Z Gastroenterol 2022; 60(01): e3
DOI: 10.1055/s-0041-1740653
Abstracts | GASL

In vitro and in silico characterization of a novel NR1H4/FXR mutation causing Progressive Familial Intrahepatic Cholestasis Type 5

Jan Stindt
1   Clinic for Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany
,
Annika Behrendt
2   Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Düsseldorf, Germany
,
Amelie Stalke
3   Department of Human Genetics, Hannover Medical School, Hannover, Germany; Department for Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany
,
Carola Dröge
1   Clinic for Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany
,
Eva-Doreen Pfister
4   Department for Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany
,
Ulrich Baumann
4   Department for Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany
,
Holger Gohlke
2   Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Düsseldorf, Germany
,
Tom Lüdde
1   Clinic for Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany
,
Verena Keitel-Anselmino
5   Clinic for Gastroenterology, Hepatology and Infectious Diseases, University Hospital Magdeburg, Otto von Guericke University, Magdeburg, Germany
› Author Affiliations
› Further Information
Also available ateRef
 
 

    Background Mutations in NR1H4/FXR underlie Progressive Familial Intrahepatic Cholestasis Type 5 (PFIC5). FXR is a member of the nuclear receptor family that, heterodimerized with RXRα, transactivates the ABCB11/BSEP promoter. PFIC5-associated NR1H4 mutations result in reduced or absent BSEP expression. Consequently, PFIC5 is a rare, low-γGT intrahepatic cholestasis to be considered in absence of disease-causing ATP8B1/FIC1 and ABCB11/BSEP mutations. Here, we describe and characterize a novel NR1H4/FXR mutation causing PFIC5 in a patient.

    Methods NR1H4 cDNA obtained from human liver was cloned into a mammalian expression plasmid and the mutation introduced via site-directed mutagenesis. Expression and localization of FXR was analyzed via immunofluorescence (IF) and western blot (WB). HEK293 cells were transfected with an ABCB11-promoter-driven luciferase reporter and expression constructs for RXRα and FXR variants, then stimulated with 9-cis-retinoic acid and obeticholic acid before used in luciferase assays. Impact of the mutation on FXR function was investigated by in silico structural analysis.

    Results and Conclusion Since our patient had normal γGT, strongly elevated serum bile acids and absent BSEP expression, yet no ABCB11 or ATP8B1 mutation, NR1H4 was sequenced yielding the homozygous c.887C>T;p.(T296I) mutation. T296I was introduced into both liver-relevant FXR isoforms (α1/α2). By IF, both wildtype and mutant FXR were localized to the nucleus in stimulated HEK293 cells co-transfected with RXRα. WB demonstrated similar expression levels of WT and T296I. However, T296I failed to induce substantial ABCB11 promoter transactivation. Preliminary in silico analysis suggested altered interaction of the mutated side chain with the α-helix-forming part of the activation function.

    Lecture Session III Metabolism (incl. NAFLD)

    28/01/2022, 17.35 pm – 18.20 pm, Lecture Hall


     

    Publication History

    Article published online:
    26 January 2022

    © 2022. Thieme. All rights reserved.

    Georg Thieme Verlag
    Rüdigerstraße 14, 70469 Stuttgart, Germany