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.

Progressive familial intrahepatic cholestasis-outcome and time to transplant after biliary diversion according to genetic subtypes.

Background: Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous disease characterized by progressive cholestasis in early childhood. Surgical therapy aims at preventing bile absorption either by external or internal biliary diversion (BD). Several different genetic subtypes encode for defects in bile transport proteins, and new subtypes are being discovered ongoingly. Overall, the literature is scarce, however, accumulating evidence points to PFIC 2 having a more aggressive course and to respond less favorable to BD. With this knowledge, we aimed to retrospectively analyze the long-term outcome of PFIC 2 compared to PFIC 1 following BD in children at our center. Methods: Clinical data and laboratory findings of all children with PFIC, who were treated and managed in our hospital between 1993 and 2022, were analyzed retrospectively. Results: Overall, we treated 40 children with PFIC 1 (n = 10), PFIC 2 (n = 20) and PFIC 3 (n = 10). Biliary diversion was performed in 13 children (PFIC 1, n = 6 and 2, n = 7). Following BD, bile acids (BA) (p = 0.0002), cholesterol (p < 0.0001) and triglyceride (p < 0.0001) levels significantly decreased only in children with PFIC 1 but not in PFIC 2. Three out of 6 children (50%) with PFIC 1 and 4 out of 7 children (57%) with PFIC 2 required liver transplantation despite undergoing BD. On an individual case basis, BA reduction following BD predicted this outcome. Of the 10 children who had PFIC 3, none had biliary diversion and 7 (70%) required liver transplantation. Conclusion: In our cohort, biliary diversion was effective in decreasing bile acids, cholesterol levels as well as triglycerides in the serum only in children with PFIC 1 but not PFIC 2. On an individual case level, a decrease in BA following BD predicted the need for liver transplantation.

Vasor: Accurate prediction of variant effects for amino acid substitutions in multidrug resistance protein 3.

The phosphatidylcholine floppase multidrug resistance protein 3 (MDR3) is an essential hepatobiliary transport protein. MDR3 dysfunction is associated with various liver diseases, ranging from severe progressive familial intrahepatic cholestasis to transient forms of intrahepatic cholestasis of pregnancy and familial gallstone disease. Single amino acid substitutions are often found as causative of dysfunction, but identifying the substitution effect in in vitro studies is time and cost intensive. We developed variant assessor of MDR3 (Vasor), a machine learning-based model to classify novel MDR3 missense variants into the categories benign or pathogenic. Vasor was trained on the largest data set to date that is specific for benign and pathogenic variants of MDR3 and uses general predictors, namely Evolutionary Models of Variant Effects (EVE), EVmutation, PolyPhen-2, I-Mutant2.0, MUpro, MAESTRO, and PON-P2 along with other variant properties, such as half-sphere exposure and posttranslational modification site, as input. Vasor consistently outperformed the integrated general predictors and the external prediction tool MutPred2, leading to the current best prediction performance for MDR3 single-site missense variants (on an external test set: F1-score, 0.90; Matthew's correlation coefficient, 0.80). Furthermore, Vasor predictions cover the entire sequence space of MDR3. Vasor is accessible as a webserver at https://cpclab.uni-duesseldorf.de/mdr3_predictor/ for users to rapidly obtain prediction results and a visualization of the substitution site within the MDR3 structure. The MDR3-specific prediction tool Vasor can provide reliable predictions of single-site amino acid substitutions, giving users a fast way to initially assess whether a variant is benign or pathogenic.

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.

Targeting FXR in Cholestasis.

The farnesoid X receptor (FXR, NR1H4) is a bile acid (BA)-activated transcription factor, which is essential for BA homeostasis. FXR and its hepatic and intestinal target genes, small heterodimer partner (SHP, NR0B2) and fibroblast growth factor 15/19 (Fgf15 in mice, FGF19 in humans), transcriptionally regulate BA synthesis, detoxification, secretion, and absorption in the enterohepatic circulation. Furthermore, FXR modulates a large variety of physiological processes, such as lipid and glucose homeostasis as well as the inflammatory response. Targeted deletion of FXR renders mice highly susceptible to cholic acid feeding resulting in cholestatic liver injury, weight loss, and increased mortality. Combined deletion of FXR and SHP spontaneously triggers early-onset intrahepatic cholestasis in mice resembling human progressive familial intrahepatic cholestasis (PFIC). Reduced expression levels and activity of FXR have been reported in human cholestatic conditions, such as PFIC type 1 and intrahepatic cholestasis of pregnancy. Recently, two pairs of siblings with homozygous FXR truncation or deletion variants were identified. All four children suffered from severe, early-onset PFIC and liver failure leading to death or need for liver transplantation before the age of 2. These findings underscore the central role of FXR as regulator of systemic and hepatic BA levels. Therefore, targeting FXR has been exploited in different animal models of both intrahepatic and obstructive cholestasis, and the first FXR agonist obeticholic acid (OCA) has been approved for the treatment of primary biliary cholangitis (PBC). Further FXR agonists as well as a FGF19 analogue are currently tested in clinical trials for different cholestatic liver diseases. This chapter will summarize the current knowledge on the role of FXR in cholestasis both in rodent models and in human diseases.

A rare cause of a cholestatic jaundice in a North African teenager.

We report an unusual case of intermittent episodes of cholestasis in a young patient. The cholestatic attacks were preceded in each case by an infection and subsequent antibiotic therapies. After ruling out many possible causes of cholestatic hepatitis, the differential diagnoses were a benign recurrent intrahepatic cholestasis or a drug-induced liver injury. We discuss here the diagnostic approach and interpretation of the genetic analysis.

Bile acid receptors in the biliary tree: TGR5 in physiology and disease.

Bile salts represent signalling molecules with a variety of endocrine functions. Bile salt effects are mediated by different receptor molecules, comprising ligand-activated nuclear transcription factors as well as G protein-coupled membrane-bound receptors. The farnesoid X receptor (FXR) and the plasma membrane-bound G protein-coupled receptor TGR5 (Gpbar-1) are prototypic bile salt receptors of both classes and are highly expressed in the liver including the biliary tree as well as in the intestine. In liver, TGR5 is localized in different non-parenchymal cells such as sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells and small and large cholangiocytes. Through TGR5 bile salts can mediate choleretic, cell-protective as well as proliferative effects in cholangiocytes. A disturbance of these signalling mechanisms can contribute to the development of biliary diseases. In line with the important role of TGR5 for bile salt signalling, TGR5 knockout mice are more susceptible to cholestatic liver damage. Furthermore, in absence of TGR5 cholangiocyte proliferation in response to cholestasis is attenuated and intrahepatic and extrahepatic bile ducts show increased cell damage, underscoring the role of the receptor for biliary physiology. Decreased TGR5 expression may also contribute to the development or progression of cholangiopathies like primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) since reduced TGR5-dependent cell-protective mechanisms such as bicarbonate secretion renders cholangiocytes more vulnerable towards bile salt toxicity. Nevertheless, TGR5 overexpression or constant stimulation of the receptor can promote cholangiocyte proliferation leading to cyst growth in polycystic liver disease or even progression of cholangiocarcinoma. Not only the stimulation of TGR5-mediated pathways by suitable TGR5 agonists but also the inhibition of TGR5 signalling by the use of antagonists represent potential therapeutic approaches for different types of biliary diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Partial external biliary diversion in bile salt export pump deficiency: Association between outcome and mutation.

AIM: To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency. METHODS: Mutations in the gene encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney (HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatography-tandem mass spectrometry. Wild-type and mutant BSEP transport of [3H]-labeled taurocholate (TC) and taurochenodeoxycholate (TCDC) was assessed by vesicular transport assays. RESULTS: A girl (at 2 mo) presented with pruritus, jaundice and elevated serum bile salts (BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919del and the nonsense mutation p.R1235X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy (age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives (< 5%). The patients' native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919del and p.G1032R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively. CONCLUSION: In summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2 (PFIC-2).

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.

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.

Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency.

UNLABELLED: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. CONCLUSIONS: PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.

Two Case Reports of Successful Treatment of Cholestasis With Steroids in Patients With PFIC-2.

Mutations in the gene encoding the canalicular bile salt export pump (BSEP) can result in progressive familial intrahepatic cholestasis type 2 (PFIC-2). Treatment options are limited, and PFIC-2 often necessitates liver transplantation. We report on a young woman and a boy who clinically presented with PFIC-2 phenotypes and dramatically improved with steroid treatment. Gene sequencing of ABCB11 encoding for BSEP revealed 2 relevant mutations in both patients. The young woman was compound heterozygous for p.T919del and p.R1235X. At the age of 5 years, partial biliary diversion was performed and rescued liver function but left serum bile salt levels elevated. At age 23 she developed systemic lupus erythematosus. Unexpectedly, steroid therapy normalized serum bile salt levels, with a strong correlation with the steroid dose. She is currently in clinical remission. The boy was compound heterozygous for the ABCB11 mutations c.150+3A>C and p.R832C and presented with intractable pruritus. When he developed colitis, he was treated with steroids. The pruritus completely disappeared and relapsed when steroids were withdrawn. To date, with low-dose budesonide, the boy has been symptom-free for >3 years. In conclusion, the clinical courses suggest that patients with BSEP deficiency and residual BSEP activity may benefit from steroid-based therapy, which represents a new treatment option.

Autoimmune BSEP disease: disease recurrence after liver transplantation for progressive familial intrahepatic cholestasis.

Severe cholestasis may result in end-stage liver disease with the need of liver transplantation (LTX). In children, about 10 % of LTX are necessary because of cholestatic liver diseases. Apart from bile duct atresia, three types of progressive familial intrahepatic cholestasis (PFIC) are common causes of severe cholestasis in children. The three subtypes of PFIC are defined by the involved genes: PFIC-1, PFIC-2, and PFIC-3 are due to mutations of P-type ATPase ATP8B1 (familial intrahepatic cholestasis 1, FIC1), the ATP binding cassette transporter ABCB11 (bile salt export pump, BSEP), or ABCB4 (multidrug resistance protein 3, MDR3), respectively. All transporters are localized in the canalicular membrane of hepatocytes and together mediate bile salt and phospholipid transport. In some patients with PFIC-2 disease, recurrence has been observed after LTX, which mimics a PFIC phenotype. It could be shown by several groups that inhibitory anti-BSEP antibodies emerge, which most likely cause disease recurrence. The prevalence of severe BSEP mutations (e.g., splice site and premature stop codon mutations) is very high in this group of patients. These mutations often result in the complete absence of BSEP, which likely accounts for an insufficient auto-tolerance against BSEP. Although many aspects of this "new" disease are not fully elucidated, the possibility of anti-BSEP antibody formation has implications for the pre- and posttransplant management of PFIC-2 patients. This review will summarize the current knowledge including diagnosis, pathomechanisms, and management of "autoimmune BSEP disease."

A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.

Genetic variations of bile salt transporters.

Bile salt transporters directly or indirectly influence biological processes through physicochemical or signalling properties of bile salts. The coordinated action of uptake and efflux transporters in polarized epithelial cells of the liver, biliary tree, small intestine and kidney determine bile salt concentrations in different compartments of the body. Genetic variations of bile salt transporters lead to clinical relevant phenotypes of varying severity ranging from a predisposition for drug-induced liver injury to rapidly progressing end-stage liver disease. This review focuses on the impact of genetic variations of bile salt transporters including BSEP, NTCP, ASBT and OSTα/ß and discusses approaches for transporter analysis.

A novel mutation within a transmembrane helix of the bile salt export pump (BSEP, ABCB11) with delayed development of cirrhosis.

BACKGROUND & AIMS: The bile salt export pump (BSEP, ABCB11) is essential for bile salt secretion at the canalicular membrane of liver cells. Clinical phenotypes associated with BSEP mutations are commonly categorized as benign recurrent intrahepatic cholestasis (BRIC-2) or progressive familial intrahepatic cholestasis (PFIC-2). METHODS: The molecular basis of BSEP-associated liver disease in a sibling pair was characterized by immunostaining, gene sequencing, bile salt analysis and recombinant expression in mammalian cells and yeast for localization and in vitro activity studies respectively. RESULTS: Benign recurrent intrahepatic cholestasis was considered in a brother and sister who both suffered from intermittent cholestasis since childhood. Gene sequencing of ABCB11 identified the novel missense mutation p.G374S, which is localized in the putative sixth transmembrane helix of BSEP. Liver fibrosis was present in the brother at the age of 18 with progression to cirrhosis within 3 years. Immunofluorescence of liver tissue showed clear canalicular BSEP expression; however, biliary concentration of bile salts was drastically reduced. In line with these in vivo findings, HEK293 cells showed regular membrane targeting of human BSEP(G374S), whereas in vitro transport measurements revealed a strongly reduced transport activity. CONCLUSIONS: The novel mutation p.G374S impairs transport function without disabling membrane localization of BSEP. While all other known BSEP mutations within transmembrane helices are associated with PFIC-2, the new p.G374S mutation causes a transitional phenotype between BRIC-2 and PFIC-2.

The bile salt export pump (BSEP) in health and disease.

The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases.