ABCB4 p.Thr201Met
| Predicted by SNAP2: | A: D (59%), C: D (71%), D: D (80%), E: D (80%), F: D (85%), G: D (53%), H: D (71%), I: D (63%), K: D (85%), L: D (80%), M: N (66%), N: D (75%), P: D (85%), Q: N (57%), R: D (71%), S: N (72%), V: D (80%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, 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: N, V: D, W: D, Y: D, |
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[hide] Functional analysis of ABCB4 mutations relates cli... Gut. 2015 Jan;64(1):147-55. doi: 10.1136/gutjnl-2014-306896. Epub 2014 Mar 4. Gordo-Gilart R, Andueza S, Hierro L, Martinez-Fernandez P, D'Agostino D, Jara P, Alvarez L
Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity.
Gut. 2015 Jan;64(1):147-55. doi: 10.1136/gutjnl-2014-306896. Epub 2014 Mar 4., [PMID:24594635]
Abstract [show]
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.
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No. Sentence Comment
9 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.
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ABCB4 p.Thr201Met 24594635:9:132
status: NEW11 Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees.
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ABCB4 p.Thr201Met 24594635:11:49
status: NEW42 Plasmids and site-directed mutagenesis The plasmid pReceiver-M02-MDR3, containing the full open reading frame of ABCB4 (Capital Biosciences, Rockville, Maryland, USA) was used as a template for introducing the substitutions G68R, T201M, P352L, D459H, S978P and E1118K, by site-directed mutagenesis using the QuickChange II system (Agilent Technologies, Santa Clara, California, USA).
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ABCB4 p.Thr201Met 24594635:42:230
status: NEW110 Mutants T201M, P479 L, S978P and E1118 K exhibited surface expression comparable to that of wild-type MDR3.
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ABCB4 p.Thr201Met 24594635:110:8
status: NEW117 MDR3 was detected as a 140-150 kDa band, Table 2 ABCB4 mutations and MDR3 immunohistochemical analysis Mutation allele 1 Mutation allele 2 Patient n Nucleotide change Predicted effect Nucleotide change Predicted effect MDR3 staining 1 c.3559C>T p.R1187X c.3633+2 T>A Splicing defect ABSENT 2 c.202G>A p.G68R c.202G>A p.G68R ABSENT 3 c.202G>A p.G68R c.202G>A p.G68R NA 4 c.1375G>C p.D459H c.1436C>T p.P479L FAINT 5 c.602C>T p.T201M c.3352G>A p.E1118K NORMAL 6 c.2932T>C p.S978P ND NORMAL Immunostaining was carried out on liver biopsy specimens in patients 2-6 and in hepatectomy specimens in patient 1.
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ABCB4 p.Thr201Met 24594635:117:425
status: NEW121 (B) Normal canalicular staining in the tissue of patient 5, who is compound heterozygous for two missense mutations (T201M and E1118K).
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ABCB4 p.Thr201Met 24594635:121:117
status: NEW128 By contrast, expression levels of mutants G68R and D459H were markedly decreased (15% and 20% of wild-type, respectively), consistent with the fact that ER retention usually leads to a premature degradation of the proteins.32 T201M and P479L mutations also caused a significant reduction in the expression of MDR3 (60% and 65% of wild-type, respectively).
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ABCB4 p.Thr201Met 24594635:128:226
status: NEW138 The T201M, P479L and E1118K mutations reduced the release of labelled PC in the presence of the bile salt.
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ABCB4 p.Thr201Met 24594635:138:4
status: NEW140 At the time point of 3 h, the percentage of [3 H]-choline phospholipids effluxed in cells expressing mutants T201M, P479L and E1118K was 27%, 18% and 39% of that in cells expressing the wild-type protein, respectively (figure 4B).
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ABCB4 p.Thr201Met 24594635:140:109
status: NEW159 It seems likely that these mutations lead to misfolded proteins which, typically, exhibit abnormal trafficking and are prematurely degraded by the ER-associated protein degradation.32 This would raise the possibility that these mutants could be rescued to the cell surface by pharmacological chaperones, as has been shown for various other misfolded mutant membrane proteins.37 38 The T201M, P479L, S978P and E1118K mutations affect PC-translocating activity of MDR3, but to different extents.
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ABCB4 p.Thr201Met 24594635:159:385
status: NEW162 (A) Mock-transfected AD-293 cells (control) and cells transfected with wild-type ABCB4 and mutant T201M were stained for MDR3 and analysed by confocal immunofluoresce microscopy.
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ABCB4 p.Thr201Met 24594635:162:98
status: NEW186 binding and ATP hydrolysis.43 Of note, the equivalent residue in mouse Abcb1, S975, directly interacts with specific substrates, as revealed by crystallographic studies.41 Furthermore, a number of mutations within this transmembrane helix has been shown to dramatically affect ATPase activity of ABCB1.44 These observations concur with the complete loss of function of MDR3 caused by mutation S978P .
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ABCB4 p.Thr201Met 24594635:186:45
status: NEW187 PC-translocation activity of the MDR3 mutant T201M was 27% of reference activity for the wild-type MDR3.
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ABCB4 p.Thr201Met 24594635:187:45
status: NEW190 It must be taken into account that the T201M and P479L mutations also lowered the MDR3 protein levels, although the extent to which these levels were decreased did not parallel with the reductions in MDR3 activity obtained in the PC efflux assays.
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ABCB4 p.Thr201Met 24594635:190:39
status: NEW189 It must be taken into account that the T201M and P479L mutations also lowered the MDR3 protein levels, although the extent to which these levels were decreased did not parallel with the reductions in MDR3 activity obtained in the PC efflux assays.
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ABCB4 p.Thr201Met 24594635:189:39
status: NEW