Genetic cholestasis. Peter Jansen Liver Center Academic Medical Center Amsterdam

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1 Genetic cholestasis Peter Jansen Liver Center Academic Medical Center Amsterdam

2 Genetic cholestasis Proteins and genes Genetic cholestatic diseases Lessons from ko/mutant models A new MDR3 phenotype A new mechanism Peroxisomes

3 Phosphatidylcholine Conjugated bile salts Bilirubine glucuronides MDR3 BSEP MRP2 FIC1 MDR1 ABCG2,BCRP ABCG5/G8 CyA, FK 506, protease inhibitors, steroids Cholesterol 17β-estradiolglucuronide protoporhyrin MRP3 Phosphatidylserine CFTR FIC1 OSTαβ ASBT ABCG5/G8 MRP2 FIC1 MDR1 INTESTINE

4 MRP3 MRP1 MRP4 Bili conjugates LTC4 MDR3 OA conjugates BSEP MRP2 BS sulfates OSTαβ BCRP E2G MDR1 BCRP Bile Salts ABCG5/G8 FIC1 MRP3 CFTR FIC1 Basolateral expression during cholestasis OSTαβ ASBT ABCG5/G8 MRP2 FIC1 MDR1 INTESTINE

5 Drugs Bile acids Oxysterols Fatty acids CAR PXR FXR VDR LXR PPARα UGT1A1 MRP2 MRP4 MRP3 BSEP OSTαβ FAS SHP Cyp7A1 ASBT MDR3 NTCP

6 PXR PXR CAR CAR FXR FXR VDR VDR LXR LXR PPAR PPARα Drugs Drugs Bile acids Bile acids Cholesterol Cholesterol Fatty acids Fatty acids UGT1A1 UGT1A1 MRP2 MRP2 MDR3 MDR3 NTCP NTCP ASBT ASBT MRP4 MRP4 MRP3 MRP3 SULT2A SULT2A OST OSTαβ αβ Cyp7A1 Cyp7A1 SHP SHP BSEP BSEP FAS FAS

7 ABC transport proteins and genes Constitutive Regulation Adaptation Gene ABCB11 ABCB4 ABC G5/G8 ABCC2 ABCB1 ATP8B1 FXR PXR CAR ABCC3 ABCC4 ABCC1 ABCG2 OSTαβ Protein BSEP MDR3 ABC G5/G8 MRP2 MDR1 FIC1 FXR PXR CAR MRP3 MRP4 MRP1 BCRP OSTαβ

8 Knockout/mutant models as predictors of disease Gene Protein Knockout mouse phenotype Control conditions Stressed (BDL, cholate/drug exposure) Constitutive ABCB11 ABCB4 BSEP MDR2 None Mild fibrosis Mild cholestasis (protected in BDL?) Severe fibrosis, inflammation, HCC ABCG5/G8 ABC G5/G8 None Reduced cholesterol secretion ABCC2 MRP2 Jaundice ABCB1 MDR1 Colitis Altered drug metabolism ATP8B1* FIC1 Increased BA Increased BA Regulation FXR PXR Mild steatosis Colitis Less necrosis in BDL More necrosis but lower BA in BDL CAR None More necrosis but lower BA in BDL Adaptation ABCC3 ABCC4 MRP3 MRP4 None None BDL, reduced hyperbilirubinemia BDL, more necrosis ABCC1 MRP1 None Sensitivity towards xenotoxicants ABCG2 BCRP Phototoxicity Sensitivity towards xenotoxicants Phototoxicity

9 CONCLUSIONS 1 In ko/mutant models, phenotypes expressed under conditions of stress Analogues of stress in humans? Cholestasis Inflammation Pregnancy Drugs

10 Progressive familial intrahepatic cholestasis PFIC type 1 ATP8B1/FIC1 Biopsy:bland cholestasis IH: FIC1 absent Low GGT Intermittent cholestasis, severe pruritus Age of onset: 0-1 year Biliary diversion transplantation BRIC type 1 ICP type 2 ABCB11/BSEP Neonatal hepatitis, cholestasis, portal fibrosis IH: BSEP absent Low GGT Permanent cholestasis, severe pruritus Age of onset: 0-1 year Biliary diversion transplantation BRIC type 2 HCC and CC < age 5 yrs type 3 ABCB4/MDR3 Bile duct proliferation, portal fibrosis IH: MDR 3 absent High GGT Permanent cholestasis, severe pruritus Age of onset: 1-5 year Ursodeoxycholic acid, transplantation Gallstones ICP

11 Diagnosis of PFIC Symptoms and signs of cholestasis Exclude bile duct dilatation Liver biopsy Histology Immune histology Genetics

12 MDR 3 genotype/phenotype

13 MDR3 phenotype Progressive Familial Intrahepatic Cholestasis (PFIC type 3) Gallstones Intrahepatic Cholestasis of Pregnancy (ICP) HCC or CC?

14 Progressive Familial Intrahepatic Cholestasis MDR3/ABCB4 -/- : del/insertion/nonsense -/- : missense +/- : missense 2006: Data de Vree and literature

15 Gallstones Intrahepatic cholestasis of pregnancy MDR3/ABCB4 - /- : homozygous mutations +/- : heterozygous mutations 2006: Data de Vree and literature

16 New MDR3 family: patient history Index case: cholestasis age 12, liver biopsy refused; genetic analysis, 2005: urso: improvement of cholestasis Brother: itching and cholestasis age 25; liverbiopsy: cholestasis, ductopenia, portal fibrosis Sister: hepatic fibrosis age 13, end stage cirrhosis age 17, liver transplantation 1992, rejection (2x) deceased

17 Brother: liver biopsy age 25 Paucity of bile ducts Cholestasis Portal fibrosis

18 Sister: liver explant age 22 Portal-portal fibrosis Paucity of bile ducts.

19 New MDR3 family Compound heterozygous MDR3/ABCB4 +/- : nonsense: frameshift +/- : missense 2006: Data de Vree

20 79 A>G: codon 27, exon/intron boundary nr 1: splice donor site frame shift NH2 terminus truncation. PFIC3-mutation 959 C>T: codon 320, Ser 320 Phe change transmembrane 5: highly conserved region Cholelithiasis, ICP-mutation Vanishing bile duct syndroom

21 A new mechanism

22 BA NTCP BSEP Bile Acids MDR3 Phospholipids Cholesterol ABCG5/G8 LIVER BA FIC1? OSTαβ ASBT BA INTESTINE

23 FIC1 G308V mutants have unaffected bile salt output WT FIC1 * * * * *p<0.05 Paulusma ea Hepatology 2006;44:

24 Absence of FIC1 results in taurocholate-induced phosphatidylserine extraction from the canalicular membrane 100 *p<0.05 % of total lipid 10 * * Wildtype FIC1 mutant 1 PE PS PC SM lysopc Paulusma ea Hepatology 2006;44:

25 Taurocholate-induced biliary output of canalicular ectoenzymes is increased in FIC 1 mutants 2500 * *p< Output (% of wild type) * Wild type Atp8b1 mutant 0 ALP APN Paulusma ea Hepatology 2006;44:

26 FIC 1 PS Cell PS PS GGT GGT PS PC Chol Bile

27 FIC 1 Cell PS PS, Proteins (GGT) PS PC Chol Bile

28 FIC 1 PS BSEP PS PC Chol

29 Peroxisomes

30 Human and rat bile acid-coa:amino acid N-acyltransferase relative mrna level B hbaat 0 brain heart kidney liver lung sk. muscle pancreas placenta relative mrnalevel rbaat brain heart kidney liver lung sk. muscle spleen testis Human and rat bile acid-coa:amino acid N-acyltransferase are liver-specific peroxisomal enzymes; implications for intracellular bile salt transport Antonella Pellicoro1, Fiona van den Heuvel1, Mariska Geuken, Han Moshage1, Peter L.M. Jansen2 and Klaas Nico Faber1.

31 Native hbaat co-localizes with catalase BAAT Catalase Combined A B C D E F G H Human and rat bile acid-coa:amino acid N-acyltransferase are liver-specific peroxisomal enzymes; implications for intracellular bile salt transport Antonella Pellicoro1, Fiona van den Heuvel1, Mariska Geuken, Han Moshage1, Peter L.M. Jansen2 and Klaas Nico Faber1.

32 Acidic pathway Neutral pathway Endoplasmic Reticulum Re-activation unconjugated bile acids Peroxisome VLCA-CoAS* BAAT Glyco/Tauro conjugates Portal blood NTCP conjugated bile acids BSEP Bile

33 Familial hypercholanemia (FHC) Bile acid-coa:amino acid N-acyltransferase defect Symptoms: elevated serum bile acid concentrations itching fat malabsorption normal γgt (?) PFIC TYPE 5? IH: anti-hbaat antibodies

34 CONCLUSIONS 2 Genetic cholestatic diseases with well characterized genotypes and phenotypes: PFIC types 1, 2, 3, (4, 5?) MDR3 deficiency as cause of VBD? FUTURE How is BSEP function affected by FIC1 deletion? Role of MRP3, MRP4 and NHR genes as disease modifier genes in PSC, PBC, NAFLD, DILI Role of peroxisomes

35 Amsterdam Frank Schaap Vasilis Triantis Paula Jansen Marleen de Vree Ronal Oude Elferink Bert Groen Coen Paulusma International Richard Thompson Etienne Sokal Emmanuel Jacquemin Groningen Antonella Pellicoro Laura Conde de la Rosa Hans Blokzijl Jacqueline Plass Jenny Ros Tera Vos Han Moshage Klaas Nico Faber Michael Muller

36 Falk Symposium XX International Bile Acid Meeting June Amsterdam