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Eur J Hum Genet. 2014 May;22(5):633-9. doi: 10.1038/ejhg.2013.214. Epub 2013 Sep 18.

Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction.

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Medical Genetics Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy.
Department of Pediatrics, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
1] Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy [2] CEINGE, Advanced Biotechnologies scarl, Naples, Italy.
IRCCS Azienda Ospedaliera-Universitaria San Martino - IST. Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico & Fondazione D'Amico per la Ricerca sulle Malattie Renali, Milan, Italy.
CRIBENS-Laboratory of Cellular Biochemistry and Molecular Biology, Catholic University of the Sacred Heart, Milan, Italy.
Human Genetics Laboratory, EO Ospedali Galliera, Genoa, Italy.


The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

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