ABCB4 p.Ser978Pro
| Predicted by SNAP2: | A: N (57%), C: D (53%), D: D (80%), E: N (57%), F: N (61%), G: N (61%), H: D (71%), I: D (63%), K: D (63%), L: N (57%), M: N (72%), N: N (78%), P: D (80%), Q: D (59%), R: D (75%), T: N (82%), V: D (66%), W: D (80%), Y: D (53%), |
| Predicted by PROVEAN: | A: N, 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, T: 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
11 Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees.
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ABCB4 p.Ser978Pro 24594635:11:63
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.Ser978Pro 24594635:42:251
status: NEW79 Two of these variants, P479L and S978P , have been described previously in a child with PFIC319 and in an adult patient with cholangiocarcinoma,13 respectively; the other six mutations are novel.
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ABCB4 p.Ser978Pro 24594635:79:33
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.Ser978Pro 24594635:110:23
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.Ser978Pro 24594635:117:471
status: NEW127 The changes S978P and E1118 K displayed protein levels comparable to the wild-type MDR3 (figure 3C).
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ABCB4 p.Ser978Pro 24594635:127:12
status: NEW141 As mock-transfected cells, cells transfected with the S978P mutant yielded identical curves of radioactivity release in the presence or absence of NaTC, thus suggesting that this mutation completely abolishes MDR3 activity (figure 4A).
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ABCB4 p.Ser978Pro 24594635:141:54
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.Ser978Pro 24594635:159:399
status: NEW175 Along these lines, it has recently been reported that the mutation of another amino acid at the Q-loop of the NBD1 of MDR3, L481R, lowers MDR3 activity.26 The S978P mutation completely abrogated the PC translocation capacity of MDR3.
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ABCB4 p.Ser978Pro 24594635:175:159
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.Ser978Pro 24594635:186:393
status: NEW197 In patient 6, a single heterozygous mutation, S978P , was detected despite extensive analysis by sequencing and MLPA.
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ABCB4 p.Ser978Pro 24594635:197:46
status: NEW199 Indeed, the presence of the deleterious S978P mutation alone is not expected to trigger a progressive form of cholestatic liver disease in this patient, and a 'second hit` leading to hepatocellular damage can be suspected.
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ABCB4 p.Ser978Pro 24594635:199:40
status: NEW174 Along these lines, it has recently been reported that the mutation of another amino acid at the Q-loop of the NBD1 of MDR3, L481R, lowers MDR3 activity.26 The S978P mutation completely abrogated the PC translocation capacity of MDR3.
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ABCB4 p.Ser978Pro 24594635:174:159
status: NEW185 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.Ser978Pro 24594635:185:393
status: NEW196 In patient 6, a single heterozygous mutation, S978P , was detected despite extensive analysis by sequencing and MLPA.
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ABCB4 p.Ser978Pro 24594635:196:46
status: NEW198 Indeed, the presence of the deleterious S978P mutation alone is not expected to trigger a progressive form of cholestatic liver disease in this patient, and a 'second hit` leading to hepatocellular damage can be suspected.
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ABCB4 p.Ser978Pro 24594635:198:40
status: NEW