Background & Aim: The canalicular bile salt export pump (BSEP/ABCB11) of hepatocytes is the main adenosine triphosphate (ATP)-binding cassette (ABC) transporter responsible for bile acid secretion. Mutations in ABCB11 cause several cholestatic diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) often lethal in absence of liver transplantation. We investigated in vitro the effect and potential rescue of a BSEP mutation by ivacaftor, a clinically approved cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) potentiator. Methods: The p.T463I mutation, identified in a PFIC2 patient and located in a highly conserved ABC transporter motif, was studied by 3D structure modelling. The mutation was reproduced in a plasmid encoding a rat Bsep-green fluorescent protein. After transfection, mutant expression was studied in Can 10 cells. Taurocholate transport activity and ivacaftor effect were studied in Madin-Darby canine kidney (MDCK) clones co-expressing the rat sodium-taurocholate co-transporting polypeptide (Ntcp/Slc10A1). Results: As the wild-type protein, Bsep T463I was normally targeted to the canalicular membrane of Can 10 cells. As predicted by 3D structure modelling, taurocholate transport activity was dramatically low in MDCK clones expressing Bsep T463I. Ivacaftor treatment increased by 1.7-fold taurocholate transport activity of Bsep T463I (P < .0001), reaching 95% of Bsep wt activity. These data suggest that the p.T463I mutation impairs ATP-binding, resulting in Bsep dysfunction that can be rescued by ivacaftor. Conclusion: These results provide experimental evidence of ivacaftor therapeutic potential for selected patients with PFIC2 caused by ABCB11 missense mutations
Background: Cholestasis is a frequent and severe condition during childhood. Genetic cholestatic diseases represent up to 25% of pediatric cholestasis. Molecular analysis by targeted-capture next generation sequencing (NGS) has recently emerged as an efficient diagnostic tool. The objective of this study is to evaluate the use of NGS in children with cholestasis. Methods: Children presenting cholestasis were included between 2015 and 2020. Molecular sequencing was performed by targeted capture of a panel of 34 genes involved in cholestasis and jaundice. Patients were classified into three categories: certain diagnosis; suggested diagnosis (when genotype was consistent with phenotype for conditions without any available OMIM or ORPHANET-number); uncertain diagnosis (when clinical and para-clinical findings were not consistent enough with molecular findings). Results: A certain diagnosis was established in 169 patients among the 602 included (28.1%). Molecular studies led to a suggested diagnosis in 40 patients (6.6%) and to an uncertain diagnosis in 21 patients (3.5%). In 372 children (61.7%), no molecular defect was identified. Conclusions: NGS is a useful diagnostic tool in pediatric cholestasis, providing a certain diagnosis in 28.1% of the patients included in this study. In the remaining patients, especially those with variants of uncertain significance, the imputability of the variants requires further investigations.
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