Clinical Features and Genotype‐Phenotype Correlations in Children With Progressive Familial Intrahepatic Cholestasis Type 3 Related to <i>ABCB4</i> Mutations

Colombo, Carla; Vajro, Pietro; Degiorgio, D.; Coviello, Domenico; Costantino, Lucy; Tornillo, Luigi; Motta, Valentina; Consonni, Dario; Maggiore, Giuseppe

doi:10.1097/mpg.0b013e3181f50363

Cited by 64 publications

(60 citation statements)

References 31 publications

(45 reference statements)

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“…In this study, besides reporting the identification of a novel ABCB4 mutation (p.(L481R)) within the Q-loop in three siblings suffering from juvenile cholelithiasis, we demonstrate that this mutation, stably transfected in HUH28 cells, as well as the mutation p.(Y403H), previously described by our group, 14 do not prevent protein targeting to the plasma membrane but induce an abnormal efflux of PC and Chol.…”

Section: Discussionsupporting

confidence: 60%

“…9 The pathogenetic potential of both mutations is suggested by segregation analysis. The p.(L481R) mutation was identified in three out of seven affected siblings belonging to family A, whereas the already characterized p.(Y403H) mutation 6,14 was identified in four affected members belonging to other two families. According to what is generally described, [13][14][15] the genotypes reported here show that (i) two mutant ABCB4 alleles are associated with severe early-onset of intrahepatic cholestasis characterized by a rapid course toward terminal liver failure.…”

Section: Discussionmentioning

confidence: 89%

“…The nucleotide change c.1207T4C that encodes the mutant protein characterized by p.(Y403H) was previously described at homozygous state in the child of family B and at heterozygous state in the other members of family B and C. 14 To evaluate the expression of MDR3 in vitro, we used two plasmids: one containing the full-length cDNA of ABCB4 wild type on a pcDNA3.1/ Hygro( þ ) backbone (pcDNA3.1/Hygro( þ )/ABCB4) and the second containing the b-galactosidase gene (pcDNA3.1.V5/His-lacZ). They were kindly provided by Professor K Ueda (Graduate School of Agriculture, Kyoto University, Kyoto, Japan) and Dr I Garagiola (Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca 0 Granda, Milan, Italy), respectively.…”

Section: Materials and Methods Subjectsmentioning

confidence: 95%

“…Families B and C have been described previously. 14 Genetic analysis, DNA cloning and site-directed mutagenesis Genomic DNA samples were isolated from blood lymphocytes and mutation analysis was performed as previously described; 6 the NCBI transcript sequence used as reference to describe our nucleotide changes was the ABCB4 isoform A, NM_000443. To exclude that the new nucleotide change identified in family A was a nonpathogenetic polymorphism, we searched this variant in 224 chromosomes of 112 healthy Caucasians 14 and in the NCBI-SNP database (http://www.ncbi.nlm.nih.gov/SNP).…”

Section: Materials and Methods Subjectsmentioning

confidence: 99%

“…10 In the following years, a growing number of ABCB4 mutations have been identified also in adult subjects affected by juvenile cholelithiasis, 11 drug-induced cholestasis 12 and idiopathic cholestasis of the adulthood. 13 Recently, our group highlighted that the simple heterozygous ABCB4 genotype could be a discrete risk factor for a wide range of hepatobiliary diseases, including the PFIC-3 phenotype with variable disease expressivity, 14 primary biliary cirrhosis and primary sclerosing cholangitis (Crosignani A, personal communication). To date, 146 disease-associated ABCB4 mutations are reported in the Human Genome Mutation Database (http://www.hgmd.org/; release date 14 December 2012); of them, 87 (about 60%) are predicted to substitute a single amino acid and 30 of these (about 34%) fall within the N-terminal NBD.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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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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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 89%

Section: Materials and Methods Subjectsmentioning

confidence: 95%

Section: Materials and Methods Subjectsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4

et al. 2014

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1ABCB4 flops phosphatidylcholine into the bile canaliculus to protect the biliary tree from the detergent activity of bile salts. Homozygous-null ABCB4 mutations cause the childhood liver disease, progressive familial intrahepatic cholestasis, but cause and effect is less clear, with many missense mutations linked to less severe cholestatic diseases. ABCB4 S320F , in particular, is described in 13 patients, including in heterozygosity with ABCB4 A286V , ABCB4 A953D, and null mutants, whose symptoms cover the spectrum of cholestatic disease. We sought to define the impact of these mutations on the floppase, explain the link with multiple conditions at the molecular level, and investigate the potential for reversal. ABCB4 S320F , ABCB4 A286V, and ABCB4 A953D expression was engineered in na€ ıve cultured cells. Floppase expression, localization, and activity were measured by western blot, confocal microscopy, and lipid transport assays, respectively. ABCB4 S320F was fully active for floppase activity but expression at the plasma membrane was reduced to 50%. ABC-B4 A286V expressed and trafficked efficiently but could not flop lipid, and ABCB4 A953D expressed poorly and was impaired in floppase activity. Proteasome inhibition stabilized nascent ABCB4 S320F and ABCB4 A953D but did not improve plasma membrane localization. Cyclosporin-A improved plasma membrane localization of both ABCB4 S320F and ABCB4 A953D

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Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations

et al. 2014

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The ABCB4 transporter mediates phosphatidylcholine (PC) secretion at the canalicular membrane of hepatocytes and its genetic defects cause biliary diseases. Whereas ABCB4 shares high sequence identity with the multidrug transporter, ABCB1, its N-terminal domain is poorly conserved, leading us to hypothesize a functional specificity of this domain. A database of ABCB4 genotyping in a large series of patients was screened for variations altering residues of the N-terminal domain. Identified variants were then expressed in cell models to investigate their biological consequences. Two missense variations, T34M and R47G, were identified in patients with low-phospholipid-associated cholelithiasis or intrahepatic cholestasis of pregnancy. The T34M and R47G mutated proteins showed no or minor defect, respectively, in maturation and targeting to the apical membrane, in polarized Madin-Darby Canine Kidney and HepG2 cells, whereas their stability was similar to that of wild-type (WT) ABCB4. By contrast, the PC secretion activity of both mutants was markedly decreased. In silico analysis indicated that the identified variants were likely to affect ABCB4 phosphorylation. Mass spectrometry analyses confirmed that the N-terminal domain of WT ABCB4 could undergo phosphorylation in vitro and revealed that the T34M and R47G mutations impaired such phosphorylation. ABCB4-mediated PC secretion was also increased by pharmacological activation of protein kinases A or C and decreased by inhibition of these kinases. Furthermore, secretion activity of the T34M and R47G mutants was less responsive than that of WT ABCB4 to protein kinase modulators. Conclusion: We identified diseaseassociated variants of ABCB4 involved in the phosphorylation of its N-terminal domain and leading to decreased PC secretion. Our results also indicate that ABCB4 activity is regulated by phosphorylation, in particular, of N-terminal residues. (HEPATOLOGY 2014;60:610-621)

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Clinical Features and Genotype‐Phenotype Correlations in Children With Progressive Familial Intrahepatic Cholestasis Type 3 Related to ABCB4 Mutations

Cited by 64 publications

References 31 publications

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

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

Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4

Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations

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