ABCMdb

ABCB11 p.Glu297Gly

ClinVar:	c.890A>G , p.Glu297Gly D , Pathogenic
Reviews:	p.Glu297Lys ! p.Glu297Gly D
Predicted by SNAP2:	A: D (75%), C: D (71%), D: D (59%), F: D (80%), G: D (85%), H: D (66%), I: D (80%), K: D (91%), L: D (85%), M: D (80%), N: D (80%), P: D (91%), Q: N (97%), R: D (85%), S: D (80%), T: D (80%), V: D (75%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D,

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[hide] BSEP: function and role in progressive familial in... Semin Liver Dis. 2001 Nov;21(4):545-50. Thompson R, Strautnieks S
BSEP: function and role in progressive familial intrahepatic cholestasis.
Semin Liver Dis. 2001 Nov;21(4):545-50., [PMID:11745042]

Abstract [show]
Secretion of bile acids is the major driving force for bile flow in mammals. The recently described adenosine triphosphate (ATP)-dependent bile acid transporter, bile salt export pump (BSEP), formerly called sister of p-glycoprotein, is responsible for active transport of bile acids across the hepatocyte canalicular membrane into bile. It is now recognized that mutations in the gene encoding this protein (ABCB11) are responsible for a subgroup of infants and children with progressive familial cholestasis (PFIC-2), a cholestatic disorder causing extreme pruritus, growth failure, and progression to cirrhosis in the first decade of life. Understanding the structure and function of BSEP has improved our understanding of the mechanisms underlying bile secretion. Determining genotype/phenotype relationships in patients with mutations in this gene are currently ongoing.

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

[hide] Bile salt transporters: molecular characterization... Physiol Rev. 2003 Apr;83(2):633-71. Trauner M, Boyer JL
Bile salt transporters: molecular characterization, function, and regulation.
Physiol Rev. 2003 Apr;83(2):633-71., [PMID:12663868]

Abstract [show]
Molecular medicine has led to rapid advances in the characterization of hepatobiliary transport systems that determine the uptake and excretion of bile salts and other biliary constituents in the liver and extrahepatic tissues. The bile salt pool undergoes an enterohepatic circulation that is regulated by distinct bile salt transport proteins, including the canalicular bile salt export pump BSEP (ABCB11), the ileal Na(+)-dependent bile salt transporter ISBT (SLC10A2), and the hepatic sinusoidal Na(+)- taurocholate cotransporting polypeptide NTCP (SLC10A1). Other bile salt transporters include the organic anion transporting polypeptides OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 MRP2,3 (ABCC2,3). Bile salt transporters are also present in cholangiocytes, the renal proximal tubule, and the placenta. Expression of these transport proteins is regulated by both transcriptional and posttranscriptional events, with the former involving nuclear hormone receptors where bile salts function as specific ligands. During bile secretory failure (cholestasis), bile salt transport proteins undergo adaptive responses that serve to protect the liver from bile salt retention and which facilitate extrahepatic routes of bile salt excretion. This review is a comprehensive summary of current knowledge of the molecular characterization, function, and regulation of bile salt transporters in normal physiology and in cholestatic liver disease and liver regeneration.

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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

[hide] Two common PFIC2 mutations are associated with the... Hepatology. 2005 Apr;41(4):916-24. Hayashi H, Takada T, Suzuki H, Akita H, Sugiyama Y
Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11.
Hepatology. 2005 Apr;41(4):916-24., [PMID:15791618]

Abstract [show]
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. 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. In the present study, we examined the transport activity and cellular localization of these two mutants in human embryonic kidney 293 and Madin-Darby canine kidney II cells, respectively. 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. 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. The inhibition of proteasome function by MG132 resulted in the cellular accumulation of the core glycosylation form of the two mutants. In contrast, transport studies for taurocholate and glycocholate with membrane vesicles isolated from complementary DNA-transfected cells indicated that both mutations did not significantly affect the transport function of BSEP per se. In conclusion, E297G and D482G mutations result in impaired membrane trafficking, whereas the transport functions of these mutants remain largely unchanged.

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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

[hide] Benign recurrent intrahepatic cholestasis associat... J Clin Gastroenterol. 2006 Feb;40(2):171-5. Kubitz R, Keitel V, Scheuring S, Kohrer K, Haussinger D
Benign recurrent intrahepatic cholestasis associated with mutations of the bile salt export pump.
J Clin Gastroenterol. 2006 Feb;40(2):171-5., [PMID:16394881]

Abstract [show]
A young patient with recurrent attacks of intrahepatic cholestasis is described. On the basis of clinical presentation, laboratory findings and genetic analysis, the diagnosis of benign recurrent intrahepatic cholestasis type 2 (BRIC-2) was established. By the use of BSEP-specific antibodies, almost complete absence of BSEP from the canalicular membrane of liver cells was detected in the patient. Two different BSEP mutations were found. One mutation (E186G) had been described in one BRIC-2 case; the second mutation (V444A) is more frequent and has been linked to intrahepatic cholestasis of pregnancy. It is concluded that this form of compound heterozygosity of the BSEP gene reduces the amount of BSEP protein due to protein instability or mis-targeting, which is the underlying reason for reduced bile salt excretion and cholemia.

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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

[hide] Cholestasis and cholestatic syndromes. Curr Opin Gastroenterol. 2006 May;22(3):209-14. Rutherford AE, Pratt DS
Cholestasis and cholestatic syndromes.
Curr Opin Gastroenterol. 2006 May;22(3):209-14., [PMID:16550034]

Abstract [show]
PURPOSE OF REVIEW: This review highlights recent advances in understanding the regulation of bile acid transport in cholestasis and in the pathogenesis, outcomes, epidemiology, and treatment of a variety of cholestatic liver diseases and their associated complications. RECENT FINDINGS: Highlights include additional understanding of the role of the nuclear receptors farsenoid X receptor, pregnane X receptor, and constitutive androstane receptor in bile acid homeostasis, new understanding of the pathogenesis of primary biliary cirrhosis, familial intrahepatic cholestasis, biliary atresia, and primary sclerosing cholangitis, and clinical trials of therapies for intrahepatic cholestasis of pregnancy, primary biliary cirrhosis, and primary sclerosing cholangitis. SUMMARY: Our understanding of the molecular mechanisms, epidemiology and pathogenesis of cholestasis continues to advance. These advances will hopefully lead to more effective therapies for specific cholestatic conditions.

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

[hide] Genetic variability, haplotype structures, and eth... Drug Metab Dispos. 2006 Sep;34(9):1582-99. Epub 2006 Jun 8. Lang T, Haberl M, Jung D, Drescher A, Schlagenhaufer R, Keil A, Mornhinweg E, Stieger B, Kullak-Ublick GA, Kerb R
Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11).
Drug Metab Dispos. 2006 Sep;34(9):1582-99. Epub 2006 Jun 8., [PMID:16763017]

Abstract [show]
Biliary excretion of bile salts and other bile constituents from hepatocytes is mediated by the apical (canalicular) transporters P-glycoprotein 3 (MDR3, ABCB4) and the bile salt export pump (ABCB11). Mutations in ABCB4 and ABCB11 contribute to cholestatic diseases [e.g., progressive familial intrahepatic cholestasis 2 (PFIC2), PFIC3, and intrahepatic cholestasis of pregnancy], and our objective was to establish genetic variability and haplotype structures of ABCB4 and ABCB11 in healthy populations of different ethnic backgrounds. All coding exons, 5 of 6 noncoding exons, 50 to 300 base pairs of the flanking intronic regions, and 2.5 to 2.8 kilobase pairs of the promoter regions of ABCB4 and ABCB11 were sequenced in 159 and 196 DNA samples of Caucasian, African-American, Japanese, and Korean origin. In total, 76 and 86 polymorphisms were identified in ABCB4 and ABCB11, respectively; among them, 14 and 28 exonic polymorphisms, and 8 and 10 protein-altering variants, of which 4 were predicted to have functional consequences. Both genes showed substantial ethnic differences with respect to allele number, frequency of common and population-specific sites, and patterns of linkage disequilibrium. Population genetic analysis suggested some selective pressure against changes in the protein, supporting the important endogenous role of these transporters. Haplotype variability was greater in ABCB11 than in ABCB4. An ABCB11 promoter haplotype was associated with significant decrease of activity compared with wild type. Our results contribute to a better understanding of the molecular basis and of ethnic differences in drug response, and provide a valuable tool for future research on the heredity of cholestatic liver injury.

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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

[hide] Hepatocellular carcinoma in ten children under fiv... Hepatology. 2006 Aug;44(2):478-86. Knisely AS, Strautnieks SS, Meier Y, Stieger B, Byrne JA, Portmann BC, Bull LN, Pawlikowska L, Bilezikci B, Ozcay F, Laszlo A, Tiszlavicz L, Moore L, Raftos J, Arnell H, Fischler B, Nemeth A, Papadogiannakis N, Cielecka-Kuszyk J, Jankowska I, Pawlowska J, Melin-Aldana H, Emerick KM, Whitington PF, Mieli-Vergani G, Thompson RJ
Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency.
Hepatology. 2006 Aug;44(2):478-86., [PMID:16871584]

Abstract [show]
Hepatocellular carcinoma (HCC) is rare in young children. We attempted to see if immunohistochemical and mutational-analysis studies could demonstrate that deficiency of the canalicular bile acid transporter bile salt export pump (BSEP) and mutation in ABCB11, encoding BSEP, underlay progressive familial intrahepatic cholestasis (PFIC)--or "neonatal hepatitis" suggesting PFIC--that was associated with HCC in young children. We studied 11 cases of pediatric HCC in the setting of PFIC or "neonatal hepatitis" suggesting PFIC. Archival liver were retrieved and immunostained for BSEP. Mutational analysis of ABCB11 was performed in leukocyte DNA from available patients and parents. Among the 11 nonrelated children studied aged 13-52 months at diagnosis of HCC, 9 (and a full sibling, with neonatal hepatitis suggesting PFIC, of a tenth from whom liver was not available) had immunohistochemical evidence of BSEP deficiency; the eleventh child did not. Mutations in ABCB11 were demonstrated in all patients with BSEP deficiency in whom leukocyte DNA could be studied (n = 7). These mutations were confirmed in the parents (n = 14). With respect to the other 3 children with BSEP deficiency, mutations in ABCB11 were demonstrated in all 5 parents in whom leukocyte DNA could be studied. Thirteen different mutations were found. In conclusion, PFIC associated with BSEP deficiency represents a previously unrecognized risk for HCC in young children. Immunohistochemical evidence of BSEP deficiency correlates well with demonstrable mutation in ABCB11.

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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

[hide] The bile salt export pump. Pflugers Arch. 2007 Feb;453(5):611-20. Epub 2006 Oct 19. Stieger B, Meier Y, Meier PJ
The bile salt export pump.
Pflugers Arch. 2007 Feb;453(5):611-20. Epub 2006 Oct 19., [PMID:17051391]

Abstract [show]
Canalicular secretion of bile salts mediated by the bile salt export pump Bsep constitutes the major driving force for the generation of bile flow. Bsep is a member of the B-family of the super family of ATP-binding cassette transporters and is classified as ABCB11. Bsep has a narrow substrate specificity, which is largely restricted to bile salts. Bsep is extensively regulated at the transcriptional and posttranscriptional level, which directly modulates canalicular bile formation. Pathophysiological alterations of Bsep by either inherited mutations or acquired processes such as inhibition by drugs or disease-related down regulation may lead to a wide spectrum of mild to severe forms of liver disease. Furthermore, many genetic variants of Bsep are known, some of which potentially render individuals susceptible to acquired forms of liver disease.

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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

[hide] Prediction of drug-induced intrahepatic cholestasi... Expert Opin Drug Saf. 2007 Jan;6(1):71-86. Sakurai A, Kurata A, Onishi Y, Hirano H, Ishikawa T
Prediction of drug-induced intrahepatic cholestasis: in vitro screening and QSAR analysis of drugs inhibiting the human bile salt export pump.
Expert Opin Drug Saf. 2007 Jan;6(1):71-86., [PMID:17181454]

Abstract [show]
Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure-activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure-activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

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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

[hide] 4-phenylbutyrate enhances the cell surface express... Hepatology. 2007 Jun;45(6):1506-16. Hayashi H, Sugiyama Y
4-phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps.
Hepatology. 2007 Jun;45(6):1506-16., [PMID:17538928]

Abstract [show]
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. 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. The dysfunctional localization is probably attributable to the retention of BSEP in endoplasmic reticulum (ER) followed by proteasomal degradation. 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. 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. The prolonged half-life of cell surface-resident BSEP with 4PBA treatment was responsible for this result. Moreover, treatment of Sprague-Dawley rats with 4PBA resulted in an increase in BSEP expression at the canalicular membrane, which was accompanied by an increase in the biliary excretion of [(3)H]taurocholic acid (TC). 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. 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.

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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

[hide] Levels of plasma membrane expression in progressiv... Am J Physiol Cell Physiol. 2007 Nov;293(5):C1709-16. Epub 2007 Sep 13. Lam P, Pearson CL, Soroka CJ, Xu S, Mennone A, Boyer JL
Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases.
Am J Physiol Cell Physiol. 2007 Nov;293(5):C1709-16. Epub 2007 Sep 13., [PMID:17855769]

Abstract [show]
Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. 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. Bile salt transport was retained in all but the E297G mutant. 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 > N591S > R1050C approximately A570T approximately E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.

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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

[hide] Phenotypic differences in PFIC2 and BRIC2 correlat... Am J Physiol Gastrointest Liver Physiol. 2008 Jan;294(1):G58-67. Epub 2007 Oct 18. Kagawa T, Watanabe N, Mochizuki K, Numari A, Ikeno Y, Itoh J, Tanaka H, Arias IM, Mine T
Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells.
Am J Physiol Gastrointest Liver Physiol. 2008 Jan;294(1):G58-67. Epub 2007 Oct 18., [PMID:17947449]

Abstract [show]
Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. 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). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. 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 > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

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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

[hide] Update on progressive familial intrahepatic choles... J Pediatr Gastroenterol Nutr. 2008 Mar;46(3):241-52. Alissa FT, Jaffe R, Shneider BL
Update on progressive familial intrahepatic cholestasis.
J Pediatr Gastroenterol Nutr. 2008 Mar;46(3):241-52., [PMID:18376240]

Abstract [show]
Three distinct forms of familial intrahepatic cholestasis are the result of mutations in the ATP8B1, ABCB11, and ABCB4 genes. The pathophysiologies of the latter 2 of these diseases are well characterized and are the result of abnormalities in canalicular excretion of bile acids and phospholipids, respectively. The molecular pathophysiology of the systemic disease associated with mutations in ATP8B1 remains unclear. In all of these diseases, wide variations in clinical phenotypes have been observed. The variability can be ascribed at least in part to predicted genotype:phenotype correlations. Disease- and genotype-specific prognoses and therapeutic approaches may exist, although much more information needs to be ascertained before clinicians can confidently make decisions based on genetic information.

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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

[hide] Diagnosis of BSEP/ABCB11 mutations in Asian patien... J Pediatr. 2008 Dec;153(6):825-32. Epub 2008 Aug 9. Chen HL, Liu YJ, Su YN, Wang NY, Wu SH, Ni YH, Hsu HY, Wu TC, Chang MH
Diagnosis of BSEP/ABCB11 mutations in Asian patients with cholestasis using denaturing high performance liquid chromatography.
J Pediatr. 2008 Dec;153(6):825-32. Epub 2008 Aug 9., [PMID:18692205]

Abstract [show]
OBJECTIVE: To determine if specific mutations were present in Asian patients with progressive familial intrahepatic cholestasis (PFIC) type 2 caused by defects in bile salt export pump (BSEP), encoded by ABCB11. STUDY DESIGN: A combination of denaturing high-performance liquid chromatography (DHPLC) and direct sequencing was used to screen ABCB11 mutations in 18 Taiwanese patients with low gamma-glutamyltransferase PFIC or benign recurrent intrahepatic cholestasis (BRIC). Polymorphisms were also analyzed in patients with PFIC (n = 21), neonatal cholestasis (n = 23), and control subjects (n = 88). RESULTS: Seven mutations in 4 of 16 patients with PFIC from different families were detected by DHPLC, including M183V, V284L, R303K, R487H, W493X, G1004D, and 1145delC. G1004D was found in a patient with BRIC. L827I was found in another patient with neonatal cholestasis. Absent or defective BSEP staining was found in the liver of patients with mutations. Polymorphisms V444A and A865V, with an allele frequencies 75.6% and 0.6%, respectively, were found in our population. No differences were found between patients with cholestasis and control subjects. CONCLUSIONS: One-fourth of Taiwanese patients with PFIC/BRIC had compound heterozygous or single heterozygous ABCB11 mutations without hot spots. All of the mutations were different from those detected in Western countries.

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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

[hide] Degradation of the bile salt export pump at endopl... Hepatology. 2008 Nov;48(5):1558-69. Wang L, Dong H, Soroka CJ, Wei N, Boyer JL, Hochstrasser M
Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II.
Hepatology. 2008 Nov;48(5):1558-69., [PMID:18798335]

Abstract [show]
The bile salt export pump (Bsep) represents the major bile salt transport system at the canalicular membrane of hepatocytes. When examined in model cell lines, genetic mutations in the BSEP gene impair its targeting and transport function, contributing to the pathogenesis of progressive familial intrahepatic cholestasis type II (PFIC II). PFIC II mutations are known to lead to a deficiency of BSEP in human hepatocytes, suggesting that PFIC II mutants are unstable and degraded in the cell. To investigate this further, we have characterized the impact of several PFIC II mutations on the processing and stability of rat Bsep. G238V, D482G, G982R, R1153C, and R1286Q all retain Bsep to the endoplasmic reticulum (ER) to different extents. Except for R1153C, the PFIC II mutants are degraded with varying half-lives. G238V and D482G are partially misfolded and can be stabilized by low temperature and glycerol. The proteasome provides the major degradation pathway for the PFIC II mutants, whereas the lysosome also contributes to the degradation of D482G. The PFIC II mutants appear to be more heavily ubiquitinated compared with the wild-type (wt) Bsep, and their ubiquitination is increased by the proteasome inhibitors. Overexpression of several E3 ubiquitin ligases, which are involved in ER-associated degradation (ERAD), lead to the decrease of both mutant and wt Bsep. Gene knockdown studies showed that the ERAD E3s Rma1 and TEB4 contribute to the degradation of G238V, whereas HRD1 contributes to the degradation of a mutant lacking the lumenal glycosylation domain (DeltaGly). Furthermore, we present evidence that G982R weakly associates with various components of the ER quality control system. These data together demonstrate that the PFIC II mutants except R1153C and DeltaGly are degraded by the ERAD pathway.

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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

[hide] Short-chain ubiquitination is associated with the ... Mol Pharmacol. 2009 Jan;75(1):143-50. Epub 2008 Oct 1. Hayashi H, Sugiyama Y
Short-chain ubiquitination is associated with the degradation rate of a cell-surface-resident bile salt export pump (BSEP/ABCB11).
Mol Pharmacol. 2009 Jan;75(1):143-50. Epub 2008 Oct 1., [PMID:18829893]

Abstract [show]
The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We demonstrated previously that 4-phenylbutyrate (4PBA) treatment, a U.S. Food and Drug Administration-approved drug for the treatment of urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. 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. Therefore, to help the development of the medical treatment of cholestasis, we investigated the underlying mechanism by which 4PBA and PFIC2-type mutations affect the BSEP degradation from cell surface, focusing on short-chain ubiquitination. In Madin-Darby canine kidney II (MDCK II) cells expressing BSEP and rat canalicular membrane vesicles, the molecular mass of the mature form of BSEP/Bsep shifted from 170 to 190 kDa after ubiquitin modification (molecular mass, 8 kDa). 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. Moreover, biotin-labeling studies using MDCK II cells demonstrated that the degradation of cell-surface-resident chimeric protein fusing ubiquitin to BSEP was faster than that of BSEP itself. In conclusion, BSEP/Bsep is modified with two to three ubiquitins, and its ubiquitination is modulated by 4PBA treatment and PFIC2-type mutations. Modulation of short-chain ubiquitination can regulate the change in the degradation rate of cell-surface-resident BSEP by 4PBA treatment and PFIC2-type mutations.

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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

[hide] Prenatal diagnosis of progressive familial intrahe... J Gastroenterol Hepatol. 2008 Sep;23(9):1390-3. Chen ST, Chen HL, Su YN, Liu YJ, Ni YH, Hsu HY, Chu CS, Wang NY, Chang MH
Prenatal diagnosis of progressive familial intrahepatic cholestasis type 2.
J Gastroenterol Hepatol. 2008 Sep;23(9):1390-3., [PMID:18853996]

Abstract [show]
BACKGROUND AND AIM: Progressive familial intrahepatic cholestasis type 2 (PFIC2) results from genetic defects of the hepatobiliary bile salt export pump (BSEP, ABCB11) at chromosome 2q24. Patients with progressive cholestasis and liver cirrhosis usually need liver transplantation in the first decade. Mutations in ABCB11 are also associated with benign recurrent intrahepatic cholestasis type 2 and intrahepatic cholestasis of pregnancy in adult patients. We aimed to make the prenatal diagnosis of PFIC2. METHODS: Genetic diagnosis was performed by genomic DNA analysis. Prenatal genetic diagnosis was made by fetal amniotic DNA and chorionic DNA analysis. RESULTS: We report on two families of PFIC2 with inherited compound heterozygous mutations of ABCB11 (M183V and R303K in Family 1, V284L and 1145delC in Family 2) from the parents. An infant with heterozygous M183V mutation was later born healthy in Family 1. A fetus with compound heterozygous missense mutation V284L and 1145delC was terminated in Family 2. CONCLUSION: Prenatal diagnosis of PFIC2 was helpful to prevent further affected children in families with this fatal disease.

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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

[hide] Bile composition in Alagille Syndrome and PFIC pat... BMC Gastroenterol. 2008 Oct 20;8:47. Emerick KM, Elias MS, Melin-Aldana H, Strautnieks S, Thompson RJ, Bull LN, Knisely A, Whitington PF, Green RM
Bile composition in Alagille Syndrome and PFIC patients having Partial External Biliary Diversion.
BMC Gastroenterol. 2008 Oct 20;8:47., [PMID:18937870]

Abstract [show]
BACKGROUND: Partial External Biliary Diversion (PEBD) is a surgical intervention to treat children with Progressive Familial Intrahepatic Cholestasis (PFIC) and Alagille syndrome (AGS). PEBD can reduce disease progression, and examining the alterations in biliary lipid composition may be a prognostic factor for outcome. METHODS: Biliary lipid composition and the clinical course of AGS and PFIC patients were examined before and after PEBD. RESULTS: Pre-PEBD bile from AGS patients had greater chenodeoxycholic/cholic acid (CDCA/CA), bile salt, cholesterol and phospholipid concentrations than PFIC patients. AGS patients, and PFIC patients with familial intrahepatic cholestasis 1 (FIC1) genotype, responded better to PEBD than PFIC patients with bile salt export protein (BSEP) genotype. After successful PEBD, AGS patients have higher biliary lipid concentrations than PFIC patients and PEBD also increases biliary phospholipid concentrations in FIC1 patients. CONCLUSION: Both AGS and FIC1 patients can benefit from PEBD, and preserved biliary phospholipid concentrations may be associated with better outcomes post-PEBD.

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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

[hide] Contribution of variant alleles of ABCB11 to susce... Gut. 2009 Apr;58(4):537-44. Epub 2008 Nov 5. Dixon PH, van Mil SW, Chambers J, Strautnieks S, Thompson RJ, Lammert F, Kubitz R, Keitel V, Glantz A, Mattsson LA, Marschall HU, Molokhia M, Moore GE, Linton KJ, Williamson C
Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy.
Gut. 2009 Apr;58(4):537-44. Epub 2008 Nov 5., [PMID:18987030]

Abstract [show]
BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) has a complex aetiology with a significant genetic component. ABCB11 encodes the bile salt export pump (BSEP); mutations cause a spectrum of cholestatic disease, and are implicated in the aetiology of ICP. 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. PCR primers were used to amplify and sequence patient and control DNA. The molecular basis for the observed phenotypes was investigated in silico by analysing the equivalent residues in the structure of the homologous bacterial transporter Sav1866. RESULTS: E297G was observed four times and D482G once. N591S was present in two patients; D676Y and G855R were not observed. The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p<0.001)). In addition, CC homozygotes were more likely to have ICP than TT homozygotes: OR 2.8 (95% CI 1.7 to 4.4 p<0.0001). Structural analyses suggest that E297G and D482G destabilize the protein fold of BSEP. The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. CONCLUSIONS: Heterozygosity for the common ABCB11 mutations accounts for 1% of European ICP cases; these two mutants probably reduce the folding efficiency of BSEP. N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. The V444A polymorphism is a significant risk factor for ICP in this population.

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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

[hide] Missense mutations and single nucleotide polymorph... Hepatology. 2009 Feb;49(2):553-67. Byrne JA, Strautnieks SS, Ihrke G, Pagani F, Knisely AS, Linton KJ, Mieli-Vergani G, Thompson RJ
Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing.
Hepatology. 2009 Feb;49(2):553-67., [PMID:19101985]

Abstract [show]
The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and single-nucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency.

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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

[hide] Progressive familial intrahepatic cholestasis. Orphanet J Rare Dis. 2009 Jan 8;4:1. Davit-Spraul A, Gonzales E, Baussan C, Jacquemin E
Progressive familial intrahepatic cholestasis.
Orphanet J Rare Dis. 2009 Jan 8;4:1., [PMID:19133130]

Abstract [show]
Progressive familial intrahepatic cholestasis (PFIC) refers to heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. The exact prevalence remains unknown, but the estimated incidence varies between 1/50,000 and 1/100,000 births. Three types of PFIC have been identified and related to mutations in hepatocellular transport system genes involved in bile formation. PFIC1 and PFIC2 usually appear in the first months of life, whereas onset of PFIC3 may also occur later in infancy, in childhood or even during young adulthood. Main clinical manifestations include cholestasis, pruritus and jaundice. PFIC patients usually develop fibrosis and end-stage liver disease before adulthood. Serum gamma-glutamyltransferase (GGT) activity is normal in PFIC1 and PFIC2 patients, but is elevated in PFIC3 patients. Both PFIC1 and PFIC2 are caused by impaired bile salt secretion due respectively to defects in ATP8B1 encoding the FIC1 protein, and in ABCB11 encoding the bile salt export pump protein (BSEP). Defects in ABCB4, encoding the multi-drug resistant 3 protein (MDR3), impair biliary phospholipid secretion resulting in PFIC3. Diagnosis is based on clinical manifestations, liver ultrasonography, cholangiography and liver histology, as well as on specific tests for excluding other causes of childhood cholestasis. MDR3 and BSEP liver immunostaining, and analysis of biliary lipid composition should help to select PFIC candidates in whom genotyping could be proposed to confirm the diagnosis. Antenatal diagnosis can be proposed for affected families in which a mutation has been identified. Ursodeoxycholic acid (UDCA) therapy should be initiated in all patients to prevent liver damage. In some PFIC1 or PFIC2 patients, biliary diversion can also relieve pruritus and slow disease progression. However, most PFIC patients are ultimately candidates for liver transplantation. Monitoring of hepatocellular carcinoma, especially in PFIC2 patients, should be offered from the first year of life. Hepatocyte transplantation, gene therapy or specific targeted pharmacotherapy may represent alternative treatments in the future.

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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

[hide] Recent insights into the function and regulation o... Curr Opin Lipidol. 2009 Jun;20(3):176-81. Stieger B
Recent insights into the function and regulation of the bile salt export pump (ABCB11).
Curr Opin Lipidol. 2009 Jun;20(3):176-81., [PMID:19684528]

Abstract [show]
PURPOSE OF REVIEW: Generation of bile is an important function of the liver. Its impairment can be caused by inherited mutations or by acquired factors and leads to cholestasis. Bile salts are an important constituent of bile and are secreted by the bile salt export pump (BSEP) from hepatocytes. RECENT FINDINGS: Significant progress was made in the understanding of mechanisms and consequences of malfunctioning BSEP. This information was gained from extensive characterization of patients with inherited BSEP deficiency and the subsequent characterization of the identified mutations in heterologous expression systems. Furthermore and importantly, clinical evidence shows that patients with severe BSEP deficiency are at risk to develop hepatocellular carcinoma. Bile salts are now recognized to be important in the modulation of whole body energy homeostasis. Because BSEP is the rate-limiting step in hepatocellular bile salt transport, it controls the spill over of bile salts into the systemic circulation. Therefore, an indirect role of BSEP in energy homeostasis becomes more and more likely. SUMMARY: In summary, knowledge on the physiologic and pathophysiologic role of BSEP is rapidly progressing. It can be anticipated that the next major step in better understanding BSEP should come from information on structure-function relationship. However, given the difficulty in structure determination of mammalian transporters, this will require major efforts.

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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

[hide] Novel ABCB11 mutations in a Thai infant with progr... World J Gastroenterol. 2009 Sep 14;15(34):4339-42. Treepongkaruna S, Gaensan A, Pienvichit P, Luksan O, Knisely AS, Sornmayura P, Jirsa M
Novel ABCB11 mutations in a Thai infant with progressive familial intrahepatic cholestasis.
World J Gastroenterol. 2009 Sep 14;15(34):4339-42., 2009-09-14 [PMID:19750581]

Abstract [show]
Progressive familial intrahepatic cholestasis (PFIC) type 2 is caused by mutations in ABCB11, which encodes bile salt export pump (BSEP). We report a Thai female infant who presented with progressive cholestatic jaundice since 1 mo of age, with normal serum gamma-glutamyltransferase. Immunohistochemical staining of the liver did not demonstrate BSEP along the canaliculi, while multidrug resistance protein 3 was expressed adequately. Novel mutations in ABCB11, a four-nucleotide deletion in exon 3, c.90_93delGAAA, and a single-nucleotide insertion in exon 5, c.249_250insT, were identified, with confirmation in her parents. These mutations were predicted to lead to synthesis of truncated forms of BSEP. Immunostaining and mutation analysis thus established the diagnosis of PFIC type 2.

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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

[hide] ABCB11 gene mutations in Chinese children with pro... Liver Int. 2010 Jul;30(6):809-15. Epub 2009 Oct 21. Liu LY, Wang ZL, Wang XH, Zhu QR, Wang JS
ABCB11 gene mutations in Chinese children with progressive intrahepatic cholestasis and low gamma glutamyltransferase.
Liver Int. 2010 Jul;30(6):809-15. Epub 2009 Oct 21., [PMID:19845854]

Abstract [show]
BACKGROUND: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe autosomal recessive liver disorder of childhood that can cause cholestasis and progress to end-stage liver disease. ABCB11 gene mutations causing PFIC2 have been reported in some population groups, but not in mainland Chinese. AIMS: To elucidate the existence of and characterize ABCB11 gene mutations in mainland Chinese with progressive intrahepatic cholestasis and low gamma glutamyltransferase (GGT). METHODS: Twenty-four children presenting with progressive intrahepatic cholestasis and low GGT were admitted to a tertiary paediatric hospital in eastern China from January 2004 to July 2007. All encoding exons and flanking areas of the ABCB11 gene were sequenced. Hepatic histopathology results were obtained by review of the medical record. RESULTS: Twelve novel mutations of ABCB11 gene were found in seven patients: three nonsense mutations, six missense mutations, two splicing mutations and one intronic mutation. Giant cell transformation of hepatocytes was demonstrated in all the four patients with ABCB11 mutations and four of 12 patients without mutations in coding sequences of ABCB11 gene who received liver needle biopsy. CONCLUSIONS: ABCB11 gene mutations play an important role in Chinese patients with progressive intrahepatic cholestasis and low GGT. The characteristics of ABCB11 gene mutations in Chinese are different from other population groups. Histological examination may be helpful in diagnosis of PFIC2.

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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

[hide] Polymorphic variants in the human bile salt export... Pharmacogenet Genomics. 2010 Jan;20(1):45-57. Ho RH, Leake BF, Kilkenny DM, Meyer Zu Schwabedissen HE, Glaeser H, Kroetz DL, Kim RB
Polymorphic variants in the human bile salt export pump (BSEP; ABCB11): functional characterization and interindividual variability.
Pharmacogenet Genomics. 2010 Jan;20(1):45-57., [PMID:20010382]

Abstract [show]
OBJECTIVES: Our aims were to identify and functionally characterize coding region nonsynonymous single nucleotide polymorphisms in the hepatic efflux transporter, bile salt export pump (BSEP; ABCB11), and to assess interindividual variability in BSEP expression. METHODS: We identified 24 single nucleotide polymorphisms, including nine nonsynonymous variants, in ABCB11 from genomic DNA of approximately 250 ethnically diverse healthy individuals using denaturing high-performance liquid chromatography analysis and DNA sequencing. Wild type and variant BSEP were generated and functionally characterized for taurocholate transport activity in vitro in HeLa cells using a recombinant vaccinia-based method. BSEP expression was assessed by real-time mRNA analysis, western blot analysis, and immunofluorescence confocal microscopy. RESULTS: For the most part, polymorphisms were rare and ethnic-dependent. In vitro functional studies revealed several rare variants, including 616A>G, 1674G>C, 1772A>G, and 3556G>A, to be associated with significantly impaired taurocholate transport activity while the 890A>G variant trended towards impaired function but was not statistically significant. The 3556G>A variant was associated with reduced cell surface to total protein expression compared with wild-type BSEP. Expression of BSEP by mRNA and protein analysis was determined from a bank of human liver samples. Wide interindividual variability was noted in both mRNA (19-fold) and protein (31-fold) expression levels. The common variant 1331T>C was associated with significantly reduced hepatic BSEP mRNA levels. CONCLUSION: Accordingly, our study indicates there are functionally relevant polymorphisms in ABCB11 which may be of potential relevance in the predisposition to acquired liver disorders such as drug-induced cholestasis.

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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

[hide] Role of the bile salt export pump, BSEP, in acquir... Drug Metab Rev. 2010 Aug;42(3):437-45. Stieger B
Role of the bile salt export pump, BSEP, in acquired forms of cholestasis.
Drug Metab Rev. 2010 Aug;42(3):437-45., [PMID:20028269]

Abstract [show]
Generation of bile is a key function of the liver. Its impairment leads to accumulation of cytotoxic bile salts in hepatocytes and, consequently, to liver disease. The bile salt export pump, BSEP, is critically involved in the secretion of bile salts into bile. Its function can be disturbed or abolished by inherited mutations. This will lead to progressive intrahepatic cholestais and severe liver disease. In addition to mutations, BSEP can be inhibited by acquired factors, such as xenobiotics or drugs, aberrant bile salt metabolites, or pregnancy. This inhibition will lead to acquired cholestasis. Some drugs are now known to be competitive inhibitors of Bsep. In addition, a polymorphism in the gene coding for BSEP has been identified as a potential susceptibility factor for acquired cholestasis.

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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

[hide] PFIC2 and ethnicity-specific bile salt export pump... Liver Int. 2010 Jul;30(6):777-9. Epub 2010 Mar 8. Ananthanarayanan M, Li Y
PFIC2 and ethnicity-specific bile salt export pump (BSEP, ABCB11) mutations: where do we go from here?
Liver Int. 2010 Jul;30(6):777-9. Epub 2010 Mar 8., [PMID:20214736]

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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

[hide] ATP8B1 and ABCB11 analysis in 62 children with nor... Hepatology. 2010 May;51(5):1645-55. Davit-Spraul A, Fabre M, Branchereau S, Baussan C, Gonzales E, Stieger B, Bernard O, Jacquemin E
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.
Hepatology. 2010 May;51(5):1645-55., [PMID:20232290]

Abstract [show]
Progressive familial intrahepatic cholestasis (PFIC) types 1 and 2 are characterized by normal serum gamma-glutamyl transferase (GGT) activity and are due to mutations in ATP8B1 (encoding FIC1) and ABCB11 (encoding bile salt export pump [BSEP]), respectively. Our goal was to evaluate the features that may distinguish PFIC1 from PFIC2 and ease their diagnosis. We retrospectively reviewed charts of 62 children with normal-GGT PFIC in whom a search for ATP8B1 and/or ABCB11 mutation, liver BSEP immunostaining, and/or bile analysis were performed. Based on genetic testing, 13 patients were PFIC1 and 39 PFIC2. The PFIC origin remained unknown in 10 cases. PFIC2 patients had a higher tendency to develop neonatal cholestasis. High serum alanine aminotransferase and alphafetoprotein levels, severe lobular lesions with giant hepatocytes, early liver failure, cholelithiasis, hepatocellular carcinoma, very low biliary bile acid concentration, and negative BSEP canalicular staining suggest PFIC2, whereas an absence of these signs and/or presence of extrahepatic manifestations suggest PFIC1. The PFIC1 and PFIC2 phenotypes were not clearly correlated with mutation types, but we found tendencies for a better prognosis and response to ursodeoxycholic acid (UDCA) or biliary diversion (BD) in a few children with missense mutations. Combination of UDCA, BD, and liver transplantation allowed 87% of normal-GGT PFIC patients to be alive at a median age of 10.5 years (1-36), half of them without liver transplantation. CONCLUSION: PFIC1 and PFIC2 differ clinically, biochemically, and histologically at presentation and/or during the disease course. A small proportion of normal-GGT PFIC is likely not due to ATP8B1 or ABCB11 mutations.

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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

[hide] The bile salt export pump: clinical and experiment... Semin Liver Dis. 2010 May;30(2):125-33. Epub 2010 Apr 26. Lam P, Soroka CJ, Boyer JL
The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease.
Semin Liver Dis. 2010 May;30(2):125-33. Epub 2010 Apr 26., [PMID:20422495]

Abstract [show]
The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.

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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

[hide] Genetic determinants of drug-induced cholestasis a... Semin Liver Dis. 2010 May;30(2):147-59. Epub 2010 Apr 26. Pauli-Magnus C, Meier PJ, Stieger B
Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy.
Semin Liver Dis. 2010 May;30(2):147-59. Epub 2010 Apr 26., [PMID:20422497]

Abstract [show]
Intrahepatic cholestasis of pregnancy and drug-induced cholestasis are two clinically important forms of acquired cholestatic liver disease. The understanding of the underlying mechanisms of acquired cholestasis has recently made considerable progress by the identification of canalicular ATP-binding cassette (ABC) transporters as likely targets for these forms of cholestasis. Cholestasis of pregnancy is linked to estrogen and progesterone metabolites. These metabolites have been shown to impair the bile salt export pump (BSEP) function by an indirect mechanism. In addition, genetic variants (as well as mutants) of the genes coding for the phosphatidylcholine translocator MDR3 and BSEP and for the farnesoid X receptor, which is critical in the transcriptional activation of MDR3 ( ABCB4) and BSEP ( ABCB11) have been associated with intrahepatic cholestasis of pregnancy. The pathogenesis of drug-induced liver injury encompasses a wide spectrum of mechanisms, some of which are still poorly understood. BSEP is now known to be subject to drug inhibition in susceptible patients. Information on genetic factors rendering individuals susceptible to inhibition of BSEP by drugs or their metabolites is still scarce. Besides rare mutations that have been linked to drug-induced cholestasis, the common p.V444A polymorphism of BSEP has been identified as a potential risk factor. In this review, the authors summarize key concepts of physiology of bile formation, diagnostic principles to indentify these forms of acquired cholestasis, as well as pathogenetic mechanisms leading to intrahepatic cholestasis of pregnancy or drug-induced cholestasis. In addition, they review the current knowledge on genetic susceptibility factors for these two forms of cholestasis.

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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

[hide] Cholestatic liver disease in children. Curr Gastroenterol Rep. 2010 Feb;12(1):30-9. Santos JL, Choquette M, Bezerra JA
Cholestatic liver disease in children.
Curr Gastroenterol Rep. 2010 Feb;12(1):30-9., [PMID:20425482]

Abstract [show]
Inherited syndromes of intrahepatic cholestasis and biliary atresia are the most common causes of chronic liver disease and the prime indication for liver transplantation in children. Our understanding of the pathogenesis of these diseases has increased substantially by the discovery of genetic mutations in children with intrahepatic cholestasis and the findings that inflammatory circuits are operative at the time of diagnosis of biliary atresia. Building on this solid foundation, recent studies provide new insight into genotype-phenotype relationships and how mutations produce altered bile composition and cholestasis. New evidence exists that although liver transplantation is curative for patients with end-stage liver disease owing to cholestasis, some patients may develop recurrence of cholestasis because of the emergence of autoantibodies that disrupt canalicular function in the new graft. Progress is also evident in biliary atresia, with recent studies identifying candidate modifier genes and directly implicating lymphocytes and inflammatory signals in the pathogenesis of bile duct injury and obstruction.

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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

[hide] Analysis of gene mutations in children with choles... J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):488-93. Matte U, Mourya R, Miethke A, Liu C, Kauffmann G, Moyer K, Zhang K, Bezerra JA
Analysis of gene mutations in children with cholestasis of undefined etiology.
J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):488-93., [PMID:20683201]

Abstract [show]
BACKGROUND: The discovery of genetic mutations in children with inherited syndromes of intrahepatic cholestasis allows for diagnostic specificity despite similar clinical phenotypes. Here, we aimed to determine whether mutation screening of target genes could assign a molecular diagnosis in children with idiopathic cholestasis. PATIENTS AND METHODS: DNA samples were obtained from 51 subjects with cholestasis of undefined etiology and surveyed for mutations in the genes SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 by a high-throughput gene chip. Then, the sequence readouts for all 5 genes were analyzed for mutations and correlated with clinical phenotypes. Healthy subjects served as controls. RESULTS: Sequence analysis of the genes identified 14 (or 27%) subjects with missense, nonsense, deletion, and splice site variants associated with disease phenotypes based on the type of mutation and/or biallelic involvement in the JAG1, ATP8B1, ABCB11, or ABCB4 genes. These patients had no syndromic features and could not be differentiated by biochemical markers or histopathology. Among the remaining subjects, 10 (or approximately 20%) had sequence variants in ATP8B1 or ABCB11 that involved only 1 allele, 8 had variants not likely to be associated with disease phenotypes, and 19 had no variants that changed amino acid composition. CONCLUSIONS: Gene sequence analysis assigned a molecular diagnosis in 27% of subjects with idiopathic cholestasis based on the presence of variants likely to cause disease phenotypes.

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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

[hide] Follow-up in children with progressive familial in... J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):494-9. Arnell H, Papadogiannakis N, Zemack H, Knisely AS, Nemeth A, Fischler B
Follow-up in children with progressive familial intrahepatic cholestasis after partial external biliary diversion.
J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):494-9., [PMID:20683202]

Abstract [show]
OBJECTIVES: The aim of this study was to examine whether reversion of histological fibrosis followed partial external biliary diversion (PEBD) in patients with progressive familial intrahepatic cholestasis (PFIC); whether the duration of cholestatic episodes after PEBD influenced the evolution of fibrosis; and whether genotyping was helpful in predicting outcome of PEBD. PATIENTS AND METHODS: Children with PFIC who underwent PEBD were investigated with genetic, biochemical, and anthropometric standard methods. Serial liver specimens were assessed histologically without knowledge of genotype and outcome. Findings were evaluated in the contexts of the total duration of cholestasis and the clinical outcome after PEBD. RESULTS: From a total of 18 children with PFIC, 13 underwent PEBD, and 12 of these (among them 10 with identified ABCB11 mutations) were amenable for clinical and histological follow-up. When compared with baseline at PEBD, statistically significant reductions were found in histological cholestasis 1 and 3 years after PEBD, and in fibrosis 5 and >10 years after PEBD. The relative duration of cholestatic episodes after PEBD was positively correlated with the severity of fibrosis. Children homozygous for the missense mutation c.890A>G in ABCB11 responded well to PEBD. CONCLUSIONS: Biliary diversion should be regarded as the first choice of surgical treatment in noncirrhotic patients with severe ABCB11 disease and may also be efficacious in other forms of PFIC.

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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

[hide] The role of the sodium-taurocholate cotransporting... Handb Exp Pharmacol. 2011;(201):205-59. Stieger B
The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation.
Handb Exp Pharmacol. 2011;(201):205-59., [PMID:21103971]

Abstract [show]
Bile formation is an important function of the liver. Bile salts are a major constituent of bile and are secreted by hepatocytes into bile and delivered into the small intestine, where they assist in fat digestion. In the small intestine, bile salts are almost quantitatively reclaimed and transported back via the portal circulation to the liver. In the liver, hepatocytes take up bile salts and secrete them again into bile for ongoing enterohepatic circulation. Uptake of bile salts into hepatocytes occurs largely in a sodium-dependent manner by the sodium taurocholate cotransporting polypeptide NTCP. The transport properties of NTCP have been extensively characterized. It is an electrogenic member of the solute carrier family of transporters (SLC10A1) and transports predominantly bile salts and sulfated compounds, but is also able to mediate transport of additional substrates, such as thyroid hormones, drugs and toxins. It is highly regulated under physiologic and pathophysiologic conditions. Regulation of NTCP copes with changes of bile salt load to hepatocytes and prevents entry of cytotoxic bile salts during liver disease. Canalicular export of bile salts is mediated by the ATP-binding cassette transporter bile salt export pump BSEP (ABCB11). BSEP constitutes the rate limiting step of hepatocellular bile salt transport and drives enterohepatic circulation of bile salts. It is extensively regulated to keep intracellular bile salt levels low under normal and pathophysiologic situations. Mutations in the BSEP gene lead to severe progressive familial intrahepatic cholestasis. The substrates of BSEP are practically restricted to bile salts and their metabolites. It is, however, subject to inhibition by endogenous metabolites or by drugs. A sustained inhibition will lead to acquired cholestasis, which can end in liver injury.

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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

[hide] Living-related liver transplantation for siblings ... Am J Transplant. 2011 Feb;11(2):394-8. doi: 10.1111/j.1600-6143.2010.03397.x. Epub 2011 Jan 10. Shimizu H, Migita O, Kosaki R, Kasahara M, Fukuda A, Sakamoto S, Shigeta T, Uemoto S, Nakazawa A, Kakiuchi T, Arai K
Living-related liver transplantation for siblings with progressive familial intrahepatic cholestasis 2, with novel genetic findings.
Am J Transplant. 2011 Feb;11(2):394-8. doi: 10.1111/j.1600-6143.2010.03397.x. Epub 2011 Jan 10., [PMID:21219577]

Abstract [show]
Progressive familial intrahepatic cholestasis is a syndrome of severe cholestasis progressing to biliary cirrhosis and liver failure that develops in childhood. This report describes two siblings with PFIC-2 who underwent living-related liver transplantation from their genetically proven heterozygous parents. Both patients had normal gamma-glutamyl transpeptidase levels, but showed severe pruritus with sleep disturbance, cholestasis, jaundice and growth failure. Genetic testing of each patient revealed two missense mutations of the bile salt export pump, S901R and C1083Y, which have not previously been associated with PFIC-2. Usual medical treatment failed to improve their clinical symptoms, and the two siblings underwent living-related liver transplantation from their heterozygous parents. The transplants improved their clinical symptoms significantly, and the patients have since shown age-appropriate growth. Electron microscopic findings of the explanted liver of the younger sister revealed dense and amorphous bile, which is characteristic of PFIC-2. In the cases presented here, living-related liver transplantation from a heterozygous donor was associated with better quality of life and improvement of growth, and thus appears to be a feasible option for PFIC-2 patients. Mutation analysis is a useful tool to help decide the course of treatment of PFIC.

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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

[hide] Progressive familial intrahepatic cholestasis (PFI... Semin Liver Dis. 2011 Feb;31(1):3-10. Epub 2011 Feb 22. Morotti RA, Suchy FJ, Magid MS
Progressive familial intrahepatic cholestasis (PFIC) type 1, 2, and 3: a review of the liver pathology findings.
Semin Liver Dis. 2011 Feb;31(1):3-10. Epub 2011 Feb 22., [PMID:21344347]

Abstract [show]
Progressive familial intrahepatic cholestatic diseases encompass a group of autosomal recessive hereditary diseases, which usually present in infancy or childhood, with cholestasis of hepatocellular origin. The currently preferred nomenclature for the three PFIC disorders that have been characterized to date is FIC1 deficiency, BSEP deficiency, and MDR3 deficiency, relating to mutations in the specific genes involved in bile acid formation and transport. Since the first description of these diseases, extensive clinical, biochemical, and molecular studies have increased our understanding of the features specific to each one of them. This review focuses mainly on the liver histology, summarizing their characteristic pathologic features, the correlation to specific genotypes, and complications arising with disease progression.

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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

[hide] Morphologic findings in progressive familial intra... Am J Surg Pathol. 2011 May;35(5):687-96. Evason K, Bove KE, Finegold MJ, Knisely AS, Rhee S, Rosenthal P, Miethke AG, Karpen SJ, Ferrell LD, Kim GE
Morphologic findings in progressive familial intrahepatic cholestasis 2 (PFIC2): correlation with genetic and immunohistochemical studies.
Am J Surg Pathol. 2011 May;35(5):687-96., [PMID:21490445]

Abstract [show]
Progressive familial intrahepatic cholestasis, type 2 (PFIC2), characterized by cholestasis in infancy that may progress to cirrhosis, is caused by mutation in ABCB11, which encodes bile salt export pump (BSEP). We correlated histopathologic, immunohistochemical, and ultrastructural features in PFIC2 with specific mutations and clinical course. Twelve patients with clinical PFIC2 and ABCB11 mutations were identified, and 22 liver biopsy and explant specimens were assessed. All had hepatocellular cholestasis; most had canalicular bile plugs. At least 1 specimen from every patient had centrizonal/sinusoidal fibrosis, often with periportal fibrosis. Neonatal hepatitis-like features (inflammation, giant cells, necrosis) varied. In 2 of the 5 patients with paired specimens obtained >6 months apart, lobular and portal fibrosis worsened. Transmission electron microscopy (EM) in all 9 patients studied showed canalicular dilatation, microvilli loss, abnormal mitochondrial internal structure, and varying intracanalicular accumulation of finely granular bile. Canalicular staining for BSEP was absent in 10 patients and present in 2 patients, 1 of whom had intermittent symptoms. ABCB11 sequencing of all patients identified 6 novel and 10 previously described mutations, with nonsense, missense, and/or noncoding mutations in the 10 patients without immunohistochemically demonstrable BSEP. Missense and/or noncoding mutations were identified in the 2 patients with demonstrable BSEP, whose clinical course was more indolent. Mutations ending ABCB11 transcription appear linked, through hepatocellular necrosis and fibrosis, to worse outcome. In conclusion, light microscopy and electron microscopy findings in clinical PFIC2 can support diagnosis, but are variable and nonspecific. Therefore, no correlation between specific mutations and histopathology is yet possible.

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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

[hide] A gene encoding a liver-specific ABC transporter i... Nat Genet. 1998 Nov;20(3):233-8. Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N, Arnell H, Sokal E, Dahan K, Childs S, Ling V, Tanner MS, Kagalwalla AF, Nemeth A, Pawlowska J, Baker A, Mieli-Vergani G, Freimer NB, Gardiner RM, Thompson RJ
A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.
Nat Genet. 1998 Nov;20(3):233-8., [PMID:9806540]

Abstract [show]
The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and gamma-glutamyltranspeptidase (gammaGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

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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

[hide] Protein quality control at the plasma membrane. Curr Opin Cell Biol. 2011 Aug;23(4):483-91. Epub 2011 May 14. Okiyoneda T, Apaja PM, Lukacs GL
Protein quality control at the plasma membrane.
Curr Opin Cell Biol. 2011 Aug;23(4):483-91. Epub 2011 May 14., [PMID:21571517]

Abstract [show]
Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined.

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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

[hide] Xenobiotic, bile acid, and cholesterol transporter... Pharmacol Rev. 2010 Mar;62(1):1-96. Epub 2010 Jan 26. Klaassen CD, Aleksunes LM
Xenobiotic, bile acid, and cholesterol transporters: function and regulation.
Pharmacol Rev. 2010 Mar;62(1):1-96. Epub 2010 Jan 26., [PMID:20103563]

Abstract [show]
Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology.

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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

[hide] Prolonged cholestasis triggered by hepatitis A vir... Ann Hepatol. 2012 Sep;11(5):710-4. Krawczyk M, Grunhage F, Langhirt M, Bohle RM, Lammert F
Prolonged cholestasis triggered by hepatitis A virus infection and variants of the hepatocanalicular phospholipid and bile salt transporters.
Ann Hepatol. 2012 Sep;11(5):710-4., [PMID:22947535]

Abstract [show]
Hepatitis A virus (HAV) infection resolves in most patients uneventfully within weeks from the onset of the disease. In rare cases, however, it may relapse or cause prolonged cholestasis. Here we present a case of a 36-year-old female patient who developed severe pruritus and jaundice three weeks after initially uncomplicated hepatitis A. A relapse of the infection was excluded. Since therapy with colestyramin, antihistaminics, naloxon and ursodeoxycholic acid (UDCA) did not improve symptoms, we decided to perform plasma absorption and to start rifampicin therapy. Under these measures, pruritus and jaundice, as well as serum bilirubin levels improved gradually and after four plasmapheresis sessions we were able to discharge the patient. Genetic testing showed the presence of two procholestatic polymorphisms, the c.3084 [GG] variant within the gene encoding the hepatocanalicular bile salt transporter ABCB11 and the c.711 [AT] variant of the phosphatidylcholine floppase ABCB4. We speculate that this compound ABCB4-ABCB11 genotype led to a severe intrahepatic cholestasis in the setting of HAV infection. In conclusion, our case suggests that polymorphisms within the hepatocanalicular transporters may contribute to a more pronounced course of HAV infection. Although dedicated studies in large cohorts of patients are needed to confirm this observation, we speculate that patients carrying procholestatic hepatobiliary transporter variants may benefit from vaccination against hepatitis A.

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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

[hide] Familial cholestasis: progressive familial intrahe... Best Pract Res Clin Gastroenterol. 2010 Oct;24(5):541-53. van der Woerd WL, van Mil SW, Stapelbroek JM, Klomp LW, van de Graaf SF, Houwen RH
Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy.
Best Pract Res Clin Gastroenterol. 2010 Oct;24(5):541-53., [PMID:20955958]

Abstract [show]
Progressive familial intrahepatic cholestasis (PFIC) type 1, 2 and 3 are due to mutations in ATP8B1, ABCB11 and ABCB4, respectively. Each of these genes encodes a hepatocanalicular transporter, which is essential for the proper formation of bile. Mutations in ABCB4 can result in progressive cholestatic disease, while mutations in ATP8B1 and ABCB11 can result both in episodic cholestasis, referred to as benign recurrent intrahepatic cholestasis (BRIC) type 1 and 2, as well as in progressive cholestatic disease. This suggests a clinical continuum and these diseases are therefore preferably referred to as ATP8B1 deficiency and ABCB11 deficiency. Similarly PFIC type 3 is designated as ABCB4 deficiency. Heterozygous mutations in each of these transporters can also be associated with intrahepatic cholestasis of pregnancy. This review summarizes the pathophysiology, clinical features and current as well as future therapeutic options for progressive familial- and benign recurrent intrahepatic cholestasis as well as intrahepatic cholestasis of pregnancy.

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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

[hide] Liver disease associated with canalicular transpor... J Hepatol. 2010 Feb;52(2):258-71. Epub 2009 Nov 21. Stapelbroek JM, van Erpecum KJ, Klomp LW, Houwen RH
Liver disease associated with canalicular transport defects: current and future therapies.
J Hepatol. 2010 Feb;52(2):258-71. Epub 2009 Nov 21., [PMID:20034695]

Abstract [show]
Bile formation at the canalicular membrane is a delicate process. This is illustrated by inherited liver diseases due to mutations in ATP8B1, ABCB11, ABCB4, ABCC2 and ABCG5/8, all encoding hepatocanalicular transporters. Effective treatment of these canalicular transport defects is a clinical and scientific challenge that is still ongoing. Current evidence indicates that ursodeoxycholic acid (UDCA) can be effective in selected patients with PFIC3 (ABCB4 deficiency), while rifampicin reduces pruritus in patients with PFIC1 (ATP8B1 deficiency) and PFIC2 (ABCB11 deficiency), and might abort cholestatic episodes in BRIC (mild ATP8B1 or ABCB11 deficiency). Cholestyramine is essential in the treatment of sitosterolemia (ABCG5/8 deficiency). Most patients with PFIC1 and PFIC2 will benefit from partial biliary drainage. Nevertheless liver transplantation is needed in a substantial proportion of these patients, as it is in PFIC3 patients. New developments in the treatment of canalicular transport defects by using nuclear receptors as a target, enhancing the expression of the mutated transporter protein by employing chaperones, or by mutation specific therapy show substantial promise. This review will focus on the therapy that is currently available as well as on those developments that are likely to influence clinical practice in the near future.

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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

[hide] Receptor for activated C-kinase 1 regulates the ce... Hepatol Res. 2009 Nov;39(11):1091-107. Epub 2009 Aug 6. Ikebuchi Y, Takada T, Ito K, Yoshikado T, Anzai N, Kanai Y, Suzuki H
Receptor for activated C-kinase 1 regulates the cellular localization and function of ABCB4.
Hepatol Res. 2009 Nov;39(11):1091-107. Epub 2009 Aug 6., [PMID:19674157]

Abstract [show]
Aim: Multidrug resistance protein 3 (MDR3/ABCB4), located on the bile canalicular membrane of hepatocytes, is responsible for the translocation of phosphatidylcholine across the plasma membrane, and its hereditary defect causes liver disorders, such as progressive familial intrahepatic cholestasis type 3. We aimed to identify the proteins responsible for the surface expression of human ABCB4. Methods: We performed yeast two-hybrid screening with the cytoplasmic linker region of ABCB4 against a human liver cDNA library. This screening allowed us to identify the receptor for activated C-kinase 1 (RACK1) as a novel binding partner of ABCB4. The association of RACK1 with the linker region of ABCB4 was further confirmed by GST-pulldown assay, although we could not find out the interaction of full length of ABCB4 and RACK1 in co-immunoprecipitation assay in HeLa cells. Results: Down-regulation of endogenous RACK1 expression by siRNA in HeLa cells resulted in the localization of ABCB4 in the cytosolic compartment as well as reduced protein expression of ABCB4, although mRNA expression and the protein stability of ABCB4 were not affected by the suppression of endogenous RACK1. Similar alterations in cellular localization of ABCB4 were also found by suppressing endogenous RACK1 expression in HepG2 cells. Consequently, ABCB4-mediated phosphatidylcholine translocation activity was significantly reduced when endogenous RACK1 expression was suppressed in HeLa cells. In contrast, the membrane surface localization and the protein expression of ABCB1 were not affected by the suppression of endogenous RACK1 expression. Conclusion: These results suggest that RACK1 may have a functional significance as a regulatory cofactor of ABCB4 and is indispensable for the plasma membrane localization and translocation function of ABCB4.

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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

[hide] Combined mutations of canalicular transporter prot... Gastroenterology. 2006 Aug;131(2):624-9. Keitel V, Vogt C, Haussinger D, Kubitz R
Combined mutations of canalicular transporter proteins cause severe intrahepatic cholestasis of pregnancy.
Gastroenterology. 2006 Aug;131(2):624-9., [PMID:16890614]

Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder that usually develops in the third trimester of pregnancy and persists until delivery. The cause of ICP remains elusive, but there is evidence that mutations in the canalicular ABC transporter phospholipid flippase (MDR3) and in the bile salt export pump (BSEP) can predispose for the development of ICP. MDR3 and BSEP were investigated by gene sequencing and immunofluorescence microscopy in a patient with severe ICP of early onset. ICP was diagnosed in a patient in the first trimester of pregnancy with severe pruritus, elevated levels of bile salts, and 48-fold elevation of transaminase levels. A liver biopsy specimen showed diminished canalicular expression of the bile salt export pump BSEP, while the expression and localization of the phospholipid flippase MDR3 was normal. Gene sequencing revealed a homozygous MDR3 gene mutation (S320F). The patient was also homozygous for the common BSEP polymorphism V444A. Treatment with ursodeoxycholate normalized transaminase levels but could not prevent further elevation of bile salt levels and preterm delivery. The combined homozygous alterations of the canalicular transporters may explain the early onset and severity of ICP in this patient. The common BSEP polymorphism V444A accounts for the reduced canalicular BSEP expression. Reduced bile salt secretion through BSEP may explain the persistence of elevated bile salt levels and incomplete efficacy of ursodeoxycholate treatment.

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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

[hide] Apical/basolateral surface expression of drug tran... Pharm Res. 2005 Oct;22(10):1559-77. Epub 2005 Sep 22. Ito K, Suzuki H, Horie T, Sugiyama Y
Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport.
Pharm Res. 2005 Oct;22(10):1559-77. Epub 2005 Sep 22., [PMID:16180115]

Abstract [show]
It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug-drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood-tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin-Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.

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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

[hide] Enterohepatic bile salt transporters in normal phy... Gastroenterology. 2004 Jan;126(1):322-42. Kullak-Ublick GA, Stieger B, Meier PJ
Enterohepatic bile salt transporters in normal physiology and liver disease.
Gastroenterology. 2004 Jan;126(1):322-42., [PMID:14699511]

Abstract [show]
The vectorial transport of bile salts from blood into bile is essential for the generation of bile flow, solubilization of cholesterol in bile, and emulsification of lipids in the intestine. Major transport proteins involved in the enterohepatic circulation of bile salts include the hepatocellular bile salt export pump (BSEP, ABCB11), the apical sodium-dependent bile salt transporter (ASBT, SLC10A2) in cholangiocytes and enterocytes, the sodium-dependent hepatocyte bile salt uptake system NTCP (SLC10A1), the organic anion transporting polypeptides OATP-C (SLC21A6), OATP8 (SLC21A8) and OATP-A (SLC21A3), and the multidrug resistance protein MRP3 (ABCC3). Synthesis and transport of bile salts are intricately linked processes that undergo extensive feedback and feed-forward regulation by transcriptional and posttranscriptional mechanisms. A key regulator of hepatocellular bile salt homeostasis is the bile acid receptor/farnesoid X receptor FXR, which activates transcription of the BSEP and OATP8 genes and of the small heterodimer partner 1 (SHP). SHP is a transcriptional repressor that mediates bile acid-induced repression of the bile salt uptake systems rat Ntcp and human OATP-C. A nuclear receptor that activates rodent Oatp2 (Slc21a5) and human MRP2 (ABCC2) is the pregnane X receptor/steroid X receptor PXR/SXR. Intracellular trafficking and membrane insertion of bile salt transporters is regulated by lipid, protein, and extracellular signal-related kinases in response to physiologic stimuli such as cyclic adenosine monophosphate or taurocholate. Finally, dysfunction of individual bile salt transporters such as BSEP, on account of genetic mutations, steric inhibition, suppression of gene expression, or disturbed signaling, is an important cause of cholestatic liver disease.

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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

[hide] Functional analysis of nonsynonymous single nucleo... Pharmacogenet Genomics. 2008 Sep;18(9):823-33. Kobayashi K, Ito K, Takada T, Sugiyama Y, Suzuki H
Functional analysis of nonsynonymous single nucleotide polymorphism type ATP-binding cassette transmembrane transporter subfamily C member 3.
Pharmacogenet Genomics. 2008 Sep;18(9):823-33., [PMID:18698235]

Abstract [show]
OBJECTIVES: The multidrug resistance-associated protein 3/ATP-binding cassette transmembrane transporter subfamily C member 3 (MRP3/ABCC3) plays an important role in exporting endogenous and xenobiotic anionic substrates, including glucuronide conjugates of xenobiotics, from hepatocytes into the blood circulation. This excretory function of ABCC3 becomes very apparent particularly under cholestatic conditions, since ABCC3 is induced when the biliary excretion pathway is impaired. In this study, we analyzed the functional properties of 11 nonsynonymous single nucleotide polymorphisms (SNPs) in the ABCC3 gene found in the public SNP database. METHODS: HeLa and Sf9 insect cells were used to analyze the protein expression and transport function, respectively. RESULTS: After transient transfection of cDNA into HeLa cells, it was found that R1381S ABCC3 exhibits intracellular accumulation of immature protein, the localization of which was mostly merged with a marker for the endoplasmic reticulum. Two kinds of SNPs type ABCC3 (S346F and S607N) lost their transport activity for [H]estradiol-17beta-D-glucuronide in membrane vesicles from Sf9 cells infected with the recombinant baculoviruses, although the band length and the amount of protein expression remained normal. In contrast, the cellular localization, protein expression and function of other eight kinds of SNPs type ABCC3 (G11D, R99Q, V765L, P920S, R923Q, R1286G, R1348C, and Q1365R ABCC3) remained normal. CONCLUSION: The results of this study suggest that the possession of R1381S, S346F, and S607N types of ABCC3 sequences may be a possible risk factor for the acquisition of hepatotoxicity, due to their poor ability to transport toxic compounds across the sinusoidal membrane.

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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

[hide] Description of two new ABCB11 mutations responsibl... Can J Gastroenterol. 2011 Jun;25(6):311-4. Beausejour Y, Alvarez F, Beaulieu M, Bilodeau M
Description of two new ABCB11 mutations responsible for type 2 benign recurrent intrahepatic cholestasis in a French-Canadian family.
Can J Gastroenterol. 2011 Jun;25(6):311-4., [PMID:21766090]

Abstract [show]
Benign recurrent intrahepatic cholestasis is a rare clinical entity that is caused by mutations in the canalicular transport genes. The present report describes two individuals from the same family whose symptoms were typical of the clinical characteristics of type 2 benign recurrent intrahepatic cholestasis. Sequencing of the ABCB11 gene revealed two previously unreported mutations that predict the absence of expression of the protein. The clinical presentation of the current cases are discussed, as are the differential diagnosis and genetic characteristics of the hereditary cholestatic disorders, overemphasizing the possibility of making a definite genetic diagnosis.

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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

[hide] Progressive familial intrahepatic cholestasis. Clin Res Hepatol Gastroenterol. 2012 Sep;36 Suppl 1:S26-35. doi: 10.1016/S2210-7401(12)70018-9. Jacquemin E
Progressive familial intrahepatic cholestasis.
Clin Res Hepatol Gastroenterol. 2012 Sep;36 Suppl 1:S26-35. doi: 10.1016/S2210-7401(12)70018-9., [PMID:23141890]

Abstract [show]
Progressive familial intrahepatic cholestasis (PFIC) refers to a heterogeneous group of autosomal-recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. The exact prevalence remains unknown, but the estimated incidence varies between 1/50,000 and 1/100,000 births. Three types of PFIC have been identified and associated with mutations in hepatocellular transport-system genes involved in bile formation. PFIC1 and PFIC2 usually appear in the first months of life, whereas onset of PFIC3 may arise later in infancy, in childhood or even during young adulthood. The main clinical manifestations include cholestasis, pruritus and jaundice. PFIC patients usually develop fibrosis and end-stage liver disease before adulthood. Serum gamma-glutamyltransferase (GGT) activity is normal in PFIC1 and PFIC2 patients, but is elevated in PFIC3 patients. Both PFIC1 and PFIC2 are caused by impaired bile salt secretion due to defects in ATP8B1 encoding the FIC1 protein and in ABCB11 encoding bile salt export pump (BSEP) protein, respectively. Defects in ABCB4, encoding multidrug resistance 3 protein (MDR3), impair biliary phospholipid secretion, resulting in PFIC3. Diagnosis is based on clinical manifestations, liver ultrasonography, cholangiography and liver histology, as well as on specific tests to exclude other causes of childhood cholestasis. MDR3 and BSEP liver immunostaining, and analysis of biliary lipid composition should help to select PFIC candidates for whom genotyping could be proposed to confirm the diagnosis. Antenatal diagnosis may be proposed for affected families in which a mutation has been identified. Ursodeoxycholic acid (UDCA) therapy should be initiated in all patients to prevent liver damage. In some PFIC1 and PFIC2 patients, biliary diversion may also relieve pruritus and slow disease progression. However, most PFIC patients are ultimately candidates for liver transplantation. Monitoring of liver tumors, especially in PFIC2 patients, should be offered from the first year of life. Hepatocyte transplantation, gene therapy and specific targeted pharmacotherapy may represent alternative treatments in the future.

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

[hide] FIC1 and BSEP defects in Taiwanese patients with c... J Pediatr. 2002 Jan;140(1):119-24. Chen HL, Chang PS, Hsu HC, Ni YH, Hsu HY, Lee JH, Jeng YM, Shau WY, Chang MH
FIC1 and BSEP defects in Taiwanese patients with chronic intrahepatic cholestasis with low gamma-glutamyltranspeptidase levels.
J Pediatr. 2002 Jan;140(1):119-24., [PMID:11815775]

Abstract [show]
To elucidate the frequency of FIC1 (ATP8B1) and BSEP (ABCB11) mutations in Taiwanese children with chronic intrahepatic cholestasis with low gamma-glutamyltranspeptidase (GGT) levels, we assessed 13 unrelated patients with infantile onset chronic intrahepatic cholestasis. Liver complementary DNA sequencing was performed in 7 infants for mutation analyses of FIC1 and BSEP genes. Two distinct liver histologic features were found. Group 1 (n = 5) was characterized by bland cholestasis and group 2 (n = 8) by giant cell transformation. Group 2 patients were associated with higher transaminase levels, alpha-fetoprotein levels, and early mortality. Novel FIC1 mutations were found in all 4 patients tested in group 1, including a 74-bp deletion, a 98-bp deletion, a nonsense, and 2 missense mutations. BSEP mutations were found in 2 of the 3 patients in group 2, including 2 missense mutations and a 1-bp deletion. Phenotypic characterization is useful to differentiate FIC1- from BSEP-related disease.

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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

[hide] Impaired expression and function of the bile salt ... J Hepatol. 2005 Sep;43(3):536-43. Noe J, Kullak-Ublick GA, Jochum W, Stieger B, Kerb R, Haberl M, Mullhaupt B, Meier PJ, Pauli-Magnus C
Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis.
J Hepatol. 2005 Sep;43(3):536-43., [PMID:16039748]

Abstract [show]
BACKGROUND/AIMS: Inherited dysfunction of the bile salt export pump BSEP (ABCB11) causes a progressive and a benign form of familial intrahepatic cholestasis, denominated as PFIC2 and BRIC2, respectively. We functionally characterized novel ABCB11 mutations encountered in two patients with a PFIC2 and a BRIC2 phenotype, respectively. METHODS: BSEP expression was determined in liver biopsies by immunohistochemistry. ABCB11 mutations were functionally characterized by taurocholate transport in SF9 cells transfected with human ABCB11. RESULTS: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((+3)A > C) 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. Hepatic BSEP expression was absent in PFIC2 and preserved in BRIC2. In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. CONCLUSIONS: The intron 4 (+3)A > C, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. By combining functional in-vitro characterization with immunohistochemical detection of variant BSEP we provide direct evidence for the role of ABCB11 mutations in the pathogenesis of different forms of intrahepatic cholestasis.

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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

[hide] Severe bile salt export pump deficiency: 82 differ... Gastroenterology. 2008 Apr;134(4):1203-14. doi: 10.1053/j.gastro.2008.01.038. Epub 2008 Jan 18. Strautnieks SS, Byrne JA, Pawlikowska L, Cebecauerova D, Rayner A, Dutton L, Meier Y, Antoniou A, Stieger B, Arnell H, Ozcay F, Al-Hussaini HF, Bassas AF, Verkade HJ, Fischler B, Nemeth A, Kotalova R, Shneider BL, Cielecka-Kuszyk J, McClean P, Whitington PF, Sokal E, Jirsa M, Wali SH, Jankowska I, Pawlowska J, Mieli-Vergani G, Knisely AS, Bull LN, Thompson RJ
Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families.
Gastroenterology. 2008 Apr;134(4):1203-14. doi: 10.1053/j.gastro.2008.01.038. Epub 2008 Jan 18., [PMID:18395098]

Abstract [show]
BACKGROUND & AIMS: Patients with severe bile salt export pump (BSEP) deficiency present as infants with progressive cholestatic liver disease. We characterized mutations of ABCB11 (encoding BSEP) in such patients and correlated genotypes with residual protein detection and risk of malignancy. METHODS: Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by single-strand conformation polymorphism analysis and sequencing of ABCB11. Genotypes sorted by likely phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical outcome. RESULTS: Eighty-two different mutations (52 novel) were identified in 109 families (9 nonsense mutations, 10 small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions, 45 missense changes). In 7 families, only a single heterozygous mutation was identified despite complete sequence analysis. Thirty-two percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% of European families (52/89). On immunohistochemical analysis (88 patients), 93% had abnormal or absent BSEP staining. Expression varied most for E297G and D482G, with some BSEP detected in 45% of patients (19/42) with these mutations. Hepatocellular carcinoma or cholangiocarcinoma developed in 15% of patients (19/128). Two protein-truncating mutations conferred particular risk; 38% (8/21) of such patients developed malignancy versus 10% (11/107) with potentially less severe genotypes (relative risk, 3.7 [confidence limits, 1.7-8.1; P = .003]). CONCLUSIONS: With this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable BSEP is typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those carrying 2 null mutations, is essential.

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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

[hide] Short- and medium-chain fatty acids enhance the ce... Biochim Biophys Acta. 2010 Sep;1801(9):1005-12. doi: 10.1016/j.bbalip.2010.04.002. Epub 2010 Apr 14. Kato T, Hayashi H, Sugiyama Y
Short- and medium-chain fatty acids enhance the cell surface expression and transport capacity of the bile salt export pump (BSEP/ABCB11).
Biochim Biophys Acta. 2010 Sep;1801(9):1005-12. doi: 10.1016/j.bbalip.2010.04.002. Epub 2010 Apr 14., [PMID:20398791]

Abstract [show]
The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously reported that 4-phenylbutyrate (4PBA), an approved drug for urea cycle disorders, is a promising agent for intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of BSEP. In the present study, we searched for effective compounds other than 4PBA by focusing on short- and medium-chain fatty acids, which have similar characteristics to 4PBA such as their low-molecular-weight and a carboxyl group. In transcellular transport studies using Madin-Darby canine kidney (MDCK) II cells, all short- and medium-chain fatty acids tested except for formate, acetate, and hexanoic acid showed more potent effects on wild type (WT) BSEP-mediated [3H]taurocholate transport than did 4PBA. The increase in WT BSEP transport with butyrate and octanoic acid treatment correlated with an increase in its expression at the cell surface. Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. The prolonged half-life of cell surface-resident WT BSEP was responsible for this increased octanoic acid-stimulated transport, but not for that of butyrate. In conclusion, short- and medium-chain fatty acids have potent effects on the increase in WT and PFIC2-type BSEP-mediated transport in MDCK II cells. Although both short- and medium-chain fatty acids enhance the transport capacity of WT and PFIC2-type BSEP by inducing those expressions at the cell surface, the underlying mechanism seems to differ between fatty acids.

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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

[hide] Differences in presentation and progression betwee... J Hepatol. 2010 Jul;53(1):170-8. doi: 10.1016/j.jhep.2010.01.034. Epub 2010 Apr 13. Pawlikowska L, Strautnieks S, Jankowska I, Czubkowski P, Emerick K, Antoniou A, Wanty C, Fischler B, Jacquemin E, Wali S, Blanchard S, Nielsen IM, Bourke B, McQuaid S, Lacaille F, Byrne JA, van Eerde AM, Kolho KL, Klomp L, Houwen R, Bacchetti P, Lobritto S, Hupertz V, McClean P, Mieli-Vergani G, Shneider B, Nemeth A, Sokal E, Freimer NB, Knisely AS, Rosenthal P, Whitington PF, Pawlowska J, Thompson RJ, Bull LN
Differences in presentation and progression between severe FIC1 and BSEP deficiencies.
J Hepatol. 2010 Jul;53(1):170-8. doi: 10.1016/j.jhep.2010.01.034. Epub 2010 Apr 13., [PMID:20447715]

Abstract [show]
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC) with normal serum levels of gamma-glutamyltranspeptidase can result from mutations in ATP8B1 (encoding familial intrahepatic cholestasis 1 [FIC1]) or ABCB11 (encoding bile salt export pump [BSEP]). We evaluated clinical and laboratory features of disease in patients diagnosed with PFIC, who carried mutations in ATP8B1 (FIC1 deficiency) or ABCB11 (BSEP deficiency). Our goal was to identify features that distinguish presentation and course of these two disorders, thus facilitating diagnosis and elucidating the differing consequences of ATP8B1 and ABCB11 mutations. METHODS: A retrospective multi-center study was conducted, using questionnaires and chart review. Available clinical and biochemical data from 145 PFIC patients with mutations in either ATP8B1 (61 "FIC1 patients") or ABCB11 (84 "BSEP patients") were evaluated. RESULTS: At presentation, serum aminotransferase and bile salt levels were higher in BSEP patients; serum alkaline phosphatase values were higher, and serum albumin values were lower, in FIC1 patients. Elevated white blood cell counts, and giant or multinucleate cells at liver biopsy, were more common in BSEP patients. BSEP patients more often had gallstones and portal hypertension. Diarrhea, pancreatic disease, rickets, pneumonia, abnormal sweat tests, hearing impairment, and poor growth were more common in FIC1 patients. Among BSEP patients, the course of disease was less rapidly progressive in patients bearing the D482G mutation. CONCLUSIONS: Severe forms of FIC1 and BSEP deficiency differed. BSEP patients manifested more severe hepatobiliary disease, while FIC1 patients showed greater evidence of extrahepatic disease.

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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

[hide] Discovery and structural development of small mole... Bioorg Med Chem. 2012 May 1;20(9):2940-9. doi: 10.1016/j.bmc.2012.03.016. Epub 2012 Mar 14. Misawa T, Hayashi H, Sugiyama Y, Hashimoto Y
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.
Bioorg Med Chem. 2012 May 1;20(9):2940-9. doi: 10.1016/j.bmc.2012.03.016. Epub 2012 Mar 14., [PMID:22464344]

Abstract [show]
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). 4-Phenylbutyric acid (4-PBA) enhances the cell surface expression and transport capacity of E297G BSEP, but has a relatively high dose (1mM or more) is required to show the effect. 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.

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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

[hide] E297G mutated bile salt export pump (BSEP) functio... Bioorg Med Chem Lett. 2012 Jun 15;22(12):3962-6. doi: 10.1016/j.bmcl.2012.04.099. Epub 2012 Apr 30. Misawa T, Hayashi H, Makishima M, Sugiyama Y, Hashimoto Y
E297G mutated bile salt export pump (BSEP) function enhancers derived from GW4064: structural development study and separation from farnesoid X receptor-agonistic activity.
Bioorg Med Chem Lett. 2012 Jun 15;22(12):3962-6. doi: 10.1016/j.bmcl.2012.04.099. Epub 2012 Apr 30., [PMID:22583617]

Abstract [show]
Bile salt export pump (BSEP) is a member of the ATP-binding cassette transmembrane transporter family and mediates biliary excretion of bile acids from hepatocytes. Several BSEP mutants, including Glu297Gly (E297G) and Asp482Gly (D482G), cause progressive familial intrahepatic cholestasis type 2. We previously found that compounds based on GW4064, a representative farnesoid X receptor (FXR) agonist, enhanced E297G BSEP transport activity. 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.

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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

[hide] The bile salt export pump (BSEP) in health and dis... Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):536-53. doi: 10.1016/j.clinre.2012.06.006. Epub 2012 Jul 12. Kubitz R, Droge C, Stindt J, Weissenberger K, Haussinger D
The bile salt export pump (BSEP) in health and disease.
Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):536-53. doi: 10.1016/j.clinre.2012.06.006. Epub 2012 Jul 12., [PMID:22795478]

Abstract [show]
The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases.

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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

[hide] Hepatobiliary transport in health and disease. Clin Lipidol. 2012 Apr;7(2):189-202. Chan J, Vandeberg JL
Hepatobiliary transport in health and disease.
Clin Lipidol. 2012 Apr;7(2):189-202., [PMID:22859919]

Abstract [show]
Bile salts, cholesterol and phosphatidylcholine are secreted across the canalicular membrane of hepatocytes into bile by ATP-binding cassette (ABC) transporters. Secretion of bile salts by ABCB11 is essential for bile flow and for absorption of lipids and fat-soluble vitamins. ABCG5 and ABCG8 eliminate excess cholesterol and sterols from the body by secreting them into bile. There are two mechanisms to protect the canalicular membrane from solubilization by bile salts; ABCB4 secretes phosphatidylcholine into bile to form mixed micelles with bile salts, and ATP8B1 maintains the canalicular membrane in a liquid-ordered state. Three different forms of progressive familial intrahepatic cholestasis (PFIC) disorders, PFIC1, PFIC2 and PFIC3, are caused by mutations in ATP8B1, ABCB11 and ABCB4, respectively. Sitosterolemia is caused by mutations in ABCG5 and ABCG8. This article reviews the physiological roles of these canalicular transporters, and the pathophysiological processes and clinical features associated with their mutations.

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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

[hide] Biosynthesis and trafficking of the bile salt expo... Mol Aspects Med. 2014 Jun;37:3-14. doi: 10.1016/j.mam.2013.05.001. Epub 2013 May 15. Soroka CJ, Boyer JL
Biosynthesis and trafficking of the bile salt export pump, BSEP: therapeutic implications of BSEP mutations.
Mol Aspects Med. 2014 Jun;37:3-14. doi: 10.1016/j.mam.2013.05.001. Epub 2013 May 15., [PMID:23685087]

Abstract [show]
The bile salt export pump (BSEP, ABCB11) is the primary transporter of bile acids from the hepatocyte to the biliary system. This rate-limiting step in bile formation is essential to the formation of bile salt dependent bile flow, the enterohepatic circulation of bile acids, and the digestion of dietary fats. Mutations in BSEP are associated with cholestatic diseases such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis, and intrahepatic cholestasis of pregnancy. Development of clinical therapies for these conditions necessitates a clear understanding of the cell biology of biosynthesis, trafficking, and transcriptional and translational regulation of BSEP. This chapter will focus on the molecular and cell biological aspects of this critical hepatic membrane transporter.

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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

[hide] Differential roles of ubiquitination in the degrad... Mol Pharmacol. 2014 Mar;85(3):482-91. doi: 10.1124/mol.113.091090. Epub 2013 Dec 30. Aida K, Hayashi H, Inamura K, Mizuno T, Sugiyama Y
Differential roles of ubiquitination in the degradation mechanism of cell surface-resident bile salt export pump and multidrug resistance-associated protein 2.
Mol Pharmacol. 2014 Mar;85(3):482-91. doi: 10.1124/mol.113.091090. Epub 2013 Dec 30., [PMID:24378332]

Abstract [show]
We previously showed that ubiquitination, a reversible post-translational modification, facilitates degradation of cell surface-resident bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2), ABC transporters that are expressed at the canalicular membrane (CM) of hepatocytes. In the current study, its underlying mechanism was investigated by evaluating the role of ubiquitination in the processes of internalization and subsequent degradation of cell surface-resident BSEP and MRP2. Cell surface biotinylation analysis using Flp-In T-REx 293 cells showed that ectopic expression of Ub(Delta)(GG), which is ubiquitin (Ub) lacking the two C-terminal glycines essential for the Ub conjugation reaction, inhibited the internalization of 3x FLAG-BSEP, but not of MRP2, and the degradation of the internalized MRP2, but not of the internalized 3x FLAG-BSEP. Its inhibitory effect on BSEP internalization was also indicated by a time-lapse imaging analysis using the rat hepatoma cell line McA-RH7777 in which Ub(Delta)(GG) delayed the loss of fluorescence from photoactivated Dronpa-BSEP on the CM. The effect of Ub(Delta)(GG) on BSEP internalization in these experiments was abrogated by treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and the introduction of a Y1311A mutation into BSEP. This mutation eliminates the ability of BSEP to interact with the AP2 adaptor complex, an adaptor protein required for cargo selection in clathrin-mediated endocytosis. In conclusion, our data suggest that ubiquitination facilitates clathrin-mediated endocytosis of BSEP and the degradation of internalized MRP2, leading to the degradation of the cell surface-resident form of both transporters.

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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

[hide] Small intestinal bacterial overgrowth in patients ... Acta Biochim Pol. 2014;61(1):103-7. Epub 2014 Mar 17. Lisowska A, Kobelska-Dubiel N, Jankowska I, Pawlowska J, Moczko J, Walkowiak J
Small intestinal bacterial overgrowth in patients with progressive familial intrahepatic cholestasis.
Acta Biochim Pol. 2014;61(1):103-7. Epub 2014 Mar 17., [PMID:24644547]

Abstract [show]
BACKGROUND & AIMS: To date, no studies concerning the presence of small intestinal bacterial overgrowth in patients with progressive familial intrahepatic cholestasis were published. Based upon characteristic of progressive familial intrahepatic cholestasis one can expect the coexistence of small intestinal bacterial overgrowth. The aim of the study was to assess the incidence of small intestinal bacterial overgrowth in patients with progressive familial intrahepatic cholestasis. METHODS: 26 patients aged 8 to 25 years with progressive familial intrahepatic cholestasis were included in the study. Molecular analysis of ABCB11 gene was performed in the vast majority of patients. In all patients Z-score for body weight and height, biochemical tests (bilirubin, bile acid concentration, fecal fat excretion) were assessed. In all patients hydrogen-methane breath test was performed. RESULTS: On the basis of first hydrogen-methane breath test, diagnosis of small intestinal bacterial overgrowth was confirmed in 9 patients (35%), 5 patients (19%) had borderline results. The second breath test was performed in 10 patients: in 3 patients results were still positive and 2 patients had a borderline result. The third breath test was conducted in 2 patients and positive results were still observed. Statistical analysis did not reveal any significant correlations between clinical, biochemical and therapeutic parameters in patients with progressive familial intrahepatic cholestasis and coexistence of small intestinal bacterial overgrowth. CONCLUSIONS: Our results suggest that small intestinal bacterial overgrowth is frequent in patients with progressive familial intrahepatic cholestasis. Moreover, it seems that this condition has the tendency to persist or recur, despite the treatment.

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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

[hide] Differential effects of membrane cholesterol conte... Mol Pharmacol. 2014 Jun;85(6):909-20. doi: 10.1124/mol.114.092262. Epub 2014 Apr 7. Guyot C, Hofstetter L, Stieger B
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).
Mol Pharmacol. 2014 Jun;85(6):909-20. doi: 10.1124/mol.114.092262. Epub 2014 Apr 7., [PMID:24711118]

Abstract [show]
Rat canalicular membranes contain microdomains enriched in cholesterol and ATP-binding cassette transporters. Cholesterol is known to regulate the activity of transporters. Here, we investigated the effect of membrane cholesterol on the transport kinetics of multidrug resistance-associated protein 2 (MRP2) and of bile salt export pump (BSEP) variants and mutants. MRP2 and BSEP were expressed with baculoviruses in insect cells, followed by vesicle isolation from control and cholesterol-loaded cells (1 mM cholesterol@randomly methylated-beta-cyclodextrin) for transport assays. We found that cholesterol stimulates MRP2 transport activity for substrates of different molecular weights: estradiol-17-beta-glucuronide (E17betaG), prostaglandin E2 (PGE2), cholecystokinin 8 (CCK8), and vasopressin displayed an increase of Vmax and a variable decrease of Km. Kinetics of E17betaG showed a sigmoidal shape and a mild cooperativity in Hanes-Woolf plots in control membranes. High cholesterol content shifted E17betaG to Michaelis-Menten kinetics. PGE2/glutathione transport followed Michaelis-Menten kinetics irrespective of cholesterol. The MRP2 substrates CCK8 and vasopressin exhibited Michaelis-Menten kinetics independent of membrane cholesterol content. Transport of ochratoxin A was ATP-dependent but was neither mediated by MRP2 nor stimulated by cholesterol. Transport of the two most common BSEP variants p.444V/A showed Michaelis-Menten kinetics irrespective of membrane cholesterol, whereby cholesterol leads to an increased Vmax while Km remains unchanged. 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. Hence, changing membrane cholesterol content modulates BSEP and MRP2 transport kinetics differently. Cholesterol increases the transport rates of BSEP and MRP2, but with the latter, may also modify the binding site as for E17betaG.

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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

[hide] Diagnosis of ABCB11 gene mutations in children wit... Mol Med Rep. 2014 Sep;10(3):1264-74. doi: 10.3892/mmr.2014.2349. Epub 2014 Jun 20. Hu G, He P, Liu Z, Chen Q, Zheng B, Zhang Q
Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis and direct sequencing.
Mol Med Rep. 2014 Sep;10(3):1264-74. doi: 10.3892/mmr.2014.2349. Epub 2014 Jun 20., [PMID:24969679]

Abstract [show]
Intrahepatic cholestasis represents a heterogeneous group of disorders that begin during childhood, most commonly manifesting as neonatal cholestasis, and lead to ongoing liver dysfunction in children and adults. For children, inherited pathogenic factors of cholestasis have gained increasing attention owing to the rapid development of molecular biology technology. However, these methods have their advantages and disadvantages in terms of simplicity, sensitivity, specificity, time required and expense. In the present study, an effective, sensitive and economical method is recommended, termed high-resolution melting (HRM) analysis and direct sequencing, based on general polymerase chain reaction, to detect mutations in diseasecausing genes. As one type of inherited intrahepatic cholestasis, progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by pathogenic mutations in the ABCB11 gene, HRM was used to detect mutations in the ABCB11 gene in the present study, and the diagnosis for PFIC2 was made by comprehensive analysis of genetic findings and clinical features. Furthermore, the characteristics of mutations and single nucleotide polymorphisms (SNPs) in the ABCB11 gene were elucidated. A total of 14 types of mutations/polymorphisms were identified in 20 patients from mainland China, including six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), one nonsense mutation (p.R928X) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Five mutations were novel. The majority of the mutations were different from those detected in other population groups. A total of 4/20 patients (1/5) were diagnosed to be PFIC2 by combining genetic findings with the clinical features. Polymorphisms V444A and A1028A, with an allele frequency of 74.5 and 67.2%, respectively, were highly prevalent in the mainland Chinese subjects. No differences were found between the patients with cholestasis and the control subjects. Efficient genetic screening facilitates the clinical diagnosis of genetic disorders. The present study demonstrated that HRM analysis was efficient and effective in detecting mutations and expanded the known spectrum of ABCB11 gene mutations.

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

[hide] Genetic variations of bile salt transporters. Drug Discov Today Technol. 2014 Jun;12:e55-67. doi: 10.1016/j.ddtec.2014.03.006. Kubitz R, Droge C, Kluge S, Stindt J, Haussinger D
Genetic variations of bile salt transporters.
Drug Discov Today Technol. 2014 Jun;12:e55-67. doi: 10.1016/j.ddtec.2014.03.006., [PMID:25027376]

Abstract [show]
Bile salt transporters directly or indirectly influence biological processes through physicochemical or signalling properties of bile salts. The coordinated action of uptake and efflux transporters in polarized epithelial cells of the liver, biliary tree, small intestine and kidney determine bile salt concentrations in different compartments of the body. Genetic variations of bile salt transporters lead to clinical relevant phenotypes of varying severity ranging from a predisposition for drug-induced liver injury to rapidly progressing end-stage liver disease. This review focuses on the impact of genetic variations of bile salt transporters including BSEP, NTCP, ASBT and OSTalpha/beta and discusses approaches for transporter analysis.

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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

[hide] [Development of new therapeutic strategy for trans... Yakugaku Zasshi. 2014;134(10):1007-11. Hayashi H
[Development of new therapeutic strategy for transporter-related diseases].
Yakugaku Zasshi. 2014;134(10):1007-11., [PMID:25274209]

Abstract [show]
Significant technological advances in gene sequence analysis and construction of genetically-modified animals during the last two decades made it possible to reveal that many transporters are associated with diseases. The bile salt export pump (BSEP/ABCB11), a member of the family of ATP-binding cassette transporters, is localized on the canalicular membrane of hepatocytes and predominantly mediates the biliary excretion of bile salts. A hereditary defect of BSEP results in severe cholestasis called progressive familial intrahepatic cholestasis type 2 (PFIC2). Without liver transplantation, this disease progresses to liver failure and death before adulthood; therefore the development of new, less invasive medical therapy for PFIC2 is of the highest priority. We have previously shown that in many cases of PFIC2 patients, the dysfunction of BSEP is attributable to decreased BSEP expression on the hepatocanalicular membrane and that 4-phenylbutyrate (4PB), an approved drug for urea cycle disorder, may be a compound with potential to restore BSEP expression. This drug inhibits ubiquitination of cell surface-resident BSEP and thereby its clathrin-mediated endocytosis through the AP2 adaptor complex, leading to increase in BSEP expression on the canalicular membrane. Clinical studies to investigate the efficacy of 4PB in the treatment of PFIC2 revealed that 4PB therapy biochemically and histologically improved liver function without any side effect. Therefore, 4PB therapy may become the preferred choice, instead of liver transplantation, for PFIC2 patients. The strategy employed and findings in this study would be valuable for the drug development of transporter-related disorders.

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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

[hide] Autoimmune BSEP disease: disease recurrence after ... Clin Rev Allergy Immunol. 2015 Jun;48(2-3):273-84. doi: 10.1007/s12016-014-8457-4. Kubitz R, Droge C, Kluge S, Stross C, Walter N, Keitel V, Haussinger D, Stindt J
Autoimmune BSEP disease: disease recurrence after liver transplantation for progressive familial intrahepatic cholestasis.
Clin Rev Allergy Immunol. 2015 Jun;48(2-3):273-84. doi: 10.1007/s12016-014-8457-4., [PMID:25342496]

Abstract [show]
Severe cholestasis may result in end-stage liver disease with the need of liver transplantation (LTX). In children, about 10 % of LTX are necessary because of cholestatic liver diseases. Apart from bile duct atresia, three types of progressive familial intrahepatic cholestasis (PFIC) are common causes of severe cholestasis in children. The three subtypes of PFIC are defined by the involved genes: PFIC-1, PFIC-2, and PFIC-3 are due to mutations of P-type ATPase ATP8B1 (familial intrahepatic cholestasis 1, FIC1), the ATP binding cassette transporter ABCB11 (bile salt export pump, BSEP), or ABCB4 (multidrug resistance protein 3, MDR3), respectively. All transporters are localized in the canalicular membrane of hepatocytes and together mediate bile salt and phospholipid transport. In some patients with PFIC-2 disease, recurrence has been observed after LTX, which mimics a PFIC phenotype. It could be shown by several groups that inhibitory anti-BSEP antibodies emerge, which most likely cause disease recurrence. The prevalence of severe BSEP mutations (e.g., splice site and premature stop codon mutations) is very high in this group of patients. These mutations often result in the complete absence of BSEP, which likely accounts for an insufficient auto-tolerance against BSEP. Although many aspects of this "new" disease are not fully elucidated, the possibility of anti-BSEP antibody formation has implications for the pre- and posttransplant management of PFIC-2 patients. This review will summarize the current knowledge including diagnosis, pathomechanisms, and management of "autoimmune BSEP disease."

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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

[hide] Successful pregnancy after ileal exclusion in prog... Ann Hepatol. 2015 Jul-Aug;14(4):550-2. Czubkowski P, Jankowska I, Pawlowska J
Successful pregnancy after ileal exclusion in progressive familial intrahepatic cholestasis type 2.
Ann Hepatol. 2015 Jul-Aug;14(4):550-2., [PMID:26019043]

Abstract [show]
Progressive familial intrahepatic cholestasis type 2 (PFIC 2) results from mutations in ABCB11 gene coding bile salt export pump (BSEP). Medical treatment is usually unsuccessful and surgery intervention is necessary. Partial external biliary diversion (PEBD) is regarded as the first choice of surgical treatment. Ileal exclusion (IE) is an alternative operation if external stoma is not tolerated; however, a favorable outcome is uncertain. In chronic liver diseases pregnancy brings additional risk of deterioration of liver function and generally is not recommended. We present the first case report of successful pregnancy in a genetically confirmed PFIC 2 patient after surgical conversion from PEBD to IE.

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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

[hide] The intrahepatic expression levels of bile acid tr... J Gastroenterol. 2015 Nov 25. Okushin K, Tsutsumi T, Enooku K, Fujinaga H, Kado A, Shibahara J, Fukayama M, Moriya K, Yotsuyanagi H, Koike K
The intrahepatic expression levels of bile acid transporters are inversely correlated with the histological progression of nonalcoholic fatty liver disease.
J Gastroenterol. 2015 Nov 25., [PMID:26601667]

Abstract [show]
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) presents as a spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter is progressive, and its pathogenesis remains poorly understood. Recently, bile acid (BA) metabolism has become a therapeutic focus in NASH patients. The aim of the present study was to explore changes in bile acid metabolism in NAFLD patients in the context of disease progression. METHODS: We prospectively enrolled patients with clinically suspected NAFLD. Patients taking ursodeoxycholic acid were excluded. The intrahepatic expression levels of genes associated with BA metabolism were determined by quantitative PCR and immunohistochemistry. RESULTS: Seventy-eight patients (male:female = 49:29) histologically diagnosed with NAFLD were analyzed. The expression levels of farnesoid X receptor, liver receptor homolog 1, and small heterodimer partner, key proteins in BA synthesis, significantly decreased as the NAFLD activity score (NAS) increased in either males or females. The levels of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of BA synthesis, were not changed. Notably, the expression levels of a main export transporter, bile salt export pump (BSEP), significantly decreased as the NAS and the each NAS component increased in both genders. The decreases of BSEP levels were also observed by immunohistochemistry, particularly in areas with pronounced fatty changes in cases with high NAS. CONCLUSIONS: The expression levels of the BA export transporter BSEP were inversely correlated with NAS in NAFLD patients. Such down-regulation may cause excessive BA levels in hepatocytes, leading to cell injury. Our findings may afford new insights into the pathogenesis of NASH.

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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