ABCB4 p.Asp459His
| Predicted by SNAP2: | A: D (71%), C: D (75%), E: D (71%), F: D (85%), G: D (80%), H: D (80%), I: D (85%), K: D (85%), L: D (85%), M: D (80%), N: N (72%), P: D (80%), Q: D (75%), R: D (85%), S: D (71%), T: D (80%), V: D (80%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: 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] 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.Asp459His 24594635:9:146
status: NEW10 G68R and D459H also led to retention of the protein in endoplasmic reticulum.
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ABCB4 p.Asp459His 24594635:10:9
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.Asp459His 24594635:42:244
status: NEW111 By contrast, mutations G68R and D459H resulted in intracellular retention of the protein, with predominant localisation in the endoplasmic reticulum (ER), as revealed by colocalisation with the ER marker calnexin (figure 2).
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ABCB4 p.Asp459His 24594635:111:32
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.Asp459His 24594635:117:382
status: NEW122 (C) Faint staining in patient 4, who is compound heterozygous for two missense mutations (D459H and P479L).
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ABCB4 p.Asp459His 24594635:122:90
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.Asp459His 24594635:128:51
status: NEW142 The same pattern was obtained with mutants G68R and D459H (data not shown), consistent with their absence of expression at the plasma membrane.
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ABCB4 p.Asp459His 24594635:142:52
status: NEW158 We found that changes G68R and D459H caused a marked reduction in MDR3 expression with an accompanying retention of the protein in the ER.
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ABCB4 p.Asp459His 24594635:158:31
status: NEW