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Pharmacological Premature Termination Codon Readthrough of ABCB11 in Bile Salt Export Pump Deficiency: An In Vitro Study
Author(s) -
Amzal Rachida,
Thébaut Alice,
Lapalus Martine,
Almes Marion,
Grosse Brigitte,
Mareux Elodie,
ColladoHilly Mauricette,
DavitSpraul Anne,
Bidou Laure,
Namy Olivier,
Jacquemin Emmanuel,
Gonzales Emmanuel
Publication year - 2021
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.31476
Subject(s) - bile salt export pump , hek 293 cells , ursodeoxycholic acid , nonsense mutation , bile acid , biology , microbiology and biotechnology , progressive familial intrahepatic cholestasis , chemistry , biochemistry , phenotype , gene , missense mutation , medicine , transplantation , transporter , liver transplantation
Background and Aims Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X, and p.E1302X) identified in patients with PFIC2. Approach and Results The ability of G418, gentamicin, and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full‐length protein was studied in human embryonic kidney 293 (HEK293), HepG2, and Can 10 cells using immunodetection assays. The function of the gentamicin‐induced full‐length protein was studied by measuring the [ 3 H]‐taurocholate transcellular transport in stable Madin‐Darby canine kidney clones co‐expressing Na+‐taurocholate co‐transporting polypeptide (Ntcp). Combinations of gentamicin and chaperone drugs (ursodeoxycholic acid, 4‐phenylbutyrate [4‐PB]) were investigated. In NIH3T3, aminoglycosides significantly increased the readthrough level of all mutations studied, while PTC124 only slightly increased the readthrough of p.E1302X. Gentamicin induced a readthrough of p.R415X, p.R470X, p.R1057X, and p.R1090X in HEK293 cells. The resulting full‐length proteins localized within the cytoplasm, except for Bsep R1090X , which was also detected at the plasma membrane of human embryonic kidney HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4‐PB and ursodeoxycholic acid significantly increased the canalicular proportion of full‐length Bsep R1090X protein in Can 10 cells. In Madin‐Darby canine kidney clones, gentamicin induced a 40% increase of the Bsep R1090X [ 3 H]‐taurocholate transport, which was further increased with additional 4‐PB treatment. Conclusion This study constitutes a proof of concept for readthrough therapy in selected patients with PFIC2 with nonsense mutations.