Purpose: Progressive familial intrahepatic cholestasis (PFIC) is a rare genetic autosomal recessive disease caused by mutations in ATP8B1, ABCB11 or ABCB4. Mutational analysis of these genes is a reliable approach to identify the disorder.

Methods: We collected and analyzed relevant data related to clinical diagnosis, biological investigation, and molecular determination in nine children carrying these gene mutations, who were from unrelated families in South China.

Results: Of the nine patients (five males, four females) with PFIC, one case of PFIC1, four cases of PFIC2, and four cases of PFIC3 were diagnosed. Except in patient no. 8, jaundice and severe pruritus were the major clinical signs in all forms. γ-glutamyl transpeptidase was low in patients with PFIC1/PFIC2, and remained mildly elevated in patients with PFIC3. We identified 15 different mutations, including nine novel mutations (p.R470HfsX8, p.Q794X and p.I1170T of ABCB11 gene mutations, p.G319R, p.A1047P, p.G1074R, p.T830NfsX11, p.A1047PfsX8 and p.N1048TfsX of ABCB4 gene mutations) and six known mutations (p.G446R and p.F529del of ATP8B1 gene mutations, p.A588V, p.G1004D and p.R1057X of ABCB11 gene mutations, p.P479L of ABCB4 gene mutations). The results showed that compared with other regions, these three types of PFIC genes had different mutational spectrum in China.

Conclusion: The study expands the genotypic spectrum of PFIC. We identified nine novel mutations of PFIC and our findings could help in the diagnosis and treatment of this disease.

Keywords: Gene; Intrahepatic cholestasis; Mutation; Phenotype.

Low phospholipid-associated cholelithiasis (LPAC) syndrome is characterized by early intrahepatic and symptomatic gallstones leading to cholangitis, acute pancreatitis and biliary colic. It has been associated with loss of function variants in the ABCB4 gene. ABCB4 encodes for a phospholipid translocator at the canalicular membrane of the hepatocyte, which "flops" phosphatidylcholine into bile. The autosomal recessive form is the most common, although autosomal dominant forms have also been described. We report the first family with autosomal dominant LPAC syndrome due to heterozygosity of the loss of function mutation c.2932T>C in ABCB4, identified by targeted next generation sequencing.

Several ABC transporters of the human liver are responsible for the secretion of bile salts, lipids and cholesterol. Their interplay protects the biliary tree from the harsh detergent activity of bile salts. Among these transporters, ABCB4 is essential for the translocation of phosphatidylcholine (PC) lipids from the inner to the outer leaflet of the canalicular membrane of hepatocytes. ABCB4 deficiency can result in altered PC to bile salt ratios, which led to intrahepatic cholestasis of pregnancy, low phospholipid associated cholelithiasis, drug induced liver injury or even progressive familial intrahepatic cholestasis type 3. Although PC lipids only account for 30-40% of the lipids in the canalicular membrane, 95% of all phospholipids in bile are PC lipids. We discuss this discrepancy in the light of PC synthesis and bile salts favoring certain lipids. Nevertheless, the in vivo extraction of PC lipids from the outer leaflet of the canalicular membrane by bile salts should be considered as a separate step in bile formation. Therefore, methods to characterize disease causing ABCB4 mutations should be considered carefully, but such an analysis represents a crucial point in understanding the currently unknown transport mechanism of this ABC transporter.

Intrahepatic cholestasis of pregnancy (ICP) is a relatively benign cholestatic pathology of the liver developing in II or III trimester of pregnancy and characterized by itchy skin and enhanced serum bile acid levels. The cause of ICP is unknown; it may have a multifactor nature involving genetic (ABCB4, EXR, ABCC2 genes), hormonal (estrogens, progesterone), and environmental factors. As a rule, ICP first manifests itself on weeks 28-30 of pregnancy in the form of pruritus especially pronounced at night time. Almost half of the patients develop jaundice, usually within 1-4 weeks after appearance of pruritus. The enhanced serum bile acid level is sometimes the first or the sole laboratory sign of the disease. Ursodeoxycholic acid is currently the drug of choice for the treatment of ICP due to its confirmed effectiveness and safety.

The low phospholipid-associated cholelithiasis (LPAC) syndrome was reported in European adults with cholelithiasis and a mutation of the ATP-binding cassette subfamily B member 4 (ABCB4). The ABCB4 encodes multidrug resistance 3, which is a phospholipid translocator. Reduced phospholipid transport can lead to the formation of biliary cholesterol stones. Here, we describe a 31-year-old Japanese man diagnosed with recurrent biliary colic. Although he recovered quickly after endoscopic treatment for the most recent presentation, he had a family history of similar problems. His mother had required endoscopic treatment for choledocholithiasis and his maternal aunt had died at age 29 years because of liver failure (etiology unknown). We, therefore, performed genetic analysis, which revealed a heterozygous ABCB4^C717S. LPAC syndrome was diagnosed and the patient has received ursodeoxycholic acid for 2 years with no recurrence. The same variant was identified in the patient's mother, who was subsequently found to have a left intrahepatic calculus requiring left-sided lobectomy. She has received ursodeoxycholic acid for 1 year with no recurrence. ABCB4^C717S is a novel pathogenic variant, and this is the first patient diagnosed with LPAC syndrome in Japan. We should consider LPAC syndrome in young adults with recurrent cholesterol gallstones to ensure early therapy.

A 40-year old woman presented with symptomatic intrahepatic gallstones in one liver segment only four years after cholecystectomy for cholelithiasis. Multiple small, yellow and round calculi were completely removed from the intrahepatic bile ducts via ERCP. The young age of the patient, recurrence of gallstones after cholecystectomy and intrahepatic gallstones suggested a subtype of the low-phospholipid associated cholelithiasis syndrome, a monogenic form of cholesterol cholelithiasis due to variations of the ABCB4 gene that encodes the canalicular phospholipid transporter MDR3.

Background and aims: Low phospholipid-associated cholelithiasis syndrome (LPAC) is characterized by recurrent symptomatic cholelithiasis in young adults associated with ABCB4 gene mutations. Current diagnosing criteria are complex and heterogeneous, making this a largely underdiagnosed entity. Also, although recommended, genetic testing is not necessary for the diagnosis and its real advantages are not clear. The aim of our study was to explore the prevalence of ABCB4 mutations in symptomatic patients with cholelithiasis before the age of 30.

Methods: We conducted a multicentric prospective cohort study including patients with symptomatic cholelithiasis presenting before 30 years of age in 4 Portuguese centres between January 2017 and December 2019. ABCB4 gene was analyzed by next generation sequencing (NGS) including all exons and flanking regions. In 17/32 patients ABCB11 and ATP8B1 variants were also analyzed by NGS.

Results: Thirty-two patients were included (75% females, median age of symptom onset was 23 ± 5 years). We found that 8/32 (25%) patients had mutations in ABCB4 gene, 3/17 (18%) in ATP8B1 gene and 1/17 (6%) in ABCB11 gene. 44% (8/18) of patients with LPAC syndrome criteria had identified variants, while the prevalence of mutations in patients with symptoms onset before 30 as sole criteria was 29%.

Conclusion: Our results suggest that LPAC should be systematically suspected and investigated in patients with symptomatic cholelithiasis before age of thirty, but genetic testing should only be attempted in patients complying with the more stringent LPAC criteria.

Keywords: ABCB4/MDR3 gene; cholelithiasis; low phospholipid-associated cholestasis and cholelithiasis syndrome (LPAC); young adults.

Obstetric cholestasis (OC) is a cholestatic disorder with a prominent genetic background including variation in diverse hepatobiliary lipid transporters, such as ABCB4 (phospholipids) and ABCB11 (bile salts). Given a marked hepatocellular dysfunction in an OC patient indicated by>> 40-fold rise in alanine aminotransferase activity and minor gamma-glutamyl transpeptidase increases, we performed genotyping of candidate gene variants associated with adult cholestatic phenotypes. Genetic analysis revealed the heterozygous ABCB4 mutation p.R590Q, the ABCB11 variant p.V444A and the lithogenic ABCG8 variant p.D19H. Aggregation of multiple hepatobiliary transporter variants is rare in OC, and may cooperate to negatively modulate hepatobiliary transport capacities.

Background and aims: ABCB4 deficiency may lead to progressive familial intrahepatic cholestasis type 3 (PFIC3), biliary cirrhosis, low-phospholipid-associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), oral contraceptive-induced cholestasis (CIC) or may remain asymptomatic. The long-term course, quality of life and histology were investigated in ABCB4 deficiency.

Methods: Adult carriers of ABCB4 gene variants from two regional academic centers were analyzed by history taking, electronic patient files, physical examination, blood analysis, abdominal ultrasound (US) and liver elastography. Patients completed a 36-Item-Short-Form-Health-Survey (SF-36) for quality of life and a Visual Analogue Scale (VAS) for pruritus. Available liver specimens were re-classified according to the Nakanuma scoring system, so far validated for primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) only. Quality-of-life-data were compared to published data of patients with PBC, PSC and the general population.

Results: Sixty-seven patients were identified, 64 (96%) were alive at time of analysis, 62 (93%) were (at some time) treated with ursodeoxycholic acid. Two patients died of cholangio-carcinoma, one of decompensated biliary cirrhosis. Three additional deaths of cholangiocarcinoma were reported in first degree relatives. Transplant-free survival was 91% (median follow-up 14 years). Liver stiffness was normal (<6.3 kPa) in 75%, intrahepatic stones were detected at US in 33% and micro-calcifications in 22% of cases. Quality of life (n=48) was lower than in the general population particularly in energy/fatigue and general health domains and comparable to that in PSC. Staging according to Nakanuma in 15 specimens reflected the clinical course.

Conclusions: ABCB4 deficiency has a mild clinical course, but impaired quality of life and limited risk of cholangiocarcinoma. The Nakanuma scoring system appears feasible for histological evaluation in ABCB4 deficiency.

Keywords: ABCB4 deficiency; intrahepatic cholestasis of pregnancy (ICP); low phospholipid-associated cholelithiasis (LPAC); persistent hepatocellular secretory failure (PHSF); quality of life.

The widely used fungicide triadimefon (TDF) has been detected in aquatic environments, and appears to disrupt steroid homeostasis; however, the toxic effects on fish reproduction triggered by TDF via the key receptor signaling pathways remain largely unknown. The present study showed that TDF (0.069, 0.138, 0.690 mg/L) exposure not only caused disordered germ cell maturation, but also decreased spawned egg production. In order to better understand this reproductive inhibition, we investigated the effects of TDF based on quantitative PCR, Western blot and mass spectrometry methodology in zebrafish. Due to the preferential accumulation of TDF in the liver, a general pattern of up-regulation of genes involved in biotransformation pathway was observed. A significant increase in abcb4 expression appeared to be responsible for TDF excretion. TDF-induced receptors (AhR2 and PXR) changed many genes involved in steroid metabolism, and subsequent disruptions in steroid homeostasis, which might be the key biological pathway in TDF reproductive toxicity. However, due to the different metabolic demands, the transcript profiles involved in steroid metabolism in zebrafish exhibited a sex-specific expression pattern. For example, the increase in gene expression of ahr2 was accompanied by a reduction in the rate of E2 biosynthesis resulting from the diminished cyp19a1a expression, and in turn led to down-regulation of esr1 and vtg1 in the liver, supporting the anti-estrogenic effect of TDF in male fish. In contrast, the increase in E2 production was accompanied by an increase in Esr1 protein expression caused by TDF and paralleled the increase in ahrr1 expression, suggesting that TDF may induce estrogenic activity through AhR-ER interactions in females. In addition, over-induction of cyp3a65 activity mediated through pxr, which helped to accelerate the transformation from TDF to triadimenol in the liver, appeared to elevate T metabolite rate in females. The down-regulation of fshβ transcript in males further suggested that TDF might adversely affect normal gametogenesis and induce reproductive toxicity.

Gallstones develop in the gallbladder or the bile ducts. According to their chemical composition, gallstones can be divided into cholesterol stones, which are common, and the rare bile pigment stones. Altogether, up to 20 % of all adults develop gallstones and more than 20 % of them symptoms or complications. Female sex, age, pregnancy, physical inactivity, obesity, overnutrition and genetic factors such as ABCB4 deficiency of the hepatic lecithin transporter are kown risk factors for gallstone formation. In about one half of all patients biliary symptoms precede the three common and potentially life-threatening complications (acute cholecystitis, acute cholangitis and biliary pancreatitis). Although our knowledge about the genetics and pathophysiology of gallstones has improved, current treatment algorithms are predominantly invasive (ERC and surgery). Thus, better strategies are needed to prevent the formation of gallstones in general.

Studies on the bioavailability of organic contaminants adsorbed to nanomaterials are increasing. In this study, we investigated the interaction between fluoxetine (FLX) and three multiwalled carbon nanotubes (MWCNTs) with different functional groups in zebrafish (Danio rerio) embryos, focusing on the FLX accumulation, the formation of the metabolite norfluoxetine (NFLX), and the active defence system responses. The accumulation of FLX in zebrafish was intensified by MWCNTs (46-99%), which simultaneously facilitated the formation of the metabolite NFLX by 23-167%. The consistent enhancement revealed that the absorbed FLX is bioavailable in zebrafish. Moreover, the coexisting MWCNTs further promoted the influences of FLX on the active defence system in zebrafish (e.g. antioxidant and metabolic function), eliciting the defence function. The influences of MWCNTs on the bioavailability of FLX in zebrafish could be ordered as OH-MWCNTs>> COOH-MWCNTs>> pristine MWCNTs. The release of FLX from MWCNTs in biofluids may partially contribute to these significant alterations. In particular, MWCNTs themselves may also modulate the bioavailability of FLX in zebrafish by downregulating the gene expression of membrane ATP-binding cassette transporter (abcb4). These findings demonstrated that MWCNTs increased the bioavailability of FLX in zebrafish, especially the functionalized MWCNTs. The production of metabolites may be a useful bio-endpoint to evaluate the bioavailability of adsorbed contaminants on nanomaterials.

Chronic inflammation precedes the majority of hepatocellular carcinoma (HCC) cases. We investigated the chemopreventive potential of S-adenosylmethionine (SAM), an essential donor for all methylation reactions in the cell, at the late precancerous stage of HCC development using the Mdr2-knockout (Mdr2-KO, Abcb4-/-) mice, a model of inflammation-mediated hepatocarcinogenesis. Previously, we revealed down-regulation of the genes regulating SAM metabolism in the liver of these mice at the precancerous stages. Now, we have supplied Mdr2-KO mice at the late precancerous stage with SAM during either a short-term (17 days) or a long-term (51 days) period and explored the effects of such supplementation on tumor development, DNA methylation and gene expression in the liver. The short-term SAM supplementation significantly decreased the number of small tumor nodules, proliferating hepatocytes and the total DNA methylation level, while it increased expression of the tumor suppressor proteins Mat1a and p21. Surprisingly, the long-term SAM supplementation did not affect tumor growth and hepatocyte proliferation, while it increased the total liver DNA methylation. Our results demonstrate that the short-term SAM supplementation in the Mdr2-KO mice inhibited liver tumor development potentially by increasing multiple tumor suppressor mechanisms resulting in cell cycle arrest. The long-term SAM supplementation resulted in a bypass of the cell cycle arrest in this HCC model by a yet unknown mechanism.

Cadmium (Cd), which seriously affects plant growth and crop production, is harmful to humans. Previous studies revealed ryegrass (Lolium multiflorum Lam.) exhibits Cd tolerance, and may be useful as a potential hyperaccumulator because of its wide distribution. In this study, the physiological and transcriptional responses of two ryegrass cultivars [i.e., high (LmHC) and low (LmLC) Cd tolerance] to Cd stress were investigated and compared. The Cd tolerance of LmHC was greater than that of LmLC at various Cd concentrations. The uptake of Evans blue dye revealed that Cd-induced root cell mortality was higher in LmLC than in LmHC after a 12-h Cd treatment. Furthermore, the content and influx rate of Cd in LmLC roots were greater than in LmHC roots under Cd stress conditions. The RNA sequencing and quantitative real-time PCR data indicated that the Cd transport regulatory genes (ABCG37, ABCB4, NRAMP4, and HMA5) were differentially expressed between the LmLC and LmHC roots. This expression-level diversity may contribute to the differences in the Cd accumulation and translocation between LmLC and LmHC. These findings may help clarify the physiological and molecular mechanisms underlying ryegrass responses to Cd toxicity. Additionally, ryegrass may be able to hyperaccumulate toxic heavy metals during the phytoremediation of contaminated soil.

Keywords: Cd influx and Cd accumulation; Cd tolerance; Cd transport regulatory genes; Ryegrass.

During the last 11 years, advances in molecular genetics have changed our approach to children with intrahepatic cholestasis. Progress in identification of mutated genes now allows genetic diagnosis for several forms of cholestasis previously grouped into PFIC (progressive familial intrahepatic cholestasis). Three distinct forms: PFIC1, PFIC2, and PFIC3 are the result of mutations in the ATP8B1, ABCB11, and ABCB4 genes. The diagnosis is supported on clinical, biochemical and histological features. The therapeutic goals in theses diseases are alleviate symptoms and improve quality of life. Inborn errors of bile acid synthesis represent a subset of familial intrahepatic cholestasis. Replacement therapy with ursodeoxycholic acid and cholic acid avoids progression of the liver injury.

Background: Intrahepatic cholestasis of pregnancy (ICP) can cause premature delivery and stillbirth. Previous studies have reported that mutations in ABC transporter genes strongly influence the transport of bile salts. However, to date, their effects are still largely elusive.

Methods: A whole-exome sequencing (WES) approach was used to detect novel variants. Rare novel exonic variants (minor allele frequencies: MAF < 1%) were analyzed. Three web-available tools, namely, SIFT, Mutation Taster and FATHMM, were used to predict protein damage. Protein structure modeling and comparisons between reference and modified protein structures were performed by SWISS-MODEL and Chimera 1.14rc, respectively.

Results: We detected a total of 2953 mutations in 44 ABC family transporter genes. When the MAF of loci was controlled in all databases at less than 0.01, 320 mutations were reserved for further analysis. Among these mutations, 42 were novel. We classified these loci into four groups (the damaging, probably damaging, possibly damaging, and neutral groups) according to the prediction results, of which 7 novel possible pathogenic mutations were identified that were located in known functional genes, including ABCB4 (Trp708Ter, Gly527Glu and Lys386Glu), ABCB11 (Gln1194Ter, Gln605Pro and Leu589Met) and ABCC2 (Ser1342Tyr), in the damaging group. New mutations in the first two genes were reported in our recent article. In addition, compared to the wild-type protein structure, the ABCC2 Ser1342Tyr-modified protein structure showed a slight change in the chemical bond lengths of ATP ligand-binding amino acid side chains. In placental tissue, the expression level of the ABCC2 gene in patients with ICP was significantly higher (P < 0.05) than that in healthy pregnant women. In particular, the patients with two mutations in ABC family genes had higher average values of total bile acids (TBA), aspartate transaminase (AST), direct bilirubin (DBIL), total cholesterol (CHOL), triglycerides (TG) and high-density lipoprotein (HDL) than the patients who had one mutation, no mutation in ABC genes and local controls.

Conclusions: Our present study provide new insight into the genetic architecture of ICP and will benefit the final identification of the underlying mutations.

Keywords: ABC transporter genes; ABCC2 gene; Gene expression; Genetic variants; ICP; Ser1342Tyr mutation; TBA; WES.

Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes.

Biliary lipids primarily consist of bile salts, phospholipids, and cholesterol. Bile salts have potent detergent properties and deleterious effects on the cell membrane and are cytotoxic to hepatocytes. We have previously reported that phosphatidylcholine (PC), the predominant bile phospholipid, protects hepatocytes from the cytotoxicity of bile salts, whereas cholesterol reverses the cytoprotective effects of PC against bile salts. ABCB4, a member of the ATP-binding cassette transporter family, secretes biliary phospholipids, especially PC, from the hepatocytes into the bile. Using Abcb4 knockout mice and HEK293 cells that stably expressed ABCB4, we examined the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate, and hyodeoxycholate on the ABCB4-mediated efflux of PC. We observed that the biliary secretion of PC in wild-type mice significantly increased following infusion of all the tested bile salts, especially taurohyodeoxycholate. On the other hand, the biliary secretion of PC in Abcb4 knockout mice was not affected by the bile salt infusions. The results also demonstrated that the efflux of PC from ABCB4-expressing HEK293 cells was significantly stimulated by taurohyodeoxycholate, which has a strong potential to form mixed micelles with PC. Furthermore, the results of our study emphasized the possibility that the specific interactions of bile salts with ABCB4 are necessary for the release of PC molecules from the binding pocket of ABCB4 into the aqueous environment. Further understanding of this mechanism will aid in the development of novel therapeutic agents for cholestatic liver diseases.

Keywords: ATP-binding cassette transporter B4 (ABCB4); bile salt; phosphatidylcholine.

ATP-binding cassette (ABC) transporters are the members of the efflux pumps that are responsible for the removal of cytotoxic substances by active transport. ABCB11, the bile salt efflux pump of hepatocytes, coordinates cellular excretion of numerous conjugated bile salts into the bile canaliculi, whereas ABCB4 acts as an ATP-dependent floppase translocating phosphatidylcholine from the inner to the outer leaflet of the bile canalicular membrane. Loss of functional ABCB11 and ABCB4 proteins causes early-onset refractory cholestasis or cholangiopathy. In this study, we investigated the expression and localization pattern of ABCB11 and ABCB4 using immunohistochemistry and RNA profiling in liver samples from patients with different types and stages of chronic cholestatic liver disease, with emphasis on primary sclerosing cholangitis (PSC), compared to a variety of cholestatic and noncholestatic hepatopathies. Therefore, ABCB11 and ABCB4 expressions were investigated on formalin-fixed and paraffin-embedded (FFPE) material in a patient cohort of total 43 patients with or without cholestatic liver diseases, on protein level using immunohistochemistry and on RNA level using nanoString technology. Intriguingly, our results demonstrated increased expression of ABCB11 and ABCB4 on protein as well as RNA level in PSC, and the expression pattern correlated with disease progression. We concluded from our study that patients with PSC demonstrate altered expression levels and pattern of ABCB11 and ABCB4 which correlated with disease progression; thereby, ABCB11 and ABCB4 analysis may be a useful tool for assessment of disease stages in PSC.

Chronic cholestasis results from bile secretory defects or impaired bile flow with few effective medical therapies available. Thyroid hormone triiodothyronine and synthetic thyroid hormone receptor agonists, such as GC-1, are known to impact lipid and bile acid (BA) metabolism and induce hepatocyte proliferation downstream of Wnt/β-catenin signaling after surgical resection; however, these drugs have yet to be studied as potential therapeutics for cholestatic liver disease. Here, GC-1 was administered to ATP binding cassette subfamily B member 4 (Abcb4^-/-; Mdr2-/-) knockout (KO) mice, a sclerosing cholangitis model. KO mice fed GC-1 diet for two and four weeks had decreased serum alkaline phosphatase but increased serum transaminases compared to KO alone. KO mice on GC-1 also had higher levels of total liver BA due to alterations in expression of BA detoxification, transport, and synthesis genes, with the net result being retention of BA in the hepatocytes. Interestingly, GC-1 does not induce hepatocyte proliferation or Wnt/β-catenin signaling in KO mice, likely a result of decreased thyroid hormone receptor beta (Thrb; TRβ) expression without Mdr2. Therefore, though GC-1 treatment induces a mild protection against biliary injury in the early stages of treatment, it comes at the expense of hepatocyte injury and is suboptimal due to lower expression of TRβ. Thus, thyromimetics may have limited therapeutic benefits in treating cholestatic liver disease.

Background: Hyperbilirubinemia is a predictor of severe drug-induced liver injury (DILI). Hepatobiliary ATP-binding cassette (ABC) transporters play an important role in the transportation of many drugs and bilirubin, however little is known about these transporters and the risk of DILI. The aim of this study was to explore associations between genetic variations in important ABC transporters and susceptibility to DILI, with a particular focus on hyperbilirubinemia.

Methods: A total of 200 patients with DILI and 200 healthy controls were enrolled as the training dataset. Another 106 patients with DILI were recruited as the validation dataset. They were genotyped for ABCB11 (BSEP) rs2287622, ABCB1 (MDR1) rs1128503, rs1045642, ABCB4 (MDR3) rs2230028, ABCC2 (MRP2) rs1885301, rs717620, rs2273697, rs3740066 and rs8187710 using polymerase chain reaction-based TaqMan genotyping assays.

Results: There were no statistical differences in any of the nine ABC transporter single nucleotide polymorphisms between the DILI and control groups. However, in the DILI group, the patients with hyperbilirubinemia had a higher frequency of the ABCC2 rs717620 C/T and T/T genotypes than those without hyperbilirubinemia (44.2% vs. 20.2%, p = 0.001). After adjusting for other confounding factors, the ABCC2 rs717620 T variant was still associated with an increased risk of hyperbilirubinemia (adjusted OR: 3.83, 95% CI: 1.73-8.48, p = 0.001). This association was confirmed by the validation dataset (adjusted OR: 3.92, 95% CI: 1.42-10.81, p = 0.015). We also found that the mortality group had higher frequencies of the ABCC2 (MRP2) rs717620 C/T and T/T genotypes than the survival group (50.0% vs. 27.9%, p = 0.048).

Conclusion: Carriage of the ABCC2 (MRP2) rs717620 T variant may increase the risk of hyperbilirubinemia and mortality in patients with DILI. Screening for this variant may help to prevent and mitigate drug-induced hyperbilirubinemia.

Background: Heterozygous ABCB4 variants are not routinely tested in adults with cholestasis because of their supposed rarity and high costs.

Methods: Nineteen adult patients presenting with unexplained cholestasis, and/or recurrent gallstones were included; genotyping was not done in five due to lack of health insurance approval.

Results: heterozygous ABCB4 variants were identified in seven patients, followed by cascade testing of 12 family members: one patient underwent liver transplantation at age 40 for end-stage liver disease; one had compensated cirrhosis; all symptomatic adults had gallstones, including four with low phospholipid-associated cholelithiasis; four had intrahepatic cholestasis of pregnancy; all children and one 54-year old female were asymptomatic. Genotype: Families A and C: c.2211G>A (p.Ala737=) combined with c.959C>T (p.Ser320Phe) in one subject; Family B: c.1130T>C (p.Ile377Thr); Family D: large deletion removing ABCB4 exons 1-4 plus ABCB1, RUNDC3B, SLC25A40, DBF4, ADAM22 exons 1-3; Family E: c.1565T>C (p.Phe522Ser) ; Family F: c.1356+2T>C combined with c.217C>G (p.Leu73Val). All patients responded to ursodeoxycholic acid.

Conclusions: We found ABCB4 variants in half of the adults with unexplained cholestasis and/or recurrent gallstones presenting at our center, suggesting that this condition is underdiagnosed and undertreated, with serious consequences not only for the patients and their families, but also in terms of healthcare costs.

Keywords: ABCB4; Adult-onset cholestasis; Gallstones; Ursodeoxycholic acid.