ATP8B1 deficiency; Pathophysiology and treatment of a cholestatic syndrome with extrahepatic symptoms

ATP8B1 deficiency is a severe and clinically highly variable hereditary disorder that is primarily characterized by intrahepatic cholestasis. It generally presents as a permanent disorder, progressive familial intrahepatic cholestasis type 1 (PFIC1), or with intermittent cholestasis (benign recurrent intrahepatic cholestasis type 1 (BRIC1)). Currently there is no effective medical therapy, and most patients need invasive surgery such as partial biliary drainage (PBD) or liver transplantation. The exact pathogenesis of ATP8B1 deficiency remains largely unknown, which complicates the development of effective therapy. ATP8B1 is thought to act as an aminophospholipid flippase, maintaining an asymmetric distribution of aminophospholipids in the plasma membrane. This is essential for the resistance of the canalicular membrane to the detergent effect of bile salts and proper function of the transporters located at this site. While this may explain the cholestasis associated with ATP8B1 deficiency, the function of ATP8B1 in extrahepatic tissues remains enigmatic. Therefore the aim of this thesis was to study the pathophysiology of extrahepatic symptoms of ATP8B1 deficiency and to contribute to the development of non-surgical therapies. In the first part of this thesis we studied the role of ATP8B1 in the intestine and inner ear by using both cellular and animal models. We generated a loss-of-function model through stable transfection of short hairpin-RNA directed against ATP8B1 in the Caco-2 cell line, which resembles intestinal epithelial cells. This resulted in severely compromised bile salt absorption, which was probably due to decreased expression of the bile salt transporter ABST at the apical plasma membrane. Further analysis of this knockdown cell line by cDNA microarray studies, revealed that especially genes encoding plasma membrane proteins were differentially expressed, which points to a general disturbance in apical membrane formation. In addition, we tested hearing capacity in patients and mice with ATP8b1 deficiency using standard audiometry, and found significant hearing loss. The Atp8b1 protein was specifically localized in the stereocilia of the cochlear hair cells and the Atp8b1G308V/G308V mutation was associated with progressive degeneration of these hair cells. Here too a disturbance of the structural integrity of the apical membrane due to ATP8B1 deficiency seems to underlie an extrahepatic symptom. In the second part of the thesis we aimed to further develop effective therapy for ATP8B1 deficiency. We report a complete and long-lasting disappearance of pruritus and normalization of serum bile salt concentrations in cholestatic BRIC1 patients within 24 hours after endoscopic nasobiliary drainage (NBD). In addition, we investigated the molecular consequences of seven disease-causing ATP8B1 mutations in vitro. Five mutations resulted in (partial) retention of ATP8B1 in the endoplasmic reticulum and degradation, indicating protein folding defects. Because protein misfolding can potentially be ameliorated by pharmacological chaperones, we subsequently assessed the effect of 4-phenylbutyrate (4-PBA), a clinically approved pharmacological chaperone. Treatment with 4-PBA partially restored defects in expression and localization of ATP8B1 mutants and in particular of ATP8B1-I661T, the most frequently identified mutation in BRIC1. We therefore propose that treatment with pharmacological chaperones may represent an effective therapeutic strategy to ameliorate the recurrent attacks of cholestasis in BRIC1 patients