the improvement of bile lithogenicity and its potential influence on the transcription of lipid-related genes. METHODS: Gallstone-susceptible mouse models were induced by feeding with a lithogenic diet (LD) for 8 weeks. Bile and liver tissues were obtained from these mouse models after 0, 4 and 8 weeks. Bile lipids were measured enzymatically, and the cholesterol saturation index (CSI) was calculated to evaluate the bile lithogenicity by using Carey's critical tables. Real-time polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of farnesoid X receptor (FXR), liver X receptor (LXR), adenosine triphosphate-binding cassette subfamily G member 5/8 (ABCG5/8), cholesterol 7-α hydroxylase (CYP7A1), oxysterol 7-α hydroxylase (CYP7B1), sterol 27-α hydroxylase (CYP27A1), peroxisome proliferator-activated receptor alpha (PPAR-α) and adenosine triphosphate-binding cassette subfamily B member 11 (ABCB11). RESULTS: The rate of gallstone formation was 100% in the 4-week group but only 30% in the UDCA-treated group. The UDCA-treated group had a significantly lower CSI compared with other groups. Of special note, the data on the effects of UDCA showed higher expression levels of ABCG8, ABCB11 and CYP27A1, as well as lower expression levels of LXR and PPAR-α, compared to the model control group. CONCLUSIONS: UDCA exhibits tremendously potent activity in restraining lipid accumulation, thus reversing the lithogenic effect and protecting hepatocytes from serious pathological damage. The abnormal expression of ABCG8, CYP7A1, CYP27A1, LXR and PPAR-α might lead to high lithogenicity of bile. These results are helpful in exploring new lipid metabolism pathways and potential targets for the treatment of cholesterol stones and for providing some basis for the study of the pathogenesis and genetic characteristics of cholelithiasis. Research on the mechanism of UDCA in improving lipid metabolism and bile lithogenicity may be helpful for clinical treatment and for reducing the incidence of gallstones.

Oxysterol/chitotriosidase based selective screening for Niemann-Pick type C in infantile cholestasis syndrome patients.

BACKGROUND: Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disease (1 per 100 000 newborns) caused by NPC proteins impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. To date the NP-C diagnostics is usually based on cholesterol detection in fibroblasts using an invasive and time-consuming Filipin staining and we need more arguments to widely introduce oxysterols as a biomarkers in NP-C. METHODS: Insofar as NP-C represents about 8% of all infant cholestases, in this prospective observational study we tried to re-assess the specificity plasma oxysterol and chitotriosidase as a biochemical screening markers of NP-C in children with cholestasis syndrome of unknown origin. For 108 patients (aged from 2 weeks to 7 years) the levels of cholestane-3ß,5α,6ß-triol (C-triol) and chitotriosidase (ChT) were measured. For patients with elevated C-triol and/or ChT the NPC1 and NPC2 genes were Sanger-sequenced and 47 additional genes (from the custom liver damage panel) were NGS-sequenced. RESULTS: Increased C-triol level (> 50 ng/ml) was detected in 4 (of 108) infants with cholestasis syndrome of unknown origin, with following molecular genetic NP-C diagnosis for one patient. Plasma cholesterol significantly correlates with C-triol (p < 0.05). NGS of high C-triol infants identified three patients with mutations in JAG1 (Alagille syndrome) and ABCB11 (Byler disease) genes. Increased ChT activity was detected in 8 (of 108) patients with various aetiologies, including NP-C, Byler disease and biliary atresia. CONCLUSION: Combined analysis of ChT activity and C-triol levels is an effective method for identifying NP-C.

Comprehensive bile acid profiling in hereditary intrahepatic cholestasis: Genetic and clinical correlations.

BACKGROUND & AIMS: Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. METHODS: We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. RESULTS: The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89 ± 1.07 min) and the undiagnosed cholestasis group (11.46 ± 1.07 min) were lower than those of healthy controls (13.69 ± 0.77 min) (both p < 0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro-LCA, glyco-LCA and hyocholic acid was identified that could differentiate the ABCB11-mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p < 0.0001) and, in non-ABCB11-mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p < 0.0000001). CONCLUSIONS: Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.

Splicing analysis of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase.

AIM: The aim of this study was to analyze the pathogenicity of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase. METHODS: The enrolled variants were identified in ABCB11 between October 2009 and June 2016. The effects on pre-RNA splicing were analyzed by in silico tools and minigene splicing assay. RESULTS: There were three intronic (c.908 + 5G > A, c.2815-8A > G, and c.612-15_-6del10bp) and two synonymous (c.1809G > A, p.K603 K and c.2418C > T, p.G806G) variants with unknown significance identified in ABCB11 of five ABCB11 heterozygotes. Parental studies were carried out for four patients, and revealed that the variants with unknown significance were compound heterozygous with other pathogenic variants. The five variants with unknown significance had minor allele frequency <0.1% or were absent from controls, and had positive prediction results by in silico tools. The effects on pre-RNA splicing were further confirmed by minigene splicing assay. c.908 + 5A caused abnormal splicing in at least 78.5 ± 3.8% of products using a cryptic splice site (ss) 22 nucleotides (nt) upstream of the wild-type (WT) 5'ss. Seven nucleotides of intron 22 upstream of the WT 3'ss was retained for all products from c.2815-8G. c.612-15_-6del caused exon 8 skipping in 24.8 ± 7.7% of products, and 55 nt of exon 8 downstream of the WT 3'ss removal in remaining products. c.1809A led to exon 15 skipping. c.2418 T removed exon 20 and 62 nt of exon 21 downstream of the WT 3'ss by using a cryptic ss. CONCLUSIONS: We successfully identified five pathogenic synonymous or intronic variants with some common features. These features might help to choose the right variant for further functional assay.

Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17ß-D-glucuronide-induced cholestasis in rats.

Estradiol-17ß-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100 nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100 nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

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.

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.

Lipid flopping in the liver.

Bile is synthesized in the liver and is essential for the emulsification of dietary lipids and lipid-soluble vitamins. It is a complex mixture of amphiphilic bile acids (BAs; which act as detergent molecules), the membrane phospholipid phosphatidylcholine (PC), cholesterol and a variety of endogenous metabolites and waste products. Over the last 20 years, the combined effort of clinicians, geneticists, physiologists and biochemists has shown that each of these bile components is transported across the canalicular membrane of the hepatocyte by its own specific ATP-binding cassette (ABC) transporter. The bile salt export pump (BSEP) ABCB11 transports the BAs and drives bile flow from the liver, but it is now clear that two lipid transporters, ABCB4 (which flops PC into the bile) and the P-type ATPase ATP8B1/CDC50 (which flips a different phospholipid in the opposite direction) play equally critical roles that protect the biliary tree from the detergent activity of the bile acids. Understanding the interdependency of these lipid floppases and flippases has allowed the development of an assay to measure ABCB4 function. ABCB4 harbours numerous mis-sense mutations which probably reflects the spectrum of liver disease rooted in ABCB4 aetiology. Characterization of the effect of these mutations at the protein level opens the possibility for the development of personalized prognosis and treatment.

Characterization and treatment of persistent hepatocellular secretory failure.

BACKGROUND & AIMS: Hepatocellular secretory failure induced by drugs, toxins or transient biliary obstruction may sometimes persist for months after removal of the initiating factor and may then be fatal without liver transplantation. We characterized patients with severe persistent hepatocellular secretory failure (PHSF) and treated them with the pregnane X receptor (PXR) agonist, rifampicin. We also studied the effect of rifampicin on PXR-dependent expression of genes involved in biotransformation and secretion in vitro. METHODS: Thirteen patients (age 18-81 years, 6 male) with hepatocellular secretory failure that persisted after removal of the inducing factor (drugs/toxin: 9) or biliary obstruction (4) were identified over 6 years. Six of these patients were screened for ATP8B1 or ABCB11 mutations. All were treated with rifampicin (300 mg daily) for 1-10 weeks. Expression of genes involved in biotransformation and secretion was determined by rtPCR in human hepatocytes and intestinal cells incubated with rifampicin (10 µmol/L). RESULTS: Serum bilirubin of patients with PHSF ranged from 264 to 755 µmol/L. Normal γGT was found in 10/13 patients of whom 3/6 tested positive for ATP8B1/ABCB11 mutations. Serum bilirubin declined to <33 µmol/L after 1-10 weeks of rifampicin treatment. In vitro, rifampicin PXR-dependently upregulated biotransformation phase 1 (CYP3A4), phase 2 (UGT1A1) and phase 3 (MRP2) enzymes/carriers as well as the basolateral bile salt exporter OSTß. CONCLUSION: Persistent hepatocellular secretory failure may develop in carriers of transporter gene mutations. In severe cases, rifampicin may represent an effective therapeutic option of PHSF. PXR-dependent induction of CYP3A4, UGT1A1, MRP2 and OSTß could contribute to the anticholestatic effect of rifampicin in PHSF.

Paediatric hepatocellular carcinoma due to somatic CTNNB1 and NFE2L2 mutations in the setting of inherited bi-allelic ABCB11 mutations.

Hepatocellular carcinoma (HCC) rarely occurs in childhood. We describe a patient with new onset of pruritus at 8 months of age who at 17 months of age was found to have a 2.5 cm HCC. To delineate the possible genetic basis of this tumour, we performed whole exome sequencing (WES) of the germline DNA and identified two novel predictably deleterious missense mutations in ABCB11, encoding bile salt export pump (BSEP), confirmed in the parental DNA as bi-allelic and inherited. Although inherited ABCB11 mutations have previously been linked to HCC in a small number of cases, the molecular mechanisms of hepatocellular carcinogenesis in ABCB11 disease are unknown. WES of the HCC tissue uncovered somatic driver mutations in the beta-catenin (CTNNB1) and nuclear-factor-erythroid-2-related-factor-2 (NFE2L2) genes. Moreover, clonality analysis predicted that the CTNNB1 mutation was clonal and occurred earlier during carcinogenesis, whereas the NFE2L2 mutation was acquired later. Interestingly, background liver parenchyma showed no inflammation or fibrosis and BSEP expression was preserved. This is the first study to identify somatic CTNNB1 and NFE2L2 mutations in early childhood arisen in the setting of inherited bi-allelic ABCB11 mutations. Rapid WES analysis expedited this child's diagnosis and treatment, and likely improved her prognosis.