ABCMdb

ABCB11 p.Glu297Gly

None has been submitted yet.

Publications

PMID: 11745042 [PubMed] Thompson R et al: "BSEP: function and role in progressive familial intrahepatic cholestasis."

No. Sentence Comment

91 Two mutations, E297G and D482G, are "common" and have been found in 25 and 16 European families, respectively. Login to comment

PMID: 12663868 [PubMed] Trauner M et al: "Bile salt transporters: molecular characterization, function, and regulation."

No. Sentence Comment

631 G238V, E297G, G982R, R1153C, and R1268Q mutations prevent the protein from trafficking to the apical membrane, whereas the G238V mutant seems to be rapidly degraded by proteasomes. Login to comment

PMID: 15791618 [PubMed] Hayashi H et al: "Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11."

No. Sentence Comment

1 However, the mechanisms for the deficiency in the function of two mutations (E297G and D482G), which are frequently found in European patients, have not yet been identified. Login to comment
3 Introduction of E297G and D482G mutations into the human BSEP gene by site-directed mutagenesis resulted in a significant reduction in the BSEP expression level, which was associated with impaired membrane trafficking. Login to comment
4 Most of the D482G BSEP and some of the E297G BSEP underwent only core glycosylation and appeared to be predominantly located in the endoplasmic reticulum. Login to comment
7 In conclusion, E297G and D482G mutations result in impaired membrane trafficking, whereas the transport functions of these mutants remain largely unchanged. Login to comment
9 T he efficient biliary excretion of monovalent bile acids is mediated by the bile salt export pump (BSEP/ABCB11), an ATP-binding cassette transmembrane transporter located on the bile canalicular membrane.1 The function of BSEP/ABCB11 has been clarified by examining the adenosine triphosphate (ATP)- dependent transport of monovalent bile acids (such as taurocholic acid) in isolated bile canalicular membrane vesicles and/or membrane vesicles isolated from cells transfected with the complementary DNA (cDNA) for BSEP.2,3 Many studies have also been performed in patients and it has been shown that the hereditary defect in the expression of BSEP results in the acquisition of progressive familial intrahepatic cholestasis type 2 (PFIC2).4,5 Genomic analysis of PFIC2 patients has revealed that many kinds of missense, premature termination, frame shift, and splicing junction mutations are associated with the BSEP gene.4 Among these, E297G and D482G, two missense mutations in the second intracellular loop and in the first ATP-binding domain, respectively, are frequently observed in PFIC2 patients. Login to comment
21 916 was shown that the introduction of some mutations in the consensus region (such as E297G) into the rat and mouse Bsep genes resulted in altered cellular distribution of the Bsep. Login to comment
28 We focused particularly on E297G and D482G mutations, which are frequently found in European PFIC2 patients.6 Materials and Methods Materials. Login to comment
44 For the determination of BSEP messenger RNA (mRNA) levels, MDCK II cells were seeded 24 hours before infection at a density of 1.3 ϫ 106 cells per 10-cm dish and were infected with recombinant adenoviruses containing wild-type, E297G, and D482G BSEP cDNA at a multiplicity of infection (MOI) of 50. Login to comment
67 To determine the localization of BSEP, MDCK II cells were seeded on glass coverslips 24 hours before infection at a density of 3 ϫ 105 cells/well in 12-well plates and were infected with recombinant adenoviruses at 25 MOI (wild-type and E297G BSEP) or 250 MOI (D482G BSEP). Login to comment
74 To examine the cellular localization and transport function of the two mutants (E297G and D482G), we constructed recombinant adenoviruses containing wild-type and mutated BSEP cDNA. Login to comment
88 In addition, E297G BSEP was detected as approximately 170 kDa and approximately 150 kDa bands. Login to comment
90 These results suggest that D482G BSEP and some of the E297G BSEP molecules are present as immature endoplasmic reticulum (ER)-resident forms. Login to comment
95 E297G BSEP was located in both the intracellular compartment and apical membrane at 25 MOI (see Fig. 3A). Login to comment
99 Cell surface BSEP was detected in wild-type and E297G-expressing MDCK II cells as the mature form, whereas no D482G BSEP molecules were detectable on the cell surface (Fig. 3B). Login to comment
102 It has been reported that proteasomes play an important role in the degradation of incompletely folded and misfolded protein retained in the ER.17,18 Because the expression levels of E297G and D482G BSEP were lower than that of the wild-type BSEP-and because it is possible that these mutants are localized in the ER-proteasomes may be responsible for their degradation. Login to comment
103 To examine this hypothesis, cells were treated with 5 ␮mol/L MG132, a specific proteasome inhibitor, for 8 hours, and its effects on E297G and D482G BSEP were examined via Western blot anasysis. Login to comment
104 Western blot analysis showed that MG132 treatment caused the accumulation of immature forms (150 kDa), particularly in E297G and D482G BSEP-expressing MDCK II cells (Fig. 4). Login to comment
106 It was found that MG132 treatment resulted in an increase in the number of wild-type, E297G, and D482G BSEP-expressing cells, and the degree of increase was particularly high for the latter two; in the absence of MG132, the number of E297G BSEP-expressing cells after adenovirus infection was only approximately 25% of those expressing wild-type BSEP, and only a minimal number of cells expressed D482G BSEP. Login to comment
107 In contrast, in the presence of MG132, the number of cells expressing E297G and D482G BSEP was almost the same as that in wild-type BSEP-expressing cells after infection of adenoviruses at the same MOI (data not shown). Login to comment
108 In the presence of MG132, E297G and D482G BSEP were predominantly expressed in the intracellular compartment, which is the same as that observed under control conditions (see Fig. 3A). Login to comment
115 (A) Results of Western blot analysis with crude membrane fraction prepared from MDCK II cells. MDCK II cells were infected with recombinant adrenoviruses at 250 MOI 48 hours before the experiments. Crude membrane fractions prepared from GFP (control), wild-type, E297G, and D482G BSEP-expressing MDCK II cells (80, 40, 80, and 80 ␮g protein, respectively) were analyzed. Login to comment
120 Before the transport experiments, the expression level of BSEP was compared among the isolated membrane vesicles expressing wild-type, E297G, and D482G BSEP. Login to comment
121 D482G BSEP, as well as wild-type and E297G BSEP, were detected as approximately 170-kDa bands in isolated membrane vesicles (Fig. 5A). Login to comment
122 The band density of the 170-kDa form of E297G and D482G in the isolated membrane vesicles was approximately 25% and 10% of wild-type BSEP, respectively (Fig. 5B). Login to comment
124 The ATP-dependent uptake of taurocholate and glycocholate by wild-type, E297G and D482G BSEP-expressing isolated membrane vesicles was much higher than that by GFP-expressing vesicles (Fig. 6A). Login to comment
125 By normalizing the BSEP expression levels in the isolated membrane vesicles based on the results of Western blot analysis (see Fig. 5B), it was demonstrated that the transport of taurocholate and glycocholate mediated per unit mass of E297G and D482G BSEP molecules was not significantly different from that by wild-type BSEP (Figs. 5B and 6B). Login to comment
127 Wild-type, E297G, and D482G BSEP-mediated initial ATP-dependent uptake rates were saturable with apparent Km values of 4.61 Ϯ 0.91, 5.41 Ϯ 0.27, and 14.3 Ϯ 2.0 ␮mol/L, respectively (Fig. 6C). Login to comment
128 The maximum taurocholate transport velocity (Vmax) for wild-type, E297G, and D482G BSEP were 2310 Ϯ 220, 485 Ϯ 15, and 761 Ϯ 60 pmol/min/mg isolated membrane vesicle protein, respectively. Login to comment
132 MDCK II cells were infected with the recombinant adenoviruses at 25 MOI (wild-type and E297G BSEP) or 250 MOI (D482G BSEP) 48 hours before the experiments. Login to comment
140 mass of E297G and D482G BSEP molecules was calculated to be 94% and 329%, respectively, of wild-type BSEP. Login to comment
141 Discussion In the present study, we analyzed the consequence of two frequently found PFIC2 mutations (E297G and D482G) in vitro to investigate the pathogenesis of PFIC2. Login to comment
143 Although quantitative PCR showed no difference in mRNA levels between the wild-type and two mutated BSEP (see Fig. 1), Western blot analysis indicated reduced expression of D482G and E297G BSEP (see Fig. 2A). Login to comment
144 In addition, the molecular weight of most of the D482G BSEP and some of the E297G molecules was approximately 150 kDa (see Fig. 2A). Login to comment
147 This suggestion is also consistent with the immunofluorescence observations which indicated that D482G and E297G BSEP molecules are located intracellularly (see Fig. 3A). Login to comment
148 The results of the surface biotinylation study also suggested that the D482G BSEP and core glycosylated form of E297G BSEP are located intracellularly (see Fig. 3B). Login to comment
149 In addition, the results of the MG132 treatment experiment suggested that E297G and D482G BSEP molecules are degraded by the proteasome pathway (see Fig. 4). Login to comment
151 Although the immunohistochemical studies indicate a loss of BSEP on the canalicular membrane in PFIC2 patients with the E297G mutation, we found that some E297G BSEP with a molecular mass of approximately 170 kDa were expressed on the apical membrane (see Fig. 3B). Login to comment
152 At the present moment, we do not know the reason why some E297G BSEP molecules are trafficked in a normal manner. Login to comment
154 Alternatively, it is also possible that the reduction in the expression level on the apical membrane of E297G BSEP (approximately 25% of wild-type BSEP; see Fig. 3B) may be related to the pathogenesis of PFIC2. Login to comment
156 Western blot analysis of the isolated membrane vesicles indicated the presence of approximately 170-kDa molecules for wild-type, E297G, and D482G BSEP. Login to comment
157 Moreover, it was found that the amount of 150-kDa molecules for E297G and D482G BSEP in the isolated membrane vesicles was lower than that in the crude membrane fraction. Login to comment
163 Effects of proteasome inhibitors on the localization of BSEP in MDCK II cells. MDCK II cells were infected with recombinant adenoviruses at 250 MOI 48 hours before the experiments. Crude membrane fractions prepared from GFP (control), wild-type, E297G, and D482G BSEP-expressing MDCK II cells (80, 40, 80, and 80 ␮g protein, respectively), treated with or without 5 ␮mol/L MG132 for 8 hours before the preparation of crude membrane, were subjected to Western blot analysis. Login to comment
165 Functional analysis using these membrane vesicles indicated that the transport of taurocholate and glycocholate in E297G and D482G BSEP-expressing isolated membrane vesicles was significantly higher than that in control isolated membrane vesicles (see Fig. 6A). Login to comment
166 Based on the hypothesis that the transport activity per unit mass of BSEP molecules can be calculated by considering the BSEP expression level in the isolated membrane vesicles, it was found that the transport of taurocholate and glycocholate mediated per unit mass of E297G and D482G BSEP molecules was not reduced compared with wild-type BSEP (see Figs. 5B and 6B). Login to comment
167 The Km values for taurocholate of wild-type, E297G, and D482G BSEP molecules were consistent with previous observations obtained using membrane vesicles isolated from human wild-type BSEP cDNA-infected Sf9 and Sf High Five cells.2,3 Based on these results, it appears that the transport function of two mutated BSEP molecules (E297G and D482G) per se remains normal (see Fig. 6). Login to comment
168 These results suggest that, if E297G and D482G are matured and consequently expressed on the membrane surface, these mutated transporters are able to transport BSEP substrates. Login to comment
174 5 ␮g (upper lane), 15 ␮g (middle lane) and 30 ␮g (lower lane) protein of isolated membrane vesicles prepared from GFP (control), wild-type, E297G and D482G BSEP-expressing HEK 293 cells were subjected to Western blot analysis. Login to comment
176 The results for wild-type (F), E297G (E), and D482G (■) BSEP are shown. Login to comment
177 (C) Results of Western blot analysis using crude membrane fraction and isolated membrane vesicles prepared from GFP (control), wild-type, E297G, and D482G BSEP-expressing HEK 293 cells (50, 20, 75, and 75 ␮g protein, respectively). Login to comment
179 membranevesiclesisolatedfromSf9cells.7 Incontrast,inthe study with mouse Bsep gene, it was found that D482G mutation resulted in impaired canalicular trafficking in HepG2 cells, although this mutation did not affect the transport of taurocholate examined using membrane vesicles isolated from Sf21 cells.8 Concerning E297G mutation, it has been shownthatE297GratBsepiswidelydistributedthroughout the cytoplasm, and the studies using isolated membrane vesicles indicated that this mutation resulted in the loss of taurocholate transport activity.7 The differences among these results involving the previous mutated rat Bsep, mouse Bsep, and the present mutated human BSEP may be explained by considering the species difference in the Bsep/BSEP sequence, although the homology of the amino acid sequence around E297G and D482G is quite high as far as human BSEPandratandmouseBsepareconcerned.Forexample,it is still possible that the introduction of the D482G mutation to human BSEP, but not rat Bsep, results in a conformational change in human BSEP so that D482G BSEP is bound to some molecular chaperons. Login to comment
182 One of the methods being explored as a potential treatment of CFTR ⌬F508 patients is to administer drugs (such as sodium 4-phenylbutyrate) which are capable of trafficking the mutated protein to the apical membrane by inhibiting the binding to the molecular chaperons in the ER.21-23 After clarifying the mechanism for the intracellular retention of E297G and D482G BSEP, it is possible to identify agents able to target these mutated BSEP molecules to the apical surface. Login to comment
184 In conclusion, the results of the present in vitro study suggest that E297G and D482G, which are frequently observed PFIC2 mutations, cause deficient BSEP maturation, although the transport functions of these mutants per se remain almost normal. Login to comment

PMID: 16394881 [PubMed] Kubitz R et al: "Benign recurrent intrahepatic cholestasis associated with mutations of the bile salt export pump."

No. Sentence Comment

72 Mutations connected to BRIC-2 involve less important structures than PFIC-2 mutations.5 Only the mutation E297G was detected in patients with either PFIC-2 or BRIC-2.5,24 Nine of the 10 BRIC-2 patients who have been described so far by van Mil et al5 had homozygous or compound heterozygous BSEP mutations. Login to comment
80 This might include protein misfolding and increased ER-associated degradation of BSEP as shown for some frequent BSEP mutations underlying PFIC-2.28,29 Other possibilities of reduced protein amounts of BSEP are an increased retention of BSEP within the secretory pathway11 or an increased vesicular retrieval of BSEP from the canalicular membrane as described in animal models of cholestasis.30,31 Targeting defects might be even more important than alteration in transporter activity as shown for the most frequent PFIC-2 mutations E297G and D482G.32 Cholestatic episodes of the patient were triggered by infections.16,33,34 These infections might induce cell stress,35,36 which might be the reason for protein instability and improper trafficking. Login to comment

PMID: 16550034 [PubMed] Rutherford AE et al: "Cholestasis and cholestatic syndromes."

No. Sentence Comment

38 Of these, two missense mutations (E297G and D482G) are present in 30% of European PFIC2 families. Login to comment

PMID: 16763017 [PubMed] Lang T et al: "Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11)."

No. Sentence Comment

286 A splicing mutation (ϩ3)AϾC (intron 4) combined with a frameshift mutation in exon 22 (p.K930X), resulting in a PFIC2 phenotype and two nonsynonymous variants in exon 9 (p. E297G) and in exon 12 (p.R432T), were encountered in the patient exhibiting a BRIC (benign recurrent intrahepatic cholestasis) phenotype. Login to comment

PMID: 16871584 [PubMed] Knisely AS et al: "Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency."

No. Sentence Comment

66 Patients, Clinical Courses, and ABCB11 / BSEP Mutations Patient/Gender; Origins Sibling(s) With PFIC Age, PFIC Manifest Intervention(s) Age, HCC Diagnosed Outcome to Date Nucleotide Changes Predicted Consequences A/Male; Northern European Caucasian 0 Cholestasis from 3 wk Hepatocyte infusion, 16 mo 21 mo (incidental in explant; AFP 199, nl Ͻ 7), at LT Healthy (2 y, 8 mo after LT) 1939delA/IVS16-8TϾG (compound heterozygote) 647K then VFTSLX/ splice site disruption B/Female; Northern European Caucasian 0 Cholestasis from 2 wk, hospitalized for evaluation aged 12 wk None 28 mo, at open biopsy; AFP not determined Palliative care only; death aged 33 mo IVS18ϩ1GϾA/74CϾA (compound heterozygote) Splice site disruption/ S25X C/Male; Northern European Caucasian 0 Cholestasis from birth None 23 mo (AFP 30k, nl Ͻ 5; liver mass); histologic diagnosis at necropsy, 24 mo Palliative care only; death aged 24 mo 1445AϾG/3691CϾT (compound heterozygote) D482G/R1231W D/Male; Northern European Caucasian 1 Cholestasis from 3 wk Partial external biliary diversion (PEBD), 11 mo; decreased pruritus temporarily, clinical-laboratory test results and growth no better 22 mo (AFP 158k, nl Ͻ 15); liver mass; lung and bone lesions; chemotherapy given; histologic diagnosis at LT, 25 mo Death, 6 d after LT (sepsis; no HCC at necropsy) 890AϾG/890AϾG (homozygote) E297G/E297G E/Male; Northern European Caucasian 1 Growth failure from 6 mo; diagnosed 9.5 mo PEBD, 10 mo; no response 29 mo (incidental in explant; AFP 6.4k, nl Ͻ 9), at LT Healthy (5 y, 10 mo after LT) IVS7ϩ1GϾA/890AϾG (compound heterozygote) Splice site disruption/ E297G F/Male; Northern European Caucasian 1 Cholestasis from 6 wk None 16 mo (clinically unsuspected), at necropsy; AFP not determined Death with metastatic HCC in lungs IVS9ϩ1GϾT/not known Splice site disruption/ not known G/Female; Arabic 3 Cholestasis from 6 wk None 15 mo (AFP 11k, nl Ͻ 7; liver mass); histologic diagnosis at LT, 16 mo Healthy (1 y, 7 mo after LT) 1416TϾA/1416TϾA (homozygote) Y472X/Y472X H/Male; Northern European Caucasian 0 Evaluation at 6 mo for jaundice and growth failure PEBD, 32 mo; excellent response (pruritus resolved, bile salts in serum normal range, growth resumed) 52 mo (marked increase in abdominal size; tumor metastasized at diagnosis; AFP 2x106, nl Ͻ 9), at open biopsy Palliative care only; death 3 wk after diagnosis 890 AϾG/ IVS13del-13ˆ-8 (compound heterozygote) E297G/splice site disruption I/Male; Central Asian Caucasian 0 Cholestasis from birth None 13 mo (incidental in explant; AFP 831, nl Ͻ 23), at LT Healthy (1 y, 11 mo after LT) IVS19ϩ2TϾC (homozygote) Splice site disruption J/Male; Central Asian Caucasian 0 Cholestasis from 1 wk None 14 mo (AFP 4k, nl Ͻ 23; liver mass), at biopsy; confirmed at LT, 15 mo Healthy (1 y, 2 mo after LT) 2316TϾG (homozygote) Y772X K/Male; Northern European Caucasian 3 Cholestasis from 3 mo None 26 mo (marked increase in abdominal size; tumor metastasized at diagnosis; AFP not measured), at open biopsy Death with metastatic HCC in lungs, aged 27 mo None sought None predicted Fig. 1. Login to comment
139 The common European missense mutation 890AϾG (E297G)8 was present in 3 patients and their parents (patients D, E, and H). Login to comment
140 Except for 890AϾG (E297G), none of the changes found was present in 500 control individuals representative of all major populations (University of California San Francisco Pharmacogenetics Project24; http://pharmacogenetics.ucsf.edu). Login to comment
177 Among the missense changes is 1 in exon 27 (patient C); those in patients D, E, and H (890AϾG [E297G], exon 9) and the other mutation in patient C (1445AϾG [D482G], exon 14) have been described previously.8 The 890AϾG (E297G) and 1445AϾG (D482G) mutations reportedly affect BSEP transport activity,21,33 with altered trafficking of BSEP to the canalicular membrane.33-35 The mutation in patient C, 3691CϾT (R1231W), is predicted to alter an amino acid residue between the ATP-binding cassette signature motif and the Walker B motif of BSEP. Login to comment
185 Also of interest is the presence among our patients of the 890AϾG (E297G) mutation (patient D, homozygous; patients E and H, heterozygous) and the 1445AϾG (D482G) mutation (patient C, heterozy- gous). Login to comment
187 Our patients D, E, and H, all with 890AϾG (E297G) mutation, came to PEBD. Login to comment

PMID: 17051391 [PubMed] Stieger B et al: "The bile salt export pump."

No. Sentence Comment

160 Their bile flow rate is slightly but not significantly lower in comparison to controls, but the total bile salt output into bile is massively reduced and their liver bile salt concen- S114R G238V V284L* C336S D482G R487H S593R E636G G982R G1004D R1153CD R1268Q E186G E297G R432T I498T I498T T923P A926P R1050C R1128H S194P G260D N519S A1228V V444A K461E M677V R698H PFIC2 BRIC2 acquired cholestasis SNP Fig. 2 Putative secondary structure of Bsep (NT-005403) generated with the TOPO program (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl). Login to comment

PMID: 17181454 [PubMed] Sakurai A et al: "Prediction of drug-induced intrahepatic cholestasis: in vitro screening and QSAR analysis of drugs inhibiting the human bile salt export pump."

No. Sentence Comment

120 H2N COOH S56L G238V G260D C336S L339V V444A K461E D482G T923P K930X G982R R1090X R1153C Outside Inside R1268Q A1228VE1186K R1128H R1057X R1050C A926P A865V R698H E636G M677V S593R E592Q N591S R575XA570T Q558H I498T R432T R415Q R299K E297G V284A I206V S194P E186G cholestasis Expert Opin. Drug Saf. (2007) 6(1) Table 1. Login to comment
121 Nonsynonymous polymorphisms and mutations in the ABCB11 gene NCBI No. Exon Nucleotide Amino acid alteration Phenotype Ref. Position Alteration rs11568361 5 167 C→T Ser56Leu - [102] - 5 341 G→C Ser114Arg PFIC2 [47]* - 6 557 A→G Glu186Gly BRIC2 [45,48] - 6 580 T→C Ser194Pro - [44] rs11568358 7 616 A→G Ile206Val - [102] - 7 695 T→del Frame shift at position 232 PFIC2 [47] - 7 713 G→T Gly238Val PFIC2 [47] - 8 779 G→A Gly260Asp - [44] - 8 851 T→C Val284Ala - [44] rs11568372 8 890 A→G Glu297Gly PFIC2/BRIC2 [35,43,45,47,102] rs2287617 8 896 G→A Arg299Lys - [102] - 8 908 G→del Frame shift at position 303 PFIC2 [35] - 9 1007 G→C Cys336Ser PFIC2 [47] - 9 1015 C→G Leu339Val - [46] - 11 1244 G→A Arg415Gln - [39] - 11 1294 G→C Arg432Thr BRIC2 [43] rs2287622 12 1331 T→C Val444Ala ICP/PFIC2? Login to comment

PMID: 17538928 [PubMed] Hayashi H et al: "4-phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps."

No. Sentence Comment

1 We previously reported that E297G and D482G BSEP, which are frequently found mutations in European patients, result in impaired membrane trafficking, whereas both mutants retain their transport function. Login to comment
3 Because sodium 4-phenylbutyrate (4PBA) has been shown to restore the reduced cell surface expression of mutated plasma membrane proteins, in the current study, we investigated the effect of 4PBA treatment on E297G and D482G BSEP. Login to comment
4 Transcellular transport and cell surface biotinylation studies using Madin-Darby canine kidney (MDCK) II cells demonstrated that 4PBA treatment increased functional cell surface expression of wild-type (WT), E297G, and D482G BSEP. Login to comment
7 Conclusion: 4PBA treatment with a clinically achievable concentration enhances the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells, and also induces functional BSEP expression at the canalicular membrane and bile acid transport via canalicular membrane in vivo. Login to comment
8 4PBA is a potential pharmacological agent for treating not only PFIC2 patients with E297G and D482G mutations but also other cholestatic patients, in whom the BSEP expression at the canalicular membrane is reduced. Login to comment
21 1506 ing junction mutations in the BSEP gene.1 Among them, E297G and D482G, two missense mutations in the second intracellular loop and in the first ATP-binding domain, respectively, are frequently observed in PFIC2 patients. Login to comment
23 However, both mutated BSEPs per se exhibit normal transport functions.10 Accordingly, restoration of the reduced cell surface expression of these mutated BSEPs is an important task for achieving the therapeutic goal for PFIC2 patients with E297G and D482G mutations. Login to comment
24 Sodium 4-phenylbutyrate (4PBA) has been shown to be capable of restoring the reduced cell surface expression of cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) with the deletion of phenylalanine at 508 (CFTR ⌬F508).11 CFTR ⌬F508 is the most common mutation in cystic fibrosis patients12 and has similar features to E297G and D482G BSEP in that this mutated molecule accumulates in the endoplasmic reticulum (ER), followed by degradation in the proteasomes, but maintains its normal function as a chloride channel.13-15 4PBA is a nontoxic butyrate analog that was originally approved for clinical use as an ammonia scavenger in subjects with urea cycle disorders.16 Clinical trials of this agent in cystic fibrosis patients with ⌬F508 demonstrated that CFTR function in the nasal epithelia is induced by 4PBA therapy.17 Considering that a surgical procedure such as liver transplantation is the only therapy to cure PFIC2, this compound may offer a new medical therapy for PFIC2. Login to comment
26 We evaluated the effectiveness and mechanism of action of 4PBA by biological and transport functional experiments with Madin-Darby canine kidney (MDCK) II cells expressing wild-type (WT), E297G, and D482G BSEP. Login to comment
36 The BD Adeno-X Adenoviral Expression System (BD Biosciences, Palo Alto, CA) was used to establish the human WT, E297G, and D482G BSEP recombinant adenoviruses as previously described.10 The virus titer was checked with an Adeno-X Rapid Titer Kit (Clontech). Login to comment
39 MDCK II cells were seeded in six-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 200 multiplicity of infection (MOI). Login to comment
47 After 2 days` culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 50 MOI. Cells were cultured for 24 hours after infection and subsequently treated with 1 mM 4PBA. Then, 24 hours after treatment, the transcellular transport assays were performed as described.19 The apparent efflux clearance across the apical membrane (PSapical) was calculated by dividing the steady-state rate for the transcellular transport of [3H]TC determined over 2 hours by the cellular concentration of [3H]TC determined at the end of the experiments (2 hours). Login to comment
49 MDCK II cells were seeded in 6-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 200 MOI. Cells were cultured for 24 hours after infection and subsequently treated with 1 mM 4PBA. Then, 24 hours after treatment, cell surface biotinylation was performed as described.10 When the degradation rate of the cell surface-resident protein was examined, biotinylated MDCK II cells were incubated for various periods at 37°C, with or without 1 mM 4PBA, before solubilization. Login to comment
52 MDCK II cells were seeded in six-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 50 MOI. Cells were cultured for 24 hours after infection and subsequently treated with 1 mM 4PBA. Then, 24 hours after treatment, RNA was isolated using ISOGEN (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer`s instructions. Login to comment
57 MDCK II cells were seeded in 6-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, and D482G BSEP at 200 MOI. Cells were cultured for 36 hours after infection and subsequently treated, with or without 5 ␮g/ml Act D (Sigma, St. Louis, MO), to inhibit mRNA synthesis and, with or without 20 ␮g/ml CHX (Sigma, St. Louis, MO), to inhibit protein synthesis. Login to comment
59 Subsequently, 6 hours (E297G, D482G BSEP) or 8 hours (WT) after 4PBA treatment, the crude membrane fraction was prepared as described previously.19 The specimens were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment
61 MDCK II cells were seeded in 6-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, and D482G BSEP at 200 MOI. Cells were cultured for 12 hours after infection, and subsequently treated, with or without 5 ␮g/ml brefeldin A (BFA) (Sigma, St. Louis, MO), to inhibit the translocation of BSEP from ER to the Golgi complex. Login to comment
82 MDCK II cells expressing WT, E297G, and D482G were treated with 4PBA for various periods and at various concentrations (Fig. 1A,B). Login to comment
84 4PBA treatment altered the expression level of the mature form of not only E297G and D482G BSEP but also WT BSEP in a concentration-dependent and time-dependent manner, the optimal condition being 1 mM for 24 hours. Login to comment
85 The expression level of the mature form of WT, E297G, and D482G BSEP was increased 2.5-fold to 3-fold in response to 1 mM 4PBA treatment for 24 hours, which is a clinically achievable concentration.21-23 We then examined the increase in cell surface expression of BSEP by 4PBA treatment using transcellular transport assay and cell surface biotinylation methods. Login to comment
87 Vectorial transport of [3H]TC in the apical direction was observed in MDCK II monolayers expressing WT, E297G, and D482G BSEP, and hardly detected in MDCK II monolayers expressing GFP. Login to comment
88 Similar to hepatocyte, coexpression of Naϩ-taurocholate cotransporting polypeptide (NTCP), basolateral uptake transporter, and BSEP, apical efflux transporter, is needed to detect the vectorial transport of [3H]TC in the apical direction in LLC-PK1 and MDCK monolayers.19,24 The finding that the expression of BSEP alone is sufficient to induce the vectorial transport of [3H]TC in the apical direction suggests that MDCK II cells endogenously express uptake transporter for bile acids in the basolateral membrane as suggested.25 The basal-to-apical flux of [3H]TC across MDCK II monolayers expressing WT, E297G, and D482G BSEP was increased 1.5-fold, 2.5-fold, and 3-fold, respectively, by 4PBA treatment under optimal conditions (1 mM, 24 hours), whereas the increase in the basal-to-apical flux of [3H]TC was not detected in MDCK II cells expressing GFP. Login to comment
93 MDCK II cells expressing WT, E297G, and D482G BSEP were treated for the indicated periods with 1 mM 4PBA before the preparation of crude membrane fractions. Prepared specimens (40 ␮g ) were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment
95 MDCK II cells expressing WT, E297G, and D482G BSEP were treated with the indicated 4PBA concentration for 24 hours before the preparation of crude membrane fractions. Prepared specimens (40 ␮g) were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment
97 PSapical was increased in MDCK II cells expressing WT, E297G, and D482G BSEP, but not affected in MDCK II cells expressing GFP by 4PBA treatment. Login to comment
98 BSEP-dependent PSapical (PSapical, BSEP) in MDCK II cells expressing WT, E297G, and D482G BSEP, which was calculated by subtracting the PSapical value in MDCK II cells expressing GFP from that in MDCK II cells expressing WT, E297G, and D482G BSEP, was enhanced 1.7-, 3.0-, and 2.8-fold, respectively, by 4PBA treatment under optimal conditions (1 mM, 24 hours). Login to comment
100 Cell surface expression of WT, E297G, and D482G BSEP was increased 1.8-fold, 3.1-fold, and 2.6-fold, respectively, by 4PBA treatment under optimal conditions (1 mM, 24 hours), whereas cell surface expression of exogenously expressing P-gp and endogenously expressing DPPIV was not affected (Fig. 2D). Login to comment
101 The increase in cell surface expression of BSEP by 4PBA treatment was to an equivalent degree to that in PSapical, BSEP, suggesting that 4PBA treatment with a clinically achievable concentration can enhance the transport capacity of BSEP in MDCK II cells expressing WT, E297G, and D482G BSEP by increasing the cell surface expression of WT, E297G, and D482G BSEP. Login to comment
104 A possible mechanism is that 4PBA treatment promotes transcription or translation of WT, E297G, and D482G BSEP and consequently increases the cell surface expression of WT, E297G, and D482G BSEP by mass action. Login to comment
107 WT, E297G, and D482G BSEP mRNA expression levels were slightly increased by 4PBA treat- Fig. 2. Login to comment
109 MDCK II cells expressing WT, E297G, D482G BSEP, and GFP were treated for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment
112 Transcellular transport of 1 ␮M [3H]TC across MDCK II monolayers expressing WT, E297G, D482G BSEP, and GFP was examined as a function of time. Login to comment
118 The clearance of the transport of [3H]TC across the apical membrane of MDCK II monolayers expressing WT, E297G, D482G BSEP, and GFP (PSapical) was determined as described in Materials and Methods. Login to comment
123 ment, but the difference was not statistically significant [P ϭ 0.10 (WT), 0.20 (E297G, D482G)] (Fig. 3A). Login to comment
126 The inhibition of transcription with Act D and translation with CHX did not affect the 4PBA-mediated increase in the mature form of BSEP in MDCK II cells expressing WT, E297G, and D482G BSEP. Login to comment
127 These results indicate that a post-translational mechanism mainly contributes to the 4PBA-mediated increase in the cell surface expression of WT, E297G, and D482G BSEP. Login to comment
135 MDCK II cells expressing WT, E297G, and D482G BSEP were treated for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment
140 MDCK II cells expressing WT, E297G, and D482G BSEP were treated for 6 hours (E297G, D482G BSEP) or 8 hours (WT BSEP), with or without 1 mM 4PBA, in the presence or absence of 5 ␮g/ml Act D (B), and 20 ␮g/ml CHX (C) before the preparation of crude membrane fractions. Prepared specimens (40 ␮g) were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment
144 MDCK II cells expressing WT, E297G, and D482G BSEP were treated for 12 hours, with or without 1 mM 4PBA, in the presence or absence of 5 ␮g/ml BFA. Login to comment
151 The expression level of the immature ER-resident form of WT, E297G, and D482G BSEP was almost identical in non- and 4PBA-treated MDCK II cells just after BFA washout (Fig. 4A; 0 hours), and that of the mature form was similar in non- and 4PBA-treated MDCK II cells until 3 hours after BFA washout, at which time the mature form of BSEP linearly increased. Login to comment
153 This result suggests that 4PBA treatment does not promote WT, E297G, and D482G BSEP maturation, but stabilizes the mature form of these proteins. Login to comment
156 To examine whether 4PBA treatment can inhibit the cell surface-resident BSEP from entering the degradation pathway, we measured the degradation rate of the cell surface-resident BSEP using biotin-labeling methods in MDCK II cells expressing WT, E297G, and D482G BSEP. Login to comment
157 The half-life of cell surface-resident WT and E297G BSEP was prolonged 1.8- and 2.5-fold, respectively, by 4PBA treatment, whereas those of exogenously expressing P-gp and endogenously expressing DPPIV was not affected (Fig. 5A,B). Login to comment
158 The degradation rate of cell surface-resident D482G BSEP could not be determined under the same conditions as WT and E297G because of its low expression level. Login to comment
161 (A) The degradation rate of cell surface-resident WT and E297G BSEP. Login to comment
162 MDCK II cells expressing WT and E297G BSEP were treated for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment
165 (B) Quantification of band density indicating WT and E297G BSEP in (A). Login to comment
166 The intensity of the band indicating WT and E297G BSEP was quantified by Image Gauge software and expressed as a percentage of the BSEP present at 0 hours, respectively. Closed and open circles represent remaining cell surface WT BSEP or E297G BSEP in MDCK II cells treated, with and without 4PBA, respectively. Login to comment
168 By the same methods as WT and E297G BSEP, the densitometric analysis was also performed for exogenously expressing P-gp and endogenously expressing DPPIV. Login to comment
181 The half-life of the cell surface-resident D482G BSEP was prolonged 3.3-fold by 4PBA treatment such as WT and E297G BSEP. Login to comment
182 The prolonged half-life of the cell surface-resident WT, E297G, and D482G BSEP is consistent with the result of the BFA washout study, which suggests that 4PBA treatment stabilizes the mature form of BSEP (Fig. 4A,B). Login to comment
184 4PBA treatment can increase the functional cell surface expression of WT, E297G, and D482G BSEP in MDCK II cells (Fig. 2). Login to comment
205 Discussion We previously reported that E297G and D482G BSEP, which are frequently found mutants in European patients, result in impaired membrane trafficking, whereas the transport function of both mutated BSEPs remains almost normal.10 Restoration of the reduced cell surface expression of these mutated BSEPs is a therapeutic goal for PFIC2 patients with E297G and D482G mutations, because both mutated BSEPs per se exhibit normal transport functions. Login to comment
206 In the current study, we analyzed the potential therapeutic effect of 4PBA against PFIC2 by examining the effect of 4PBA treatment on the cell surface expression of E297G and D482G BSEP. Login to comment
208 Initially, we examined the effect of 4PBA treatment on WT, E297G, and D482G BSEP using MDCK II cells. Login to comment
210 Together with the finding that the increase in PSapical, BSEP by 4PBA treatment correlates with the cell surface expression of WT, E297G, and D482G BSEP (Fig. 2C), 4PBA treatment with a clinically achievable concentration can enhance the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells. Login to comment
214 Furthermore, the expression levels of the immature ER-resident form of WT, E297G, and D482G BSEP were almost identical in non- and 4PBA-treated MDCK II cells just after BFA washout (Fig. 4A; 0 hours). Login to comment
215 These results indicate that a post-translational mechanism mainly contributes to the 4PBA-mediated increase in the cell surface expression of WT, E297G, and D482G BSEP. Login to comment
216 Although we investigated whether 4PBA treatment promotes the maturation of WT, E297G, and D482G BSEP as the possible post-translational mechanism, such an effect of 4PBA treatment was not observed in the BFA washout study (Fig. 4A,B). Login to comment
217 However, the results of this study showed that 4PBA treatment could stabilize the mature form of WT, E297G, and D482G BSEP (Fig. 4A,B). Login to comment
230 E297G, and D482G BSEP (Fig. 5A-E). Login to comment
231 Because cell surface-resident BSEP constitutively cycles between the canalicular membrane and the intracellular compartment, and is finally removed from this cycle to the degradation pathway,30,31 4PBA treatment may interrupt the internalization process or promote the recycling process and, consequently, increase the cell surface expression of WT, E297G, and D482G BSEP. Login to comment
233 Although the molecular mechanism of this effect remains unknown, taking into the consideration that the prolonged half-life with 4PBA treatment was only detected in cell surface-resident WT, E297G, and D482G BSEP, but not for other membrane proteins such as P-gp and DPPIV (Fig. 5B,E), 4PBA treatment possibly induces a specific interaction of WT, E297G, and D482G BSEP with adaptor proteins. Login to comment
238 If the prolonged half-life of cell surface-resident Bsep with 4PBA treatment is responsible for this result in vivo as well as in vitro, the protective effect of 4PBA treatment against hepatocellular damage by increasing biliary excretion of bile acids and lowering intrahepatic bile acids may be remarkably observed in PFIC2 patients with E297G and D482G mutations. Login to comment
249 In conclusion, we demonstrated that 4PBA treatment with a clinically achievable concentration induces the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells and also enhances functional Bsep expression at the canalicuar membrane and bile acid transport via canalicular membrane in vivo. Login to comment
251 These results suggest that 4PBA is a potential pharmacological agent not only for PFIC2 patients with E297G and D482G mutations but also for other cholestatic patients, in whom the BSEP expression at the canalicular membrane is reduced. Login to comment

PMID: 17855769 [PubMed] Lam P et al: "Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases."

No. Sentence Comment

9 Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Login to comment
10 Bile salt transport was retained in all but the E297G mutant. Login to comment
11 Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT Ͼ N591S Ͼ R1050C ϳ A570T ϳ E297G ϾϾ D482G. Login to comment
32 These studies suggest that two PFIC2 mutations, D482G and E297G, lead to reduced total protein expression presumably due to folding, processing, and/or trafficking defects (8, 19, 22, 30). Login to comment
39 0363-6143/07 $8.00 Copyright (c) 2007 the American Physiological Societyhttp://www.ajpcell.org C1709 trast to these conclusions, we find that all five green fluorescent protein (GFP)-tagged mutant proteins, including D482G and E297G, traffic to the cell surface as detected by confocal microscopy and by biotinylation of plasma membrane Bsep when expressed in MDCK and HEK293 cells, respectively. Login to comment
41 Since ATPase and bile salt transport activity when expressed in Sf9 cells are also normal (with the exception of the E297G mutant) the severity of the clinical phenotype correlates most closely with mutation-induced defects in cell surface expression of the mature protein. Login to comment
45 All point mutations (D482G, E297G, A570T, R1050C, N591S) were introduced with the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Login to comment
64 PFIC2 (D482G, E297G), BRIC2 (A570T, R1050C), and ICP (N591S) mutations that are investigated in this study are indicated in italics. Login to comment
94 Sf9 cells infected with recombinant virus (mock, WT, D482G, E297G, A570T, R1050C, and N591S) were harvested, and cell membranes were prepared as described previously (23). Login to comment
107 The subcellular distributions of D482G, E297G, A570T, R1050C, and N591S were examined first because intracellular accumulation of misfolded proteins has been shown for many diseases. Login to comment
118 The level of cell surface expression of Bsep protein was quantitated by densitometry and determined to be in the following order: WT (100%) Ͼ N591S (75.6 Ϯ 15.6%) Ͼ E297G (38.5 Ϯ 12.6%) ϳ R1050C (35.6 Ϯ 14.5%) ϳ A570T (29.5 Ϯ 8.8%) ϾϾ D482G (5.7 Ϯ 2.3%). Login to comment
120 The total expression (cell surface and intracellular proteins) also showed changes similar to those in cell surface expression: WT (100%) Ͼ N591S (73.5 Ϯ 4.3%) Ͼ E297G (33.7 Ϯ 20.4%) ϳ R1050C (26.7 Ϯ 16.0%) ϳ A570T (11.9 Ϯ 5.2%) ϾϾ D482G (2.5 Ϯ 2.0%) (Fig. 4B). Login to comment
151 The E297G mutation reduced the taurocholate uptake of Bsep to a level similar to membrane vesicles from mock-treated cells. Login to comment
152 Although the mutations have significant effects on Bsep protein expression, the catalytic functions of the protein are maintained in all mutants except the E297G-expressing membrane vesicles. Login to comment
155 Cells were transiently transfected with pEGFPN1 (control), rat Bsep-GFP [wild type (WT)], D482G-GFP, E297G-GFP, A570T-GFP, R1050C-GFP, and N591S-GFP constructs. Login to comment
169 Cells were transfected with rat Bsep-GFP (WT), D482G-GFP, E297G-GFP, A570T-GFP, R1050C-GFP, and N591S-GFP constructs and were examined for GFP by confocal laser microscopy. Login to comment
171 D482G, E297G, A570T, and R1050C mutants partially colocalized with recycling endosome marker Rab11. Login to comment
176 D4, D482G; E2, E297G; A5, A570T; R10, R1050C; N5, N591S. Login to comment
183 P Ͻ 0.05, D482G, E297G, A570T, R1050C, and N591S compared with WT control. Login to comment
193 With the exception of the E297G variant, all of the studied variants Fig. 5. Login to comment
210 In contrast, in membrane vesicles from HEK293 cells that overexpress the human E297G variant, taurocholate uptake activity is retained with the same transport efficiency as WT Bsep (8). Login to comment
211 In addition, in patients the E297G mutation has been associated with different clinical phenotypes as well as in healthy relatives (9, 19, 28). Login to comment
212 On the basis of this study and others, it appears that the biochemical data in the human HEK293 cells indicate that E297G mutation results in some protein expression and has residual bile acid transport activity. Login to comment
213 However, the absence of bile acid transport activity in Sf9 membrane vesicles expressing the E297G variant suggests that the mechanism for the E297G-associated defect is likely to be more complex. Login to comment
228 Bsep function [ATPase, taurocholate (TCA) transport] is retained in Bsep mutants except E297G mutant in Sf9 cells. Login to comment
229 A: immunoblot of the isolated Sf9 cell membrane proteins with anti-Bsep polyclonal antibody: GFP control expressed in HEK293 cells, WT expressed in HEK293 cells, rat liver homogenate (L), pFastBac1-Gus control (C), wild type (WT), D482G (D4), E297G (E2), A570T (A5), R1050C (R10), N591S (N5). Login to comment
231 B: TCA (200 ␮M) stimulates and orthovanadate inhibits ATPase activity in membrane vesicles containing comparable levels of WT or D482G, E297G, A570T, R1050C, and N591S mutant Bsep. Login to comment
234 C: except for the E297G mutant, Bsep WT and mutant-expressing Sf9 cells demonstrate marked ATP stimulated [3 H] TCA in the same membrane vesicles as in Fig. 6C. Login to comment
237 **P Ͻ 0.05 for E297G compared with WT control. Login to comment

PMID: 17947449 [PubMed] Kagawa T et al: "Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells."

No. Sentence Comment

13 The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Login to comment
16 BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. Login to comment
19 In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C Ͼ D482G Ͼ E297G Ͼ K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. Login to comment
29 Of these, D482G and E297G mutations are most frequent (35). Login to comment
30 Studies of TC transport activity and intracellular trafficking by D482G and E297G mutants have reported inconstant results. Login to comment
139 The positions of 8 PFIC2 mutations (E297G, K461E, D482G, G982R, R1057C, R1153C, 3767-3768insC, and R1268Q) and 2 BRIC2 mutations (A570T and R1050C) are indicated by ଝ and ଙ, respectively. Login to comment
164 The E297G mutation revealed ϳ10.2 Ϯ 6.6% TC transport activity of WT (Fig. 6A). Login to comment
168 The E297G mutant was expressed at 16.1 Ϯ 6.8% of that of WT, whereas expression of the other mutants, except for R1057X, was at trace level (Fig. 6, B and C). Login to comment
184 Subcellular distribution study revealed that E297G, R1057X, A570T, and R1050C mutants were predominantly located along the apical membrane, whereas the other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, and 3767-3768insC) remained intracellular (Fig. 7). Login to comment
186 Two PFIC2 mutants (D482G and E297G) were expressed at 10-30% of WT, trafficked correctly, and exhibited 10-30% activity. Login to comment
244 In the eight PFIC2 mutants studied, D482G and E297G were predominantly distributed in the apical membrane and exhibited TC transport activity (D482G: 32.3% and E297G: 10.2% of WT). Login to comment
247 Our observation that the D482G and E297G mutants exhibited less impaired transport activity than other mutants is in agreement with the clinical phenotype. Login to comment
248 The response to biliary diversion is better in PFIC2 patients carrying D482G and E297G mutations than in those with other mutations (27). Login to comment
288 Furthermore the E297G mutation, which is responsible for PFIC2, also occurs in BRIC2 patients (37). Login to comment
290 From the view of maintenance of TC transport activity, the mutants could be aligned in the following order: A570T and R1050C Ͼ D482G Ͼ E297G Ͼ K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. Login to comment
295 When we were preparing the manuscript, a paper appeared in which 4-phenylbutyrate (4PBA) was demonstrated to extend the half-life of cell surface-resident WT, E297G, and D482G BSEP by 1.8-, 2.5-, and 3.3-fold, respectively, in MDCK II cells (7). Login to comment

PMID: 18376240 [PubMed] Alissa FT et al: "Update on progressive familial intrahepatic cholestasis."

No. Sentence Comment

187 E297G is the most common mutation in individuals of European descent, accounting for approximately 30% of BSEP mutations in European series. Login to comment
192 The E297G and D482G mutations may yield proteins that are functional but do not traffic appropriately to the canalicular membrane. Login to comment
194 Even more subtle mutations of ABCB11 were described in patients with an intermittent form of disease (BRIC2; see below), these mutations include E186G, A570T, T923P, A926P, R1050C, R1128H, V444A, and E297G. Login to comment
195 The E297G mutation was previously described in patients with BSEP disease, and V444A was also found in intrahepatic cholestasis of pregnancy (69,70). Login to comment

PMID: 18692205 [PubMed] Chen HL et al: "Diagnosis of BSEP/ABCB11 mutations in Asian patients with cholestasis using denaturing high performance liquid chromatography."

No. Sentence Comment

28 In a recent study analyzing patients of mostly European origin, 82 mutations that occurred throughout the protein were detected in 109 families.13 Some mutations were found in a number of affected families of European descent, such as E297G and D482G.4,13-14 No such hot spots have been found in patients with ABCB11 mutations in other ethnic backgrounds, especially in Asian populations. In this study, we developed a method to perform ABCB11 genomic analysis more efficiently by first amplifying all of the ABCB11 exons, and then subjecting the exons to denaturing high performance liquid chromatography (DHPLC) analysis, followed by confirmation using direct sequencing. Login to comment
119 E297G and D482G were present in 58% of European families with PFIC, and the 2 mutations were proposed to originate from Northern Europe and Central/Eastern Europe, respectively.13 From our present data, there is no evidence that these mutations have spread to east Asia. Login to comment

PMID: 18798335 [PubMed] Wang L et al: "Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II."

No. Sentence Comment

31 Inhibition of proteasomes also stabilized Bsep G238V, E297G, and D482G when examined in Madin-Darby canine kidney (MDCK) cells and human embryonic kidney (HEK) cells.8,10,13 These findings suggest that the proteasome plays a major role in the degradation of these BSEP mutants in PFIC II patients. Login to comment

PMID: 18829893 [PubMed] Hayashi H et al: "Short-chain ubiquitination is associated with the degradation rate of a cell-surface-resident bile salt export pump (BSEP/ABCB11)."

No. Sentence Comment

2 On the other hand, BSEP mutations, E297G and D482G, found in progressive familial intrahepatic cholestasis type 2 (PFIC2), reduced it by shortening the degradation rate of cell-surface-resident BSEP. Login to comment
5 Ubiquitination susceptibility of BSEP/Bsep was reduced in vitro and in vivo by 4PBA treatment and, conversely, was enhanced by BSEP mutations E297G and D482G. Login to comment
20 1 Copyright (c) 2009 The American Society for Pharmacology and Experimental Therapeutics 49288/3415636 Mol Pharmacol 75:143-150, 2009 Printed in U.S.A. degradation from the endoplasmic reticulum (ER) are responsible for the reduced cell-surface expression of BSEP in PFIC2 patients with E297G and D482G mutations (Hayashi and Sugiyama, 2007), both of which are the most frequently found in patients with PFIC2 (Strautnieks et al., 2008). Login to comment
42 The BD Adeno-X Adenoviral Expression System (BD Biosciences) was used to create BSEP, E297G BSEP, D482G BSEP, HA-BSEP, HA-BSEP-Ub⌬GG , and HA-BSEP-Ub⌬GG/I44A recombinant adenoviruses as described previously (Hayashi et al., 2005a). Login to comment
60 After a 24-h culture, confluent cells were infected with recombinant adenovirus containing cDNAs for BSEP, E297G BSEP, D482G BSEP, HA-BSEP, and GFP at a multiplicity of infection of 200. Login to comment
112 We reported previously that E297G and D482G, frequent mutations in PFIC2 patients, shorten the half-life of cell-surface-resident BSEP by approximately 1.5-and 4-fold, respectively, and, conversely, 4PBA treatment prolongs cell-surface-resident BSEP 2-fold (Hayashi and Sugiyama, 2007). Login to comment
113 To explore a possible correlation between the half-life of cell-surface-resident BSEP and the short-chain ubiquitination susceptibility of BSEP, mutated BSEP was immunoprecipitated from MDCK II cells expressing E297G BSEP and D482G BSEP, and the immunoprecipitates were subjected to Western blot analysis for Ub and BSEP (Fig. 2A). Login to comment
114 BSEP, E297G BSEP, and Bsep were also immunoprecipitated from MDCK II cells expressing BSEP and E297G BSEP after 4PBA treatment and rCMVs prepared from 4PBA-treated SD rats, and the immunoprecipitates were subjected to Western blot analysis for Ub and BSEP (Fig. 3, A-C). Login to comment
115 Quantitative densitometry analysis revealed that the ratio of the short-chain ubiquitinated BSEP, PFIC2-type mutated BSEPs (Figs. 2A and 3, A and B, arrow) to the mature form of BSEP, PFIC2-type mutated BSEPs (Figs. 2A and 3, A and B, filled arrowhead) was significantly greater, 6- and 30-fold by E297G and D482G mutations, respectively, than that in wild-type BSEP (Fig. 2B), and was reduced in a time-dependent manner after 4PBA treatment in vitro (Fig. 3, D and E). Login to comment
120 A, short-chain ubiquitination susceptibility of PFIC2-type mutated BSEPs, E297G BSEP and D482G BSEP. Login to comment
126 Open, gray, and closed columns represent the ratio of band density indicating the short-chain ubiquitinated BSEP to that indicating the mature form of BSEP in MDCK II cells expressing BSEP, E297G BSEP, and D482G BSEP, respectively. Each bar represents the mean Ϯ S.E., n ϭ 3 to 4. Asterisks represent statistically significant differences between BSEP and mutated BSEP, ‫,ء‬ P Ͻ 0.05, and ‫,ءء‬ P Ͻ 0.01. Login to comment
136 A and B, short-chain ubiquitination susceptibility of BSEP (A) and E297G BSEP (B) in 4PBA-treated MDCK II cells. Login to comment
141 Bsep was immunoprecipitated from solubilized rCMVs prepared from 4PBA-treated SD rats with anti-rBsep antibody. Immunoprecipitates were separated by 6% SDS-PAGE and subjected to Western blot analysis. D and E, quantification of the short-chain ubiquitinated BSEP and E297G BSEP normalized with regard to the mature form of BSEP and E297G BSEP in A and B. Login to comment
148 We have found previously that shortening the half-life of cell-surface-resident BSEP is partly responsible for the reduced cell surface expression of BSEP in patients with PFIC2 with E297G and D482G mutations (Hayashi and Sugiyama, 2007). Login to comment

PMID: 18853996 [PubMed] Chen ST et al: "Prenatal diagnosis of progressive familial intrahepatic cholestasis type 2."

No. Sentence Comment

103 Some common mutations have been found in European patients, such as E297G and D482G.15 There is no hotspot found in Asian patients reported from Taiwan and Japan.9,16 In this study, the four mutations found in the two families had not been found in European or Japanese patients. Login to comment

PMID: 18937870 [PubMed] Emerick KM et al: "Bile composition in Alagille Syndrome and PFIC patients having Partial External Biliary Diversion."

No. Sentence Comment

58 The sequenced mutations included: patient 8 (611+1 G>A/890 A>G (heterozygote), E297G); patient 9 (IVS13del-13^-8/890 A>G (heterozygote), E297G) and patient 10 (1460 G>C (homozygote), R487P). Login to comment

PMID: 18987030 [PubMed] Dixon PH et al: "Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy."

No. Sentence Comment

2 Methods: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T.C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. Login to comment
5 Results: E297G was observed four times and D482G once. Login to comment
9 Structural analyses suggest that E297G and D482G destabilise the protein fold of BSEP. Login to comment
18 ABCC2 (encoding MRP2) variation has also been implicated in ICP in a South American cohort,23 but this has not been replicated to date in European populations.24 BSEP is a high affinity liver-specific transporter, which is responsible for the export of conjugated bile acids into the bile canaliculus.25-27 It is a member of the ABC transporter family of proteins, of which there are 48 members in the human genome and whose functions include the transport of a range of substances including lipids, drugs, cholesterol and bile salts.28 Two mutant alleles of ABCB11, namely E297G and D482G, have been found frequently in European families and one or both are present in 58%.29 The function of BSEP and the role of ABCB11 mutations in PFIC and BRIC indicate that variation in this gene could be involved in the aetiology of ICP. Login to comment
21 BSEP is homologous with the bacterial multidrug resistance protein (Sav1866) for which two structures have recently been determined at high resolution by x ray crystallography.35 36 We therefore used this structure to consider the possible effects of E297G, D482G, N591S and V444A on BSEP, all of which were identified in patients with ICP, to provide insights into potential mutational mechanisms. Login to comment
48 RESULTS Sequencing DNA sequence was generated for the UK ICP cohort (333 patients) for exons 9 and 14 which contain the common European mutations E297G and D482G, together with exons 15, 17 and 21 containing the previously described ICP-linked mutation (N591S) and the two DIC-linked mutations (D676Y and G855R), respectively. Login to comment
49 Analysis of this sequence revealed that three patients were heterozygous for E297G. Login to comment
51 One carrier (patient number 1, table 1) with a family history of ICP had a maternal sample available which was sequenced and demonstrated to be heterozygous for the E297G mutation. Login to comment
54 In this cohort, a single occurrence each of E297G and D482G was identified together with two occurrences of N591S. Login to comment
77 S363 occupies the position of V444 in Sav1866. S363 hydrogen-bonds with D341 in an adjacent antiparallel b-strand within the Sav1866 NBD (fig 2C), but as it is only one of many bonds between the Table 1 Clinical features of ICP patients with heterozygous ABCB11 mutations Patient No Cohort Biochemistry (highest level measured) Other maternal clinical findings Fetal and labour complications Family history of ICPBile acids* ALT* GGT* E297G 1 UK 32 133 27 - - Yes 2{ UK 41 166 16 - M, H No 3{ UK 118 229 NP G, C Pr No 4 CE 41 82 NP - - No D482G 5 CE 74 98 15 J, P, BR - No N591S 6 CE 270 195 29 - - - 7 CE 32 60 11 - - - *Normal ranges: bile acids, ,14 mmol/l; alanine aminotransferase (ALT), (31 IU/l; c-glutamyl transferase (GGT), ,30 IU/l; bilirubin (BR), ,17 mmol/l. Login to comment
88 Indeed, in the sequence of MJ0796 from Methanococcus jannaschii this position is occupied by a serine, suggesting that the serine side chain is likely to be tolerated as a replacement for N591 in BSEP.38 DISCUSSION We report here the identification of seven heterozygous carriers of BSEP (ABCB11) mutations (four E297G, one D482G and two N591S) in a large cohort of patients with ICP. Login to comment
91 To understand the effects of PFIC and BRIC mutations, several groups have studied the effects of the E297G and D482G mutations on BSEP expression, transport, localisation, function and stability, in vitro. Login to comment
96 Similar data, implicating primarily a defect in protein folding, have also been reported for the E297G mutation40 41 43 44 However, one study42 observed that while E297G in rat Bsep is expressed at the plasma membrane of HEK293 cells at roughly 40% of the level of the wild-type protein, its ATPase activity (measured in membrane vesicles prepared from insect cells) was uncoupled from the transport of taurocholate. Login to comment
97 Other studies report low transport activity for the E297G mutant of rat40 41 and human44 BSEP. Login to comment
98 However, one study concluded, after a large correction for the low level of expression, that the E297G mutant in human BSEP could drive wild-type levels of taurocholate transport activity.43 The reason for this discrepancy is not clear but may be related to the protein background, expression system or experimental methodology used by the different groups. Login to comment
103 Table 3 Allelic analysis for the V444A polymorphism associated with cholestasis Allele OR (95%CI) p Value C vs T 1.70 (1.4 to 2.1) ,0.001 Table 4 Genotypic analysis for the V444A polymorphism associated with cholestasis OR (95% CI) p Value CC vs CT 1.9 (1.3 to 2.6) ,0.001 CC vs TT 2.8 (1.7 to 4.4) ,0.001 CC and CT vs TT 1.9 (1.3 to 2.9) 0.001 Biliary tract Gut 2009;58:537-544. doi:10.1136/gut.2008.159541 Figure 2 The structure of Sav1866 suggests a molecular mechanism for the bile salt export pump (BSEP) mutations E297G and D482G (A) Schematic representation of the drug exporter Sav1866 with two bound ADPs shown as spheres (pdb 2HYD). Login to comment
115 The N-terminal Biliary tract Gut 2009;58:537-544. doi:10.1136/gut.2008.159541 of the E297G mutant is likely to be less stable, resulting in reduced expression at the plasma membrane, but, because of the position of this residue at the interface between the domains, mutant protein at the cell surface may also be deficient in communication between the bile acid-binding sites and the ATP catalytic sites, offering a molecular explanation for the uncoupling observed in one of the studies.42 However, the underlying codon change in the D482G mutant may also impair RNA splicing,45 raising the possibility that the phenotypic effect of the 482G codon may be mediated earlier in the gene expression pathway. Login to comment
122 Our results suggest that the common E297G and D482G mutations of this gene do not play a major role in ICP predisposition in our population. Login to comment
123 Examination of clinical information for the E297G and N591S carriers failed to identify any factor to distinguish them from the general ICP population. Login to comment
127 The 444A allele may contribute to susceptibility in a considerably higher number of cases of ICP than the E297G and D482G mutations. Login to comment

PMID: 19101985 [PubMed] Byrne JA et al: "Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing."

No. Sentence Comment

7 Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Login to comment
67 ABCB11 Missense Mutations and SNPs Functionally Analyzed in This Study Exon Nucleotide Change Predicted Protein Effect Location in Protein Associated Phenotype Prevalence or Frequency* Any Defect(s) Identified Reference 4 c.149TϾC L50S NH2 term PFIC 1 family (het) Immature protein 31 5 c.270TϾC F90F EC1 SNP 2.7%-7.7% 43, 45 6 c.403GϾA E135K EC1 BRIC 1 family (het) Reduced levels of mature protein † 6 c.409GϾA E137K EC1 BRIC / ICP 1 family (het) Immature protein ‡ 7 c.500CϾT A167V TM2 PFIC 1 family (hom) Mild exon skipping beta 7 c.557AϾG E186G IC1 BRIC 2 families (both het) Moderate exon skipping; greatly reduced levels of mature protein 8, 37 7 c.580TϾC S194P IC1 SNP-PSC 1.1% 43 7 c.593TϾC L198P IC1 BRIC / ICP / DC 1 family (het) Greatly reduced levels of mature protein # 8 c.713GϾT G238V EC2 PFIC 1 family (hom) 29 8 c.725CϾT T242I TM4 PFIC 1 family (het) 31 8 c.779GϾA G260D TM4 SNP-PBC 0.8% 43 9 c.850GϾC V284L IC2 PFIC 1 family (het) No protein 28 9 c.851TϾC V284A IC2 SNP 0.5% Increased levels of mature protein 43, 45† 9 c.889GϾA E297K IC2 Prolonged NNH 1 family (het) Moderate differential splicing; immature protein ‡ 9 c. 890AϾG E297G IC2 PFIC, BRIC PFIC, 45 families (14 hom, 31 het) BRIC, 4 families (2 hom, 2 het) Greatly reduced levels of mature protein 7, 8, 12, 29-32, 35 10 c.936GϾT Q312H IC2 PFIC 1 family (het) ‡ 10 c.937CϾA R313S IC2 PFIC 1 family (het) 31 10 c.957AϾG G319G TM5 SNP 1.5 - 7.5% Mild exon skipping 42, 43, 45 10 c.980GϾA G327E TM5 PFIC 1 family (het) 31 10 c.1007GϾC C336S TM5 PFIC 1 family (het) 29 11 c.1168GϾC A390P NBF PFIC, BRIC 2 families (both het) Immature protein 31; # 12 c.1129GϾA G410D NBF PFIC 1 family (het) 31 12 c.1238TϾG L413W NBF PFIC 1 family (het) Greatly reduced levels of mature protein 31 12 c.1244GϾA R415Q NBF SNP-ICP 1.3% 42 12 c.1295GϾC R432T NBF BRIC 1 family (het) Reduced levels of mature protein 12 13 c.1331CϾT A444V NBF SNP, ICP, CC, DC, BRIC 43-60% Increased levels of mature protein 8, 28, 37, 39-45 13 c.1381AϾG K461E WA PFIC 1 family (hom) Immature protein 7 13 c.1388CϾT T463I WA PFIC 1 family (het) Mild exon skipping 31 13 c.1396CϾA Q466K Adj WA PFIC 1 family (het) 31 13 c.1409GϾA R470Q Adj WA PFIC 2 families (1 het, 1 consanguineous) Immature protein 31 14 c.1442TϾA V481E NBF1 PFIC 1 family (het) 31 14 c.1445AϾG D482G NBF1 PFIC 22 families (16 het, 6 hom) Severe differential splicing; immature protein 7, 30-32 14 c.1468AϾG N490D NBF1 PFIC 1 family (het) Greatly reduced levels of mature protein; reduction in bile salt transport 31 14 c.1493TϾC I498T NBF1 PFIC / BRIC 1 family (het) 38 14 c.1530CϾA T510T NBF1 SNP-PBC 0.7% 43 14 c.1535TϾC I512T NBF1 PFIC 1 family (het) 31 14 c.1544AϾC N515T NBF1 PFIC 1 family (het) 31, 32 14 c.1440GϾA R517H NBF1 PFIC 1 family (het) No protein 31, 32 14 c.1605CϾT A535A NBF1 SNP 0.3% Slightly reduced levels mature protein 39, 45 14 c.1621AϾC I541L NBF1 PFIC 3 families (1 het, 2 consanguineous) No protein 31-33 15 c.1643TϾA F548Y Adj ABCm PFIC 1 family (het) 31, 32 15 c.1685GϾA G562D ABCm PFIC 1 family (het) 31 15 c.1708GϾA A570T Adj ABCm/WB PFIC, BRIC PFIC, 1 family Greatly reduced levels of mature protein; reduction in bile salt transport 8, 31 Table 1. Login to comment
103 On changing the wild-type exon 9 nucleotide sequence to the sequence of E297G (c.890AϾG), the cryptic donor splice site disappears. Login to comment
106 Additional analysis of the exon 9 variant sequences to identify changes to ESE or ESS motifs was performed using RESCUE-ESE and ESE FINDER 2.0 or the FAS-ESS programme, respectively.47-49,52,53 An SC35 site was identified, which was maintained irrespective of which nucleotide form was analyzed, but the presence of sequence coding for either E297K or E297G resulted in the introduction of an ESS hexamer. Login to comment
142 The mutations N490D (c.1468AϾG; Fig. 5A), R1128H (c.3383GϾA; Fig. 5B), E297G (c. 890AϾG), and A570T (c.1708GϾA; Fig. 5D), and E186G (c.557AϾG; Fig. 5E) resulted in significantly reduced levels of mature protein, and the L198P (c.593TϾC) variant was barely detectable (Fig. 5C). Login to comment
150 The addition of glycerol and incubation at a reduced temperature are conditions that promote, in cultured cells, the correct processing of membrane proteins trapped in the ER or Golgi.54-56 In an initial experiment, the effects of glycerol alone, incubation at 28°C alone, or glycerol and 28°C in combination on wild-type BSEP, R948C (c.2842CϾT), and E297G (c. 890AϾG) were assessed. Login to comment
152 There was no such effect by any treatment for R948C, but for E297G, incubation at 28°C alone or in combination with 10% glycerol gave the same levels of 160 kDa BSEP as the wild-type protein. Login to comment
179 (A) ABCB11 cDNA constructs encoding the mutants R948C (c.2842CϾT) and E297G (c.890AϾG) were transfected into CHO-K1 cells and assessed for the effect of 5% or 10% glycerol, incubation at 28°C, or incubation with 10% glycerol at 28°C for the appearance of mature 160 kDa BSEP form. Login to comment
219 These include the PFIC-associated mutations E297G (c.890AϾG; Fig. 6A), R1128C (c.3382CϾT) and R1231Q (c.3692GϾA; Fig. 6C), and R1268Q (c.3892GϾA; Fig. 6.d), the BRIC-associated mutations R1128H (c.3383GϾA) and R1050C (c.3148CϾT; Fig. 6C), and E297K (c.889GϾA; Fig. 6D), as well as A570T (c.1708GϾA), which can be associated with either form of disease. Login to comment
220 A recent study introduced E297G and D482G into human BSEP and assessed their trafficking in MDCK cells.66 The addition of sodium 4-phenylbutyrate prolonged the half-life of both mutant BSEP forms and resulted in increased functional expression of the proteins. Login to comment
221 This concurs with the data presented here, which show reduced levels of mature BSEP when E297G and D482G were expressed in vitro (Fig. 5D). Login to comment
222 Furthermore, the addition of 10% glycerol and incubation at 28°C resulted in a substantial and a marginal increase in mature E297G and D482G protein, respectively (Fig. 6A, D). Login to comment
225 However, if there were some wild-type splicing present, these agents could allow a partial rescue of phenotype, taking into account the slight defect in protein function previously determined for this mutant protein.15 Because there are no observed splicing defects associated with the nucleotide change associated with E297G, such therapies are possible in the future for this mutation. Login to comment

PMID: 19133130 [PubMed] Davit-Spraul A et al: "Progressive familial intrahepatic cholestasis."

No. Sentence Comment

88 Missense mutations are also common defects [25] that either affect protein processing and trafficking (i.e. p.E297G, p.D482G) [26,27] or disrupt functional domains and protein structure. Login to comment
180 Preliminary data suggest that PFIC2 patients with p.D482G or p.E297G mutations may respond well to biliary diversion [60]. Login to comment

PMID: 19684528 [PubMed] Stieger B et al: "Recent insights into the function and regulation of the bile salt export pump (ABCB11)."

No. Sentence Comment

36 This study [13 ] also revealed that the common E297G and D482G mutant forms of BSEP varied most in their expression level between patients. Login to comment
37 This is of interest, as E297G and D482G have been shown to display residual [14] and normal [15] transport activity, respectively. Login to comment
81 Interestingly, it was demonstrated that levels of the E297G and the D482G mutants of BSEP could be increased at the plasma membrane, if cells were treated with 4-phenylbutyrate [53 ]. Login to comment

PMID: 19750581 [PubMed] Treepongkaruna S et al: "Novel ABCB11 mutations in a Thai infant with progressive familial intrahepatic cholestasis."

No. Sentence Comment

87 E297G and D482G are the two most common mutations in persons of European descent, and account for approximately 58% of BSEP mutations in European studies[7] . Login to comment

PMID: 19845854 [PubMed] Liu LY et al: "ABCB11 gene mutations in Chinese children with progressive intrahepatic cholestasis and low gamma glutamyltransferase."

No. Sentence Comment

89 Common mutations, such as E297G and D482G detected in western population (23), were not found in Chinese, either from Taiwan (9, 10) or from mainland China. Login to comment

PMID: 20010382 [PubMed] Ho RH et al: "Polymorphic variants in the human bile salt export pump (BSEP; ABCB11): functional characterization and interindividual variability."

No. Sentence Comment

145 Another rare BSEP variant, 890A > G (Glu297Gly), tended to have impaired taurocholate transport function, but did not reach statistical significance (P = 0.051). Login to comment
156 Calnexin, an intracellular resident Table 1 Single-nucleotide polymorphisms in ABCB11 Allele frequencies (%) SNP rs number Amino acid change African-American European-American Asian-American Mexican-American Pacific Islanders 108T > C rs3815675 Synonymous 1.5 1.5 25 5.0 21.4 167C > T rs11568361 Ser56Leu 0.5 0 0 0 0 174C > T rs11568362 Synonymous 0.5 0 0 0 0 270T > C rs414877 Synonymous 3.0 3.5 5.0 0 7.1 402C > T rs11568377 Synonymous 3.5 0 0 0 0 585G > C rs11568365 Synonymous 0.5 0 0 0 0 616A > G rs11568357 Ile206Val 2.5 0 0 0 0 696G > T rs11568358 Synonymous 0 0.5 0 0 0 807T > C rs2287616 Synonymous 2.0 0.5 23.3 5.0 21.4 890A > G rs11568372 Glu297Gly 0 0.5 0 0 0 957A > G rs7563233 Synonymous 31.5 0.5 0 15.0 0 1281C > T rs11568360 Synonymous 0.5 0 0 0 0 1331T > C rs2287622 Val444Ala 53.0 57.1 66.7 50.0 92.9 1671C > T rs11568368 Synonymous 0 0.5 0 0 0 1674G > C rs11568369 Gln558His 0 0.5 0 0 0 1772A > G rs11568367 Asn591Ser 0 0.5 0 0 0 1774G > C rs11568370 Glu592Gln 0 0.5 0 0 0 1791G > T rs11568371 Synonymous 0 0.5 0 0 0 2029A > G rs11568364 Met677Val 15.0 5.5 1.7 5.0 0 2412A > G rs11568373 Synonymous 8.0 0 0 5.0 0 3084A > G rs97692 Synonymous 28.6 54.6 63.3 37.5 21.4 3258A > G rs11568359 Synonymous 7.0 0 0 0 0 3435A > G rs11568366 Synonymous 1.0 0 0 0 0 3556G > A rs1521808 Glu1186Lys 2.5 0 0 0 0 Allele frequencies for single-nucleotide polymorphisms (SNPs) in ABCB11 were determined from a DNA panel of ethnically defined healthy individuals - African-Americans (n = 100), European-Americans (n = 100), Asian-Americans (n = 30), Mexican-Americans (n = 10) and Pacific Islanders (n = 7). Login to comment

PMID: 20028269 [PubMed] Stieger B et al: "Role of the bile salt export pump, BSEP, in acquired forms of cholestasis."

No. Sentence Comment

72 Some mutations (e.g., E297G) are found in patients with severe, as well as with benign, BSEP deficiency syndrome (Pauli-Magnus etal., 2005). Login to comment
73 Functional characterization of the E297G variant of BSEP revealed residual transport activity (Noe etal., 2005). Login to comment
78 For example, in a patient with benign recurrent intrahepatic cholestasis type 2, the two compound heterozygous mutations, E297G and R432T, were identified. Login to comment

PMID: 20214736 [PubMed] Ananthanarayanan M et al: "PFIC2 and ethnicity-specific bile salt export pump (BSEP, ABCB11) mutations: where do we go from here?"

No. Sentence Comment

29 It is interesting that common ABCB11 mutations found in Western populations such as E297G and D482G were not found in either the current or the Chen study. Login to comment

PMID: 20232290 [PubMed] Davit-Spraul A et al: "ATP8B1 and ABCB11 analysis in 62 children with normal gamma-glutamyl transferase progressive familial intrahepatic cholestasis (PFIC): phenotypic differences between PFIC1 and PFIC2 and natural history."

No. Sentence Comment

97 6† p.R415X p.R415X na na PFIC2 no. Login to comment
99 9† p.E297G p.E297G 0.80 BSEP À PFIC2 no. 10a p.R1128C p.R1128C 0.10 BSEP À PFIC2 no. Login to comment
101 11† p.R1128C p.R1128C na na PFIC2 no. Login to comment
104 14b† p.I420T p.I1061VfsX34 na na PFIC2 no. 15*,‡ p.A167T p.G1058HfsX38 0.5 BSEP À PFIC2 no. 16* p.R1231W p.I528X na na PFIC2 no. 17 p.M62K p.I112T þ p.R698H 0.10 BSEP À PFIC2 no. 18* p.E297G p.H484RfsX5 0.16 BSEP À PFIC2 no. 19* p.E297G p.I610GfsX45 0.23 BSEP À PFIC2 no. Login to comment
107 24† p.R1153C c.3213 14 A>G 0.13 BSEP À PFIC2 no. 25* p.G982R p.Q101DfsX8 0.10 BSEP À PFIC2 no. 26* p.N591S þ p.V597V nf 0.39 BSEP À PFIC2 no. 27* p.G982R p.R1001R na BSEP À PFIC2 no. 28 p.L232CfsX9 nf na BSEP À PFIC2 no. 29 p.W114R nf 0.50 BSEP À PFIC2 no. Login to comment
110 35† p.E297G p.S699P na na PFIC2 no. Login to comment
217 *Patient harboring heterozygous BSEP mutation p.E297G. Login to comment
218 †Patient harboring homozygous BSEP mutation p.E297G. Login to comment
224 The only PFIC2 child with a total success of BD harbored homozygous mutation p.E297G and two PFIC2 children with a partial failure of BD were heterozygous for p.E297G mutation. Login to comment
261 This lower BD response rate than in other reports might be due to a more advanced stage of liver fibrosis before BD was performed, because an absence of cirrhosis was previously thought to be predictive of success, and/or to how BD success or failure was defined.14,22,28-30 For example, pruritus relief was considered a BD success in a recent series and likely equals BD partial failure in our series.30 ABCB11 genotype could also have a predictive value because the PFIC2 patient with BD success had a homozygous p.E297G mutation and two other PFIC2 patients with BD partial failure harbored on one allele the p.E297G mutation.31 This mutant is known to be associated with impaired membrane trafficking but to retain some transport activity when correctly targeted.32 The mechanism involved in the recovery of normal BA secretion after BD in patients harboring this mutant protein is unknown but could be related to canalicular targeting of the mutated BSEP. Login to comment
213 *Patient harboring heterozygous BSEP mutation p.E297G. Login to comment
214 †Patient harboring homozygous BSEP mutation p.E297G. Login to comment
220 The only PFIC2 child with a total success of BD harbored homozygous mutation p.E297G and two PFIC2 children with a partial failure of BD were heterozygous for p.E297G mutation. Login to comment
257 This lower BD response rate than in other reports might be due to a more advanced stage of liver fibrosis before BD was performed, because an absence of cirrhosis was previously thought to be predictive of success, and/or to how BD success or failure was defined.14,22,28-30 For example, pruritus relief was considered a BD success in a recent series and likely equals BD partial failure in our series.30 ABCB11 genotype could also have a predictive value because the PFIC2 patient with BD success had a homozygous p.E297G mutation and two other PFIC2 patients with BD partial failure harbored on one allele the p.E297G mutation.31 This mutant is known to be associated with impaired membrane trafficking but to retain some transport activity when correctly targeted.32 The mechanism involved in the recovery of normal BA secretion after BD in patients harboring this mutant protein is unknown but could be related to canalicular targeting of the mutated BSEP. Login to comment

PMID: 20422495 [PubMed] Lam P et al: "The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease."

No. Sentence Comment

49 Similar to the results of immunofluorescence studies in liver tissue from PFIC2 patients,47 when PFIC2 human mutations were expressed in model mammalian cell lines (MDCK, HEK293, HepG2), the proteins failed to reach or be maintained at the cell surface.54-57 When BSEP mutations that cause PFIC2 (D482G, E297G), BRIC2 (A590T, R1050C), and ICP (N591S) were compared, the clinical severity of these mutations tended to correlate inversely with the amount of protein expressed on the cell surface. Login to comment
51 For example, the PFIC2 mutant D482G`s protein half-life is short compared with the wild-type and is shortened further after ubiquitylation with E3 ubiquitin ligases.58 However, a small amount of this mutant protein can reach the plasma membrane where it is functional.58 Additional studies have shown that the resident time on the cell surface is greatly reduced with D482G and E297G mutant proteins as a result of accelerated internalization, reduced recycling, and/or targeting of endocytosed proteins for degradation.57,59 These studies suggest that the use of small molecules that modulate these pathways might be worthwhile therapeutic approaches in some of these cholestatic disorders. Login to comment
52 For example, 4-phenylbutyrate (4-PBA) can enhance cell surface expression of D482G and E297G proteins.60 Furthermore, administration of 4-PBA to normal rats enhances BSEP expression and bile salt secretion.60 Further studies are clearly needed in this area. Login to comment

PMID: 20422497 [PubMed] Pauli-Magnus C et al: "Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy."

No. Sentence Comment

79 In the same study, heterozygosity for the BSEP mutations p.E297G, p.D482G, and p.N591S formerly associated with benign and progressive forms of familial intrahepatic cholestasis type 2 were found in four, one, and two ICP patients, respectively, allowing the extrapolation that 1% of European ICP cases are caused by these mutations.105 Although the molecular and mechanistic basis for p.V444A and p.N591S were not apparent, in silico structural and functional analysis suggests that p.E297G and p.D482G destabilizes the protein fold of BSEP, leading to decreased taurocholate transport in case of p.E297G.105,106 In addition, decreased hepatic BSEP expression,107,108 and very recently, significantly reduced hepatic mRNA levels109 was reported in healthy human liver tissue carrying the alanine allele in position 444 of BSEP, which could predispose to the development of ICP by way of decreased canalicular availability of BSEP. Login to comment

PMID: 20425482 [PubMed] Santos JL et al: "Cholestatic liver disease in children."

No. Sentence Comment

67 Interestingly, 93% of the mutations produced abnormal or absent BSEP expression on liver biopsies; immunostaining identified a variable pattern of BSEP expression in patients carrying the most common E297G or D482G mutations, thus limiting the use of immunohistochemistry to reliably pinpoint BSEP deficiency. Login to comment

PMID: 20683201 [PubMed] Matte U et al: "Analysis of gene mutations in children with cholestasis of undefined etiology."

No. Sentence Comment

95 Gene sequence variants likely to cause disease phenotypes in subjects with CUEÃ Subject Age g-GTP Liver biopsy Gene Variant (allele frequency, if new) References CUE-1 1 mo 458 Not done JAG1 p.C251X New CUE-2 3.5 mo 632 Cholestasis, GCT, small bile ducts JAG1 p.V1086E New CUE-3 1.5 y 400 Pseudoacinar transformation, portal inflammation, moderate fibrosis ABCB4 p.Q945X/p.Y1171C (0%) New/new CUE-4 26 y 47 Cytoplasmic and canalicular cholestasis, portal fibrosis ATP8B1 p.N45T/p.I1050K (0%) (20)/new CUE-5 3.5 y 40 Electron microscopy consistent with Byler disease ATP8B1 c.1819þ1g>a/p.R930X Newy /(27) CUE-6 1.5 y 20 Canalicular cholestasis, portal inflammation ATP8B1 g.92918del565/g.92918del565 (13) CUE-7 1 y 44 GCT, portal inflammation, and fibrosis ABCB11 p.R928X/p.R1090X (13,28) CUE-8 2.5 y 62 Canalicular cholestasis, periportal inflammation, portal fibrosis ABCB11 p.I541T/p.I541T (12) CUE-9 5.5 y 54 Cholestasis, GCT, ductopenia, bridging fibrosis ABCB11 p.E297G/E297G (22) CUE-10 11 y 9 Minimal cholestasis; insufficient representation of portal tracts ABCB11 p.R948C/p.E1223D (0%) (12)/new CUE-11 6 mo 64 Cytoplasmic and canalicular cholestasis, GCT, fibrosis ABCB11 p.C68Y (0%)/p.R832H (0%) New, new CUE-12 2 y 42 Not done ABCB11 c.3770delA/c.3770delA Newy /newy CUE-13 19 y 29 Marked cholestasis, portal fibrosis. Login to comment

PMID: 20683202 [PubMed] Arnell H et al: "Follow-up in children with progressive familial intrahepatic cholestasis after partial external biliary diversion."

No. Sentence Comment

116 Genetic Findings and Outcome After PEBD As shown earlier, disease in patients homozygous for the missense mutation c.890A>G (p.E297G) exhibits a wide phenotypic spectrum from disease resembling ''benign`` recurrent intrahepatic cholestasis (20) to severe PFIC (18). Login to comment
117 In our group of children, outcome after PEBD was good in those homozygous for c.890A>G (p.E297G). Login to comment
122 One child homozygous for c.890A>G (p.E297G) was diagnosed with HCC at an early age. Login to comment

PMID: 21103971 [PubMed] Stieger B et al: "The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation."

No. Sentence Comment

411 Another interesting finding of this study (Strautnieks et al. 2008) is the observation that the two common E297G and D482G mutants of BSEP varied most in their expression level among the respective carriers. Login to comment
412 This is notable, as E297G and D482G variants have been demonstrated to display residual (Noe et al. 2005) or normal (Hayashi et al. 2005a) transport activity, respectively. Login to comment
488 (2008) nanotassessed Of note, it was recently demonstrated that levels of the E297G and the D482G mutants of BSEP could be increased at the apical membrane of MDCK cells, if the cells were treated with 4-phenylbutyrate (Hayashi and Sugiyama 2007) or with short-and medium-chain fatty acids (Kato et al. 2010). Login to comment

PMID: 21219577 [PubMed] Shimizu H et al: "Living-related liver transplantation for siblings with progressive familial intrahepatic cholestasis 2, with novel genetic findings."

No. Sentence Comment

104 The common mutations include E297G, R575X, R1057X, G982R, C336S, R1153C, D482G, K461E, R1153C, R1268Q, R1090X, G238V, S114R, S593R, del 695 and del 3213 (22). Login to comment

PMID: 21344347 [PubMed] Morotti RA et al: "Progressive familial intrahepatic cholestasis (PFIC) type 1, 2, and 3: a review of the liver pathology findings."

No. Sentence Comment

108 As reported by Strautnieks et al, canalicular BSEP is not detectable by IHC in cases with protein-truncating mutations.12 With other mutations, such as single copy of the E297G and D482G, the BSEP expression varies from absent to abnormal to present/ normal. Login to comment

PMID: 21490445 [PubMed] Evason K et al: "Morphologic findings in progressive familial intrahepatic cholestasis 2 (PFIC2): correlation with genetic and immunohistochemical studies."

No. Sentence Comment

143 Immunohistochemical Findings and Genetic Abnormalities Patient BSEP Mutation Type of Mutation(s) 1 Absent c.890A>G (p.E297G)* Missense5,7,10,13,16,19,20 2 Absent c.1723C>T (p.R575X) Nonsense7,19,20 c.2178+1G>T Noncoding region20 3 Present c.1708G>A (p.A570T) Missense20 c.3634G>T (p.V1212F) Missense, predicted deleterious 4 Absent c.3164T>C (p.L1055P)* Missense, predicted deleterious 5 Absent c.3692G>A (p.R1231Q) Missense20 c.2296G>A (p.G766R) Missense20 6 Absent c.2782C>T (p.R928X) Nonsense13 c.3268C>T (p.R1090X) Nonsense5,7,13 7 Present c.3347G>A (p.G1116E) Missense, predicted deleterious IVS 23-8 G-A Noncoding region 8 Absent IVS 16-8 T>Gw Noncoding region10 9 Absent c.2944G>A (p.G982R) Missense5,7,19,20 c.2296G>A (p.G766R) Missense20 10 Absent c.2944G>A (p.G982R) Missense5,7,19,20 c.2296G>A (p.G766R) Missense20 11 Absent c.319T>C (p.C107R) Missense, predicted deleterious c.611+4A>G Noncoding region 12 Absent c.1723C>T (p.R575X) Nonsense7,19,20 c.2178+1G>T Noncoding region20 *Homozygous. Login to comment

PMID: 9806540 [PubMed] Strautnieks SS et al: "A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis."

No. Sentence Comment

105 890 A→G (E297G) predicts the substitution of a glutamate by a glycine in the second intracellular loop, between transmembrane spans 4 and 5. Login to comment
142 The ABC transporter family of proteins is the largest so far iden- Table 1• BSEP mutations found in PFIC patients Nucleotide mutation Amino acid number/ Protein consequence Families mutation 1723 C→T R575X Termination codon in first B2 heterozygous nucleotide binding fold Q homozygous 3169 C→T R1057X Termination codon in second B5 heterozygous nucleotide binding fold 908 del G 303 17 novel amino acids then truncation Family 57 heterozygous 3767-3768 ins C 1256 39 novel amino acids then truncation Family 99 homozygous 890 A→G E297G Glutamate to glycine in the intracellular loop S1, S3, S4B, S5, S6, S7, 38 homozygous between transmembrane spans 4 and 5 S4A, B5, B6, B7, 53, L heterozygous 1381 A→G K461E Lysine to glutamate in first Walker A motif Family 55 homozygous 1445 A→G D482G Aspartate to glycine in first P and 52 homozygous nucleotide binding fold 2944 G→A G982R Glycine to arginine in transmembrane span 11 Family 18 homozygous 3457 C→T R1153C Arginine to cysteine in second C and D homozygous nucleotide binding fold 3803 G→A R1268Q Arginine to glutamine in second J homozygous nucleotide binding fold In each case the nucleotide position in the human coding sequence is given along with details of the predicted protein consequence. Login to comment
144 Fig. 5 The amino acid sequence in the immediate vicinity of the E297G mutation. Login to comment
164 The mutation 890 A→G (E297G) has been found in 13 families. Login to comment
236 The endonucleases used were: HphI (E297G), BpmI (K461E), FokI (D482G), AlwNI (G982R), BsrBI (R1153C) and AvaII (R1268Q). Login to comment

PMID: 21571517 [PubMed] Okiyoneda T et al: "Protein quality control at the plasma membrane."

No. Sentence Comment

31 Structural destabilization of the Pma1 and Gap1 transmembrane domains in strains defective of sphingoid base synthesis could be mechanistically similar to the farnesol-induced 484 Membranes and organelles Table 1 Peripheral protein QC substrates Membrane protein Mutation/condition Degron PM stability Related disease Reference Mammalian bile salt export pump (BSEP) E297G (Cy) D482G (Cy) 2-3 Ub # progressive familial intrahepatic cholestasis type 2 (PFIC2) [36] CFTR rDF508 (Cy) poly/multimono Ub # cystic fibrosis (CF) [44 ,27 ] D70 (Cy truncation) poly/multimono Ub # [27 ] N894D, N900D (Ex) poly/multimono Ub # [22] Na/H exchanger (NHE6) D255-256 (TM) poly/multimono Ub # Angelman syndrome [35] MLC1 multiple (TM or Cy) ND # megalencephalic leukoencephalopathy with subcortical cysts (MLC) [66] HERG low K+ Ub # type 2 long QT syndrome [37] LDL receptor high salt or low pH (Ex) ND # hypercholesterolemia [16] Dopamine D4.4 receptor M345T (TM) poly/multimono Ub # attention deficit hyperactivity disorder [28 ] Vasopressin V2 receptor W164S (TM) poly/multimono Ub # nephrogenic diabetes insipidus [28 ] alpha-2A adrenergic receptor D79N (TM) ND # cardiovascular diseases [14] N422D (TM) ND # [14] Di3loop (Cy) ND # [69] CD4tl-lm L57C (Cy) poly/multimono Ub # model protein [28 ] H+ /K+ -ATPase b subunit N99Q, N130Q, N161Q, N222Q (Ex) ND # gastric, autoimmune diseases [18] k opioid receptor N25/39Q (Ex) ND # pain control, neuronal phenotypes [19] D opioid receptor N18Q/N33Q (Ex) ND # pain control, neuronal phenotypes [20] GLUT1 N45Y, Q or D (ex) ND # GLUT1 deficiency syndrome [70] EGFR L858R (Cy), exon 19 deletion (Cy) poly/multimono Ub # cancer suspectibility [71] ErbB2 Hsp90 inhibition poly/multimono Ub # breast cancer [72] TGFBR2 Hsp90 inhibition poly/multimono Ub # tumor suspectibility [73] Yeast Pma1 Icb1-100 poly/multimono Ub # NA [29] Pma1-7 poly/multimono Ub # NA [7] Pma1-10 poly/multimono Ub # NA [52] Gap1 absence of sphingolipids poly/multimono Ub # NA [9] Abbreviations: Cy, cytosolic; Ex, extracellular; TM, transmembrane; Ub, ubiquitin; ND, not determined; #, decreasing stability; CFTR, cystic fibrosis transmembrane conductance regulator; HERG, human ether-a` -go-go related gene; LDL, low-density lipoprotein; GLUT, glucose transporter; EGFR, epidermal growth factor receptor; ErbbB2, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; TGFBR2, transforming growth factor (TGF)- beta type II; Pma1, H(+)-ATPase; Gap1, general amino acid permease. Login to comment

PMID: 20103563 [PubMed] Klaassen CD et al: "Xenobiotic, bile acid, and cholesterol transporters: function and regulation."

No. Sentence Comment

6508 Nucleotide Change Amino Acid Change In Vitro Function Protein Expression/ Localization ABCB11 BSEP N.D. G238V N.D. Intracellular A890G E297G 2 Intracellular N.D. C336S ↔ Normal G1296C R432T 2 Reduced T1331C V444A ↔ Normal/Reduced A1445G D482G 2 Normal/Reduced G2026T D676Y 2 Reduced G2563A G855R 2 Reduced G2944A G982R 2 Intracellular C3457T R1153C 2 Intracellular G3803A R1268Q 2 Intracellular searchers were able to identify functional roles for Mrp2 using rats lacking this transporter (Eisai hyperbilirubinemic rats on a Sprague-Dawley background and transport-deficient (TR-) on a Wistar background) (Paulusma et al., 1996; Ito et al., 1997). Login to comment

PMID: 22947535 [PubMed] Krawczyk M et al: "Prolonged cholestasis triggered by hepatitis A virus infection and variants of the hepatocanalicular phospholipid and bile salt transporters."

No. Sentence Comment

58 To detect genetic factors contributing to severe phenotype, we genotyped procholestatic mutations and polymorphisms in the ABCB4 (p.R590Q, c.711A>T), ABCB11 (p.E297G, p.A444V, p.D482G, c.3084A>G), ATP8B1 (p.N45T, p.E429A, p.I661T) and FXR (c.-1 G>T) genes. The genotyping was performed using PCR-based assays with 5`-nuclease and fluorescence detection (TaqMan). Login to comment
59 The variants ABCB11 p.E297G and APT8B1 p.N45T were sequenced. Login to comment
57 To detect genetic factors contributing to severe phenotype, we genotyped procholestatic mutations and polymorphisms in the ABCB4 (p.R590Q, c.711A>T), ABCB11 (p.E297G, p.A444V, p.D482G, c.3084A>G), ATP8B1 (p.N45T, p.E429A, p.I661T) and FXR (c.-1 G>T) genes. The genotyping was performed using PCR-based assays with 5`-nuclease and fluorescence detection (TaqMan). Login to comment

PMID: 20955958 [PubMed] van der Woerd WL et al: "Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy."

No. Sentence Comment

67 In more than half of the European families the missense mutations E297G and/or D482G are present. Login to comment
69 Generally missense mutations, e.g. E297G or D482G, lead to a less severe phenotype than mutations that are predicted to result in premature protein truncation or total failure of protein production [6,23,29]. Login to comment
135 The type of mutation seems to be associated with the outcome of PEBD, with better prognosis in disease caused by milder mutations, especially for the ABCB11 mutations E297G and D482G [3,23]. Login to comment

PMID: 20034695 [PubMed] Stapelbroek JM et al: "Liver disease associated with canalicular transport defects: current and future therapies."

No. Sentence Comment

287 4-PBA has been tested in vitro for the E297G and D482G mutations frequently found inABCB11deficiency.Treatmentreducedtheproteinubiquitination and increased the cell surface expression of mature ABCB11 [200- 202]. Login to comment
286 4-PBA has been tested in vitro for the E297G and D482G mutations frequently found inABCB11deficiency.Treatmentreducedtheproteinubiquitination and increased the cell surface expression of mature ABCB11 [200-202]. Login to comment
285 4-PBA has been tested in vitro for the E297G and D482G mutations frequently found inABCB11deficiency.Treatmentreducedtheproteinubiquitination and increased the cell surface expression of mature ABCB11 [200- 202]. Login to comment

PMID: 19674157 [PubMed] Ikebuchi Y et al: "Receptor for activated C-kinase 1 regulates the cellular localization and function of ABCB4."

No. Sentence Comment

13 For example, we have found that E297G and D482G mutations in ABCB11, which are frequently found in PFIC2 patients, are associated with the retention of these mutated transporters in the endoplasmic reticulum (ER), although their transport function itself remains normal;20 indeed, following treatment with sodium 4-phenylbutyrate, E297G and D482G mutants appeared on the plasma membrane and were able to excrete substrate bile salts in Madin-Darby canine kidney (MDCK) II cells.21 In addition, insertion of several kinds of mutations in the ABCG5 or ABCG8 gene, which is found in sitosterolemia patients, results in the ER localization of this heterodimer.22 Furthermore, it has been demonstrated that localization of ABCB4 on the bile canalicular membrane is less marked in patients with one of the PFIC3 mutations.15 Concerning the pathogenesis of PFIC3, Dixon et al. tried to examine the localization and function of PFIC3 mutant in HEK293T cells;23 due to the difficulties in establishing a functional assay system of ABCB4 in mammalian cells, they introduced the PFIC3 mutation to the equivalent site of MDR1/ABCB1 to examine the function and localization of the mutated protein.23,24 From this perspective, it is important to identify the mechanism for the cellular localization of membrane transporters. Login to comment

PMID: 16890614 [PubMed] Keitel V et al: "Combined mutations of canalicular transporter proteins cause severe intrahepatic cholestasis of pregnancy."

No. Sentence Comment

79 In addition to changes in protein localization, V444A may alter transport activity as described recently for 2 BRIC-associated BSEP mutations (R432T and E297G).19 Another possibility of reduced transporter activity includes an increased susceptibility of V444A toward the inhibitory effect of estrogens.20 Homozygous V444A can be expected in 25% of the population, but homozygous V444A alone cannot explain the severity of ICP in our patient. Login to comment

PMID: 16180115 [PubMed] Ito K et al: "Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport."

No. Sentence Comment

240 Seven amino acid substitutions in BSEP, linked to PFICII (G238V, E297G, C336S, D482G, G982R, R1153C, R1268Q), have been reported and have been examined using rat Bsep expressed in MDCK (128). Login to comment
241 Five of these mutations resulted in disappearance from the apical surface in MDCK cells (G238V, E297G, G982R, R1153C, R1268R) (128). Login to comment

PMID: 14699511 [PubMed] Kullak-Ublick GA et al: "Enterohepatic bile salt transporters in normal physiology and liver disease."

No. Sentence Comment

117 It is caused by mutations of the BSEP (ABCB11) gene, which is located on chromosome 2q 24.173 Children with PFIC2 do not express BSEP.174 When PFIC2-related BSEP mutations are introduced artificially into rat Bsep and expressed in Madin-Darby canine kidney and Sf9 insect cells, the G238V, E297G, G982R, R1153C, and R1268Q mutations prevent the protein from trafficking to the apical membrane, whereas the G238V mutant seems to be rapidly degraded by proteasomes.175 Whereas mutation C336S affects neither Bsep transport activity nor trafficking, mutations E297G, G982R, R1153C, and R1268Q abolish taurocholate transport activity. Login to comment
119 A clinical syndrome with recurrent intrahepatic cholestasis but normal liver architecture in an adolescent patient has been associated with compound heterozygosity for the E297G and a novel R432T mutation,176 suggesting that certain adult forms of cholestasis may also be caused by BSEP mutations and reduced transport function. Login to comment
141 Role of Bile Salt Transporters in the Pathogenesis of Liver Disease Species Transport protein Gene symbol Physiologic function Alterations in liver disease References Basolateral transport proteins Rat Ntcp Slc10a1 Naϩ-dependent hepatocellular bile salt uptake Decreased expression in rat models of cholestasis Decreased mRNA and protein levels during pregnancy, associated with decreased nuclear binding of HNF1␣ and RAR␣:RXR␣ 201,211,231 232 Human NTCP SLC10A1 Naϩ-dependent hepatocellular bile salt uptake Decreased mRNA and protein levels in human cholestatic liver disease 30,187 Decreased expression in HCC 72 Rat Oatp1 Slc21a1 Multispecific uptake of organic anions and amphipathic compounds Decreased expression in bile duct ligation and in ethinyl estradiol induced cholestasis 211,233 Oatp2 Slc21a5 Multispecific uptake of organic anions and of cardiac glycosides (digoxin) Decreased mRNA but not protein levels in carbon tetrachloride induced liver injury Decreased mRNA and protein levels in ethinylestradiol-induced cholestasis 234 126 Oatp4 Slc21a10 Multispecific uptake of organic anions and amphipathic compounds Decreased expression in bile duct ligation and sepsis 235 Human OATP-C SLC21A6 Hepatocellular uptake of bile salts and other organic anions Reduced mRNA in PSC and inflammatory cholestasis Decreased expression in HCC 29,30 217 OATP8 SLC21A8 Hepatocellular uptake of organic anions, peptides, and xenobiotics Decreased expression in HCC because of increased expression of the transcriptional repressor HNF3beta 218 Rat/human Mrp1/MRP1 ABCC1 Efflux of cytotoxic cations and non-bile salt organic anions Increased expression in hepatoma cells and sepsis 199,236 Rat/human Mrp3/MRP3 ABCC3 Efflux of organic anions, bile salts, and anticancer agents Increased expression in Eisai Hyperbilirubinemic Rats and in bile duct ligation Increased expression in Dubin-Johnson syndrome and primary biliary cirrhosis 237 61 Hepatocyte canalicular transport proteins Mouse/rat/ human Bsep/Bsep/ BSEP ABCB11 Canalicular efflux of bile salts Gene mutations and absence of the protein in patients with PFIC2, characterized by low GGT levels and reduced biliary bile acid excretion 174,238 Compound heterozygosity for the E297G/R432T mutations in a patient with recurrent intrahepatic cholestasis 176 Reduced mRNA and canalicular BSEP staining in human inflammatory cholestasis 30 Cisinhibition by cholestatic drugs such as cyclosporine A 190 Transinhibition by the cholestatic estrogen metabolite estradiol-17beta-D-glucuronide 190,239 Increased expression in C57L/J gallstone-susceptible mice, despite reduced bile salt excretory capacity 220,240 Mouse/rat/ human Mdr2/Mdr2/ MDR3 ABCB4 Biliary excretion of phospholipids Mdr2 -/- knockout mice exhibit an absence of phospholipids in bile and develop progressive liver disease with portal inflammation, bile duct proliferation and fibrosis 241 PFIC3, characterized by high GGT levels and absent lipoprotein X in serum, is caused by mutations in the MDR3 gene (chromosome 7q21) 177 MDR3 mutations in PFIC3 are associated with intrahepatic cholestasis of pregnancy 242 Rat/human Mrp2/MRP2 ABCC2 Canalicular excretion of organic anions Decreased mRNA and protein levels in bile duct ligation and endotoxinemia 200,243 Decreased canalicular density of Mrp2 transporter molecules in endotoxinemia, taurolithocholate cholestasis, and bile duct ligation 145,200,243 Mutations in the rat Mrp2 gene cause hereditary conjugated hyperbilirubinemia 244 Mutations in the human MRP2 gene cause the Dubin-Johnson syndrome with absent protein expression 181,183 MRP2 function is inhibited by anabolic 17␣-alkylated steroids 245,246 Decreased canalicular MRP2 staining in PBC and inflammatory cholestasis 30,31 Decreased mRNA levels in PSC 29 Human FIC1 ATP8B1 Putative aminophospholipid translocator P-type ATPase, positional candidate in genetic linkage analysis of PFIC1 (Byler`s disease) and BRIC 171 PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis. Login to comment

PMID: 18698235 [PubMed] Kobayashi K et al: "Functional analysis of nonsynonymous single nucleotide polymorphism type ATP-binding cassette transmembrane transporter subfamily C member 3."

No. Sentence Comment

156 A similar phenomenon that the protein accumulated in ER seems potentially functional has been reported for ABCB11 mutations, such as E297G Fig. 4 rat Abcc2-WT rat Abcc2 Calnexin rat Abcc2 Calnexin merge merge rat Abcc2-R1388S Subcellular localization of mutated rat Abcc2 in HEK293 cells. Login to comment
173 For example, E297G ABCB11 is mislocalized to ER when heterologously expressed in MDCK cells [39,40], although the patient with compound heterozygosity for E297G and R432T ABCB11 showed normal canalicular localization, and the primary cause of dysfunction of ABCB11 was attributed to the reduced transport function [41]. Login to comment

PMID: 21766090 [PubMed] Beausejour Y et al: "Description of two new ABCB11 mutations responsible for type 2 benign recurrent intrahepatic cholestasis in a French-Canadian family."

No. Sentence Comment

87 Among the two most common mutations in individuals of European descent are the E297G and D482G mutations, which collectively account for approximately 58% of all cases (9). Login to comment
86 Among the two most common mutations in individuals of European descent are the E297G and D482G mutations, which collectively account for approximately 58% of all cases (9). Login to comment

PMID: 23141890 [PubMed] Jacquemin E et al: "Progressive familial intrahepatic cholestasis."

No. Sentence Comment

151 Preliminary data suggest that PFIC2 patients with p.D482G or p.E297G mutations may respond well to biliary diversion. Login to comment

PMID: 11815775 [PubMed] Chen HL et al: "FIC1 and BSEP defects in Taiwanese patients with chronic intrahepatic cholestasis with low gamma-glutamyltranspeptidase levels."

No. Sentence Comment

111 The BSEP (ABCB11) mutation V284L found in case 7 was in the vicinity of common European mutation E297G.7 No common mutations were shared among our patients, indicating that these mutations did not come from 123 Group 1 Group 2 P (n = 5) (n = 8) value Peak bilirubin (<17.1 &#b5;mol/L) 277 615 .059 (Range) (137-573) (180-1094) Peak AST (<60 U/L) 137 876 .002* (Range) (50-384) (504-1530) Peak ALT (<50 U/L) 74 471 .001* (Range) (24-174) (237-847) Serum GGT (<94 U/L) 24 55 .018* (Range) (14-41) (23-98) Serum bile acid (<10 &#b5;mol/L) 180 71 .001* (Range) (139-214) (16-127) Serum cholesterol (<200 U/L) 103 196 .207 (Range) (74-134) (47-438) Serum triglyceride (<200 U/L) 206 209 .966 (Range) (106-323) (38-448) AFP SD score 0.57 8.76 .007* (Range) (0-1.36) (2.4-16.2) Group 1, Patients with bland cholestasis in histology; Group 2, patients with giant cell transformation in histologic features. Login to comment

PMID: 16039748 [PubMed] Noe J et al: "Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis."

No. Sentence Comment

4 Results: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((C3)AOC) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Login to comment
6 In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. Login to comment
61 The single nucleotide polymorphisms 890AOG (codon GAGOGGG) and 1294GOC (codon AGAOACA), which result in the nonsynonymous mutations E297G and R432T, respectively, were introduced into the reference h ABCB11 cDNA. Login to comment
68 Functional transport studies ATP-dependent transport of labeled bile salts was determined for the E297G and the R432T mutations by a rapid filtration assay as described [16]. Login to comment
100 The parents of the patient had normal liver enzyme levels in the absence of clinical signs of hepatopathy. 3.2.1. Sequencing Sequence analysis indicated a nonsynonymous mutation in exon 9 (891GOA) predicting the substitution of a glutamate by a glycine in position 297 (E297G) combined with a nonsynonymous mutation in exon 12 (1296GOC), predicting a substitution of an arginine by a threonine in position 432 (R432T). Login to comment
101 Analysis of the patient`s parents indicated that the E297G mutation was inherited from the mother, whereas the R432T mutation was inherited from the father (Fig. 1). Login to comment
110 Transport studies Western blot analysis detected comparable protein amounts for R432T, E297G and reference BSEP in SF-9 cell vesicles (Fig. 3). Login to comment
111 Transport capacity of the mutated constructs amounted to about 13 and 20% of reference activity for the R432T and the E297G construct, respectively (Fig. 3). Login to comment
112 Initial taurocholate transport by reference BSEP as well as by R432T and E297G mutants exhibited saturability with increasing substrate concentrations (Fig. 4). Login to comment
114 The Km values for R432T and the E297G mutant were 5.6 mmol/L and 22 mmol/L, respectively, which is comparable to the Km value of reference BSEP. Login to comment
115 In contrast, the maximum taurocholate transport velocity for the BSEP mutants was greatly reduced, with Vmax values of 53 pmol/L mg proteinK1 minK1 , and 111 pmol/ L mg proteinK1 minK1 for R432T and E297G, respectively, compared to 686 pmol/L mg proteinK1 minK1 for reference BSEP (Fig. 4). Login to comment
127 Compound heterozygosity for two nonsynonymous variants in exon 9 (E297G) and in exon 12 (R432T) was encountered in the patient exhibiting a BRIC phenotype. Login to comment
128 While the E297G site had already been associated with inherited intrahepatic cholestasis, the R432T mutation was previously undescribed in cholestatic disease. Login to comment
130 Out of the detected variants, the E297G mutation had been functionally characterized by Wang and Hayashi [12,17], who found this mutation to result in defective trafficking of the protein to the apical membrane of transfected MDCK cells, whereas in-vitro transport studies yielded discrepant results. Login to comment
133 However, it cannot completely be excluded that in our patient the R432T allele is compensating for defective BSEP expression associated with the E297G mutation. Login to comment
134 In previous studies the E297G site has been associated with different clinical phenotypes as it was found in patients with PFIC2 and BRIC2 syndrome as well as in phenotypically normal subjects, indicating variable penetrance of this mutation [5-8,11]. Login to comment
137 As our data indicate residual BSEP function for the E297G variant, it can be suspected that the E297G variant alone is not responsible for the observed phenotype in patients with progressive intrahepatic cholestasis. Login to comment
146 Kinetics of ATP-dependent transport of taurocholate by reference BSEP, R432T BSEP mutant and E297G BSEP mutant in membrane vesicles isolated from Sf9 cells. Login to comment
147 Initial uptake rates for taurocholate by reference BSEP, E297G mutant (60 s) and R432T mutant (90 s) were determined in the presence and absence of ATP (5 mmol/L). Login to comment
150 The E297G example demonstrates that homo- or heterozygosity for a nonsynonymous ABCB11 mutation does not suffice to explain a PFIC2 or BRIC2 phenotype as long as its consequences on BSEP expression and function are not established. Login to comment
154 Differences in experimental design might also explain the recent discrepancies observed with in-vitro phenotyping of the E297G mutation by different groups [12,17]. Login to comment
160 Initial data suggest that alleles with residual function such as the E297G mutant are predictive for a positive outcome of bile duct diversion [18] and the functional characterization of ABCB11 mutants might therefore help to determine patients, who will profit from such an intervention. Login to comment

PMID: 18395098 [PubMed] Strautnieks SS et al: "Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families."

No. Sentence Comment

7 Thirty-two percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% of European families (52/89). Login to comment
9 Expression varied most for E297G and D482G, with some BSEP detected in 45% of patients (19/42) with these mutations. Login to comment
77 The common mutations E297G, D482G, R575X, R1153C, and R1153H abolish HphI, FokI, FokI, BsrBI, and BsrBI sites, respectively, while G982R creates an AlwNI site. Login to comment
98 Most frequent were E297G and D482G, one or both of which were present in 58% of European families (52/89) and 15% of non-European families (3/20). Login to comment
99 E297G was detected in 34 European families (41 alleles) and on one allele in both an African-American and a South Asian family. Login to comment
112 In 23 families homozygosity was associated with known consanguinity, while in 9 families, 2 copies of either E297G or D482G were found. Login to comment
113 To assess effects of specific ABCB11 genotypes on expression of immunohistochemically detectable BSEP protein, families were grouped according to whether they carried 2 likely protein-truncating mutations, at least one missense mutation (E297G or D482G excluded), at least one copy of E297G, at least one copy of D482G, or only one identified mutation (Supplementary Tables 1A-E). Login to comment
116 Variability in BSEP expression was greatest when either of the 2 common European mutations, E297G or D482G, was present on one or both alleles (Supplementary Tables 1C-E). Login to comment
117 Twenty-nine patients with at least one copy of E297G were immunostained; BSEP staining was not detected in 16 (55%), was deficient in 12 (41%), and was normal in 1. Login to comment
142 The exception was E297G, present on a single allele in 1 of 200 European white control chromosomes (http://pharmacogenetics.ucsf.edu),39 in keeping with the high frequency of this allele among European BSEP-deficient patients. Login to comment
150 Missense Mutations in ABCB11 Nucleotide change Predicted effect Exon CpG site Location Change in: Size Charge Hyd/Pol Shape c.149Tb0e;C p.Leu50Ser 4 No NH2 term Y Y Y c.470Ab0e;G p.Tyr157Cys 6 No TM2 Y Y Y c.725Cb0e;T p.Thr242Ile 8 No TM4 Y Y c.890Ab0e;G p.Glu297Gly 9 No IC2 Y Y Y c.908Gb0e;A p.Arg303Lys 9 No IC2 c.937Cb0e;A p.Arg313Ser 10 Yes IC2 Y Y Y Y c.980Gb0e;A p.Gly327Glu 10 No TM5 Y Y Y c.1168Gb0e;C p.Ala390Pro 11 No TM/NBF Y c.1229Gb0e;A p.Gly410Asp 12 No TM/NBF Y Y c.1238Tb0e;G p.Leu413Trp 12 No TM/NBF c.1388Cb0e;T p.Thr463Ile 13 No Adj Walker A Y Y Y c.1396Cb0e;A p.Gln466Lys 13 No Adj Walker A Y c.1409Gb0e;A p.Arg470Gln 13 Yes Adj Walker A Y c.1415Ab0e;G p.Tyr472Cys 13 No Adj Walker A Y Y Y c.1442Tb0e;A p.Val481Glu 14 No NBF1 Y Y Y c.1445Ab0e;G p.Asp482Gly 14 No NBF1 Y Y c.1460Gb0e;C p.Arg487Pro 14 Yes NBF1 Y Y Y Y c.1468Ab0e;G p.Asn490Asp 14 No NBF1 Y c.1535Tb0e;C p.Ile512Thr 14 No NBF1 Y Y Y c.1544Ab0e;C p.Asn515Thr 14 No NBF1 Y Y c.1550Gb0e;A p.Arg517His 14 Yes NBF1 Y Y c.1621Ab0e;C p.Ile541Leu 14 No NBF1 c.1622Tb0e;C p.Ile541Thr 14 No NBF1 Y Y Y c.1643Tb0e;A p.Phe548Tyr 15 No Adj ABC c.1685Gb0e;A p.Gly562Asp 15 No ABC Y Y c.1708Gb0e;A p.Ala570Thr 15 Yes ABC/Walker B Y c.1763Cb0e;T p.Ala588Val 15 No Adj Walker B Y c.2272Gb0e;C p.Gly758Arg 19 No NBF/TM Y Y Y c.2296Gb0e;A p.Gly766Arg 19 Yes TM7 Y Y Y c.2494Cb0e;T p.Arg832Cys 21 Yes IC3 Y Y Y Y c.2576Cb0e;G p.Thr859Arg 21 No IC3 Y Y Y Y c.2842Cb0e;T p.Arg948Cys 23 Yes IC4 Y Y Y Y c.2935Ab0e;G p.Asn979Asp 23 No TM11 Y c.2944Gb0e;A p.Gly982Arg 23 Yes TM11 Y Y Y c.3086Cb0e;A p.Thr1029Lys 24 No TM12 Y Y Y Y c.3329Cb0e;A p.Ala1110Glu 25 Yes Adj Walker A Y Y Y c.3382Cb0e;T p.Arg1128Cys 25 Yes Adj Walker A Y Y Y Y c.3457Cb0e;T p.Arg1153Cys 26 Yes NBF2 Y Y Y Y c.3458Gb0e;A p.Arg1153His 26 Yes NBF2 Y Y c.3460Tb0e;C p.Ser1154Pro 26 No NBF2 Y c.3628Ab0e;C p.Thr1210Pro 27 No Adj ABC Y c.3691Cb0e;T p.Arg1231Trp 27 Yes ABC/Walker B Y Y c.3692Gb0e;A p.Arg1231Gln 27 Yes ABC/Walker B Y c.3724Cb0e;A p.Leu1242Ile 27 No Walker B c.3892Gb0e;A p.Gly1298Arg 28 No NBF2 Y Y Y NOTE. Login to comment
156 The intracellular loops contained 6 changes (13%), of which 3, including E297G, occurred in intracellu- Table 3. Login to comment
162 In 5 cases, the single mutations were splice-site changes or E297G/D482G. Login to comment
208 By far most common, however, were E297G and D482G, at least one of which was present in 58% (52/89) of European and in 15% (3/20) of non-European families. Login to comment
209 The population distribution of E297G, most frequent along the North European seaboard, suggests an origin in Northern Europe and spread southward through Central and Eastern Europe. Login to comment
222 BSEP deficiency represents a phenotypic continuum between progressive early-onset1 disease and remitting, and late-onset, phenotypes.3-7 Eleven different mutations have been reported in BSEP deficiency disease clinically assessed as intermediate6 or mild in severity.4,5,7 Three of these, E297G, A570T, and c.2178af9;1Gb0e;A, have also been found in progressive familial intrahepatic cholestasis. Login to comment
225 That homozygosity for E297G is seen in discrepant phenotypes strongly indicates that environment and genetic background also play roles. Login to comment
231 Residual staining was most striking in patients with E297G or D482G, with some seen in 45% of patients (19/42) carrying at least one of these alleles. Login to comment
234 Because most of these were found in combination with E297G or D482G, their individual effects could not be assessed. Login to comment

PMID: 20398791 [PubMed] Kato T et al: "Short- and medium-chain fatty acids enhance the cell surface expression and transport capacity of the bile salt export pump (BSEP/ABCB11)."

No. Sentence Comment

5 Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. Login to comment
19 We and other groups have indicated previously that the reduced cell surface expression of BSEP is responsible for the dysfunction of BSEP in PFIC2 patients with E297G or D482G mutation, both of which are observed frequently in European PFIC2 patients [12,14-17]. Login to comment
29 We previously reported that 4-phenylbutyrate (4PBA), an approved drug for treating urea cycle disorders, is a promising agent for the treatment of intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of wild type (WT), and PFIC2-type (E297G and D482G) BSEP [18]. Login to comment
34 Therefore, in this study, we investigated the potency of short-and medium-chain fatty acids against intrahepatic cholestasis by assessing the effects of these compounds on WT, E297G, and D482G BSEP expression and their transport functions using western blot analysis and transcellular transport, respectively. Login to comment
41 Generation of recombinant adenovirus The BD Adeno-X Adenoviral Expression System (BD Biosciences) was used to establish the human WT, HA-WT, E297G, D482G BSEP and BCRP recombinant adenoviruses as described previously [3,14,22,23]. Login to comment
49 After 2 days of culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP or GFP at 20 MOI. Cells were cultured for 24 h after infection and subsequently treated with media containing 4PBA, formate, acetate, propionate, butyrate, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, or decanoic acid, each at 1 mM. Then, 24 h after treatment, transcellular transport assays were performed as described [18,24]. Login to comment
51 WT, E297G, and D482G BSEP-dependent PSapical (PSapical, WT BSEP-dependent, PSapical, E297G BSEP-dependent, and PSapical, D482G BSEP-dependent) were calculated by subtracting the PSapical value in MDCK II cells expressing GFP (PSapical, GFP) from that in MDCK II cells expressing WT, E297G, and D482G BSEP (PSapical, WT BSEP, PSapical, E297G BSEP, and PSapical, D482G BSEP), respectively. Login to comment
57 Preparation of crude membrane fraction MDCKII cells were seeded in 12-well plates at a density of 2.5&#d7;105 cells per well. After 24 h of culture, confluent cells were infected by recombinant adenovirus containing cDNAs for E297G or D482G BSEP at 200 MOI. Login to comment
73 Up-regulation of WT, E297G, and D482G BSEP-mediated transport by fatty acids We previously found that 4PBA treatment induces WT, E297G, and D482G BSEP expression at the cell surface by inhibiting the degradation of cell surface-resident WT, E297G, and D482G BSEP, and which facilitates the biliary excretion of bile acids. Login to comment
74 In this study, we first examined whether short-and medium-chain fatty acids, which have similar characteristics to 4PBA such as a carboxyl group and low-molecular-weight, can increase the cell surface expression of WT, E297G, and D482G BSEP and enhance WT, E297G, and D482G BSEP-mediated bile acid transport in a manner similar to that of 4PBA. Login to comment
80 The effect of fatty acid treatment on transport function of WT, E297G, and D482G BSEP in MDCK II cells. Login to comment
81 (A-C) MDCK II cells expressing WT (A), E297G (B), D482G BSEP (C), or GFP (A-C) were treated with formate, acetate, propionate, butyrate, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, or decanoic acid, each at 1 mM, for 24 h before the experiments. Login to comment
82 Transcellular transport of 1 bc;M [3 H]TC across MDCK II monolayers expressing WT (A), E297G (B), D482G BSEP (C), or GFP (A-C) was examined. Login to comment
84 The PSapical was expressed as the percentage of PSapical in MDCK II cells expressing WT (A), E297G (B), or D482G (C) BSEP under the untreated condition. Login to comment
85 The closed and open bars represent WT (A), E297G (B), or D482G (C) BSEP- and GFP (A-C)-expressing MDCK II cells, respectively. Each bar represents the mean&#b1;S.E. of triplicate determinations. Asterisks represent statistically significant differences between the control and the compound-treated cells. Login to comment
89 Butyrate and octanoic acid, the most potent short-and medium-chain fatty acids, respectively, also enhanced PSapical of [3 H]TC in MDCK II cells expressing E297G and D482G BSEP, both of which are the most frequently observed in European PFIC2 patients (Fig. 1B and C). Login to comment
90 PSapical, E297G BSEP-dependent and PSapical, D482G BSEP-dependent substantially increased with the treatment of butyrate and octanoic acid 8.4- and 3.1-fold in MDCK II cells expressing E297G BSEP and 3.6- and 1.7-fold in that expressing D482G BSEP, respectively. Login to comment
92 Increased WT, E297G, and D482G BSEP expression after fattyacid treatment Results from a cell surface biotinylation study showed that WT BSEP expression at the cell surface of MDCK II cells increased 2.7-, and 6.1- Fig. 2. Login to comment
93 The effect of octanoic acid and butyrate treatment on expression level of WT, E297G, and D482G BSEP in MDCK II cells. Login to comment
94 (A-C) Determination of expression level of HA-WT (A), E297G (B), and D482G BSEP (C). Login to comment
95 MDCK II cells expressing HA-WT (A), E297G (B), or D482G BSEP (C) were treated with 4PBA, butyrate, or octanoic acid, each at 1 mM, for 24 h before the experiments. Login to comment
97 Quantification of band density indicating the 170 kDa forms of HA-WT (A), E297G (B), or D482G BSEP (C) was also shown (right). Login to comment
105 The expression levels of 170 kDa form of E297G and D482G BSEP in crude membrane fractions, which are considered to be the mature form located on the cell surface [14,18], were also elevated 3.1and 2.7-fold for E297G BSEP and 2.6- and 2.2-fold for D482G BSEP with the treatment of butyrate and octanoic acid, respectively (Fig. 2B and C). Login to comment
106 Since it is difficult to detect E297G and D482G in cell surface fractions due to low expression, crude membrane fractions were used instead to determine the alteration in those expression levels. Login to comment
107 The increased expression rates of WT BSEP on the cell surface and of the 170 kDa forms of E297G and D482G BSEP in crude membrane fractions induced by these three compounds correlated with those of WT, E297G, and D482G BSEP-mediated [3 H]TC transport (Fig. 3A-C). Login to comment
108 This suggests that all three compounds increase WT, E297G, and D482G BSEP-mediated transport by inducing WT, E297G, and D482G BSEP expression at the cell surface, but not by the activating WT, E297G, and D482G BSEP transport function itself. Login to comment
111 Neither compounds had any effect on these expression levels (Fig. 2D), suggesting that both compounds do indeed increase the expression of WT, E297G, and D482G BSEP with high specificity. Login to comment
119 Correlation between expression level and transport capacity of WT, E297G, and D482G BSEP under octanoic acid- and butyrate-treated conditions. Login to comment
120 The WT, E297G, and D482G BSEP-dependent clearance of [3 H]TC across the apical membrane of MDCK II monolayers (PSapical, WT BSEP-dependent (A), PSapical, E297G BSEP-dependent (B), PSapical, D482G BSEP-dependent (C)) treated with 4PBA, butyrate, or octanoic acid was determined as described in 'Materials and methods` using the results of Fig. 1. Login to comment
121 The PSapical, WT BSEP-dependent (A), PSapical, E297G BSEP-dependent (B), and PSapical, D482G BSEP-dependent (C) are expressed as the percentages of these values under the untreated condition. Login to comment
122 The expression level of the 170 kDa forms of HA-WT BSEP in cell surface fractions and E297G and D482G BSEP in crude membrane fractions are taken from those in Fig. 2A-C. Login to comment
136 We previously demonstrated that 4PBA is a potential therapeutic agent to combat intrahepatic cholestasis, which induces the cell surface expression of WT and PFIC2-type (E297G and D482G) BSEP by slowing its degradation rate from cell surface [18]. Login to comment
143 Both butyrate and octanoic acid were also effective on E297G and D482G BSEP (Figs. Login to comment
148 Although there is no evidence for the molecular mechanism responsible for the effect elicited by octanoic acid, based on our previous study of 4PBA [22], it is likely that octanoic acid reduces the susceptibility of ubiquitination of WT, E297G, and D482G BSEP, an important regulatory signal for degradation of these proteins on the cell surface [22], by inactivating the enzymes involved in the ubiquitination reaction and consequently also increases their expressions at the cell surface. Login to comment
149 Another possible explanation is that octanoic acid directly stabilizes the cell surface-resident WT, E297G, and D482G BSEP and interrupts their ubiquitinations at the cell surface. Login to comment
151 Because the mRNA expression level of WT BSEP itself was not significantly increased by butyrate treatment under the experimental conditions we used in this study (Fig. 5), it is possible that butyrate induces a gene expression that promotes the translation and/or inhibits the ER-associated protein degradation pathway, and subsequently increases the amount of WT, E297G, and D482G BSEP in Fig. 5. Login to comment
172 The rapid degradation of BSEP/Bsep from the cell surface partly contributes to the reduced BSEP/Bsep expression at the cell surface in PFIC2 patients with E297G or D482G mutations and in rat models of endotoxin- or drug-induced cholestasis [10-17], suggesting that octanoic acid may combat intrahepatic cholestasis. Login to comment
174 Since octanoic acid induced the expression of E297G and D482G BSEP, accompanied with the enhanced [3 H]TC transport by them (Fig. 3B and C), particularly, PFIC2 patients with E297G or D482G mutations are expected to benefit from its effectiveness. Login to comment
182 Considering the effectiveness on E297G and D482G BSEP, a particular benefit to PFIC2 patients with either an E297G or D482G mutation is expected. Login to comment

PMID: 20447715 [PubMed] Pawlikowska L et al: "Differences in presentation and progression between severe FIC1 and BSEP deficiencies."

No. Sentence Comment

64 We also performed exploratory analyses to evaluate whether BSEP patients carrying 1 or 2 copies of the common European D482G (c.1445A>G) and/or E297G (c.890A>G) mutations differed from other BSEP patients, and whether FIC1 patients carrying G308V differed from other FIC1 patients. Login to comment
77 One of two 'common` ABCB11 mutations (D482G or E297G) was identified on one or both alleles in 51 BSEP patients (61%) [2,12]. Login to comment
81 Since functional studies have suggested that the BSEP D482G and E297G mutations may not completely abolish protein function [9,44-47], we performed exploratory analyses stratified by mutation. Login to comment

PMID: 22464344 [PubMed] Misawa T et al: "Discovery and structural development of small molecules that enhance transport activity of bile salt export pump mutant associated with progressive familial intrahepatic cholestasis type 2."

No. Sentence Comment

0 Discovery and structural development of small molecules that enhance transport activity of bile salt export pump mutant associated with progressive familial intrahepatic cholestasis type 2 Takashi Misawa a , Hisamitsu Hayashi b , Yuichi Sugiyama b,d1; , Yuichi Hashimoto a,d1; a Institute of Molecular and Cellular Biosciencies, The University of Tokyo, 1-1-1 Bunkyo-ku, Tokyo 113-0032, Japan b Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan a r t i c l e i n f o Article history: Received 13 February 2012 Revised 5 March 2012 Accepted 5 March 2012 Available online 14 March 2012 Keywords: Bile salt export pump Bile acid Farnesoid X receptor Mutant a b s t r a c t Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by hereditary mutations of bile salt export pump (BSEP), such as E297G BSEP, which is a folding-defective mutant that is unable to traffic beyond the endoplasmic reticulum (ER). Login to comment
1 4-Phenylbutyric acid (4-PBA) enhances the cell surface expression and transport capacity of E297G BSEP, but has a relatively high dose (1 mM or more) is required to show the effect. Login to comment
2 Here, we show that bile acids possibly act as pharmacological chaperones, promoting the proper folding and trafficking of E297G BSEP. We also describe the discovery and structural development of non-steroidal compounds with potent pharmacological chaperone activity for E297G BSEP. Login to comment
8 Genomic analysis of PFIC2 patients has revealed many kinds of missense, premature termination, frame-shift and splicing-junction mutations in the BSEP gene.3 Among them, two missense mutations, E297G (Glu297Gly) and D482G (Asp482Gly), are frequently observed in PFIC2 patients.4,5 Generally, wild-type (WT) BSEP is synthesized and folded at the endoplasmic reticulum (ER). Login to comment
10 But, the E297G mutant is considered to be folding-defective (misfolded). Login to comment
12 Hayashi et al. reported that the E297G and D482G mutations cause a reduction of BSEP on plasma membrane, but importantly, they found that the transport function of both mutated BSEPs is almost normal if they localized correctly.5 Thus, the folding-defective nature, linked to abnormal trafficking (retention at ER), of E297G mutant lies at the core of the etiology of PFIC2, at least in part. Login to comment
13 If E297G BSEP could be induced to fold properly, the mutant protein could be transported to the correct localization, where it should be functional, providing a promising strategy for treatment of E297G BSEP-related PFIC2. Login to comment
14 Hayashi et al. reported that sodium 4-phenylbutylic acid (4-PBA) enhanced the cell-surface expression and transport capacity of E297G BSEP by prolonging the half-life of cell surface-resident BSEP, through modulating its ubiquitination status and AP2-mediated internalization.5-7 Although 4-PBA can be beneficial for the treatment for PFIC2, a relatively high dose (1 mM or more) is required to rescue the function of the mutant BSEP in vivo. Login to comment
16 Based on this background, we searched for novel compounds able to enhance the transport capacity of E297G BSEP at lower doses. Login to comment
21 Bioorganic & Medicinal Chemistry 20 (2012) 2940-2949 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc target protein and induces or promotes proper folding and trafficking of the protein.13 The advantages of pharmacological chaperones are considered to include high specificity and efficacy at much lower dose levels than 4-PBA.13 In the cases of some folding-defective mutant proteins, including mutated rhodopsin, it has been shown that their ligands (substrates, inhibitors, modulators, etc.) act as pharmacological chaperones.8 Therefore, we hoped that bile acids, which are substrates of BSEP, might act as pharmacological chaperones of E297G BSEP, consequently enhancing the cell-surface expression and transport capacity of E297G BSEP. Login to comment
22 Here, we show that bile acids do act as pharmacological chaperones of E297G BSEP. We also describe the discovery and structural development of non-steroidal compounds with potent pharmacological chaperone activity for E297G BSEP. Login to comment
25 Effects of bile acids on [3 H]TC accumulation To screen compounds that enhance the transport capacity of E297G BSEP, we established a functional assay system, using Madin-Darby canine kidney II (MDCKII) cells expressing Na+ -taurocholate cotransporting polypeptide (NTCP) and WT or E297G BSEP. Login to comment
26 The amount of [3 H]taurocholate ([3 H]TC) accumulation in cells expressing E297G BSEP was higher than that in cells 0.00 0.50 1.00 1.50 2.00 2.50 WT E297G CA 0uM 10uM 100uM [ 3 H]TC accumulation (BSEP cells/GFP cells) 0&#b5;M 10&#b5;M 100&#b5;M Figure 1. Concentration-dependence of the effect of cholic acid (CA) on [3 H]TC accumulation in MDCK II cells expressing wild type (WT) BSEP or E297G BSEP. Vertical scale: The amounts of [3 H]TC accumulated in the MDCK II cells transfected with WT BSEP, E297G BSEP or GFP were measured by means of radioactivity (dpm). Login to comment
28 Table 1 Decreasing effect of bile acids on [3 H]TC accumulation O O R2 R3 H R3 H H H HO R1 H H H HO R1 Compd R1 R2 R3 Accumulation of [3 H] TC (%) 100 lM 10 lM Cholic acid (CA) OH OH OH 40 73 Glycocholic acid (GC) OH OH Glycine 108 Taurocholic acid (TC) OH OH Taurine 105 Deoxycholic acid (DCA) H OH OH 65 Chenodeoxycholic acid (CDCA) b-OH H OH 46 50 Ursodeoxycholic acid (UDCA) a-OH H OH 43 76 Table 2 Decreasing effect of GW4064 and derivatives on [3 H]TC accumulation N O O N R1 R4 R1 R1 N R3 R2 O R3 Compd R1 R2 R3 R4 Accumulation of [3 H]TC (%) at 10 lM CDCA 50 GW4064 33 6g Cl Cl 3-COOH H 111 6a Cl Cl 3-COOH Me 66 6b Cl Cl 3-COOH n-Bu 49 6c Cl Cl 3-COOH Bn 31 6d Cl Cl 3-COOH Phenethyl 53 6e Cl Cl 3-COOH 1-Naphthyl 59 6f Cl Cl 3-COOH 2-Naphthyl 70 15a H Cl 3-COOH Bn 64 15b Cl H 3-COOH Bn 85 15c Me Cl 3-COOH Bn 93 15d Cl Cl 2-COOH Bn 80 15e Cl Cl 4-COOH Bn 35 5c Cl Cl 3-COOMe Bn 93 expressing WT BSEP because of the cellular efflux function of [3 H]TC by BSEP and the lower cell-surface expression of E297G BSEP compared with WT BSEP (Fig. 1).14 We first examined the effect of several BSEP substrates and other related bile acids, that is, cholic acid (CA), glycocholic acid (GC), taurocholic acid (TC), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA), on the function of E297G BSEP. Login to comment
31 0 0.5 1 1.5 2 2.5 3 3.5 4 1 2 3 4 0 1 3 10 Concentration (&#b5;M) 6c [ 3 H]TC accumulation (BSEP cells/GFP cells) 0 0.5 1 1.5 2 2.5 3 3.5 1 2 3 4 0 1 3 10 Concentration (&#b5;M) GW4064 [ 3 H]TC accumulation (BSEP cells/GFP cells) (a) (b) Figure 3. Concentration-dependence of the effects of GW4064 (a), and 6c (b) on [3 H]TC accumulation in MDCK II cells expressing E297G BSEP. Vertical scale: same as the legend of Figure 1. Login to comment
39 Effect of 4-PBA and 6c on [3 H]TC transport across the apical membrane in monolayers of MDCK II cells expressing E297G BSEP. Login to comment
43 We speculated that bile acids increased the cell-surface expression of E297G BSEP by acting as pharmacological chaperones, thereby decreasing [3 H]TC accumulation in cells expressing E297G BSEP. Login to comment
49 CDCA is an endogenous FXR ligand,15,16 and acts as a signaling molecule that governs lipid, steroid, and cholesterol homeostasis.17 FXR is involved in possesses such as glucose utilization, inflammation, and carcinogenesis.18,19 After deorphanization of FXR in 1999, extensive studies were directed to the creation of FXRligands thatcouldmodulateFXR-mediatedspecificgene expression, and several steroidal or non-steroidal FXR ligands have been reported (Fig. 2).20-22 We hypothesized that non-steroidal FXR ligands might act as a structural equivalent of CDCA and enhance the transport capacity of E297G BSEP. First, we investigated whether FXR ligands enhance the transport capacity of E297G BSEP. We found that GW4064, which is a potent non-steroidal agonist for FXR, dose-dependently decreased [3 H]TC accumulation (Fig. 3a). Login to comment
50 GW4064has theadvantagethatitsbasic architecture isdistinctfrom that of CDCA, and it shows no activity towards other nuclear receptors, including the retinoic acid receptor.20 GW4064 seemed to be superior to CDCA in its efficacy to decrease [3 H]TC accumulation in cells expressing E297G BSEP (Table 2), and was selected as a lead compound for further structural development. Login to comment
52 Structure-activity relationship of GW4064 derivatives Kainuma and co-workers reported that the double bond of GW4064 can be replaced with a carbamoyl moiety without loss of FXR activity.23 Similarly, we designed and prepared secondary amide 6g, N-methyl amide 6a, N-butyl amide 6b, N-benzyl amide 6c, N-phenethyl amide 6d, and N-naphthyl amide 6e and 6f derivatives, and examined whether these derivatives decrease [3 H]TC accumulation in cells expressing E297G BSEP. Login to comment
66 As shown in Table 2, ester derivative 5c had no effect on [3 H]TC accumulation in cells expressing E297G BSEP at 10 lM. Login to comment
68 These results indicate that a carboxyl substituent at the meta- or para-position is important for interaction with E297G BSEP. Login to comment
72 Transcellular transport assay Finally, we evaluated the effectiveness of 6c in a transcellular transport assay with MDCK II cells expressing E297G BSEP. Login to comment
77 Conclusion We focused on the idea that the transport capacity of E297G BSEP might be increased by pharmacological chaperones that promote proper trafficking of the mutant BSEP. First, we established that bile acids decrease [3 H]TC accumulation in cells expressing E297G BSEP. Login to comment
80 Further studies to examine in detail the molecular mechanism(s) of the enhancement of E297G BSEP trafficking and the effects of these compounds on other BSEP mutants are in progress. Login to comment
91 The BD Adeno-X-Adenoviral Expression System (BD Biosciences, Palo Alto, CA) was used to establish human WT and E297G BSEP recombinant adenoviruses as previously described.14 As a control, recombinant adenovirus containing green fluorescence protein (GFP) was used. Login to comment
93 Generation of recombinant adenovirus The recombinant adenoviruses containing wild-type BSEP, E297G BSEP, or GFP were constructed as described previously.14 The BSEP cDNA was amplified via polymerase chain reaction(PCR) with Ex-Taq (Takara Bio, Shiga, Japan) from the commercially available cDNA library (Clontech, PaloAlto, CA). Login to comment
103 After 2 days` culture, confluent cells were infected with recombinant adenovirus containing cDNA for WT, E297G, BSEP or GFP at 50 MOI. Cells were cultured for 24 hours after infection and subsequently treated with compounds. Login to comment
107 After 1 day of culture, confluent cells were infected with recombinant adenovirus containing cDNA for WT, E297G BSEP or GFP at 50 MOI. Cells were cultured for 24 h after infection and subsequently treated with compounds. Login to comment

PMID: 22583617 [PubMed] Misawa T et al: "E297G mutated bile salt export pump (BSEP) function enhancers derived from GW4064: structural development study and separation from farnesoid X receptor-agonistic activity."

No. Sentence Comment

1 Several BSEP mutants, including Glu297Gly (E297G) and Asp482Gly (D482G), cause progressive familial intrahepatic cholestasis type 2. Login to comment
2 We previously found that compounds based on GW4064, a representative farnesoid X receptor (FXR) agonist, enhanced E297G BSEP transport activity. Login to comment
3 Here, we conducted a structure-activity relationship analysis of GW4064 derivatives aimed at separating E297G BSEP-function-promoting activity and FXR-agonistic activity. Among newly synthesized reversed-amide derivatives of previously reported GW4064 analogs 2a-2f, we identified 7c as a selective BSEP function enhancer. Login to comment
5 Bile salt export pump (BSEP) is a member of the ATP-binding cassette transmembrane transporter family and mediates the biliary excretion of monovalent bile acids (such as taurocholate) from hepatocytes.1-3 It is well known that several BSEP mutants cause progressive familial intrahepatic cholestasis type 2 (PFIC2) which is a recessive hereditary liver disease characterized by cholestasis and jaundice in the first year of life, leading to cirrhosis and death before adulthood unless liver transplantation is carried out.4 Among the PFIC2-causing mutations, two missense mutations, that is, E297G (Glu297Gly) and D482G (Asp482Gly), are frequently observed in PFIC2 patients.5,6 The E297G mutant is considered to be folding-defective (misfolded), and as a result, this mutant BSEP is retained within the cell on endoplasmic reticulum (ER), and does not acquire the usual golgi-related glycosylation. Login to comment
6 Hayashi et al. reported that the E297G and D482G mutations cause a reduction of BSEP on plasma membrane, but importantly, they found that the transport function of both mutated BSEPs is almost normal if they are correctly localized.7 Furthermore, Hayashi et al. reported that sodium 4-phenylbutyric acid (4-PBA) enhanced the cell-surface expression and transport capacity of E297G BSEP by prolonging the half-life of cell-surface-resident BSEP, through modulating its ubiquitination status and AP2-mediated internalization.6-8 In our previous work, we found that bile acids showed moderate E297G BSEP-function-promoting activity, and chenodeoxycholic acid (CDCA) was the most potent function-promoter among the tested bile acids.9 Furthermore, compounds based on GW4064 [a representative farnesoid X receptor (FXR) agonist10 ] ( Fig. 1) enhanced the BSEP transport activity. Among the tested compounds, compound 2d possessed the most potent E297G BSEP-function-promoting activity at 10 lM (Table 2). Login to comment
9 FXR is a well-characterized member of the so-called metabolic subfamily of nuclear receptors, and is a transcriptional sensor for bile acids.12,13 It is well known that FXR-agonists such as CDCA and GW4064 increase endogenous BSEP mRNA level and lower the bile acid pool.14 We considered that the contribution of FXR-agonistic activity to the improvement of BSEP function in our assay system was negligible, because we had calculated the ratio of the amount of [3 H]taurocholate ([3 H]TC) accumulated in MDCK II cells expressing WT or E297G BSEP versus that in the MDCK II cells expressing GFP to confirm the effect of GW4064 derivatives on the function of BSEP. Login to comment
18 Structure of GW4064 and E297G BSEP function enhancers. Login to comment
19 Table 1 E297G BSEP-function-promoting activity and FXR-agonistic activity of bile acids Compd R1 R2 Accumulation of [3 H]TCa (%) at 100 lM FXR-agonistic activity EC50 c (lM) CA (3) OH OH 40b NAd DCA (4) H OH 65b 50 CDCA (5) b-OH H 46b 27 UDCA (6) a-OH H 43b NAd a The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment
22 Table 2 FXR-agonistic activity of E297G BSEP function enhancers Compd R1 R2 Accumulation of [3 H]TCa (%) at 10 lM FXR-agonistic activity EC50 c (lM) and efficacyd (%) CDCA (5) 50b 27 (41) GW4064 (1) 33b 0.07 (100) 2a 3-COOH H 111b 3.0 (61) 2b 3-COOH Me 66b 3.1 (45) 2c 3-COOH n-Bu 49b 1.1 (43) 2d 3-COOH Bn 31b 2.5 (31) 2e 3-COOH Phenethyl 53b 0.84 (30) 2f 3-COOH 1-Naphthyl 59b 3.0 (28) 2g 2-COOH Bn 80b NAe 2h 4-COOH Bn 35b 2.6 (41) 2i 3-COOMe Bn 93b NAe a The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment
29 One was an accumulation assay to evaluate the E297G BSEP-function-promoting activity, using Madin-Darby canine kidney II (MDCKII) cells expressing Na+ -taurocholate cotransporting polypeptide (NTCP) and E297G BSEP, as described previously.9 With this method, we evaluated the amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP; this accumulation is determined by several parameters, including the cellular efflux of [3 H]TC via BSEP. Login to comment
31 We previously reported that these bile acids possess moderate E297G BSEP-function-promoting activity.9 Compounds 4 and 5 showed weak FXR-agonistic activity, whereas compounds 3 and 6 showed no FXR-agonistic activity (Table 1). Login to comment
32 These results suggest that E297G BSEP-function-promoting activity can be separated from FXR-agonistic activity. Login to comment
38 Next, we investigated the FXR-agonistic activity of our previously reported E297G BSEP function enhancers derived from GW4064 (2a-2i, Table 2). Login to comment
39 E297G BSEP function enhancers 2a-f and 2h showed weak FXR-agonistic activity, while 2g and 2i were inactive. Login to comment
47 In the case of GW4064, a brief SAR report from GSK researchers disclosed that a carboxyl group is essential and is preferably located at the 3-position (meta-position) for Table 3 E297G BSEP-function-promoting activity and FXR-agonistic activity of 7a-7e Compd R Accumulation of [3 H]TCa (%) at 10 lM FXR-agonistic activity EC50 (lM) and efficacyb (%) CDCA 50 27 (41) GW4064 33 0.07 (100) 2d 31 2.5 (31) 7a H 75 NAc 7b Me 60 NAc 7c n-Bu 42 NAc 7d Bn 50 >10 7e 1-Naphthyl 59 0.41 (35) a The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment
51 Concentration dependency of the effect of 2d, 7a, and 7c-7d on [3 H]TC accumulation in MDCK II cells expressing E297G BSEP. Login to comment
52 The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment
54 ortho-Isomer 2g and ester derivative 2i showed neither E297G BSEP-function-promoting nor FXR-agonistic activity, whereas the para-isomer (2h) showed these activities with similar potency to the meta-isomer 2d. Login to comment
56 As expected, E297G BSEP function enhancers (2a-2f, 2h) also showed FXR-agonistic activity (Table 2). Login to comment
57 As shown in Table 2, substitution of the double bond at the central portion of GW4064 (1) to an amide bond, that is, compound 2d, had no effect on E297G BSEP-function-promoting activity, but decreased the potency of FXR-agonistic activity, that is, the EC50 values for GW4064 (1) and 2d are 0.07 lM and 2.5 lM, respectively. Login to comment
62 So, we speculated that the N-substituted amide-type central portion of the molecule might be the key to separating E297G BSEP-function-promoting activity from FXR-agonistic activity. Login to comment
68 We evaluated the E297G BSEP-function-promoting activity and FXR-agonistic activity of the prepared reversed-amide derivatives 7a-7e (Table 3 and Fig. 3). Login to comment
69 These compounds possessed moderate to potent E297G BSEP-function-promoting activity. Among the reversed-amide derivatives, N-butyl derivative 7c possessed the most potent activity (accumulation of [3 H]TC was reduced to 42% and 59% at 10 lM and 1 lM, respectively, Table 3 and Fig. 3). Login to comment
71 In summary, we confirmed that bile acids and 2a-2f exhibit both FXR-agonistic activity and E297G BSEP-function-promoting activity. Login to comment

PMID: 22795478 [PubMed] Kubitz R et al: "The bile salt export pump (BSEP) in health and disease."

No. Sentence Comment

176 Interestingly, the very common polymorphism p.V444A of BSEP (allele frequency > 50%), which may occur isoallelic to mutations, strongly increases ERAD, as shown in expression studies of cloned BSEP [23]; thirdly, a decrease in half-life may shorten residency of BSEP at the canalicular membrane as shown for the two common PFIC-2 mutations p.E297G and p.D482G in Madin-Darby canine kidney (MDCK) cells [134]. Login to comment
182 Mutations such as p.E186G, p.E297G, p.R432T, p.A570T, p.T923P, p.A926P, p.G1004D, p.R1050C and p.R1128H have been described in BRIC-2 patients [137,140-142] (Table 1). Login to comment
185 PFIC BRIC/NFC ICP Other liver diseases Genetic variants without disease association Missense mutations M1V C336S D549V L1055P E135K E137K T87R V43I S701P G19R W342G G556R C1083Y E137K L198P M123T S56L L712L L50S A382G G562D A1110E E186G E297G S194P Q121K A865D M62K R387H A570T S1114R L198P R415Q L198P R128H A865G C68Y A390P L581F G1116E E297G V444A G260D I206V S874P C107R G410D A588V G1116F G374S D482G E297K V284A I939M I112T L413W S593R G1116R A390P N591S V444A G295C R958Q W114R I420T I627T S1120N R432T T655I T510T G295R F959C Y157C D440E E636G R1128C V444A T655I G295S F959V A167T G455E R698C S1144R I498T D676Y R299K T965S A167V K461E S699P R1153C A570T P710P R303K F971L I182K T463I E709K R1153H T586I L827I L339V F971Y M183T Q466K G758R S1154P G648V G855R H423R L1006F M183V R470Q G766R N1173D T655I E1186K V444A N1009H G188W Y472C Y818F T1210P T923P V444D K1145N M217R V481E R832C N1211D A926P V444G I1183T R223C D482G R832H V1212F R948C A459V S226L R487H T859R R1231Q G1004D I468I G238V R487P A865V R1231W R1050C R487L T242I N490D Q869P L1242I G1116R Q546K A257G I498T G877R D1243G R1128H Q558H V284L G499E S901R R1268Q L1197G E592Q E297G I512T R948C A1283V R1231Q V597M R303G N515T N979D G1292V R616G R303K R517H G982R G1298R T619A Q312H F540L G1004D M677L R313S I541L T1029K M677V G327E I541T G1032R R696Q W330R F548Y A1044P R698H Nonsense mutations (premature stop-codons) S25X Y472X Y772X R1090X E96X W493X Q791X V1147X W330X R520X R928X Q1215X Y354X I528X Y1041X R1235X R415X R575X R1057X E1302X R470X Q702X Q1058X Table 1 (Continued) PFIC BRIC/NFC ICP Other liver diseases Genetic variants without disease association Splice site mutations 76 + 3G > T 908 + 1delG 2178 + 1G > T 3057-2A > G Q159Q 77-1G > C 908 + 1G > T 2179-2A > G 3213 + 1delG Q361Q 99-1G > T 908 + 1G > A 2343 + 1G > T 3213 + 4A > G 150 + 3A > C 1435-13 -8del 2343 + 2T > C 3213 + 5G > A 390-1G > A 2012-8T > G 2611-2A > T 611 + 1G > A 2178 + 1G > A R1001R Deletions/insertions/frame shifts Q101Dfs8X L380Wfs18X G648Vfs5X Q1058Hfs38X F959Hfs1X T127Hfs6X A382 A388del K700Sfs12X I1061Vfs34X F959Gfs48X N199Ifs14X P456Pfs24X T919del L1165del L232Cfs9X H484Rfs5X K930Efs92X A1192Efs50X R303Sfs17X I528Sfs21X K930Efs79X T1256Tfs40X V368Rfs27X I610Qfs45X K969 K972del Synonymous variants without disease association R33R F90F L232L I416I G557G I876I A1028A K1145K D36D I134I Y269Y G418G V597V G937G K1070K R52R S136S Q312Q F427F A804A Y981Y T1086T D58D V195V G319G E395E A535A G817G G1004G A1110A The overview shows ࣈ 290 known variants of BSEP on the protein level, except splice site mutations, which are shown on cDNA level. Login to comment
208 A few ''common`` BSEP mutations (including p.E297G, p.D482G and p.N591S) have been detected in ICP-patients in heterozygous form and account for about 1.4% (7/491) of ICP-patients [150]. Login to comment
209 Canalicular expression of BSEP with the ''typical`` ICP mutation p.N591S was higher than that of the BRIC-2 (p.A570 T, p.R1050 C) or PFIC-2 mutations (p.D482G, p.E297G) [144]. Login to comment

PMID: 22859919 [PubMed] Chan J et al: "Hepatobiliary transport in health and disease."

No. Sentence Comment

84 The most common mutations are E297G and D482G [26]. Login to comment
85 The disease progresses more slowly in patients with D482G mutations, and they develop cirrhosis at an older age [24]. Login to comment

PMID: 23685087 [PubMed] Soroka CJ et al: "Biosynthesis and trafficking of the bile salt export pump, BSEP: therapeutic implications of BSEP mutations."

No. Sentence Comment

136 When seven PFIC2 missense mutations were expressed in MDCK cells, five of these common mutations (G238V, E297G, G982R, R1153C and R1268Q) were unable to traffic to the apical membrane (Wang et al., 2002). Login to comment
138 Two common mutations in human BSEP, E297G and D482G, have been reported to have both reduced (Noe et al., 2005; Wang et al., 2002) and normal (Hayashi et al., 2005; Lam et al., 2007) transport activity. Login to comment
143 The variability in the clinical phenotype of the E297G mutation, on the other hand, may be due to the ability of the mRNA to be stabilized by splicing factors present in the hepatocyte (Byrne et al., 2009). Login to comment
145 We examined differences in protein maturation, plasma membrane localization and transport activity in mutants of rat Bsep representing two PFIC2 (D482G and E297G), two BRIC2 (A570T and R1050C) and one ICP (N591) mutation (Lam et al., 2007). Login to comment
146 It was found that all but the PFIC2 mutation, E297G, retained the ability to transport taurocholate. Login to comment
197 This and other studies also suggest that the residence time on the cell surface of the common D482G and E297G mutant proteins is greatly reduced due to accelerated internalization, reduced recycling or targeting of the endocytosed protein for degradation. Login to comment
200 Indeed, in vitro studies demonstrate that 4-phenylbutyrate (4-PBA) can increase the cell surface expression of PFIC2 mutant proteins D482G and E297G in MDCK cells and stimulate bile salt secretion and Bsep expression in vivo in rats (Hayashi and Sugiyama, 2007). Login to comment

PMID: 24378332 [PubMed] Aida K et al: "Differential roles of ubiquitination in the degradation mechanism of cell surface-resident bile salt export pump and multidrug resistance-associated protein 2."

No. Sentence Comment

192 The cell surface-resident forms of BSEP with E297G and D482G, both of which are the most frequently found mutations in patients with progressive familial IC type 2, a genetic disorder caused by mutations in the BSEP gene, are highly ubiquitinated (Hayashi and Sugiyama, 2009). Login to comment

PMID: 24644547 [PubMed] Lisowska A et al: "Small intestinal bacterial overgrowth in patients with progressive familial intrahepatic cholestasis."

No. Sentence Comment

42 biochemical and genetic data of the studied patients Sex ABCB11 gene defect HMBT first/ second/ third Age (y) Body mass, Z-score Height, Z-score Treatment Bilirubin (mg/dl) Bile acids (&#b5;mol/l) Fecal fat excretion (g/day) M - B/-/- 7 -0.67 -0.77 BD 0.9 1.4 10 M - B/B/N 7.11 -0.88 -1.21 BD 0.9 1.5 6.7 F - B/P/- 15 -0.41 -1.36 BD 0.6 3.6 9.9 F - B/N/- 8.11 -0.25 -1.11 IB/BD 0.9 4.1 0.5 M c.1445 A>G; p.Asp482Gly. c.1544 A>C; p.Asn515Thr B/-/- 11.4 -1.99 -3.42 BD 0.9 2.6 2.1 M c.890 A>G; p.Glu297Gly (HOM) P/N/- 19.7 0.41 0.56 IB 1.1 405 2.2 F - P/N/- 12.11 -1.31 -0.73 UDCA 1.5 21.7 1.1 F c.1445 A>G; p.Asp482Gly. Login to comment
46 c.890 A>G; p.Glu297Gly P/P/P 9.8 -1.01 -1.48 BD 0.9 4 4.5 M c.890 A>G; p.Glu297Gly (HET) N/-/- 12.5 0.83 -1.14 BD 1.1 2.1 2 M c.1445 A>G; p.Asp482Gly. Login to comment
47 c.2494 C>T; p.Arg832Cys N/-/- 11.9 -1.73 -3.27 BD 0.9 5.6 13 M c.890 A>G; p.Glu297Gly. Login to comment

PMID: 24711118 [PubMed] Guyot C et al: "Differential effects of membrane cholesterol content on the transport activity of multidrug resistance-associated protein 2 (ABCC2) and of the bile salt export pump (ABCB11)."

No. Sentence Comment

11 The transport activity of the BSEP mutants p.E297G and p.R432T increased at high cholesterol content but did not reach the capacity of normal BSEP. Login to comment
177 Finally, we studied the effect of cholesterol on two mutant forms of BSEP, p.E297G and p.R432T, found in patients with BSEP deficiency syndromes. Login to comment
262 (A, D, and G) Western blot analysis of p.444A variant (A), p.E297G (D), and p.R432T (G) mutant BSEP-expressing vesicles with and without cholesterol loading (30 mg protein per lane). Login to comment

PMID: 24969679 [PubMed] Hu G et al: "Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis and direct sequencing."

No. Sentence Comment

202 Common mutations that have been reported in European populations, such as E297G and D482G, were not detected in Chinese subjects. Login to comment

PMID: 25027376 [PubMed] Kubitz R et al: "Genetic variations of bile salt transporters."

No. Sentence Comment

89 Among these p.E297G, p.D482G and p.N591S belong to the more frequent mutations. Login to comment
90 While homozygosity of p.E297G or p.D482G is linked to PFIC-2, heterozygosity of these mutations was detected in about 1.4% of 491 patients with intrahepatic cholestasis of pregnancy (ICP) [17]. Login to comment
100 For example, the two 'BRIC-2 mutations` p.A570T and p.R1050C had lower expression levels than the 'ICP mutation` p.N591S, but higher expression levels as compared to the PFIC-2 mutations p.D482G and p.E297G [59]. Login to comment
112 The missense mutations p.G238V, p.E297G, p.G982R, p.R1153C and p.R1268Q all led to a reduced expression at the apical membrane, when expressed in MDCK cells [124]. Login to comment
117 For the two common BSEP mutations p.E297G and p.D482G it has been reported that treatment with 4-phenylbutyrate prolongs half-life at the canalicular membrane and consecutively overall expression of BSEP [29]. Login to comment
138 b Some variants (such as E297G or N591S of BSEP) may affect transport handling on different levels. Login to comment

PMID: 25274209 [PubMed] Hayashi H et al: "[Development of new therapeutic strategy for transporter-related diseases]."

No. Sentence Comment

12 BSEP Expression on the Canalicular Membrane Is Decreased in Patients with PFIC25) 1008 Vol. 134 (2014) d3b;ឋf5c;ᵨఌa2;dd;c8;fc;b7;b9;fc3;⍈f5c;ᵨఔecb;௱ıf;d30;Pde;bd2;ឋ Ĳb;ఐĴa;,[cd;be4;Ĳa;̩d;a5f;Pfd;ωc;bb3;İc;f15;İd;d77;௭௯Ĵc;Ĵb;&#ff0e;ᱯĲb; [cd;be4;Ĳa;̩d;ᑁPc6;c41;௦௷ede;ఔᕈ௳Ĵb;Kbe;<a3;Ĳe;e00;f8b;௼௱௺, BSEP Ĳe;΋a;f1d;b50;᜕ᶒĲb;d77;8e0;௱௺˿a;Kc7;௳Ĵb;e0c;c11;`e3;cbb;ឋ Kbe;<a3;Ĳe; PFIC2 İc;Me5;఑Ĵc;௺௤Ĵb;&#ff0e; 3,4) PFIC2 Ĳb;bfe;௱௺ Ĳf;,Ife;Hb6;௻Ĳf;ᑁOd1;Ḅᷚcd5;İc;Nba;acb;௱௺İa;఑ıa;,̩d;Ofb;ʲd; ఔ5d7;௫Ĳa;௫Ĵc;௾,əd;᧡ʟf;Ȍd;Ĳb;Qf4;b7b;ឋĲe;d4c;Έe;ఔfbf;Ĵb;&#ff0e; ̩d;Ofb;ʲd;Ĳf;˯f;Ḽ௳Ĵb;İb;௽௦İb;İc;Nba;b9f;௻Ĳf;Ĳa;İf;ea;b9;af;İc; ad8;௤௭௼, ad8;Ϙd;Ĳa;ɰb;⊕cbb;İc;İb;İb;Ĵb;௭௼,௯఑Ĳb;Ȃd;Kab; ᢓᑴᒐఔe00;˯f;ʞd;ᵨ௱d9a;௫Ĳa;İf;௺Ĳf;Ĳa;఑Ĳa;௤௭௼Ĳa;௽ İb;఑,<a3;ὅĲb;ᾙf53;Ḅ,[d1;\ad;ḄĲb;ɏa;ᜧĲa;ca0;>c5;ఔf37;௤Ĵb; cbb;ᷚcd5;௻௢Ĵb;&#ff0e;௱ıf;İc;௷௺,PFIC2 Ĳb;bfe;௳Ĵb;ȕb;Uac; 6c1;Ĳe;ξb;˿a;İc;ᑗʟb;௯Ĵc;௺௤Ĵb;&#ff0e; ee5;e0a;Ĳe;Pcc;ʚf;İb;఑,b46;ὅĲf;,PFIC2 <a3;ὅĲe; 60%ee5; e0a;İc;fdd;8e0;௳Ĵb; 297 ˴a;Lee;Ĳe;b0;eb;bf;df;f3;⏚İc;b0;ea;b7;f3;Ĳb; ee3;Ĵf;Ĵb;᜕ᶒf53;(E297G) ,482 ˴a;Lee;Ĳe;a2;b9;d1;e9;ae;f3; ⏚İc;b0;ea;b7;f3;Ĳb;ee3;Ĵf;Ĵb;᜕ᶒf53;(D482G)Ĳe; 2 ௸Ĳe; d2;c8; BSEP ᜕ᶒf53;˿a;Ife;d30;Pde;ఔEcb;bc9;௱,a2e;஽Ĳe; in vitro b9f; a13;ఔb9f;Abd;௱ıf;&#ff0e; 5) ıd;Ĳe;d50;ʧc;,e21;᜕ᶒİc; BSEP Ĳe;e0d;b89;b9a;ᓄĲb;2cd;İd;,BSEP Ĳe;d30;Pde;̳c;˿a;Ife;[cf;ఔf4e;e0b;௯ ıb;௺௤Ĵb;௭௼ఔ΂b;௤3fa;௱ıf;(Fig. 1) &#ff0e;ıd;Ĳe;e00;Ab9;௻, e21; BSEP ᜕ᶒf53;ఔ˿a;Ife;௯ıb;ıf;d30;Pde;İb;఑d30;Pde;̳c;d9;b7; af; eb; ఔ abf; Xfd; ௱ , f38; 〈 b9f; a13; ఔ b9f; Abd; ௱ ıf; ௼ ௭ Ĵd; , E297G, D482G İc;ᓮf4d; BSEP [cf;f53;ıf;Ĵa;Ĳe;Pc6;c41;⏚f38;〈 d3b;ឋĲb;Ĳf;f71;aff;ఔe0e;௨Ĳa;௤௭௼İc;ʔe;఑İb;௼Ĳa;௷ıf;&#ff0e; E297G, D482G ᜕ᶒఔᢝ௸ PFIC2 <a3;ὅĲe;̩d;d44;e54; ఔᵨ௤ıf;Ȃd;Kab;d44;e54;Cd3;⁐cd5;௻Ĳf;,bdb;d30;Pc6;ba1;Ꮢ̳c;e0a;Ĳb;İa; ௫Ĵb; BSEP ˿a;Ife;[cf;İc;♿℉Ĳb;f4e;e0b;௱௺İa;Ĵa;, 4) Ĵf;Ĵc;Ĵf; Ĵc;Ĳe; in vitro Ye3;᪆d50;ʧc;௼̺f;ɕd;Ĳa;Lf8;_a2;İc;a8d;ఉ఑Ĵc;௺௤ Ĵb;&#ff0e;௱ıf;İc;௷௺,eca;f8c;Ǵb;஽Ĳe; PFIC2 <a3;ὅĲe;Kc5;ɦb;˿a; Kc7;a5f;Ecb;ఔe88;e2c;௳Ĵb;e0a;௻,b46;ὅĲe;b9f; a13;cfb;Ĳf;Ϗe;e38;Ĳb;ᨵᵨ Ĳa;c4;fc;eb;௼Ĳa;Ĵb;௭௼İc;ʟf;f85;௯Ĵc;Ĵb;&#ff0e; PFIC2 cbb;ᷚUac;Ĳe;?a2;d22; b46;ὅ఑Ĳf;,in vitro b9f; a13;İb;఑,PFIC2 <a3;ὅ௻Ĳf;ɏa; İf;Ĳe;ᛊᔠ,BSEP Ĳe;d30;Pde;̳c;˿a;Ife;[cf;İc;f4e;e0b;௳Ĵb;e00;Ab9; ௻,˿a;Ife;௱௺௤Ĵb; BSEP Ĳe;Pc6;c41;⏚f38;〈d3b;ឋĲf;fdd;ᢝ ௯Ĵc;௺௤Ĵb;௭௼İb;఑,BSEP Ĳe;d30;Pde;̳c;˿a;Ife;[cf;ఔᜉ4a0; ௯ıb;Ĵb;Ab9;cd5;ad6;Ĳe;ξb;˿a;İc;,PFIC2 Ĳe;ᑁOd1;Ḅᷚcd5;Ĳe;Nba; acb;Ĳb;௸Ĳa;İc;Ĵb;௼ὃ௨ıf;&#ff0e;ᱯĲb;,Qe8;e8a;fc3;ea6;௻ BSEP Ĳe;d30;Pde;̳c;˿a;Ife;[cf;ఔ ad8;ఉĴb;Uac;4b9;ఔᨵ௳Ĵb;Ae2;b58;ȕb;Uac;6c1;ఔ Ȝc;b9a;௳Ĵb;௭௼İc;௻İd;Ĵc;௾,d4c;e08;Ḅca0;>c5;,5ca;ఁξb;˿a;ea; b9;af;ఔefd;e1b;௱ıf; PFIC2 Ĳb;bfe;௳Ĵb;Ab0;Uac;ᒛXfd;İc;b9f;Ife;5ef; Pfd;௻௢Ĵb;௼ὃ௨,BSEP Ĳe;d30;Pde;̳c;˿a;Ife;[cf;Ĳe;᜕4d5;ఔc21; fbf;İb;௸Med;᧲╹௻a55;fa1;5ef;Pfd;Ĳa;Hec;Qea;Ĳe; in vitro b9f; a13;cfb;ఔ Ecb;bc9;௱,a2e;஽Ĳe;Ae2;b58;ȕb;Uac;6c1;ఔb9;af;ea;fc;cb;f3;b0;௱ıf;&#ff0e; ıd;Ĳe;d50;ʧc;,⋋ᦪĲe;χd;ឋᓄᔠᱥĲe;Ȝc;b9a;Ĳb;ᡂȑf;௱ıf; (Fig. 2) &#ff0e;௭Ĵc;఑Ĳe;χd;ឋᓄᔠᱥĲe;௦௵,d2;c8;Ĳb;d4c;5e3; ᢗe0e;௱ıf;f8c;,BSEP İc;˿a;Ife;௳Ĵb;̩d;Qd3;Ĳb;ᑖe03;௳Ĵb;௭௼ İc;ʔe;఑İb;௼Ĳa;௷௺௤ıf;c3f;d20;b5;a4;af;eb;ᶒe38;Kc7;(urea cycle disorder; UCD ) Ĳe; cbb; ᷚ Uac; ௻ ௢ Ĵb; 4-phenylbutyrate (4PB)Ĳb;ḼLee;௱,௯఑Ĳb;Ẇa76;ఔ⍈ఉĴb;௭ ௼௼௱ıf;&#ff0e; 6) UCD <a3;ὅĲb;bfe;௳Ĵb;Qe8;e8a;ᵨ[cf;Ĳe; 4PB ఔe9;c3;c8;Ĳb;d4c; 5e3;ᢗe0e;௱ıf;௼௭Ĵd;,̩d;bdb;d30;Pc6;ba1;Ꮢ̳c;Ĳb;İa;௫Ĵb; Bsep Ĳe;˿a;Ife;[cf;ᜉ4a0;,̩d;bdb;d30;Pc6;ba1;Ꮢ̳c;ఔecb;௱ıf;Pc6;c41;⏚f38;〈 Ĳe;ea2;⍈İc;Yb3;bdf;௯Ĵc;,4PB İc; in vivo ௻ఊȜc;Ed8;Ĳe;Uac;4b9; ఔ˿a;Ife;௳Ĵb;௭௼İc;Nba;a8d;௯Ĵc;ıf;&#ff0e; 6) ıd;௭௻,b21;Ĳb;d2;c8; ௻Ĳe;Uac;4b9;ఔNba;a8d;௳Ĵb;ıf;ఉ,Ƕb;ᳮ;d4;6e1;f1a;Ĳe;ɳf;a8d;Ĳe;e0b;, UCD <a3;ὅఔbfe;c61;௼௱ıf;ec;c8;ed;b9;da;af;c6;a3;d6;Ẇa76;ఔ b9f;Abd;௱ıf;&#ff0e;UCD <a3;ὅĲf;,Nba;b9a;a3a;Aad;f8c;,4PB Ĳe;ʞd;ᵨ ఔξb;;cb;௱,ᨬd42;ḄĲb;Ĳf;̩d;Ofb;ʲd;ఔ5d7;௫Ĵb;&#ff0e;Nba;b9a;a3a;Aad;Ĳe; Lee;Ḅ௻?a1;5d6;௯Ĵc;ıf;̩d;˯f;ʳc;b5;f3;d7;eb;5ca;ఁ,̩d;Ofb;ʲd;Ĳe;ωb; Ĳb;˯f;௲Ĵb;<a3;ὅĲe;᤺3fa;̩d;İb;఑̩d;c97;̳c;ᑖ˱b;ఔabf;Xfd;௱, BSEP ˿a;Ife;[cf;ఔa55;fa1;௱ıf;௼௭Ĵd;,᤺3fa;̩d;௻d04; 3 Ǵd;Ĳe; ᜉ4a0;İc;a8d;ఉ఑Ĵc;&#f;&#ff0e; 7) ĳe;ıf;,4PB Ĳe;ʞd;ᵨξb;;cb;f8c;Ĳb;⊈ 1009 Fig. 2. Login to comment

PMID: 25342496 [PubMed] Kubitz R et al: "Autoimmune BSEP disease: disease recurrence after liver transplantation for progressive familial intrahepatic cholestasis."

No. Sentence Comment

50 It has been shown that, BSEP with the common PFIC-2-related mutation E297G is trapped in the ER in a core-glycosylated state [29]. Login to comment

PMID: 26019043 [PubMed] Czubkowski P et al: "Successful pregnancy after ileal exclusion in progressive familial intrahepatic cholestasis type 2."

No. Sentence Comment

59 Several estrogens and progesterone metabolites are able to induce trans-inhibition of BSEP and the subsequent toxicity induced by the accumulation of bile acids, which may also play a role in the etiopathogenesis of intrahepatic cholestasis of pregnancy (ICP).10,11 Mutations in MDR3 (ABCB4) gene coding transporter for phospholipids across the canalicular membrane may account for 15% of cases of ICP.12 Interestingly, a Czubkowski P, et al. , 2015; 14 (4): 550-552 few "common" BSEP mutations (including p.E297G, p.D482G, and p.N591S) have been detected in ICP-patients in heterozygous form, and common BSEP polymorphism (p.V444A) has been linked to ICP as well.13 The reoccurrence of BSEP cholestasis and development of ICP may be clinically indistinguishable, since both usually present with pruritus, elevated bile acids and aminotransferases, and normal hepatic imaging.11,12 Moreover, in ICP, mutations or polymorphisms of some hepatobiliary transport proteins may contribute to disease pathogenesis or severity, but on the other hand consideration must be given to the possibility of other rare underlying hepatic disorders that may be unmasked during pregnancy with cholestasis as its first manifestation.14 Thus, the diagnosis of ICP should be given after exclusion of preexisting liver disease.15 Pruritus remains the most important clinical symptom of PFIC-2. Login to comment

PMID: 26601667 [PubMed] Okushin K et al: "The intrahepatic expression levels of bile acid transporters are inversely correlated with the histological progression of nonalcoholic fatty liver disease."

No. Sentence Comment

143 In patients with either of the two common BSEP mutations p.E297G and p.D482G, 4-phenylbutyrate prolongs BSEP half-life in the canalicular membrane and, thus, increases the overall BSEP expression level [29]. Login to comment