Volume 135, Issue 1, Pages 131-141 (July 2008) ABCB4 Heterozygous Gene Mutations Associated With Fibrosing Cholestatic Liver Disease in Adults  Marianne Ziol, Véronique Barbu, Olivier Rosmorduc, Annonciade Frassati–Biaggi, Nathalie Barget, Brigitte Hermelin, Georges L. Scheffer, Selma Bennouna, Jean–Claude Trinchet, Michel Beaugrand, Nathalie Ganne–Carrié  Gastroenterology  Volume 135, Issue 1, Pages 131-141 (July 2008) DOI: 10.1053/j.gastro.2008.03.044 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Summary of the mutations identified in our patients with unexplained cholestasis and of the known mutations described in patients with progressive familial intrahepatic cholestasis type 3 (PFIC3), low phospholipid associated cholelithiasis (LPAC) syndrome, and intrahepatic cholestasis of pregnancy (ICP). The frequency of each of the mutations found in our study is indicated in circles. The new mutations found in our patients are mentioned in bold. Other indicated mutations have been described in references de Vree et al,5 Jacquemin et al,6 Rosmorduc et al,7,8 Gendrot et al,10 Dixon et al,11 Pauli–Magnus et al,12 Floreani et al,13 and Keitel et al.21 (Modified from Oude Elferink et al.20) Gastroenterology 2008 135, 131-141DOI: (10.1053/j.gastro.2008.03.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Evolution of serum γ-glutamyl transferase during ursodesoxycholic acid treatment (15 mg/kg/day) in patients with ABCB4 heterozygous disease-associated mutation. Despite a good compliance to treatment, cases 4 and 7 had recurrent cholelithiasis symptoms related to intrahepatic microlithiasis a few months after cholecystectomy. Evolution of serum alkaline phosphatases during treatment was similar to serum gammaglutamyl transferase (data not shown). Gastroenterology 2008 135, 131-141DOI: (10.1053/j.gastro.2008.03.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Histologic features representative of the abnormalities observed in patients with ABCB4 mutations. (A) Portal tracts were enlarged (F2 stage according to the Ishak scoring system) with accumulation of collagen (b) associated with ductular proliferation evidenced on cytokeratin 19 immunostaining (c). Portal tracts inflammatory infiltrate was moderate, and neither interlobular destructive bile duct lesions nor periportal or lobular necroinflammatory lesions were observed (a). Case 8, serial sections, hematoxylin, eosin, and saffron (a); picrosirius red (b); cytokeratin 19 (c); original magnification, ×5. (B) Activation of portal myofibroblasts and macrophage infiltration of portal tracts in patients with ABCB4 mutations. (a) Most portal tracts displayed positive cells for α-smooth muscle cell actin. Activated portal myofibroblast surrounded reactive ductules. No peculiar distribution around the interlobular bile duct was observed. (b) Portal tracts infiltrate was predominantly made of macrophages evidenced on CD68 immunostaining. Exocytosis of macrophages through an interlobular bile duct was sometimes detected (c). (C) In case 2, the shape of a cholesterol crystal (arrow) was observed on 1 section of the paraffin-embedded liver biopsy sample. The cholesterol crystal appears to be engulfed by a macrophage infiltrating the wall of the interlobular bile duct. H&E staining. Original magnification, ×100. (D) Fibrosis progression in ABCB4 mutations. Case 7 underwent a first liver biopsy for unexplained anicteric cholestasis when he was 28 years old. Fibrous expansion of most portal tracts with short fibrous septa (F2 according to Ishak scoring system17) was observed (a). Six years later, recurrence of abnormal liver enzymes after cholecystectomy leads to a second liver biopsy. Portal fibrosis had significantly progressed to bridging fibrosis (F4 according to the Ishak scoring system) associated with perisinusoidal fibrosis (b). Picrosirius red staining; original magnification, ×5. Gastroenterology 2008 135, 131-141DOI: (10.1053/j.gastro.2008.03.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Immunohistochemical detection of MDR3 in patients with anicteric cholestasis. MDR3 immune reactivity strongly outlines, as continuous pattern, the bile canaliculi network in patients without ABCB4 mutations (a–d). In contrast, case 7 (e) with both missense ABCB4 heterozygous mutation and nonsynonymous polymorphism did not disclose any staining. Cases 5 (f), 7 (g), 9 (h), and 11 (e) with missense heterozygous mutation showed a faint and discontinuous staining compared with controls. For positive control of the antigenic preservation of the tissue, immunostaining with another member of ABC transporters (MRP2-clone M2III-6 encoded by ABCC2) showed a strong canalicular staining (insets in e, f, g, h). a–h: MDR3 (P3II-26) immunostaining, original magnification, ×1000; insets in e, f, g, and h: MRP2 (M2III-6) immunostaining, original magnification, ×500. Gastroenterology 2008 135, 131-141DOI: (10.1053/j.gastro.2008.03.044) Copyright © 2008 AGA Institute Terms and Conditions