ABCB4 p.Ala544Asp
| Predicted by SNAP2: | C: D (63%), D: D (85%), E: D (80%), F: D (80%), G: D (71%), H: D (75%), I: D (75%), K: D (80%), L: D (80%), M: D (66%), N: D (71%), P: D (85%), Q: D (71%), R: D (75%), S: D (53%), T: D (53%), V: D (75%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Heterozygous MDR3 missense mutation associated wit... Hum Mol Genet. 2000 May 1;9(8):1209-17. Dixon PH, Weerasekera N, Linton KJ, Donaldson O, Chambers J, Egginton E, Weaver J, Nelson-Piercy C, de Swiet M, Warnes G, Elias E, Higgins CF, Johnston DG, McCarthy MI, Williamson C
Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.
Hum Mol Genet. 2000 May 1;9(8):1209-17., 2000-05-01 [PMID:10767346]
Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.
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No. Sentence Comment
64 Mature A544D-P-gp1 at the cell surface is functional There are no established systems for quantification of PC translocation in mammalian systems making it currently not possible to study the function of the MDR3 protein.
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ABCB4 p.Ala544Asp 10767346:64:7
status: NEW90 R123 fluorescence (Fig. 3b, upper left quadrant) consistent with cell surface expression of A544D-P-gp1 and extrusion of R123.
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ABCB4 p.Ala544Asp 10767346:90:92
status: NEW91 Further confirmation that the extrusion of R123 in the A544D-P-gp1-expressing cells is due to the function of the mutant P-gp was obtained by incubating the cells with the P-gp1-inhibitor cyclosporin A.
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ABCB4 p.Ala544Asp 10767346:91:55
status: NEW93 These data are typical of cells expressing functional P-gp1 (28) and indicate that the A544D-P-gp1 mutant is functional if it reaches the cell membrane.
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ABCB4 p.Ala544Asp 10767346:93:87
status: NEW94 Evidence that A544D-P-gp1 is a trafficking mutant The finding that substitution of an aspartic acid for an alanine in a highly conserved region of NBD1 did not alter the ability of the mature protein to transport R123 was unexpected and suggested that the equivalent A546D-MDR3 protein mutant would also be functional.
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ABCB4 p.Ala544Asp 10767346:94:14
status: NEW136 In order to gain insight into the functional consequences of this mutation, we tested the effects of the equivalent mutation to A546D in P-gp1 (A544D).
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ABCB4 p.Ala544Asp 10767346:136:144
status: NEW137 Transient expression of A544D-P-gp1 in HEK293T cells, and subsequent FACS and western analysis suggested that this mutation reduces the abundance of the protein and impairs protein trafficking to the cell Figure 4.
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ABCB4 p.Ala544Asp 10767346:137:24
status: NEW138 UIC2-PE antibody labelling of intact HEK293T cells transiently transfected with pCIneo-βgal (grey intermittent line), pMDR1-wt (black) or pMDR1-A544D (grey).
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ABCB4 p.Ala544Asp 10767346:138:150
status: NEW140 The transfected populations were observed to consist of two separate populations (M1 and M2 for pMDR1-wt, M3 and M4 for pMDR1-A544D) with different amounts of protein at the cell surface.
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ABCB4 p.Ala544Asp 10767346:140:126
status: NEW142 Western analysis of HEK293T cells transiently transfected with pCIneo- βgal (-VE), pMDR1-A544D (A544D) and pMDR1-wt (WT).
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ABCB4 p.Ala544Asp 10767346:142:95
status: NEWX
ABCB4 p.Ala544Asp 10767346:142:102
status: NEW174 FACS analysis of cells transiently transfected with pMDR1-A544D (a), pMDR1-wt (b) or pCIneo-βgal (c).
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ABCB4 p.Ala544Asp 10767346:174:58
status: NEW204 The A544D mutation was introduced into NBD1 of P-gp1 by oligonucleotide-directed mutagenesis ('Altered sites` II; Promega) of pSBH.
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ABCB4 p.Ala544Asp 10767346:204:4
status: NEW206 The nucleotide sequence of the mutated DNA was verified by automated DNA sequencing as described above, prior to subcloning the mutated EcoRI-HindIII DNA fragment back into pMDR-wt to generate pMDR-A544D.
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ABCB4 p.Ala544Asp 10767346:206:198
status: NEW