Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.

Heterozygous ABCB4 mutations in children with cholestatic liver disease.

BACKGROUND & AIMS: Monoallelic defects in ABCB4, which encodes the canalicular floppase for phosphatidylcholine MDR3, have been encountered in association with a variety of hepatobiliary disorders, particularly in adult subjects. In this study, we examined the presence of heterozygous ABCB4 variants in a cohort of children with chronic cholestasis and assessed the pathogenicity of the missense changes identified. METHODS: Sixty-seven children with chronic liver dysfunction were studied by the sequencing of ABCB4 and multiplex ligation-dependent probe amplification analysis. The molecular defects arising from missense variants were analysed in MDCK-II and AD-293 cells. RESULTS: Defects in a single allele of ABCB4 were identified in nine subjects. They included one small insertion (p.I1242Nfs), one nonsense mutation (p.R144X) and six missense changes (p.T175A, p.G228R, p.A250T, p.S320F, p.P352L and p.A934T). In four children, these defects in ABCB4 co-existed with various medical conditions. In vitro phenotyping of the six missense variants revealed that four (T175A, G228R, S320F and A934T) led to reduced MDR3 protein levels. Two mutations (G228R and A934T) resulted in trapping of the protein in the endoplasmic reticulum. Phosphatidylcholine efflux activity was decreased to 56-18% of reference levels for MDR3 mutants T175A, A250T and S320F. The G228R, P352L and A934T mutants were found to be non-functional. CONCLUSIONS: These results illustrate the varying effects of ABCB4 missense mutations and suggest that even a modest reduction in MDR3 activity may contribute or predispose to the onset of cholestatic liver disease in the paediatric age.

Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity.

OBJECTIVE: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a potentially lethal autosomal recessive liver disease associated with mutations in ABCB4, the gene encoding the canalicular translocator of phosphatidylcholine MDR3. While some affected children benefit from ursodeoxycholic acid (UDCA) therapy, others evolve to end-stage liver disease. We aimed to evaluate whether these different outcomes are related to the impact of ABCB4 mutations. DESIGN: Six children with PFIC3 were investigated by sequencing of ABCB4 exons and flanking intron-exon boundaries and by immunohistochemistry. ABCB4 missense mutations were phenotyped in vitro by assessing their effects on MDR3 expression, subcellular localisation, and phosphatidylcholine-translocating activity. The resulting data were contrasted with the clinical outcomes. RESULTS: Eight distinct ABCB4 mutations were identified: one nonsense, one splicing and six missense mutations, four of which (G68R, T201M, P479L, D459H) affected MDR3 expression level. G68R and D459H also led to retention of the protein in endoplasmic reticulum. Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees. Three children with mutations that caused a total loss of MDR3 expression/function manifested progressive liver disease refractory to UDCA treatment. This was also the case in a patient carrying two different mutations that, in combination, resulted in a 90% reduction in total MDR3 activity. A favourable response to UDCA was achieved in two patients with estimated MDR3 activities of 50% and 33%, respectively. CONCLUSIONS: These data provide experimental evidence of the correlation between the degree of MDR3 floppase activity and the clinical outcomes of PFIC3.

LPAC syndrome associated with deletion of the full exon 4 in a ABCB4 genetic mutation in a patient with hepatitis C.

Low-phospholipid-associated cholelithiasis syndrome (LPAC) is associated with ABCB4 genetic mutation. ABCB4 encodes MDR3 protein, involved in biliary phosphatidylcholine excretion.Higher prevalence in women, biliary symptoms in young adults and ursodesoxycholic acid (UDCA) response are the main features. We report the case of a 48-year-old man with hepatitis C, genotype 1b, fibrosis F3, null responder to Peg-IFN-alpha-2b/ribavirin and nephritic colic. In 2011 he developed jaundice, pruritus and epigastric pain.He showed increased serum levels of AST, ALT, GGT, bilirubin and alpha-fetoprotein, and viral load (14,600,000 IU/mL). Pancreatic- CT, endoscopic ultrasonography and echo-Doppler showed noncirrhotic chronic liver disease. The episode resolved spontaneously and one year later he suffered a similar episode. UDCA was started with excellent response. An immunohistochemistry study and sequencing of ABCB4 did not find alteration. MLPA® technique detected heterozygous deletion of the full exon 4 confirming LPAC syndrome diagnosis.