ATP8B1 deficiency constitutes a potentially lethal form of intrahepatic cholestasis. We and others have previously identified many distinct mutations in ATP8B1.1, 3, 11, 17–19 Correlations between missense mutations and phenotypes of individual patients Dactolisib cost remained limited, because most mutations are confined to only few patients and because of the high variation in penetrance and clinical presentation. Furthermore, the molecular consequence of ATP8B1 mutations remained largely unexplored. Here, we combined protein expression and localization studies with homology modeling to demonstrate the effects of selected ATP8B1 mutations on the protein

level. These studies have high relevance for the patient population affected with ATP8B1 deficiency, because three of the selected mutations—p.G308V detected in Amish families, p.D554N in Greenland Inuit, and p.I661T in most European BRIC1 patients—are the most frequently identified mutations, together affecting the vast majority of

all patients characterized with ATP8B1 deficiency. Although our data do not fully explain the large variability in clinical presentation, they demonstrate that ATP8B1 deficiency presents as a protein folding disease, for a surprisingly large majority of the selected mutations. This conclusion is supported by several lines of evidence. First, with the exception of ATP8B1 L127P, all ATP8B1 mutants displayed significantly reduced protein ALK inhibitor expression, whereas mRNA expression was unaffected. Second, the recovery of ATP8B1 mutant expression upon MG132 and epoxomycin Calpain treatment indicates that increased proteasomal degradation is a common consequence of these ATP8B1

mutations. Third, incubation at reduced temperature has been demonstrated to restore proper folding of mutated proteins, and increased ATP8B1 mutant expression was observed when cells were cultured at 30°C. Fourth, most ATP8B1 mutants showed minimal plasma membrane localization. Instead, they were retained in the ER. Fifth, homology modeling predicted significant changes in the ATP8B1 structure due to the various mutations. In conclusion, in most cases, ATP8B1 deficiency is a consequence of protein misfolding, resulting in reduced expression at the plasma membrane. In vitro, a further reduction of ATP8B1 I661T protein abundance at the cell surface occurs when cells are cultured at 40°C. This may suggest that temporary decrease in ATP8B1 abundance at the plasma membrane could trigger the onset of a cholestatic episode in BRIC1 patients afflicted with the p.I661T mutation, because patients report that episodes are sometimes preceded by fever. Current treatment of ATP8B1 deficiency has major obstacles. Reduction of the (hydrophobic) bile salt pool using ursodeoxycholate or cholestyramine is only rarely effective.2 Surgical and/or endoscopic drainage of bile salts is more successful, but involves invasive procedures.