ABCMdb

PMID: 26256905

Frider B, Castillo A, Gordo-Gilart R, Bruno A, Amante M, Alvarez L, Mathet V
Reversal of advanced fibrosis after long-term ursodeoxycholic acid therapy in a patient with residual expression of MDR3.
Ann Hepatol. 2015 Sep-Oct;14(5):745-51., [PubMed]

Sentences

No. Mutations Sentence Comment

71 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn Site-directed mutagenesis The substitutions R47Q and T82N were introduced into the plasmid pReceiver-M02-MDR3 (Capital Biosciences, Rockville, MD, USA), which contains the full open reading frame of ABCB4, by site-directed mutagenesis using the QuickChange II system (Stratagene, La Jolla, CA, USA). Login to comment 72 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn Primers used for mutagenesis were as follows (top strand shown; mutated nucleotides are in lowercase): R47Q, 5`-CATTGTT- TCaATACTCCGATTGGC-3`; T82N, 5`-GGAGAGAT- GAaTGACAAATTTG-3`. Login to comment 110 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn Effects of R47Q and T82N mutations on MDR3 subcellular localization and expression. Login to comment 120 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn Genetic analysis Sequence analysis revealed that P1 was a compound heterozygote for two missense mutations in exon 4 of ABCB4: c.140G > A and c.245C > A, (p.R47Q and p.T82N respectively; the first of them located in a cytoplasmic domain and the latter in an extracellular domain). Login to comment 122 ABCB4 p.Thr82Asn Genetic analysis of P2 showed a missense mutation in ABCB4 shared with her nephew, c.245C>A (p.T82N). Login to comment 123 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn In vitro studies of ABCB4 mutations In order to determine whether these substitutions affected subcellular localization or expression of MDR3, MDCKII and HEK293T cells were transfected with expression vectors containing wild-type or the mutated versions of MDR3 (R47Q and T82N) and were analyzed by either immunofluorescence or Western blot. Login to comment 127 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn Thus, R47Q and T82N mutations do not alter MDR3 localization, but lead to reduced protein levels. Login to comment 129 ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn In this disorder, the severity of cholestasis, the time of presentation and the favourable response to UDCA therapy seems to depend on the degree of penetrance of the genetic defect.4,6 The phenotypic characterization of the mutations found in our patient confirms this assumption, since R47Q and T82N do not impair the canalicular expression of MDR3, but they result in a dramatic reduction in the levels of the protein, a finding that correlates with data from the immunohistochemical analysis of the liver biopsy. Login to comment 131 ABCB4 p.Arg47Gln ABCB4 p.Arg47Gln ABCB4 p.Thr82Asn ABCB4 p.Thr82Asn ABCB4 p.Arg47Gly However, it was reported that mutations at R47 impair phosphorylation of N-terminal domain of MDR3, which is determinant for PC secretion.14 It was reported that R47G mutated protein has similar localization and stability to wild-type protein, but PC secretion activity B A KDa 200 150 100 MDR3 Na/K-ATPase Mock WT R47Q T82N Wild-type R47Q T82N resulted markedly decreased because of this lack of phosphorylation of neighboring residues, either Thr44 or Ser49. Login to comment 132 ABCB4 p.Thr82Asn The finding of the mutation T82N in the affected aunt with PFIC3 is a strong evidence of the association of this mutation with the cholestatic disease. Login to comment