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Variants in ABCB4 (MDR3) across the spectrum of cholestatic liver diseases in adults

Open AccessPublished:May 03, 2020DOI:https://doi.org/10.1016/j.jhep.2020.04.036

      Summary

      The ATP binding cassette subfamily B member 4 (ABCB4) gene on chromosome 7 encodes the ABCB4 protein (alias multidrug resistance protein 3 [MDR3]), a P-glycoprotein in the canalicular membrane of the hepatocytes that acts as a translocator of phospholipids into bile. Several variants in ABCB4 have been shown to cause ABCB4 deficiency, accounting for a disease spectrum ranging from progressive familial cholestasis type 3 to less severe conditions like low phospholipid-associated cholelithiasis, intrahepatic cholestasis of pregnancy or drug-induced liver injury. Furthermore, whole genome sequencing has shown that ABCB4 variants are associated with an increased incidence of gallstone disease, gallbladder and bile duct carcinoma, liver cirrhosis or elevated liver function tests. Diagnosis of ABCB4 deficiency-related diseases is based on clinical presentation, serum biomarkers, imaging techniques, liver histology and genetic testing. Nevertheless, the clinical presentation can vary widely and clear genotype-phenotype correlations are currently lacking. Ursodeoxycholic acid is the most commonly used medical treatment, but its efficacy has yet to be proven in large controlled clinical studies. Future pharmacological options may include stimulation/restoration of residual function by chaperones (e.g. 4-phenyl butyric acid, curcumin) or induction of ABCB4 transcription by FXR (farnesoid X receptor) agonists or PPARα (peroxisome proliferator-activated receptor-α)-ligands/fibrates. Orthotopic liver transplantation remains the last and often only therapeutic option in cirrhotic patients with end-stage liver disease or patients with intractable pruritus.

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      Linked Article

      • Challenges in understanding the consequences of variants in ABCB4 gene
        Journal of HepatologyVol. 74Issue 1
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          We read with interest the review by Stattermayer et al.1 which provides a summary of ABCB4 variants in liver disease and the spectrum of clinical manifestations. The authors introduce a clinical case of a male who was diagnosed with asymptomatic cryptogenic liver disease aged 19, and subsequently presented aged 38 with decompensated liver disease and was identified to have a novel homozygous frameshifting variant in ABCB4 (p.R1256fs∗39). His asymptomatic sister was found to be homozygous for the same variant in ABCB4 aged 34, and has been established on ursodeoxycholic acid with no evidence of advanced fibrosis on non-invasive measurements.
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      • Reply to: “Challenges in understanding the consequences of variants in ABCB4 gene”
        Journal of HepatologyVol. 74Issue 1
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          We thank Dr. Nayagam and his colleagues for their insightful comment on our recently published review on genetic variants in ABCB4 in adult patients with cholestatic liver disease.1 In fact, the index patient presented in the clinical vignette within this review showed a comparably mild disease course as his homozygous frameshift variant (p.R1256fs∗39) would have suggested. He became symptomatic in early adulthood and showed progression of his cholestatic liver disease with portal fibrosis to decompensated liver cirrhosis within almost 2 decades.
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      Clinical vignette

      In 1995, a 19-year-old male, asymptomatic patient (body weight: 70 kg, height: 176 cm, BMI: 22.6 kg/m2) was referred for evaluation of abnormal elevated liver tests detected at a routine laboratory check-up (alkaline phosphatase [ALP] 406 U/L [normal: 35–105 U/L]; gamma-glutamyltransferase [GGT] 270 U/L [<40 U/L]; alanine aminotransferase [ALT] 188 U/L [<35 U/L]; aspartate aminotransferase [AST] 88 U/L [<35 U/L]). Total bilirubin, prothrombin time and albumin were in the normal range. Platelet count was 171 G/L (normal range: 150–400 G/L), suggesting the absence of portal hypertension. Viral and autoimmune hepatitis, Wilson disease, and α1-antitrypsin deficiency were excluded by appropriate serologic and laboratory tests. Primary biliary cholangitis (PBC)-specific anti-mitochondrial antibodies (AMA-M2) and antinuclear antibodies (ANA: sp100, gp210) were negative. The patient denied drinking alcohol and had no drug history. No intake of hepatotoxic agents or herbal supplements was reported. Cholelithiasis or cholangiectasia were excluded by sonography. A percutaneous liver biopsy showed portal fibrosis with incipient bridging, scarce lymphomononuclear cell infiltration in the portal field, bile duct proliferation, but an absence of cholestasis or any hepatocellular damage (Fig. 1A–C). Staining for cytokeratin 7 revealed that bile ducts were present in 13 of 18 portal fields, thus excluding ductopenia. An endoscopic retrograde cholangiography at that time (1995) showed no large bile duct abnormalities.
      Figure thumbnail gr1
      Fig. 1Histological findings in liver biopsies of a patient with ABCB4 homozygous mutation.
      (A) Percutaneous liver biopsy at the time of first presentation shows periportal fibrosis with incipient bridging fibrosis (chromatrope-aniline staining). (B) Scarce lymphomononuclear cell infiltration in the portal tracts. The preexisting bile ducts are regularly configurated with discrete signs of unspecific alteration represented by segmental nuclear loss of the epithelium (black arrow, detail showed in higher magnification). (C) The liver parenchyma shows slight regenerative changes, without any inflammatory infiltrate and cholestasis. (D) The liver biopsy performed 18 years later at the time of the decompensation shows cirrhotic liver tissue (chromatrope-aniline staining) with scarce lymphomononuclear cell infiltration. (E) Preexisting bile ducts are significantly altered by reactive epithelial proliferation and segments of nuclear loss, an intraluminal cholestasis cannot be seen. Residual liver parenchyma presented in HE staining. (F) areas of pericellular fibrosis and Mallory-Denk bodies (red arrow). Intracytoplasmic bile pigment is present in some of the hepatocytes (red asterisk). ABCB4, ATP binding cassette subfamily B member 4.
      No final diagnosis was made; nevertheless, treatment with ursodeoxycholic acid (UDCA) was initiated. The patient was lost to follow-up until 2014, when he deteriorated clinically, presenting with jaundice and pruritus. A second liver biopsy showed liver cirrhosis with scarce lymphomononuclear cell infiltration (Fig. 1D–F). Preexisting bile ducts showed mild secondary alterations including reactive epithelial proliferation and segments of nuclear loss. No mechanical obstruction could be observed. Residual liver parenchyma showed pericellular fibrosis, reactive lymphocytic infiltrates, and Mallory-Denk bodies. Intracytoplasmic bile pigment was present in some of the hepatocytes. Hepatic copper content was 632 μg/g dry weight (normal: <50 μg/g), and the 24 h urinary copper excretion was 229 μg/day (both findings are common in chronic cholestatic diseases), but ATP7B mutation analysis revealed no evidence of Wilson disease. Further genetic testing identified a previously unknown homozygous variant in exon 28 of ABCB4 – c.3768_3769delAG (p.R1256Sfs∗39; reference sequence #NM_018849.2) – that caused a frameshift mutation resulting in a consecutive prolongation of 39 additional amino acids. According to in silico testing (MutationTaster, Mutalyzer), the mutation was rated as disease causing. Furthermore, a well-known per se non-disease-causing homozygous variant in ABCB11 (c.1331 T>C [p.V444A]) was identified.
      Due to decompensated liver disease (model for end-stage liver disease [MELD] score 24) with ascites and jaundice the patient was listed for orthotopic liver transplantation. Subsequently, the patient further deteriorated with oesophageal variceal haemorrhage, ascites and acute kidney injury (MELD score at admission: 31). He died several days later as a result of multiple organ failure.
      Family screening identified his clinically asymptomatic sister (34 years at diagnosis) as a homozygous carrier of the same variant in ABCB4. All routine liver parameters were in the normal range (ALP: 60 U/L, GGT: 26 U/L, AST 15 U/L [<35 U/l], ALT 24 U/L [<35 U/l]) at diagnosis. Total fasting bile acids were slightly elevated: 13 μmol/L (<10 μmol/L). Treatment with UDCA was initiated. She is now on regular follow-up for 5 years. The patient is in stable condition under UDCA treatment, with normal cholestasis markers but mildly elevated transaminases. Fasting bile acids were elevated over time and showed a fluctuating course (2017: 24.5 μmol/L, peak in 2018: 204.1 μmol/L, 2019: 34.0 μmol/L), but the patient did not complain of pruritus. Repeated MR-imaging presented no alterations of the intrahepatic bile ducts (Fig. 2A,B). Repeated transient elastography showed a liver stiffness consistent with the absence of fibrosis (2016: 4.0 kPa; 2017: 3.5 kPa; 2018: 4.3 kPa).
      Figure thumbnail gr2
      Fig. 2Imaging findings of the clinically asymptomatic sister of the index patient and of a 44-year old female patient with LPAC.
      (A) MR-cholangiography and (B) T1 weighted sequence after contrast application (Primovist®), both without any pathological findings, in the asymptomatic sister of the index patient. (C–F) Imaging findings of a 44-year old female patient with LPAC (not related) that became symptomatic at the age of 20 with recurring cholelithiasis. Cholecystectomy was performed at the age of 23. She underwent 2 ERCPs due to recurring choledocholithiasis (6 and 10 years after cholecystectomy, respectively) and was then set on UDCA treatment. Since then she is clinically asymptomatic with normal transaminases and cholestasis parameters. Nevertheless, repeated MRI showed progressive segmental cholangitis in segment VIII as well as dilated bile ducts and hepatolithiasis in segments II, III and VIII. Genetic work-up (performed at age of 40) identified a heterozygous variant (c.1634G>A) in ABCB4 and a non-disease-causing variant (c.1331T>C [p.V444A]) in ABCB11. (C) diffusion weighted MRI sequence showing segmental cholangitis in segment VIII (white circle). (D) T2 weighted haste sequence centrally showing intrahepatic cholelithiasis (white arrow). (E) T1 weighted MRI sequence after contrast application (Primovist®) centrally showing intrahepatic cholelithiasis (white arrow). (F) ultrasound study showing intrahepatic gallstones with a proximal bile duct dilatation (presenting as cystic formation with intraluminal hyperechoic foci measuring 1.4 cm in diameter, white arrow). ABCB4/11, ATP binding cassette subfamily B member 4/11; ERCP, endoscopic retrograde cholangiopancreatography; LPAC, low phospholipid-associated cholelithiasis; UDCA, ursodeoxycholic acid.
      These 2 cases highlight the phenotypic diversity of ABCB4 variants. No clear diagnosis was made at the time of referral of the index patient (age 19). Within 18 years, he progressed from cholestatic liver disease with portal fibrosis to decompensated cirrhosis. The final diagnosis was made by genetic testing. However, his sister (now at the age of 40) with the same homozygous variant has no evidence of advanced liver disease to date.

      Introduction

      Over the past 2 decades, modern genetic studies have yielded major insights into the molecular-biological mechanisms and pathophysiology of liver diseases. The fundamental technologies of genetic analysis enabled positional cloning of unidentified disease genes and simple gene tests for known single nucleotide polymorphisms (SNPs). Further, genome-wide association studies (GWAS) compared genotype frequencies across the whole genome between disease and control groups and thus identified new genetic risk factors. Next generation sequencing (NGS) made it possible to identify different variants within the exome (whole exome sequencing [WES]) or even the whole genome (whole genome sequencing [WGS]) in individual patients.
      • Karlsen T.H.
      • Lammert F.
      • Thompson R.J.
      Genetics of liver disease: from pathophysiology to clinical practice.
      ,
      • Hakim A.
      • Zhang X.
      • DeLisle A.
      • Oral E.A.
      • Dykas D.
      • Drzewiecki K.
      • et al.
      Clinical utility of genomic analysis in adults with idiopathic liver disease.
      However, despite the possibilities and limitations of state-of-the-art computational methods, the assignment of certain variants to pathogenicity remains difficult.
      • Eichler E.E.
      Genetic variation, comparative genomics and the diagnosis of disease.
      An heterogenous group of cholestatic liver diseases with autosomal-recessive inheritance was first described by Clayton et al. in 1969 as Byler's disease in a population of Amish kindred.
      • Clayton R.J.
      • Iber F.I.
      • Ruebner B.H.
      • McKusick V.A.
      Byler disease: fatal intrahepatic cholestasis in an Amish kindred.
      Later, based on the underlying genetic defect of hepatobiliary transport systems, the disease has been reclassified into 3 types and renamed progressive familial intrahepatic cholestasis (PFIC) type 1, 2 and 3
      • Bull L.N.
      • Carlton V.E.H.
      • Stricker N.L.
      • Baharloo S.
      • DeYoung J.A.
      • Freimer N.B.
      • et al.
      Genetic and morphological findings in progressive familial intrahepatic cholestasis (Byler disease [PFIC-1] and Byler syndrome): evidence for heterogeneity.
      • Bull L.N.
      • van Eijk M.J.
      • Pawlikowska L.
      • DeYoung J.A.
      • Jujin J.A.
      • Liao M.
      • et al.
      A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.
      • Trauner M.
      • Meier P.J.
      • Boyer J.L.
      Molecular pathogenesis of cholestasis.
      (see overview in Table 1). Although the exact prevalence is unknown, PFIC-1, -2, and -3 are rare diseases, with an overall estimated incidence of 1 per 50,000 to 1 per 100,000 births.
      • Srivastava A.
      Progressive familial intrahepatic cholestasis.
      Nevertheless, PFIC type 1, 2 and 3 account for up to 10–15% of neonatal cholestasis syndromes and 10–15% of childhood liver transplants.
      • Jacquemin E.
      Progressive familial intrahepatic cholestasis.
      Several variants in ABCB4 have been identified accounting for a disease spectrum ranging from PFIC-3 to less severe forms like LPAC, ICP or DILI.
      Table 1Overview of clinical, biochemical and histological features of different types of PFIC.
      PFIC-1PFIC-2PFIC-3PFIC-4PFIC-5PFIC-6
      GeneATP8B1ABCB11ABCB4TJP2NR1H4MYO5B
      Chromosomal location18q21.312q31.17q21.129q21.1112q23.118q21.1
      Gene productFIC1BSEPMDR3TJP2FXRMYO5B
      Disease onsetInfancyNeonates, early infancyLate infancy, adolescence, early adulthoodNeonatesNeonatesNeonates, early infancy
      Disease courseModerateSevereInsidiousSevereSevereMild/moderate
      End-stage liver disease1st decadeInfancy1st–2nd decadeInfancyInfancyUnknown
      Extrahepatic disease manifestationDiarrhoea, pancreatitis, hearing loss, short statureAbsentAbsentUnknownUnknownDiarrhoea, neurologic symptoms (?)
      Pruritus+++++(+)++Unknown+++
      Risk of cholelithiasis-+++UnknownUnknownUnknown
      Risk of liver tumors-+++++UnknownUnknown
      Serum ALT or AST↑↑Normal/↑↑↑Normal/↑
      Serum GGTNormalNormal↑↑NormalNormalNormal
      Serum bile acids↑↑↑↑↑↑↑(↑)↑↑(↑)↑↑(↑)
      Liver histologyCanalicular cholestasis, lobular fibrosisCanalicular cholestasis, giant-cell hepatitis, hepatocellular necrosis, portal fibrosisBiliary fibrosis, ductular proliferation, macrophagic infiltration of portal tracts, ductopenia, cholesterol crystalsGiant cell transformation, canalicular cholestasisDuctular reaction, diffuse giant cell transformation, hepatocyte ballooning, intralobular cholestasisGiant cell transformation, canalicular cholestasis
      ABCB4/11, ATP binding cassette subfamily B member 4/11; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP8B1, ATPase class I, type 8B, member 1; BSEP, bile salt export pump; CFTR, cystic fibrosis transmembrane conductance regulator; FIC1, familial intrahepatic cholestasis 1 protein; FXR, farnesoid X receptor; GGT, gamma-glutamyltransferase; MYO5B, myosin 5b; PFIC, progressive familial intrahepatic cholestasis; TJP2, tight junction protein 2.
      PFIC-1 (formerly known as Byler's disease) is associated with genetic defects in ATP8B1 on chromosome 18 (18q21) which encodes familial intrahepatic cholestasis 1 protein (FIC1), a member of the type 4 subfamily of P type adenosine triphosphatase (ATPase).
      • Bull L.N.
      • Carlton V.E.H.
      • Stricker N.L.
      • Baharloo S.
      • DeYoung J.A.
      • Freimer N.B.
      • et al.
      Genetic and morphological findings in progressive familial intrahepatic cholestasis (Byler disease [PFIC-1] and Byler syndrome): evidence for heterogeneity.
      ATP8B1 is a multispan transmembrane protein with flippase activity, that translocates aminophospholipids from the outer (exoplasmic) to the inner (endoplasmic) leaflet of the biological canalicular membrane in hepatocytes.
      • Paulusma C.C.
      • Oude Elferink R.P.J.
      • Jansen P.L.M.
      Progressive familial intrahepatic cholestasis type 1.
      PFIC-2, which was formerly known as Byler's syndrome, is caused by a gene defect in ABCB11. ABCB11 (ATP binding cassette subfamily B member 11) resides on chromosome 2 (2q31) and encodes the canalicular bile salt export pump (BSEP),
      • Strautnieks S.S.
      • Kagalwalla A.F.
      • Tanner M.S.
      • Knisely A.S.
      • Bull L.
      • Freimer N.
      • et al.
      Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24.
      the main transporter of bile acids from hepatocytes into bile.
      PFIC-3 is caused by variants in ABCB4 (ATP binding cassette subfamily B member 4) located on chromosome 7 (7q21) encoding the ABCB4 protein.
      • van der Bliek A.M.
      • Kooiman P.M.
      • Schneider C.
      • Borst P.
      Sequence of mdr3 cDNA encoding a human p-glycoprotein.
      Recently, further gene defects were detected in patients with PFIC (PFIC4: TJP2, PFIC5: FXR, PFIC6: MYO5B; Fig. 3).
      • Vitale G.
      • Gitto S.
      • Vukotic R.
      • Raimondi F.
      • Andreone P.
      Familial intrahepatic cholestasis: new and wide perspectives.
      • Gomez-Ospina N.
      • Potter C.J.
      • Xiao R.
      • Manickam K.
      • Kim M.S.
      • Kim K.H.
      • et al.
      Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis.
      • Gonzalez E.
      • Taylor S.A.
      • Davit-Spraul A.
      • Thebaut A.
      • Thomassin N.
      • Guettier C.
      • et al.
      MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease.
      Figure thumbnail gr3
      Fig. 3Molecular mechanisms underlying cholestasis associated with ABCB4 deficiency.
      ABCB4 is a transmembrane P-glycoprotein acting as a floppase transporting PC from the inner to the outer leaflet resulting in PC secretion into the bile. Variants in ABCB4 leading to inadequate phospholipid secretion may cause injury of the biliary epithelium and canalicular membrane by toxic bile acid concentrations, destabilization of micelles and thus promote cholesterol crystallisation and increased biliary lithogenicity. Additionally, increased lithogenicity in turn facilitates liver damage by obstruction of small bile ducts. Inspired and adapted from a figure of J. Stindt presented by V. Keitel at the Postgraduate Course of The Liver Meeting of the AASLD, November 9th, 2019 in Boston, MA. ABCB4/11, ATP binding cassette subfamily B member 4/11; ATP8B1, ATPase class I, type 8B, member 1; FXR, farnesoid X receptor; MYO5B, myosin 5B; PC, phosphatidylcholine; TJP2, tight junction protein 2.
      The ABCB4 protein (also known as multidrug resistance 3 protein [MDR3]) is a P-glycoprotein that acts as a phospholipid translocator in the canalicular membrane of hepatocytes, where it is responsible for secretion of phospholipids, predominantly phosphatidylcholine, into bile.
      • Oude Elferink R.P.
      • Paulusma C.C.
      Function and pathophysiological importance of ABCB4 (MDR3 P -glycoprotein).
      ,
      • Smit J.J.
      • Schinkel A.H.
      • Oude Elferink R.P.
      • Groen A.K.
      • Wagenaar E.
      • van Deemter L.
      • et al.
      Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.
      In contrast to patients with PFIC-1 or -2, who present symptoms very early in childhood (4-5 months of age), patients with PFIC-3 usually develop cholestasis in late infancy or adolescence. In some cases of PFIC-3, signs of cirrhosis and portal hypertension (e.g. gastrointestinal bleeding) might appear as the initial symptoms in older children or even young adults
      • Srivastava A.
      Progressive familial intrahepatic cholestasis.
      (Table 1).
      Genetic changes (e.g. missense variations in the coding sequence, insertion, deletion, variations in the splicing area) in the ABCB4 gene can result in either missense or nonsense variants leading to a premature stop codon, with loss of function, truncation or even complete failure of production of the protein, reflecting the wide clinical spectrum of ABCB4-associated diseases. PFIC-3 is associated with homozygous or compound-heterozygous variants with severe gene defects that usually cause either a premature truncation of the protein or a total failure of protein production, while heterozygous ABCB4 variants result in less severe clinical patterns. Milder phenotypes of PFIC-3 may present as ICP,
      • Pauli-Magnus C.
      • Lang T.
      • Meier Y.
      • Zodan-Marin T.
      • Jung D.
      • Breymann C.
      • et al.
      Sequence analysis of bile salt export pump (ABCB11) and multi drug resistance p-glycoprotein 2 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy.
      cholesterol gallstone disease (low phospholipid-associated cholelithiasis [LPAC]),
      • Rosmorduc O.
      • Hermelin B.
      • Poupon R.
      MDR3 gene defects in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis.
      drug-induced cholestasis or liver injury,
      • Lang C.
      • Meier Y.
      • Stieger B.
      • Beuers U.
      • Lang T.
      • Kerb R.
      • et al.
      Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury.
      adult idiopathic/cryptogenic cirrhosis
      • Gotthard D.
      • Runz H.
      • Keitel V.
      • Flechtenmacher C.
      • Wirtenberger M.
      • Weiss K.H.
      • et al.
      A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults.
      ,
      • Ziol M.
      • Barbu V.
      • Rosmorduc O.
      • Frassati-Biaggi A.
      • Barget N.
      • Hermelin B.
      • et al.
      ABCB4 heterozygous mutations associated with fibrosing cholestatic liver disease in adults.
      or transient neonatal cholestasis.
      • Jacquemin E.
      • de Vree J.M.
      • Cresteil D.
      • Sokal E.M.
      • Sturm E.
      • Dumont M.
      • et al.
      The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis in adulthood.
      Whole genome sequencing in a large-scale Icelandic population
      • Gudbjartsson D.F.
      • Helgasson H.
      • Gudjonsson S.A.
      • Zink F.
      • Oddson A.
      • Gylfason A.
      • et al.
      Large-scale whole genome sequencing of the Icelandic population.
      identified an association of ABCB4 mutations with higher risk of gallstone diseases, gallbladder and bile duct carcinoma, liver cirrhosis and even higher serum levels of liver-related biomarkers like AST, ALT and GGT. Mutations in ABCB4 are now recognised as a major genetic determinant of chronic liver disease.
      • Lammert F.
      • Hochrath K.
      A letter on ABCB4 from Iceland: on the highway to liver disease.
      The aim of this review is to provide a concise synopsis on the spectrum of diseases of the hepatobiliary system related to variants in the ABCB4 gene.

      Pathophysiology and clinical presentation

      The ABCB4 gene resides on chromosome 7 (7q21) and encodes the ABCB4 protein,
      • van der Bliek A.M.
      • Kooiman P.M.
      • Schneider C.
      • Borst P.
      Sequence of mdr3 cDNA encoding a human p-glycoprotein.
      a P-glycoprotein which is expressed in the canalicular membrane of hepatocytes functioning as a phospholipid translocator. It acts as a floppase, transporting lipids from the inner to the outer leaflet of the membrane, and is thereby responsible for phospholipid secretion – predominantly phosphatidylcholine – into the bile.
      • Smit A.J.
      • Timmermans-Hereijgers J.L.
      • Roelofsen B.
      • Wirtz K.W.
      • van Bitterswijk W.J.
      • Smit J.J.
      • et al.
      The human MDR3 p-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice.
      The ABCB4 protein consists of 2 cytoplasmic nucleotide binding domains (NBDs) and 2 transmembrane domains, each with 6 transmembrane segments. The NBDs are large domains that include highly conserved sequences for ATP binding, like the Walker A and B motifs, the signature sequence and the A-, D-, H- and Q-loop.
      • Zolnerciks J.K.
      • Andress E.J.
      • Nicolao M.
      • Linton K.J.
      Structure of ABC transporters.
      Clinical presentation can vary widely and clear genotype-phenotype correlations are lacking so far.
      Biliary phospholipids are responsible for neutralisation of the detergent effects of hydrophobic bile salts by formation of mixed micelles.
      • Hofmann A.F.
      Bile acid secretion, bile flow and biliary lipid secretion in humans.
      Thus, ABCB4 protein defects lead to intrahepatic cholestasis, which occurs as a direct result of injury to the biliary epithelium and canalicular membrane in the setting of a “toxic” bile acid composition.
      • De Vree J.M.
      • Jacquemin E.
      • Sturm E.
      • Cresteil D.
      • Bosma P.J.
      • Aten J.
      • et al.
      Mutations in MDR3 gene cause progressive familial intrahepatic cholestasis.
      ,
      • Smit J.J.
      • Schinkel A.H.
      • Oude Elferink R.P.
      • Groen A.K.
      • Wagenaar E.
      • van Deemter L.
      • et al.
      Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.
      Moreover, low phospholipid levels lead to destabilisation of micelles and thus promote cholesterol crystallisation, increasing biliary lithogenicity. Increased lithogenicity in turn facilitates liver damage by obstructing small bile ducts.
      • Jacquemin E.
      Progressive familial intrahepatic cholestasis.
      In contrast to cholestasis associated with variants in ATP8B1 and ABCB11, ABCB4 deficiency-related cholestasis is generally characterised by higher levels of GGT (high GGT cholestasis), which might reflect direct toxic effects to the bile duct epithelium, owing to higher free bile salt concentrations and lower phospholipid levels.
      • Jacquemin E.
      • de Vree J.M.
      • Cresteil D.
      • Sokal E.M.
      • Sturm E.
      • Dumont M.
      • et al.
      The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis in adulthood.
      ,
      • De Vree J.M.
      • Jacquemin E.
      • Sturm E.
      • Cresteil D.
      • Bosma P.J.
      • Aten J.
      • et al.
      Mutations in MDR3 gene cause progressive familial intrahepatic cholestasis.
      Nevertheless, a study by Schneider et al.
      • Schneider G.
      • Paus T.C.
      • Kullak-Ublick G.A.
      • Meier P.J.
      • Wienker T.F.
      • Lang T.
      • et al.
      Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy.
      showed that GGT is not a reliable marker to discriminate between cholestasis attributed to ATP8B1-, ABCB11- or ABCB4-associated disease. Some patients with disease-causing variants in ABCB4 may present with low or even normal GGT levels.
      Delaunay et al.
      • Delaunay J.L.
      • Durand-Schneider A.M.
      • Dossier C.
      • Falguieres T.
      • Gautherot J.
      • Davit-Spraul A.
      • et al.
      A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3.
      proposed a classification of ABCB4 variations in PFIC-3 as either nonsense variations (I) or missense variations affecting primarily the maturation (II), the activity (III) or the stability (IV) of the protein, and mutations without detectable defect (V), similar to the classifications initially proposed for cystic fibrosis.
      • Wilschanski M.
      • Durie P.R.
      Patterns of GI disease in adulthood associated with mutations in the CFTR gene.
      While patients with PFIC-3 are predominantly homozygous or compound-heterozygous with nonsense mutations, less severe clinical phenotypes of cholestatic liver disease like intrahepatic cholestasis of pregnancy (ICP) or contraceptive-induced cholestasis (CIC), LPAC, transient neonatal cholestasis, drug-induced liver injury (DILI) or adult idiopathic cirrhosis
      • Kubitz R.
      • Bode J.
      • Erhardt A.
      • Graf D.
      • Kircheis G.
      • Müller-Stöver I.
      • et al.
      Cholestatic liver disease from child to adult: the diversity of MDR3 disease.
      are linked to heterozygous variants of ABCB4, or homozygous variants that at least partially preserve protein function
      • Reichert M.C.
      • Lammert F.
      ABCB4 gene aberrations in human liver disease: an evolving spectrum.
      (Table 2). Several studies have been published to address genotype-phenotype correlations in patients with ABCB4-associated cholestatic liver diseases.
      • Jacquemin E.
      • de Vree J.M.
      • Cresteil D.
      • Sokal E.M.
      • Sturm E.
      • Dumont M.
      • et al.
      The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis in adulthood.
      ,
      • Delaunay J.L.
      • Durand-Schneider A.M.
      • Dossier C.
      • Falguieres T.
      • Gautherot J.
      • Davit-Spraul A.
      • et al.
      A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3.
      ,
      • Poupon R.
      • Rosmorduc O.
      • Boelle B.Y.
      • Chretien Y.
      • Corpechot C.
      • Chazouielleres C.
      • et al.
      Genotype-phenotype relationships in the low-phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients.
      ,
      • Colombo C.
      • Vajro P.
      • Degiorgio D.
      • Coviello D.A.
      • Constantino L.
      • Tornillo L.
      • et al.
      Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations.
      Compound-heterozygous or homozygous carriers of disease-causing mutations had higher rates of progression to cirrhosis and end-stage liver disease, whereas heterozygotes generally had less severe disease or even an absence of symptoms.
      • Schatz S.B.
      • Jüngst C.
      • Keitel-Anselmo V.
      • Kubitz R.
      • Becker C.
      • Gerner P.
      • et al.
      Phenotypic spectrum and diagnostic pitfall of ABCB4 deficiency depending on age of onset.
      Most patients with LPAC were heterozygous and had missense mutations; individuals harbouring a truncating variant had an earlier disease onset than those with missense mutations, but nevertheless the occurrence of severe liver disease was independent of genotype.
      • Davit-Spraul A.
      • Gonzales E.
      • Baussan C.
      • Jacquemin E.
      The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects.
      PFIC-3 the most severe form is characterised by cholestasis with jaundice and pruritus presenting in late infancy, adolescence or sometimes even young adulthood and progresses to cirrhosis and end-stage liver disease.
      Table 2Summary of different disease manifestations associated with ABCB4 variants.
      PFIC-3LPACICP
      Underlying genetic defect in ABCB4Homozygous, compound-heterozygousHeterozygousHeterozygous
      Age at presentationInfancy, adolescence, (early adulthood)Early adulthood (<40 years)Pregnancy (2nd/3rd trimester)
      Clinical presentationPruritusCholelithiasis, biliary colicGestational pruritus
      Disease courseInsidiousBenignBenign
      ComplicationsBiliary cirrhosis, HCC, cholangiocarcinomaJaundice, cholangitis, biliary pancreatitis, intrahepatic stonesPremature birth, foetal asphyxia, meconium-stained amniotic fluid
      TreatmentUDCA, (cholestyramine, rifampicin, fibrates), OLTUDCA, cholecystectomyUDCA
      Differential diagnosisAll causes of neonatal cholestasis, Alagille's syndrome, sclerosing cholangitis (primary/secondary), AAT-deficiencyPrimary sclerosing cholangitis, Caroli's disease (congenital dilation of intrahepatic bile ducts)Acute fatty liver of pregnancy, HELLP syndrome, Budd-Chiari-syndrome
      AAT, α1-antitrypsin; ABCB4, ATP binding cassette subfamily B member 4; HCC, hepatocellular carcinoma; HELLP, hemolysis, elevated liver enzymes, low platelets; ICP, intrahepatic cholestasis of pregnancy; LPAC, low phospholipid-associated cholelithiasis; OLT, orthotopic liver transplantation; PFIC, progressive familial intrahepatic cholestasis; UDCA, ursodeoxycholic acid.

      Progressive familial intrahepatic cholestasis type 3

      Cholestasis, characterised by jaundice and pruritus, is the clinical hallmark of all types of PFIC. While patients with PFIC-1 and -2 develop symptoms in very early infancy (PFIC-1) or even during the neonatal period (PFIC-2), the age of onset in PFIC-3 is generally later: in late infancy (around one-third of cases) or even in adolescence or young adulthood
      • Jacquemin E.
      Progressive familial intrahepatic cholestasis.
      (Table 1). Pruritus is generally less severe in PFIC-3 than in PFIC-1 or -2. Complications due to cirrhosis and portal hypertension like gastrointestinal bleeding can often be the first clinical presentation in older children and young adults. PFIC-3 usually progresses from chronic cholestasis with or without jaundice to portal hypertension and end-stage liver disease within the first to second decade of life. Patients suffering from PFIC-3 are also at an increased risk of developing cholesterol stones in intrahepatic bile ducts, as well as in the gall bladder.
      • Davit-Spraul A.
      • Gonzales E.
      • Baussan C.
      • Jacquemin E.
      The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects.
      Furthermore, the risk of hepatocellular carcinoma (HCC) in these patients is mildly increased. In contrast to PFIC-1, extrahepatic disease manifestations (diarrhoea, pancreatitis, pneumonia, hearing loss, short stature) are generally absent. In females, cholestasis and jaundice may present first in early adulthood due to hormonal changes (e.g. intake of oral contraceptives containing oestrogens or progesterone, pregnancy) and thus may be misinterpreted as ICP or CIC.
      • Ganne-Carrié N.
      • Baussan C.
      • Grando V.
      • Gaudelus J.
      • Cresteil D.
      • Jacquemin E.
      Progressive familial intrahepatic cholestasis type 3 revealed by oral contraceptive pills.
      Laboratory findings show elevated GGT activity which is a typical finding in PFIC-3 (high GGT cholestasis), but usually not in other types of PFIC. Serum ALT and AST levels are usually only mildly increased. Serum bile acids are raised but not as high as in patients with PFIC-1 or -2.

      Low phospholipid-associated cholelithiasis

      LPAC is a rare form of intrahepatic cholelithiasis that occurs in young adults and is associated with variants in the ABCB4 gene. It was first described by Rosmorduc et al.
      • Rosmorduc O.
      • Hermelin B.
      • Poupon R.
      MDR3 gene defects in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis.
      in 2001 as a peculiar form of intrahepatic cholelithiasis with gallbladder cholesterol stones in 2 men and 4 women (age between 26 and 55 years). In all 6 patients, a variant in ABCB4 was found, and all responded well to UDCA treatment. A second study
      • Rosmorduc O.
      • Hermelin B.
      • Boelle P.Y.
      • Parc R.
      • Taboury J.
      • Poupon R.
      ABCB4 gene mutation-associated cholelithiasis in adults.
      by the same authors in 2003 confirmed these observations in 32 predominantly female patients and several cases have been published since.
      LPAC should be suspected in patients with cholesterol gallbladder stones and at least 2 of the following characteristics
      • Rosmorduc O.
      • Poupon R.
      Low phospholipid associated cholelithiasis: association with mutation in the MDR3/ABCB4 gene.
      : i) onset of symptoms before the age of 40 years, ii) intrahepatic hyperechogenic foci, sludge or microlithiasis, iii) recurrence of biliary symptoms (jaundice, biliary colic, cholangitis, acute pancreatitis) after cholecystectomy. In a large study of patients fulfilling the diagnostic criteria for LPAC, ~50% of patients harboured a variant in ABCB4 (predominantly heterozygous missense variants).
      • Poupon R.
      • Rosmorduc O.
      • Boelle B.Y.
      • Chretien Y.
      • Corpechot C.
      • Chazouielleres C.
      • et al.
      Genotype-phenotype relationships in the low-phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients.
      Often, a family history of gallstones in first-degree relatives can be found. Furthermore, LPAC is more prevalent in women and shows a strong association with ICP.
      • Poupon R.
      • Rosmorduc O.
      • Boelle B.Y.
      • Chretien Y.
      • Corpechot C.
      • Chazouielleres C.
      • et al.
      Genotype-phenotype relationships in the low-phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients.
      In a small study,
      • Condat B.
      • Zanditenas D.
      • Barbu V.
      • Hauuy M.P.
      • Parfait B.
      • El Naggar A.
      • et al.
      Prevalence of low phospholipid-associated cholelithiasis in young female patients.
      almost 25% of young female patients admitted for symptomatic cholelithiasis had LPAC, which was particularly common in those with normal body weight and onset under 18 years.
      Again, low phospholipid concentrations in the bile lead to a bile composition that is supersaturated with cholesterol and hence highly lithogenic, leading to the formation of macroscopic stones. Moreover, cholesterol crystals precipitate in the bile and damage the cholangiocyte epithelium together with free (non-micellar bound) bile acids. Importantly, biliary lesions induce cholestasis with elevated GGT levels.

      Intrahepatic cholestasis of pregnancy

      ICP is defined as an acquired form of cholestasis during pregnancy in otherwise healthy women with normal medical history. It usually occurs during the second or third trimester of pregnancy, while serum concentrations of oestrogens and progesterone reach their peak. ICP represents the most common pregnancy-related liver disease.
      • Pauli-Magnus C.
      • Meier P.J.
      • Stieger B.
      Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy.
      Its incidence varies widely across different geographical regions and ethnic backgrounds, ranging between 0.05 to more than 20%.
      • Geenes V.
      • Williamson C.
      Intrahepatic cholestasis of pregnancy.
      The highest incidence rates were reported from Chile in women with Araucanian Indian descent. In Europe, the prevalence of ICP is around 2%, 15% of which are attributable to ABCB4 deficiency.
      • Bacq Y.
      Liver diseases unique to pregnancy: a 2010 update.
      Clinical symptoms quickly resolve after delivery (typically within 12 weeks post-partum), which supports the key role of female sex hormones in the pathophysiology. Women affected by ICP also show a higher susceptibility to develop cholestasis under oral contraception (CIC).
      • Jacquemin E.
      • de Vree J.M.
      • Cresteil D.
      • Sokal E.M.
      • Sturm E.
      • Dumont M.
      • et al.
      The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis in adulthood.
      Furthermore, environmental co-factors seem to play a pivotal role in ICP, as indicated by the wide variation in incidence according to different geographic regions. Besides hormonal and most likely environmental factors, genetic susceptibility accounts for higher risk of ICP. An association with transporter mutations in ATP8B1, ABCB11 and ABCB4,
      • Pauli-Magnus C.
      • Meier P.J.
      • Stieger B.
      Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy.
      has also been observed, and more than 10% of women with ICP harbour a mutated allele in ABCB4.
      • Dixon P.H.
      • Williamson C.
      The pathophysiology of intrahepatic cholestasis of pregnancy.
      A pathogenic role of ABCB4 was first discussed in a large consanguineous family with an index case suffering from PFIC-3 and several female family members with recurrent ICP episodes.
      • Jacquemin E.
      • Cresteil D.
      • Manouvrier S.
      • Boute O.
      • Hadchouel M.
      Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy.
      Diagnosis should be considered in patients with otherwise unexplained chronic cholestatic liver disease or hepatobiliary disease in younger ages, positive family history or consanguineous background.
      ICP is characterised by pruritus, raised bile salt and transaminase levels and elevated concentrations of serum bilirubin in about 25% of cases. Elevation of fasted serum bile acids above 10 μmol/L is often the first and sometimes even the only laboratory abnormality in ICP. In ICP associated with ABCB4 deficiency, serum GGT levels are elevated and may distinguish it from other forms of ICP.
      • Pauli-Magnus C.
      • Meier P.J.
      • Stieger B.
      Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy.
      Other laboratory findings include elevation of total and direct bilirubin and transaminases (2- to 10-fold increase).
      ICP is associated with an increased risk of perinatal complications, including premature delivery, respiratory distress, meconium staining of the amniotic fluid, low Apgar scores, and even stillbirth.
      • Keitel V.
      • Dröge C.
      • Stepanow S.
      • Fehm T.
      • Mayatepek E.
      • Köhrer K.
      • et al.
      Intrahepatic cholestasis of pregnancy (ICP): case report and review of literature.
      A recent meta-analysis showed that the risk of stillbirth significantly increased with a maternal serum bile acid level >100 μmol/L.
      • Ovadia C.
      • Seed P.T.
      • Sklavounos A.
      • Geenes V.
      • Di Ilio C.
      • Chambers J.
      • et al.
      Association of adverse perinatal outcomes of intrahepatic cholestasis of pregnancy with biochemical markers: results of aggregate and individual patient data meta-analyses.
      As serum bile acid levels are generally <100 μmol/L in ICP, the risk of stillbirth in these women is probably comparable to the general population, provided serum bile testing is performed until delivery.
      Although ICP is reversible after delivery, it may unmask an underlying defect associated with mutations in ABCB4 which predispose individuals to hepatobiliary diseases, cirrhosis and hepatobiliary malignancies (HCC and cholangiocarcinoma). Regular follow-up and genetic counselling should be discussed for women with ICP due to ABCB4 deficiency or other genetic factors, as ICP may have adverse long-term consequences (an increased risk of gallstones, liver cirrhosis, HCC, autoimmune-mediated and cardiovascular diseases has been reported).
      • Ropponen A.
      • Sund R.
      • Riikonen S.
      • Ylikorkala O.
      • Aittomäki K.
      Intrahepatic cholestasis of pregnancy as an indicator of liver and biliary diseases: a population-based study.
      • Wikström Shemer E.A.
      • Stephansson O.
      • Thuresson M.
      • Thorsell M.
      • Ludvigsson J.F.
      • Marschall H.U.
      Intrahepatic cholestasis of pregnancy and cancer, immune-mediated and cardiovascular diseases: a population-based cohort study.
      • Westbrook R.H.
      • Dusheiko G.
      • Williamson C.
      Pregnancy and liver disease.

      Drug-induced liver injury

      Multiple lines of evidence suggest that ABCB4 deficiency predisposes to cholestatic DILI (e.g., CIC
      • Ganne-Carrié N.
      • Baussan C.
      • Grando V.
      • Gaudelus J.
      • Cresteil D.
      • Jacquemin E.
      Progressive familial intrahepatic cholestasis type 3 revealed by oral contraceptive pills.
      ): molecular evidence from tissue culture suggests that xenobiotics that inhibit P-glycoproteins, including ABCB4, can also induce cholestasis in predisposed patients with ABCB4 aberrations.
      • Smith A.J.
      • van Helvoort A.
      • van Meer G.
      • Szabo K.
      • Welker E.
      • Szakacs G.
      • et al.
      MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping.
      Patients with 4 different non-synonymous variants in ABCB4 were found to have up to a threefold increased risk of cholestatic DILI from psychotropic drugs, proton pump inhibitors, or selected antibiotics and oral contraceptives.
      • Lang C.
      • Meier Y.
      • Stieger B.
      • Beuers U.
      • Lang T.
      • Kerb R.
      • et al.
      Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury.
      ABCB4 deficiency predisposes individuals to DILI in general and CIC in particular. Treatment with drugs that potentially inhibit the function or expression of ABCB4 (e.g. sirolimus, cyclosporine, verapamil or vinblastine) may impair biliary phosphatidylcholine excretion in patients with genetically determined ABCB4 deficiency that is otherwise clinically silent. However, canalicular ABC transporter expression is profoundly disturbed in most cases of cholestatic DILI irrespective of ABCB4 variants.
      • Zollner G.
      • Thueringer A.
      • Lackner C.
      • Fickert P.
      • Trauner M.
      Alterations of canalicular ATP-binding cassette transporter expression in drug-induced liver injury.
      Besides, despite the potential role of different ABCB4 variants in DILI, it must be emphasised that HLA haplotypes are more relevant for the pathogenesis of DILI.
      EASL Clinical Practice Guidelines
      Drug-induced liver injury.

      Diagnostic assessment

      Although syndromes associated with ABCB4 variants (or other transporters in the canalicular membrane of hepatocytes) are rare, they should be considered in patients with otherwise unexplained hepatobiliary disease, consanguineous family background, or with a family history of liver disease. Furthermore, ICP, DILI with cholestasis or occurrence of (intrahepatic) cholelithiasis in younger age groups (possibly indicative of LPAC) indicates possible genetic determinants of disease.
      Differential diagnosis primarily depends on the clinical setting. Alagille syndrome and biliary atresia should be excluded in paediatric patients. In pregnant women, other causes for intrahepatic cholestasis or acute fatty liver of pregnancy, viral hepatitis with cholestasis or HELLP syndrome (haemolysis, elevated liver enzymes, low platelets) should be considered. Differential diagnosis in adult patients includes PBC and primary sclerosing cholangitis. Besides, variants in ABCB4 may have a pivotal role as disease modifiers in patients with adult cholestatic liver disease.
      • Trauner M.
      • Fickert P.
      • Wagner M.
      MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes.
      In young adults with otherwise unexplained cholestatic liver disease, hepatic copper accumulation (like in the index patient of the clinical vignette) may lead to the incorrect diagnosis of Wilson disease.
      • Ramraj R.
      • Finegold M.J.
      • Karpen S.J.
      Progressive familial intrahepatic cholestasis type 3: overlapping presentation with Wilson disease.
      Diagnosis of ABCB4 deficiency associated disease is based on the exclusion of other causes of cholestatic liver disease by laboratory assessment, imaging studies, liver histology and genetic testing.

      Laboratory assessment

      Laboratory assessment should include routine blood tests for transaminases (ALT and AST), panels of cholestasis (AP, GGT, serum bilirubin and serum bile acids). Further, liver synthesis parameters (coagulation and serum albumin) need to be checked. Other causes of cholestatic liver disease (e.g. PBC, IgG4-associated disease, viral hepatitis) need to be ruled out by appropriate laboratory testing, imaging, or histological assessment. Cholestasis associated with ABCB4 variants may show markedly higher serum levels of GGT than other cholestatic syndromes, most likely reflecting bile duct injury due to toxic biliary constituents and/or cholesterol crystals.
      • Trauner M.
      • Fickert P.
      • Halilbasic E.
      • Moustafa T.
      Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases.
      Nevertheless, as stated,
      • Schneider G.
      • Paus T.C.
      • Kullak-Ublick G.A.
      • Meier P.J.
      • Wienker T.F.
      • Lang T.
      • et al.
      Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy.
      some patients with ABCB4 deficiency present with normal or only mildly elevated GGT levels.

      Imaging investigations

      Abdominal ultrasound is the first examination in patients presenting with clinical signs of cholestasis. Although, findings are normal or unspecific (e.g. signs of advanced disease like cirrhosis or portal hypertension) in many patients, it helps to exclude other causes of cholestasis, especially extrahepatic cholestasis. Ultrasonography may show intrahepatic hyperechogenic foci or real gallstones with dorsal acoustic shadow in patients with LPAC. Colour Doppler examination reveals “twinkling” artifacts associated with stones, which may also correspond to the “comet tail” signs formed by small stones not detected in normal ultrasound.
      • Rosmorduc O.
      • Hermelin B.
      • Poupon R.
      MDR3 gene defects in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis.
      Nevertheless, it needs to be pointed out that intrahepatic hyperechogenic foci, sludge or microlithiasis suggestive of LPAC are only observed in up to 85% of cases in patients with an established diagnosis of LPAC.
      Magnetic resonance cholangiopancreatography (MRCP) may help to rule out primary and secondary sclerosing cholangitis or extrahepatic causes of cholestasis. MRCP and CT scans can be used to detect intrahepatic stones and to document markedly dilated bile ducts (Fig. 2C–F).

      Liver histology

      Portal fibrosis and true bile ductular proliferation can be seen in PFIC-3 at disease onset as well as mild giant cell hepatitis. Giant cell hepatitis, ductular proliferation and portal fibrosis are absent in PFIC-1, whereas liver histology in PFIC-2 shows lobular and portal fibrosis and pronounced hepatocellular necrosis and giant cell hepatitis.
      • Morotti R.A.
      • Suchy F.J.
      • Magid M.S.
      Progressive familial intrahepatic cholestasis (PFIC) type 1, 2, and 3: a review of the liver pathology findings.
      Marked portal fibrosis and biliary cirrhosis can be observed in the later stages of PFIC-3. Disease-causing variants in ABCB4 lead to a defect in hepatic biliary phospholipid secretion with increased cholesterol saturation. Cholesterol precipitation may be seen in liver histology as lipid crystals
      • Wendum D.
      • Barbu V.
      • Rosmorduc O.
      • Arrive L.
      • Flejou J.F.
      • Poupon R.
      Aspects of liver pathology in adult patients with MDR3/ABCB4 gene mutations.
      and may be suggestive but not specific for ABCB4 deficiency. As all these histological findings are non-specific, biopsy specimens should be subjected to immunohistochemical staining with antibodies against ABCB11 and ABCB4 (standardised antibodies for immunohistochemistry staining against ATP8B1 are not yet available). Though absent or faint immunostaining is diagnostic for PFIC-2 or -3 respectively, normal immunostaining cannot exclude the diagnosis of PFIC, as some mutations in ABCB4 or ABCB11 are associated with only a functional defect of the gene product and show otherwise normal synthesis and expression.
      • De Vree J.M.
      • Jacquemin E.
      • Sturm E.
      • Cresteil D.
      • Bosma P.J.
      • Aten J.
      • et al.
      Mutations in MDR3 gene cause progressive familial intrahepatic cholestasis.
      Further, in a series of heterozygous patients with ABCB4 variants
      • Trauner M.
      • Fickert P.
      • Wagner M.
      MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes.
      immunostaining on paraffin sections was not efficient, as all these patients had a strong diffuse canalicular staining. Thus, immunostaining cannot be used to rule out ABCB4 deficiency.
      Ursodeoxycholic acid is the most commonly used medical treatment, but large controlled studies on its benefit are lacking.
      The presence of subtle bile duct changes in liver biopsy, such as epithelial injury (with nuclear loss see Fig. 1), atrophy and cholesterol clefts should raise the suspicion of an ABCB4 deficiency.

      Genetic testing

      Genetic testing is still the gold standard for diagnosing ABCB4 deficiency and other inherited syndromes of intrahepatic cholestasis. So far, more than 500 missense and 24 loss-of-function variants in ABCB4 are known (genome aggregation database, gnomAD: https://gnomad.broadinstitute.org). The large gene size and the lack of highly predominant mutational hotspots form obstacles in exploration of gene-gene interactions and genotype-phenotype correlations. A commercially available customised resequencing gene chip was developed that reads 5 of the most common genes (SERPINA11-antitrypsin], JAG1, ATP8B1, ABCB11 and ABCB4) responsible for intrahepatic cholestasis simultaneously in a single assay with high call rate and accuracy.
      • Liu C.
      • Aronow B.J.
      • Jegga A.J.
      • Wang N.
      • Miethke A.
      • Mourya R.
      • et al.
      Novel resequencing chip to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis.
      Recently several studies showed that in up to half of patients in whom cholestatic liver disease remained unexplained after standard diagnostic work-up, variants in genes responsible for PFIC could be detected by genetic examination (sometimes in a heterozygous state).
      • Aamann L.
      • Ørntoft N.
      • Vogel I.
      • Groenbaek H.
      • Becher N.
      • Vilstrup H.
      • et al.
      Unexplained cholestasis in adults and adolescents: diagnostic benefit of genetic examination.
      • Dröge C.
      • Bonus M.
      • Baumann U.
      • Klindt C.
      • Lainka E.
      • Kathemann S.
      • et al.
      Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants.
      • Vitale G.
      • Gitto S.
      • Raimondi F.
      • Mattiaccio A.
      • Mantovani V.
      • Vukotic R.
      • et al.
      Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencing.
      Thus, patients with otherwise unexplained cholestasis (see clinical vignette above) despite extensive diagnostic work-up (laboratory assessment, MRCP, liver histology) need to undergo genetic sequencing in ABCB4 and other genes associated with cholestatic liver disease.
      • Hakim A.
      • Zhang X.
      • DeLisle A.
      • Oral E.A.
      • Dykas D.
      • Drzewiecki K.
      • et al.
      Clinical utility of genomic analysis in adults with idiopathic liver disease.
      Stimulation/restoration of residual function by chaperones or induction of transcription by FXR or PPAR agonists may be promising future therapeutic options.

      Therapy

      Medical treatment

      Medical therapy is the first-line treatment in all patients with any kind of PFIC or ABCB4 variant associated syndrome. The main objective is to provide symptom relief in case of pruritus, to improve the nutritional status of the patient and to treat or prevent complications due to cirrhosis and portal hypertension like ascites or gastro-oesophageal haemorrhage. Simple procedures like moisturising the skin or trimming nails may be helpful against pruritus. The total daily caloric intake should be 125% of the recommended daily allowance (RDA). Dietary fat should be provided as medium chain triglycerides, which do not require bile salts for absorption. Water- and fat-soluble vitamins should be supplemented appropriately (1–2 times RDA). Furthermore, adequate exposure to sunlight and calcium supplementation is mandatory.
      • Feranchak A.P.
      • Ramirez R.O.
      • Sokol R.J.
      Medical and nutritional management of cholestasis.
      Hepatic osteodystrophy – caused by calcium and vitamin D malabsorption leading to secondary hyperparathyroidism, rickets or osteomalacia – is a significant extrahepatic expression of chronic cholestatic liver disease.
      • Gasser R.W.
      Cholestasis and metabolic bone disease - a clinical review.
      Besides, preclinical studies
      • Hochrath K.
      • Stokes C.S.
      • Geisel J.
      • Pollheimer M.J.
      • Fickert P.
      • Dooley S.
      • et al.
      Vitamin D modulates biliary fibrosis in ABCB4-deficient mice.
      • Reiter F.P.
      • Hohenester S.
      • Nagel J.M.
      • Wimmer R.
      • Artmann R.
      • Wottke L.
      • et al.
      1,25-(OH)₂-vitamin D₃ prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4(-/-) model.
      • Hochrath K.
      • Ehnert S.
      • Ackert-Bicknell C.L.
      • Lau Y.
      • Schmid A.
      • Krawczyk M.
      • et al.
      Modeling hepatic osteodystrophy in Abcb4 deficient mice.
      in Abcb4−/− mice indicate that vitamin D modulates biliary injury and fibrogenesis, and vitamin D deficiency seems to aggravate liver fibrosis in this animal model. Although the beneficial effects of vitamin D do not fully protect against liver fibrosis, the authors speculate that adequate supplementation may abate hepatic injury and confer antifibrotic effects.
      UDCA is the most commonly used medical treatment in patients with PFIC-3, LPAC or ICP. It is a hydrophilic bile acid that replaces toxic hydrophobic bile salts. Under long-term treatment it may amount to up to 40% of total serum bile salt concentration.
      • Poupon R.
      Intrahepatic cholestasis of pregnancy: from bedside to bench to bedside.
      It is also postulated to induce the expression of ABCB11 and ABCB4 and thus increase the secretion of bile acids and phospholipids into the bile.
      • Marschall H.U.
      • Wagner M.
      • Zollner G.
      • Diczfalusy U.
      • Gumhold J.
      • Silbert D.
      • et al.
      Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.
      Around 70% of patients with PFIC-3 respond to UDCA
      • Jacquemin E.
      • Hermans D.
      • Myara A.
      • Habes D.
      • Debray D.
      • Hadchouel M.
      • et al.
      Ursodeoxycholic acid therapy in pediatric patients with progressive familial intrahepatic cholestasis.
      ; nevertheless, a response to UDCA is unlikely in patients with low GGT cholestasis (approximately 35–40%)
      • Paumgartner G.
      • Beuers U.
      Ursodeoxycholic acid in cholestatic liver disease: mechanism of action and therapeutic use revisited.
      and in patients with a total defect in ABCB4 expression.
      • Jacquemin E.
      • de Vree J.M.
      • Cresteil D.
      • Sokal E.M.
      • Sturm E.
      • Dumont M.
      • et al.
      The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis in adulthood.
      UDCA is a safe drug with no major side effects or teratogenicity, allowing its use during pregnancy in patients with ICP. In a meta-analysis,
      • Bacq Y.
      • Sentilhes L.
      • Reyes H.B.
      • Glantz A.
      • Kondrackiene J.
      • Binder T.
      • et al.
      Efficacy of ursodeoxycholic acid in treating intrahepatic cholestasis of pregnancy: a meta-analysis.
      it was shown to be effective against maternal pruritus and it improved liver tests in women with ICP. However, a recent randomised placebo-controlled trial testing UDCA in ICP (PITCHES) observed no difference between UDCA- or placebo-treated women regarding adverse perinatal outcomes (perinatal death, preterm delivery, or neonatal unit admission).
      • Chappell L.C.
      • Bell J.L.
      • Smith A.
      • Linsell L.
      • Juszczak E.
      • Dixon P.H.
      • et al.
      Ursodeoxycholic acid versus placebo in women with intrahepatic cholestasis of pregnancy (PITCHES): a randomised controlled trial.
      Thus, the authors concluded that its use needs to be reconsidered in ICP. However, it is possible that the PITCHES trial population also included participants who did not suffer from ICP according to the EASL CPG, but had either pruritus without cholestasis or with an underlying chronic liver disease different from ICP, since pruritus, serum bile acids and ALT tended to decrease rather than increase in some patients – this is unexpected, as pruritus usually worsens and ALT and serum bile acids usually increase during the third trimester in patients with ICP.
      • Beuers U.
      • de Vries E.
      Reply to: “UDCA therapy in intrahepatic cholestasis of pregnancy?”.
      Based on these uncertainties in the PITCHES trial, it appears that UDCA treatment may be useless in women with pruritus during pregnancy who do not have an underlying secretory defect of the liver, but its benefit in ICP has to be evaluated in further trials.
      Rifampicin and cholestyramine are used for symptomatic treatment of cholestasis-associated pruritus. Rifampicin is the strongest inductor of CYP3A4 expression, increasing the 6-α hydroxylation of bile salts, which are thereafter glucuronidated and excreted in the urine. It induces the uridine diphosphate-glucuronosyl transferase (UGT1A1), leading to increased conjugation and excretion of bilirubin. Enhanced detoxification of bile acids and bilirubin conjugation by rifampicin together with stimulation of hepatobiliary transport systems by UDCA seem to have independent but complementary effects in patients with cholestatic diseases
      • Marschall H.U.
      • Wagner M.
      • Zollner G.
      • Diczfalusy U.
      • Gumhold J.
      • Silbert D.
      • et al.
      Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.
      and may also benefit patients with ABCB4 deficiency.
      Rifampicin may be used as a second-line treatment in patients who failed UDCA treatment and a recent study showed that it reduced serum bile acids in women with severe ICP.
      • Geenes V.
      • Chambers J.
      • Khurana R.
      • Wilksrom Shemer E.
      • Sia W.
      • Mandair D.
      • et al.
      Rifampicin in the treatment of severe intrahepatic cholestasis of pregnancy.
      Besides, cholestyramine, dexamethasone and S-adenosyl-L-methionine have been used in ICP, but due to varying results and lack of data none of them can be considered as first-line treatment.
      • Marschall H.U.
      • Wagner M.
      • Zollner G.
      • Diczfalusy U.
      • Gumhold J.
      • Silbert D.
      • et al.
      Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.
      In patients with end-stage liver disease and/or intractable pruritus orthotopic liver transplantation remains the last and often only therapeutic option.
      Fibrates, peroxisome proliferator-activated receptor (PPAR)α ligands, have been successfully used in patients with cholestasis (mostly PBC) for the last 2 decades.
      • Cuperus F.J.
      • Halilbasic E.
      • Trauner M.
      Fibrate treatment for primary biliary cirrhosis.
      Bezafibrate was recently shown to be beneficial in a randomised placebo-controlled trial in patients with PBC not responding to UDCA
      • Corpechot C.
      • Chazouillères O.
      • Rousseau A.
      • Le Gruyer A.
      • Habersetzer F.
      • Mathurin P.
      • et al.
      A placebo-controlled trial of bezafibrate in primary biliary cholangitis.
      and in patients with cholestatic pruritus.
      • De Vries E.
      • Bolier R.
      • Goet J.
      • Pares A.
      • Verbeek J.P.H.M.
      • De Vree M.
      • et al.
      Bezafibrate is more effective then placebo in pruritus of chronic cholestasis: the FITCH trial.
      One of the postulated mechanisms of action of bezafibrate involves the PPARα-dependent increase in ABCB4 expression
      • Shoda J.
      • Okada K.
      • Inada Y.
      • Kusama H.
      • Utsunomiya H.
      • Oda K.
      • et al.
      Bezafibrate induces multidrug-resistance P-Glycoprotein 3 expression in cultured human hepatocytes and humanized livers of chimeric mice.
      leading to biliary phospholipid excretion, as shown in bezafibrate-treated patients undergoing percutaneous biliary drainage.
      • Nakamuta M.
      • Fujino T.
      • Yada R.
      • Yasutake K.
      • Yoshimoto T.
      • Harada N.
      • et al.
      Therapeutic effect of bezafibrate against biliary damage: a study of phospholipid secretion via the PPARalpha-MDR3 pathway.
      Similarly, fenofibrate, another PPARα ligand, also upregulated ABCB4 in primary cultured human hepatocytes and stimulated its activity in HepG2 cells. Further, fenofibrate increased phosphatidylcholine excretion into bile canaliculi in cultured rat hepatocytes.
      • Ghonem N.S.
      • Ananthanarayanan M.
      • Soroka C.J.
      • Boyer J.L.
      Peroxisome proliferator-activated receptor α activates human multidrug resistance transporter 3/ATP-binding cassette protein subfamily B4 transcription and increases rat biliary phosphatidylcholine secretion.
      Thus, fibrates represent an interesting therapeutic strategy for patients with PFIC-3 and the entire spectrum of ABCB4 deficiency.
      The bile acid-activated nuclear farnesoid X receptor (FXR) is considered the master regulator in bile acid metabolism in the liver. FXR induces the transcription of ABCB4 and ABCB11 and thereby the export of bile acids and phospholipids from the hepatocyte into the canaliculus (Fig. 3).
      • Huang L.
      • Zhao A.
      • Lew J.L.
      • Zhang T.
      • Hrywna Y.
      • Thompson J.R.
      • et al.
      Farnesoid X receptor activates transcription of the phospholipid pump MDR3.
      This makes FXR a potential target for treatment in patients with chronic cholestatic liver disease, as reflected by its recent approval as a second-line treatment in PBC.
      • Nevens F.
      • Andreone P.
      • Mazzella G.
      • Strasser S.I.
      • Bowlus C.
      • Invernizzi P.
      • et al.
      A placebo-controlled trial of obeticholic acid in primary biliary cholangitis.
      In a recent study, the FXR agonist obeticholic acid significantly induced ABCB4 in human precision cut liver slices.
      • Ijssennagger N.
      • Janssen A.W.F.
      • Milona A.
      • Ramos Pittol J.M.
      • Hollmann D.A.A.
      • Mokry M.
      • et al.
      Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.
      Another study in an Abcb4-/- mouse model showed that dual FXR/TGR5 (membrane G protein-coupled receptor) activation improved liver injury by reducing biliary bile acid output and promoting HCO3--rich bile secretion.
      • Baghdasaryan A.
      • Claudel T.
      • Gumhold J.
      • Silbert D.
      • Adorini L.
      • Roda A.
      • et al.
      Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO⁻₃ output.
      A recent study
      • Gautherot J.
      • Claudel T.
      • Cuperus F.
      • Fuchs C.D.
      • Falguières T.
      • Trauner M.
      Thyroid hormone receptor β1 stimulates ABCB4 to increase biliary phosphatidylcholine excretion in mice.
      uncovered an additional pathway of ABCB4 regulation in mice. It showed that ABCB4 is hormonally regulated at a transcriptional level by ligands for the liver-specific thyroid hormone receptor β (currently also being assessed as a therapeutic target for non-alcoholic steatohepatitis), possibly revealing additional therapeutic opportunities for ABCB4-related liver disease.
      In the last few years, there have been several promising studies aimed at ameliorating the consequences of disease-causing variants in ABCB4. Drugs with chaperone-like activity like curcumin and 4-phenylbutyric acid (4-PBA) may enhance targeting of misfolded and intracellularly trapped mutant proteins to the plasma membrane,
      • Hayashi H.
      • Sugiyama Y.
      4-phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps.
      • van den Berghe P.V.
      • Stapelbroek J.M.
      • Krieger E.
      • de Bie P.
      • van de Graaf S.F.J.
      • de Groot R.E.A.
      • et al.
      Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.
      • van der Velden L.M.
      • Stapelbroek J.M.
      • Krieger E.
      • van den Berghe P.V.E.
      • Berger R.
      • Verhulst P.M.
      • et al.
      Folding defects in P-type ATP8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate.
      • Gordo-Gilart R.
      • Andueza S.
      • Hierro L.
      • Jara P.
      • Alvarez L.
      Functional rescue of trafficking-impaired ABCB4 mutants by chemical chaperons.
      leading to enhanced phospholipid efflux activity, though this effect was mutant-specific. In a study
      • Gonzales E.
      • Grosse B.
      • Schuller B.
      • Davit-Spraul A.
      • Conti F.
      • Guettier C.
      • et al.
      Targeted pharmacotherapy in progressive familial intrahepatic cholestasis type 2: evidence for improvement of cholestasis with 4-phenylbutyrate.
      of 4 patients with PFIC-2 and at least 1 mistrafficking variant in ABCB11, 4-PBA led to a decrease in serum bile acids and pruritus. The improvement may be a result of the ability of 4-PBA to retarget mutated ABCB11. 4-PBA (sodium phenylbutyrate) is approved for urea cycle disorders and has a wide spectrum of side effects: irregular menstrual cycles being the most common adverse effect, followed by appetite loss, body odour and dysgeusia. Laboratory alterations like acidosis or alkalosis, electrolyte changes, hypoalbuminemia and increased transaminases or ALP were also observed. However, in a small study in 7 patients with PFIC-1 and -2 treated with 4-PBA no adverse events were reported even after dose escalation.
      • Nakano S.
      • Osaka S.
      • Sabu Y.
      • Minowa K.
      • Hirai S.
      • Kondou H.
      • et al.
      Effect of food on the pharmacokinetics and therapeutic efficacy of 4-phenylbutyrate in progressive familial intrahepatic cholestasis.
      Correction and restoration of the activity of ABCB4 variants, although in vitro, provide interesting leads in the context of personalised medicine. Recently, ivacaftor – a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator – was shown to rescue defective phosphatidylcholine secretion activity.
      • Delaunay J.L.
      • Bruneau A.
      • Hoffmann B.
      • Durand-Schneider A.M.
      • Barbu V.
      • Jacquemin E.
      • et al.
      Functional defect of variants in the adenosine triphosphate-binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770).
      Similarly, another study showed that structural analogues of roscovitine (a molecule used in clinical trials in cystic fibrosis correcting the F508del variant in CFTR) were able to correct intracellular traffic of endoplasmic reticulum-retained ABCB4 variants in HepG cells.
      • Vauthier V.
      • Ben Saad A.
      • Elie J.
      • Oumata N.
      • Durand-Schneider A.M.
      • Bruneau A.
      • et al.
      Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models.

      Surgical treatment

      Liver transplantation is the last therapeutic option for patients with end-stage liver disease, HCC or those with low quality of life due to intractable pruritus despite optimal medical management. Liver transplantation improves cholestasis-associated symptoms in more than 75% of patients irrespective of PFIC subtype over a short-term follow-up of 3-5 years.
      • Englert C.
      • Grabhorn E.
      • Richter A.
      • Rogiers X.
      • Burdelski M.
      • Ganschow R.
      • et al.
      Liver transplantation in children with progressive familial intrahepatic cholestasis.
      ,
      • Hori T.
      • Egawa H.
      • Takada Y.
      • Ueda M.
      • Oike F.
      • Ogura Y.
      • et al.
      Progressive familial intrahepatic cholestasis: a single-centre experience of living-donor liver transplantation during two decades in Japan.
      In patients with LPAC, cholecystectomy may be necessary in cases with recurrent biliary colic or acute cholecystitis and cholelithiasis. Nevertheless, cholecystectomy should be deferred in those without macroscopic gallbladder stones and only symptomatic biliary sludge. Instead, UDCA treatment should be attempted prior to all surgical interventions and continued after cholecystectomy. In rare cases with intrahepatic concrements and bile duct dilations, biliary drainage or even hepatic resection may be necessary.
      • Erlinger S.
      Low phospholipid-associated cholestasis and cholelithiasis.

      Conclusions

      Variants of ABCB4 are associated with a broad spectrum of disease entities ranging from mildly elevated liver enzymes or cholestasis to progressive liver disease with decompensated biliary cirrhosis; only recently, evidence has emerged on the association of ABCB4 with hepatobiliary malignancies.
      • Gudbjartsson D.F.
      • Helgasson H.
      • Gudjonsson S.A.
      • Zink F.
      • Oddson A.
      • Gylfason A.
      • et al.
      Large-scale whole genome sequencing of the Icelandic population.
      ,
      • Reichert M.C.
      • Lammert F.
      ABCB4 gene aberrations in human liver disease: an evolving spectrum.
      Due to the low prevalence and the overlapping disease manifestations, diagnosis is often delayed for many years. Although, genotype-phenotype correlations are lacking, testing for mutations in ABCB4 should be considered in cases of chronic (cholestatic) liver disease in order to counsel individual patients and their families. In this regard, treating physicians should be aware of a standardised approach for diagnosis including laboratory, imaging, histological and molecular genetic techniques.
      So far, therapeutic options for patients are limited. Besides medical treatment with UDCA, cholestyramine or rifampicin, orthotopic liver transplantation remains the only treatment option in patients with end-stage liver disease or intractable pruritus. Nevertheless, large and especially prospective data are lacking. UDCA can often only delay biliary cirrhosis and hepatic decompensation, particularly in severe forms of ABCB4 deficiency, like PFIC-3. As effective treatment options are currently lacking, the search for new treatment approaches and identification of molecular targets for therapeutic interventions are of great interest.

      Abbreviations

      4-PBA, 4-phenylbutyric acid; ABCB4, ATP binding cassette subfamily B member 4; ABCB11, ATP binding cassette subfamily B member 11; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP8B1, ATPase class I, type 8B, member 1; BRIC, benign recurrent intrahepatic cholestasis; BSEP, bile salt export pump; CIC, contraceptive-induced intrahepatic cholestasis; CFTR, cystic fibrosis transmembrane conductance regulator; DILI, drug-induced liver injury; ERCP, endoscopic retrograde cholangiopancreatography; FIC1, familial intrahepatic cholestasis 1 protein; FXR, farnesoid X receptor; ICP, intrahepatic cholestasis of pregnancy; LPAC, low phospholipid-associated cholelithiasis; MDR3, multidrug resistance protein 3; MRCP, magnetic resonance cholangiopancreatography; MYO5B, myosin 5b; PBC, primary biliary cholangitis; PFIC, progressive familial intrahepatic cholestasis; PPARα, peroxisome proliferator-activated receptor-α; TJP2, tight junction protein 2; UDCA, ursodeoxycholic acid.

      Financial support

      The authors received no financial support to produce this manuscript.

      Authors' contributions

      A.F.S. writing of the manuscript, literature research; E.H., F.W., P.F. literature research, critical revision of the manuscript for important intellectual content; M.T. literature research, outlining and writing of the manuscript.

      Conflict of interest

      P.F. has served as a speaker and/or consultant and/or advisory board member for AbbVie, Bristol-Myer-Squibb, Gilead, and MSD and has received research funding from Gilead. M.T. served as a speaker and/or consultant and/or advisory board member for Albireo, Boehringer Ingelheim, Bristol-Myers Squibb, Falk, Genfit, Gilead, Intercept, MSD, Novartis, Phenex, Regulus and Shire, and received travel support from AbbVie, Falk, Gilead, and Intercept, as well as grants/research support from Albireo, Cymabay, Falk, Gilead, Intercept, MSD, and Takeda. He is also co-inventor of patents on the medical use of 24-norursodeoxycholic acid.
      All other authors have no conflict of interest to declare.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We would like to thank our radiologists Prof. Dr. Ahmed Ba-Ssalamah und Dr. Nina Bastati for their cooperation in discussing and sharing the MRI pictures.

      Supplementary data

      References

        • Karlsen T.H.
        • Lammert F.
        • Thompson R.J.
        Genetics of liver disease: from pathophysiology to clinical practice.
        J Hepatol. 2015; 62: 6-14
        • Hakim A.
        • Zhang X.
        • DeLisle A.
        • Oral E.A.
        • Dykas D.
        • Drzewiecki K.
        • et al.
        Clinical utility of genomic analysis in adults with idiopathic liver disease.
        J Hepatol. 2019; 70: 1214-1221
        • Eichler E.E.
        Genetic variation, comparative genomics and the diagnosis of disease.
        N Engl J Med. 2019; 381: 64-74
        • Clayton R.J.
        • Iber F.I.
        • Ruebner B.H.
        • McKusick V.A.
        Byler disease: fatal intrahepatic cholestasis in an Amish kindred.
        Am J Dis Child. 1969; 117: 112-124
        • Bull L.N.
        • Carlton V.E.H.
        • Stricker N.L.
        • Baharloo S.
        • DeYoung J.A.
        • Freimer N.B.
        • et al.
        Genetic and morphological findings in progressive familial intrahepatic cholestasis (Byler disease [PFIC-1] and Byler syndrome): evidence for heterogeneity.
        Hepatology. 1997; 1: 155-164
        • Bull L.N.
        • van Eijk M.J.
        • Pawlikowska L.
        • DeYoung J.A.
        • Jujin J.A.
        • Liao M.
        • et al.
        A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.
        Nat Genet. 1998; 18: 219-224
        • Trauner M.
        • Meier P.J.
        • Boyer J.L.
        Molecular pathogenesis of cholestasis.
        N Engl J Med. 1998; 339: 1217-1227
        • Srivastava A.
        Progressive familial intrahepatic cholestasis.
        J Clin Exp Hepatol. 2014; 4: 25-36
        • Jacquemin E.
        Progressive familial intrahepatic cholestasis.
        Clin Res Hepatol Gastroenterol. 2012; 36: 26-35
        • Paulusma C.C.
        • Oude Elferink R.P.J.
        • Jansen P.L.M.
        Progressive familial intrahepatic cholestasis type 1.
        Semin Liver Dis. 2010; 30: 117-124
        • Strautnieks S.S.
        • Kagalwalla A.F.
        • Tanner M.S.
        • Knisely A.S.
        • Bull L.
        • Freimer N.
        • et al.
        Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24.
        Am J Hum Genet. 1997; 61: 630-633
        • van der Bliek A.M.
        • Kooiman P.M.
        • Schneider C.
        • Borst P.
        Sequence of mdr3 cDNA encoding a human p-glycoprotein.
        Gene. 1988; 71: 401-411
        • Vitale G.
        • Gitto S.
        • Vukotic R.
        • Raimondi F.
        • Andreone P.
        Familial intrahepatic cholestasis: new and wide perspectives.
        Dig Liver Dis. 2019; 51: 922-933
        • Gomez-Ospina N.
        • Potter C.J.
        • Xiao R.
        • Manickam K.
        • Kim M.S.
        • Kim K.H.
        • et al.
        Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis.
        Nat Commun. 2016; 7: 10713
        • Gonzalez E.
        • Taylor S.A.
        • Davit-Spraul A.
        • Thebaut A.
        • Thomassin N.
        • Guettier C.
        • et al.
        MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease.
        Hepatology. 2017; 65: 164-173
        • Oude Elferink R.P.
        • Paulusma C.C.
        Function and pathophysiological importance of ABCB4 (MDR3 P -glycoprotein).
        Pflugers Arch. 2007; 453: 601-610
        • Smit J.J.
        • Schinkel A.H.
        • Oude Elferink R.P.
        • Groen A.K.
        • Wagenaar E.
        • van Deemter L.
        • et al.
        Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.
        Cell. 1993; 75: 451-462
        • Pauli-Magnus C.
        • Lang T.
        • Meier Y.
        • Zodan-Marin T.
        • Jung D.
        • Breymann C.
        • et al.
        Sequence analysis of bile salt export pump (ABCB11) and multi drug resistance p-glycoprotein 2 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy.
        Pharmacogenetics. 2004; 14: 91-102
        • Rosmorduc O.
        • Hermelin B.
        • Poupon R.
        MDR3 gene defects in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis.
        Gastroenterology. 2001; 120: 1459-1467
        • Lang C.
        • Meier Y.
        • Stieger B.
        • Beuers U.
        • Lang T.
        • Kerb R.
        • et al.
        Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury.
        Pharmacogenet Genomics. 2007; 17: 47-60
        • Gotthard D.
        • Runz H.
        • Keitel V.
        • Flechtenmacher C.
        • Wirtenberger M.
        • Weiss K.H.
        • et al.
        A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults.
        Hepatology. 2008; 48: 1157-1166
        • Ziol M.
        • Barbu V.
        • Rosmorduc O.
        • Frassati-Biaggi A.
        • Barget N.
        • Hermelin B.
        • et al.
        ABCB4 heterozygous mutations associated with fibrosing cholestatic liver disease in adults.
        Gastroenterology. 2008; 135: 131-141
        • Jacquemin E.
        • de Vree J.M.
        • Cresteil D.
        • Sokal E.M.
        • Sturm E.
        • Dumont M.
        • et al.
        The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis in adulthood.
        Gastroenterology. 2001; 120: 1448-1458
        • Gudbjartsson D.F.
        • Helgasson H.
        • Gudjonsson S.A.
        • Zink F.
        • Oddson A.
        • Gylfason A.
        • et al.
        Large-scale whole genome sequencing of the Icelandic population.
        Nat Genet. 2015; 47: 435-444
        • Lammert F.
        • Hochrath K.
        A letter on ABCB4 from Iceland: on the highway to liver disease.
        Clin Res Hepatol Gastroenterol. 2015; 39: 655-658
        • Smit A.J.
        • Timmermans-Hereijgers J.L.
        • Roelofsen B.
        • Wirtz K.W.
        • van Bitterswijk W.J.
        • Smit J.J.
        • et al.
        The human MDR3 p-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice.
        FEBS Lett. 1994; 354: 263-266
        • Zolnerciks J.K.
        • Andress E.J.
        • Nicolao M.
        • Linton K.J.
        Structure of ABC transporters.
        Essays Biochem. 2011; 50: 43-61
        • Hofmann A.F.
        Bile acid secretion, bile flow and biliary lipid secretion in humans.
        Hepatology. 1990; 12: 17-22
        • De Vree J.M.
        • Jacquemin E.
        • Sturm E.
        • Cresteil D.
        • Bosma P.J.
        • Aten J.
        • et al.
        Mutations in MDR3 gene cause progressive familial intrahepatic cholestasis.
        Proc Natl Acad Sci U S A. 1998; 95: 282-287
        • Schneider G.
        • Paus T.C.
        • Kullak-Ublick G.A.
        • Meier P.J.
        • Wienker T.F.
        • Lang T.
        • et al.
        Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy.
        Hepatology. 2007; 45: 150-158
        • Delaunay J.L.
        • Durand-Schneider A.M.
        • Dossier C.
        • Falguieres T.
        • Gautherot J.
        • Davit-Spraul A.
        • et al.
        A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3.
        Hepatology. 2016; 63: 1620-1631
        • Wilschanski M.
        • Durie P.R.
        Patterns of GI disease in adulthood associated with mutations in the CFTR gene.
        Gut. 2007; 56: 1153-1163
        • Kubitz R.
        • Bode J.
        • Erhardt A.
        • Graf D.
        • Kircheis G.
        • Müller-Stöver I.
        • et al.
        Cholestatic liver disease from child to adult: the diversity of MDR3 disease.
        Z Gastroenterol. 2011; 49: 728-736
        • Reichert M.C.
        • Lammert F.
        ABCB4 gene aberrations in human liver disease: an evolving spectrum.
        Semin Liver Dis. 2018; 38: 299-307
        • Poupon R.
        • Rosmorduc O.
        • Boelle B.Y.
        • Chretien Y.
        • Corpechot C.
        • Chazouielleres C.
        • et al.
        Genotype-phenotype relationships in the low-phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients.
        Hepatology. 2013; 58: 1105-1110
        • Colombo C.
        • Vajro P.
        • Degiorgio D.
        • Coviello D.A.
        • Constantino L.
        • Tornillo L.
        • et al.
        Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations.
        J Pediatr Gastroenterol Nutr. 2011; 52: 73-83
        • Schatz S.B.
        • Jüngst C.
        • Keitel-Anselmo V.
        • Kubitz R.
        • Becker C.
        • Gerner P.
        • et al.
        Phenotypic spectrum and diagnostic pitfall of ABCB4 deficiency depending on age of onset.
        Hepatol Commun. 2018; 2: 504-514
        • Davit-Spraul A.
        • Gonzales E.
        • Baussan C.
        • Jacquemin E.
        The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects.
        Semin Liver Dis. 2010; 30: 134-146
        • Ganne-Carrié N.
        • Baussan C.
        • Grando V.
        • Gaudelus J.
        • Cresteil D.
        • Jacquemin E.
        Progressive familial intrahepatic cholestasis type 3 revealed by oral contraceptive pills.
        J Hepatol. 2003; 38: 693-694
        • Rosmorduc O.
        • Hermelin B.
        • Boelle P.Y.
        • Parc R.
        • Taboury J.
        • Poupon R.
        ABCB4 gene mutation-associated cholelithiasis in adults.
        Gastroenterology. 2003; 125: 452-459
        • Rosmorduc O.
        • Poupon R.
        Low phospholipid associated cholelithiasis: association with mutation in the MDR3/ABCB4 gene.
        Orphanet J Rare Dis. 2007; 29: 29
        • Condat B.
        • Zanditenas D.
        • Barbu V.
        • Hauuy M.P.
        • Parfait B.
        • El Naggar A.
        • et al.
        Prevalence of low phospholipid-associated cholelithiasis in young female patients.
        Dig Liver Dis. 2013; 45: 915-919
        • Pauli-Magnus C.
        • Meier P.J.
        • Stieger B.
        Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy.
        Semin Liver Dis. 2010; 30: 147-159
        • Geenes V.
        • Williamson C.
        Intrahepatic cholestasis of pregnancy.
        World J Gastroenterol. 2009; 15: 2049-2066
        • Bacq Y.
        Liver diseases unique to pregnancy: a 2010 update.
        Clin Res Hepatol Gastroenterol. 2011; 35: 182-193
        • Dixon P.H.
        • Williamson C.
        The pathophysiology of intrahepatic cholestasis of pregnancy.
        Clin Res Hepatol Gastroenterol. 2016; 40: 141-153
        • Jacquemin E.
        • Cresteil D.
        • Manouvrier S.
        • Boute O.
        • Hadchouel M.
        Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy.
        Lancet. 1999; 353: 210-211
        • Keitel V.
        • Dröge C.
        • Stepanow S.
        • Fehm T.
        • Mayatepek E.
        • Köhrer K.
        • et al.
        Intrahepatic cholestasis of pregnancy (ICP): case report and review of literature.
        Z Gastroenterol. 2016; 54: 1327-1333
        • Ovadia C.
        • Seed P.T.
        • Sklavounos A.
        • Geenes V.
        • Di Ilio C.
        • Chambers J.
        • et al.
        Association of adverse perinatal outcomes of intrahepatic cholestasis of pregnancy with biochemical markers: results of aggregate and individual patient data meta-analyses.
        Lancet. 2019; 393: 899-909
        • Ropponen A.
        • Sund R.
        • Riikonen S.
        • Ylikorkala O.
        • Aittomäki K.
        Intrahepatic cholestasis of pregnancy as an indicator of liver and biliary diseases: a population-based study.
        Hepatology. 2006; 43: 723-728
        • Wikström Shemer E.A.
        • Stephansson O.
        • Thuresson M.
        • Thorsell M.
        • Ludvigsson J.F.
        • Marschall H.U.
        Intrahepatic cholestasis of pregnancy and cancer, immune-mediated and cardiovascular diseases: a population-based cohort study.
        J Hepatol. 2015; 63: 456-461
        • Westbrook R.H.
        • Dusheiko G.
        • Williamson C.
        Pregnancy and liver disease.
        J Hepatol. 2016; 64: 933-945
        • Smith A.J.
        • van Helvoort A.
        • van Meer G.
        • Szabo K.
        • Welker E.
        • Szakacs G.
        • et al.
        MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping.
        J Biol Chem. 2000; 275: 23530-23539
        • Zollner G.
        • Thueringer A.
        • Lackner C.
        • Fickert P.
        • Trauner M.
        Alterations of canalicular ATP-binding cassette transporter expression in drug-induced liver injury.
        Digestion. 2014; 90: 81-88
        • EASL Clinical Practice Guidelines
        Drug-induced liver injury.
        J Hepatol. 2019; 70: 1222-1261
        • Trauner M.
        • Fickert P.
        • Wagner M.
        MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes.
        Semin Liver Dis. 2007; 27: 77-98
        • Ramraj R.
        • Finegold M.J.
        • Karpen S.J.
        Progressive familial intrahepatic cholestasis type 3: overlapping presentation with Wilson disease.
        Clin Pediatr (Phila). 2012; 51: 689-691
        • Trauner M.
        • Fickert P.
        • Halilbasic E.
        • Moustafa T.
        Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases.
        Wien Med Wochenschr. 2008; 158: 542-548
        • Morotti R.A.
        • Suchy F.J.
        • Magid M.S.
        Progressive familial intrahepatic cholestasis (PFIC) type 1, 2, and 3: a review of the liver pathology findings.
        Semin Liver Dis. 2011; 31: 3-10
        • Wendum D.
        • Barbu V.
        • Rosmorduc O.
        • Arrive L.
        • Flejou J.F.
        • Poupon R.
        Aspects of liver pathology in adult patients with MDR3/ABCB4 gene mutations.
        Virchows Arch. 2012; 460: 291-298
        • Liu C.
        • Aronow B.J.
        • Jegga A.J.
        • Wang N.
        • Miethke A.
        • Mourya R.
        • et al.
        Novel resequencing chip to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis.
        Gastroenterology. 2007; 132: 119-126
        • Aamann L.
        • Ørntoft N.
        • Vogel I.
        • Groenbaek H.
        • Becher N.
        • Vilstrup H.
        • et al.
        Unexplained cholestasis in adults and adolescents: diagnostic benefit of genetic examination.
        Scand J Gastroenterol. 2018; 53: 305-311
        • Dröge C.
        • Bonus M.
        • Baumann U.
        • Klindt C.
        • Lainka E.
        • Kathemann S.
        • et al.
        Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants.
        J Hepatol. 2017; 67: 1253-1264
        • Vitale G.
        • Gitto S.
        • Raimondi F.
        • Mattiaccio A.
        • Mantovani V.
        • Vukotic R.
        • et al.
        Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencing.
        J Gastroenterol. 2018; 53: 945-958
        • Feranchak A.P.
        • Ramirez R.O.
        • Sokol R.J.
        Medical and nutritional management of cholestasis.
        in: Such F.J. Sokol R.J. Ballistreri W.F. Liver Disease in Children. 2nd edition. Lippincott, Williams and Wilkins, Philadelphia, PA2001: 195-238 ([chapter 10])
        • Gasser R.W.
        Cholestasis and metabolic bone disease - a clinical review.
        Wien Med Wochenschr. 2008; 158: 553-557
        • Hochrath K.
        • Stokes C.S.
        • Geisel J.
        • Pollheimer M.J.
        • Fickert P.
        • Dooley S.
        • et al.
        Vitamin D modulates biliary fibrosis in ABCB4-deficient mice.
        Hepatol Int. 2014; 8: 443-452
        • Reiter F.P.
        • Hohenester S.
        • Nagel J.M.
        • Wimmer R.
        • Artmann R.
        • Wottke L.
        • et al.
        1,25-(OH)₂-vitamin D₃ prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4(-/-) model.
        Biochem Biophys Res Commun. 2015; 459: 227-233
        • Hochrath K.
        • Ehnert S.
        • Ackert-Bicknell C.L.
        • Lau Y.
        • Schmid A.
        • Krawczyk M.
        • et al.
        Modeling hepatic osteodystrophy in Abcb4 deficient mice.
        Bone. 2013; 55: 501-511
        • Poupon R.
        Intrahepatic cholestasis of pregnancy: from bedside to bench to bedside.
        Liv Int. 2005; 25: 467-468
        • Marschall H.U.
        • Wagner M.
        • Zollner G.
        • Diczfalusy U.
        • Gumhold J.
        • Silbert D.
        • et al.
        Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.
        Gastroenterology. 2005; 129: 476-485
        • Jacquemin E.
        • Hermans D.
        • Myara A.
        • Habes D.
        • Debray D.
        • Hadchouel M.
        • et al.
        Ursodeoxycholic acid therapy in pediatric patients with progressive familial intrahepatic cholestasis.
        Hepatology. 1997; 25: 519-523
        • Paumgartner G.
        • Beuers U.
        Ursodeoxycholic acid in cholestatic liver disease: mechanism of action and therapeutic use revisited.
        Hepatology. 2002; 36: 525-531
        • Bacq Y.
        • Sentilhes L.
        • Reyes H.B.
        • Glantz A.
        • Kondrackiene J.
        • Binder T.
        • et al.
        Efficacy of ursodeoxycholic acid in treating intrahepatic cholestasis of pregnancy: a meta-analysis.
        Gastroenterology. 2012; 143: 1492-1501
        • Chappell L.C.
        • Bell J.L.
        • Smith A.
        • Linsell L.
        • Juszczak E.
        • Dixon P.H.
        • et al.
        Ursodeoxycholic acid versus placebo in women with intrahepatic cholestasis of pregnancy (PITCHES): a randomised controlled trial.
        Lancet. 2019; 394: 849-860
        • Beuers U.
        • de Vries E.
        Reply to: “UDCA therapy in intrahepatic cholestasis of pregnancy?”.
        J Hepatol. 2020; 72: 587-588
        • Geenes V.
        • Chambers J.
        • Khurana R.
        • Wilksrom Shemer E.
        • Sia W.
        • Mandair D.
        • et al.
        Rifampicin in the treatment of severe intrahepatic cholestasis of pregnancy.
        Eur J Obstet Gynecol Reprod Biol. 2015; 189: 59-63
        • Cuperus F.J.
        • Halilbasic E.
        • Trauner M.
        Fibrate treatment for primary biliary cirrhosis.
        Curr Opin Gastroenterol. 2014; 30: 279-286
        • Corpechot C.
        • Chazouillères O.
        • Rousseau A.
        • Le Gruyer A.
        • Habersetzer F.
        • Mathurin P.
        • et al.
        A placebo-controlled trial of bezafibrate in primary biliary cholangitis.
        N Engl J Med. 2018; 378: 2171-2181
        • De Vries E.
        • Bolier R.
        • Goet J.
        • Pares A.
        • Verbeek J.P.H.M.
        • De Vree M.
        • et al.
        Bezafibrate is more effective then placebo in pruritus of chronic cholestasis: the FITCH trial.
        Hepatology. 2019; 70 (9A, Abstract): S1
        • Shoda J.
        • Okada K.
        • Inada Y.
        • Kusama H.
        • Utsunomiya H.
        • Oda K.
        • et al.
        Bezafibrate induces multidrug-resistance P-Glycoprotein 3 expression in cultured human hepatocytes and humanized livers of chimeric mice.
        Hepatol Res. 2007; 37: 548-556
        • Nakamuta M.
        • Fujino T.
        • Yada R.
        • Yasutake K.
        • Yoshimoto T.
        • Harada N.
        • et al.
        Therapeutic effect of bezafibrate against biliary damage: a study of phospholipid secretion via the PPARalpha-MDR3 pathway.
        Int J Clin Pharmacol Ther. 2010; 48: 22-28
        • Ghonem N.S.
        • Ananthanarayanan M.
        • Soroka C.J.
        • Boyer J.L.
        Peroxisome proliferator-activated receptor α activates human multidrug resistance transporter 3/ATP-binding cassette protein subfamily B4 transcription and increases rat biliary phosphatidylcholine secretion.
        Hepatology. 2014; 59: 1030-1042
        • Huang L.
        • Zhao A.
        • Lew J.L.
        • Zhang T.
        • Hrywna Y.
        • Thompson J.R.
        • et al.
        Farnesoid X receptor activates transcription of the phospholipid pump MDR3.
        J Biol Chem. 2003; 278: 51085-51090
        • Nevens F.
        • Andreone P.
        • Mazzella G.
        • Strasser S.I.
        • Bowlus C.
        • Invernizzi P.
        • et al.
        A placebo-controlled trial of obeticholic acid in primary biliary cholangitis.
        N Engl J Med. 2016; 375: 631-643
        • Ijssennagger N.
        • Janssen A.W.F.
        • Milona A.
        • Ramos Pittol J.M.
        • Hollmann D.A.A.
        • Mokry M.
        • et al.
        Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.
        J Hepatol. 2016; 64: 1158-1166
        • Baghdasaryan A.
        • Claudel T.
        • Gumhold J.
        • Silbert D.
        • Adorini L.
        • Roda A.
        • et al.
        Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO⁻₃ output.
        Hepatology. 2011; 54: 1303-1312
        • Gautherot J.
        • Claudel T.
        • Cuperus F.
        • Fuchs C.D.
        • Falguières T.
        • Trauner M.
        Thyroid hormone receptor β1 stimulates ABCB4 to increase biliary phosphatidylcholine excretion in mice.
        J Lipid Res. 2018; 59: 1610-1619
        • Hayashi H.
        • Sugiyama Y.
        4-phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps.
        Hepatology. 2007; 45: 1506-1516
        • van den Berghe P.V.
        • Stapelbroek J.M.
        • Krieger E.
        • de Bie P.
        • van de Graaf S.F.J.
        • de Groot R.E.A.
        • et al.
        Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.
        Hepatology. 2009; 50: 1783-1795
        • van der Velden L.M.
        • Stapelbroek J.M.
        • Krieger E.
        • van den Berghe P.V.E.
        • Berger R.
        • Verhulst P.M.
        • et al.
        Folding defects in P-type ATP8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate.
        Hepatology. 2010; 51: 286-296
        • Gordo-Gilart R.
        • Andueza S.
        • Hierro L.
        • Jara P.
        • Alvarez L.
        Functional rescue of trafficking-impaired ABCB4 mutants by chemical chaperons.
        PLoS One. 2016; 11: e0150098
        • Gonzales E.
        • Grosse B.
        • Schuller B.
        • Davit-Spraul A.
        • Conti F.
        • Guettier C.
        • et al.
        Targeted pharmacotherapy in progressive familial intrahepatic cholestasis type 2: evidence for improvement of cholestasis with 4-phenylbutyrate.
        Hepatology. 2015; 62: 558-566
      1. (Available at:) (Accessed Feburary 14, 2020)
        • Nakano S.
        • Osaka S.
        • Sabu Y.
        • Minowa K.
        • Hirai S.
        • Kondou H.
        • et al.
        Effect of food on the pharmacokinetics and therapeutic efficacy of 4-phenylbutyrate in progressive familial intrahepatic cholestasis.
        Sci Rep. 2019; 9: 17075
        • Delaunay J.L.
        • Bruneau A.
        • Hoffmann B.
        • Durand-Schneider A.M.
        • Barbu V.
        • Jacquemin E.
        • et al.
        Functional defect of variants in the adenosine triphosphate-binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770).
        Hepatology. 2017; 65: 560-570
        • Vauthier V.
        • Ben Saad A.
        • Elie J.
        • Oumata N.
        • Durand-Schneider A.M.
        • Bruneau A.
        • et al.
        Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models.
        Sci Rep. 2019; 9: 6653
        • Englert C.
        • Grabhorn E.
        • Richter A.
        • Rogiers X.
        • Burdelski M.
        • Ganschow R.
        • et al.
        Liver transplantation in children with progressive familial intrahepatic cholestasis.
        Transplantation. 2007; 84: 1361-1363
        • Hori T.
        • Egawa H.
        • Takada Y.
        • Ueda M.
        • Oike F.
        • Ogura Y.
        • et al.
        Progressive familial intrahepatic cholestasis: a single-centre experience of living-donor liver transplantation during two decades in Japan.
        Clin Transplant. 2011; 25: 776-785
        • Erlinger S.
        Low phospholipid-associated cholestasis and cholelithiasis.
        Clin Res Hepatol Gastroenterol. 2012; 36: 36-40