Functional Variants of the Central Bile Acid Sensor FXR Identified in Intrahepatic Cholestasis of Pregnancy
2007; Elsevier BV; Volume: 133; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2007.05.015
ISSN1528-0012
AutoresSaskia W. C. van Mil, Alexandra Milona, Peter H. Dixon, Roman Müllenbach, Victoria Geenes, Jenny Chambers, Vasylyna Shevchuk, Gudrun E. Moore, Frank Lammert, Anna Glantz, Lars‐Åke Mattsson, John C. Whittaker, Malcolm G. Parker, Roger White, Catherine Williamson,
Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoBackground & Aims: Intrahepatic cholestasis of pregnancy (ICP) is characterized by liver impairment, pruritus, and elevated maternal serum bile acids. It can cause premature delivery and intrauterine death. Bile acid synthesis, metabolism, and transport are regulated by the bile acid sensor FXR, and we hypothesized that genetic variation in FXR confers susceptibility to ICP. Methods: The coding regions and intron/exon boundaries of FXR were sequenced in 92 British ICP cases of mixed ethnicity. Subsequently, a case-control study of allele frequencies of these variants in 2 independent cohorts of Caucasian ICP patients and controls was performed. Variants were cloned into an FXR expression plasmid and tested in functional assays. Results: We identified 4 novel heterozygous FXR variants (−1g>t, M1V, W80R, M173T) in ICP. W80R was not present in Caucasians and M1V was detected uniquely in 1 British case. M173T and −1g>t occur both in Caucasian cases and controls, and we found a significant association of M173T with ICP (OR, 3.2; 95% confidence interval, 1.1–11.2; P = .02) when the allele frequencies of both Caucasian cohorts were analyzed together. We demonstrate functional defects in either translation efficiency or activity for 3 of the 4 variants (−1g>t, M1V, M173T). Conclusions: This is the first report of functional variants in FXR. We propose that these variants may predispose to ICP, and because FXR has a central role in regulating bile and lipid homeostasis they may be associated with other cholestatic and dyslipidemic disorders. Background & Aims: Intrahepatic cholestasis of pregnancy (ICP) is characterized by liver impairment, pruritus, and elevated maternal serum bile acids. It can cause premature delivery and intrauterine death. Bile acid synthesis, metabolism, and transport are regulated by the bile acid sensor FXR, and we hypothesized that genetic variation in FXR confers susceptibility to ICP. Methods: The coding regions and intron/exon boundaries of FXR were sequenced in 92 British ICP cases of mixed ethnicity. Subsequently, a case-control study of allele frequencies of these variants in 2 independent cohorts of Caucasian ICP patients and controls was performed. Variants were cloned into an FXR expression plasmid and tested in functional assays. Results: We identified 4 novel heterozygous FXR variants (−1g>t, M1V, W80R, M173T) in ICP. W80R was not present in Caucasians and M1V was detected uniquely in 1 British case. M173T and −1g>t occur both in Caucasian cases and controls, and we found a significant association of M173T with ICP (OR, 3.2; 95% confidence interval, 1.1–11.2; P = .02) when the allele frequencies of both Caucasian cohorts were analyzed together. We demonstrate functional defects in either translation efficiency or activity for 3 of the 4 variants (−1g>t, M1V, M173T). Conclusions: This is the first report of functional variants in FXR. We propose that these variants may predispose to ICP, and because FXR has a central role in regulating bile and lipid homeostasis they may be associated with other cholestatic and dyslipidemic disorders. Intrahepatic cholestasis of pregnancy (ICP) affects 1 in 200 pregnancies in Caucasians.1Abedin P. Weaver J.B. Egginton E. Intrahepatic cholestasis of pregnancy: prevalence and ethnic distribution.Ethn Health. 1999; 4: 35-37Crossref PubMed Scopus (83) Google Scholar, 2Heinonen S. Kirkinen P. Pregnancy outcome with intrahepatic cholestasis.Obstet Gynecol. 1999; 94: 189-193Crossref PubMed Scopus (116) Google Scholar It presents with pruritus and liver impairment, and can be complicated by fetal distress, spontaneous prematurity, and unexplained intrauterine death.3Williamson C. Hems L.M. 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Chatterjee B. Dehydroepiandrosterone sulfotransferase is a target for transcriptional induction by the vitamin D receptor.Mol Pharmacol. 2004; 65: 720-729Crossref PubMed Scopus (138) Google Scholar Conversely, FXR activates transcription of the short heterodimer partner (SHP), which represses bile acid import (NTCP)34Denson L.A. Sturm E. Echevarria W. Zimmerman T.L. Makishima M. Mangelsdorf D.J. Karpen S.J. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp.Gastroenterology. 2001; 121: 140-147Abstract Full Text PDF PubMed Scopus (361) Google Scholar and synthesis (CYP7A1, CYP7B1).35Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.Mol Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar In the intestine, FXR acts via SHP to reduce import of bile acids into the ileocytes (ASBT)36Li H. Chen F. Shang Q. Pan L. Shneider B.L. Chiang J.Y. Forman B.M. Ananthanarayanan M. Tint G.S. Salen G. Xu G. FXR-activating ligands inhibit rabbit ASBT expression via FXR-SHP-FTF cascade.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G60-G66Crossref PubMed Scopus (65) Google Scholar and directly induces transport through the enterocyte (IBABP) and export into the peripheral blood (OSTα, OSTβ).37Landrier J.F. Eloranta J.J. Vavricka S.R. Kullak-Ublick G.A. The nuclear receptor for bile acids, FXR, transactivates the human organic solute transporter-alpha and -beta genes.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G476-G485Crossref PubMed Scopus (174) Google Scholar Thus, FXR maintains low bile acid levels in hepatocytes and enterocytes.We hypothesize that FXR dysfunction causes cholestasis; therefore, we investigated whether ICP patients harbor FXR mutations.Materials and MethodsSubjectsThe case-control study conformed to the guidelines outlined by the 1975 Declaration of Helsinki and permission was obtained from the Ethics Committees of the Hammersmith Hospitals NHS Trust, London (REC 97/5197), the Ethics Committee of the Faculty of Medicine at the University of Göteborg, and the University Hospital Aachen, Germany (AZ EK 2008). All ICP patients were diagnosed on the basis of clinical symptoms in combination with routine laboratory investigations as previously described.21Mullenbach R. Bennett A. Tetlow N. Patel N. Hamilton G. Cheng F. Chambers J. Howard R. Taylor-Robinson S.D. Williamson C. ATP8B1 mutations in British cases with intrahepatic cholestasis of pregnancy.Gut. 2005; 54: 829-834Crossref PubMed Scopus (126) Google Scholar, 38Wasmuth H.E. Glantz A. Keppeler H. Simon E. Bartz C. Rath W. Mattsson L.Å. Marschall H.U. Lammert F. Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene.Gut. 2007; 56: 265-270Crossref PubMed Scopus (129) Google Scholar British patients with FXR variants were numbered 1–32. In total, 290 British controls were recruited. Ethical approval was given to include anonymized controls who were originally recruited for a study of birth weight in consecutive pregnant women delivering at Queen Charlotte’s Hospital (n = 260). Liver function tests were available for approximately 50% of cases, and if transaminases were raised while pregnant or within 3 months of pregnancy, controls were excluded. Additionally, 30 British Caucasian controls were recruited specifically for this study at Queen Charlotte’s Hospital, and had no history of cholestasis, hepatic impairment, or itching during pregnancy. Forty-nine Swedish ICP cases were recruited previously in a prospective screen of all pregnant women and had total bile acid levels ≥40 μmol/L.38Wasmuth H.E. Glantz A. Keppeler H. Simon E. Bartz C. Rath W. Mattsson L.Å. Marschall H.U. Lammert F. Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene.Gut. 2007; 56: 265-270Crossref PubMed Scopus (129) Google Scholar Fifty-nine Swedish controls had total bile acid levels t mutation, the Kozak sequence (CCACC) of the pGL3-control vector was replaced by the Kozak sequence of wild-type FXR (TTTGG) and FXR −1g>t (TTTGT) by site-directed mutagenesis (Supplementary Table 3; see supplementary material on line at www.gastrojournal.org).Two-Dimensional Gel ElectrophoresisWe rehydrated 7-cm pH 6.2–7.5 immobiline gel (IPG) strips (GE Healthcare, Chalfont St. Giles, UK) with in vitro synthesized protein in 8 mol urea, 4% (wt/vol) CHAPS, 40 mmol Tris overnight. Isoelectric focusing was performed on a IPGphor (Pharmacia Biotech, Piscataway, NJ): 0.5 hours 100 V; 0.5 hours 200 V, gradient to 1,000 V (1 hour), gradient to 3,000 V (2 hours), gradient to 6,000 V (1 hour), gradient to 8,000 V (1 hour), 8,000 V (1 hour), and 500 V (1 hour). Strips were equilibrated in 50 mmol Tris-HCl pH 6.8, 6 mol urea, 30% glycerol, and 2% SDS containing 65 mmol DTT followed by the same buffer containing 240 mmol iodoacetamide. Strips were then transferred onto 15% polyacrylamide gels, and overlaid with 0.5% low melting point agarose in electrophoresis buffer. Gels were subjected to immunoblotting.ImmunoblottingHEK293T cells were transfected with different plasmids using Fugene 6 transfection reagent (Invitrogen, Carlsbad, CA) and harvested after 72 hours. One- and 2-dimensional SDS-page gels were electrotransferred to nitrocellulose membranes and probed with anti-FXR (1:1000, PPMX, Tokyo, Japan). The intensity of the bands was determined by ImageJ and expressed as percentage of β-actin expression.ImmunofluorescenceHEK293T cells were transfected with pcDNA3.1, FXR WT, or FXR M173T. After 48 hours, cells were fixed with 4% paraformaldehyde in PBS (20 minutes, 4°C), washed with PBS 0.1% Triton X-100, and blocked with 5% skimmed milk in PBS Triton X-100. FXR was stained with anti-FXR (1:200) and visualized with FITC-conjugated donkey anti-mouse immunoglobulin G (Jackson Laboratories Inc, West Grove, PA).Transactivation AssaysHEK293T cells were plated in 96-well plates in phenol red-free medium supplemented with 5% dextran charcoal-stripped fetal serum. Cells were transfected with pSG5-RXRα/Gal4DBD-RXRα, pGL3, or pGL3-BSEP/IBABP promoters, pCMV-Renilla, and expression vectors encoding wild-type or mutant FXRα2 as indicated. On day 3, fresh medium with or without 50 μmol/L CDCA was applied to the cells. After 24 hours, the reporter Firefly and Renilla luciferase activity were determined as previously described.39Belandia B. Parker M.G. Functional interaction between the p160 coactivator proteins and the transcriptional enhancer factor family of transcription factors.J Biol Chem. 2000; 275: 30801-30805Crossref PubMed Scopus (98) Google Scholar Transfection experiments were performed at least 3 times and results are shown as mean values of quadruplicates ± standard deviations. For the Kozak pGL3-control experiment, HEK293T cells were cotransfected with the pRL-CMV and pGL3-control plasmid containing different Kozak sequences. After 24 hours, cells were harvested and assayed for Renilla and Firefly luciferase. In all reporter assay experiments, Renilla luciferase counts were not significantly different in the experimental groups, indicating that the relative firefly luciferase expression is a good measure for the absolute activity of the different mutants.Three-Dimensional ModelingThe human FXR and RXR protein sequences were aligned with the ecdysone receptor (EcR) and ultraspiracle (USP). FXR and RXR sequence orthologs to the DNA-binding domains (DBD) of EcR and USP were superimposed onto the crystal structure of the heterodimeric EcR/USP/DNA complex40Devarakonda S. Harp J.M. Kim Y. Ozyhar A. Rastinejad F. Structure of the heterodimeric ecdysone receptor DNA-binding complex.EMBO J. 2003; 22: 5827-5840Crossref PubMed Scopus (72) Google Scholar using the 3-dimensional modeling program Yasara.Electromobility Shift AssaysBinding reactions contained 20 mmol HEPES pH 7.4, 50 mmol KCl, 1 mmol β-mercaptoethanol, 10% glycerol, 1 μg of poly(dI-dC), and 2 μL of in vitro translated FXR wild type or FXR M173T and RXRα and were preincubated at room temperature for 15 minutes before the addition of 32Barbier O. Torra I.P. Sirvent A. Claudel T. Blanquart C. Duran-Sandoval D. Kuipers F. Kosykh V. Fruchart J.C. Staels B. FXR induces the UGT2B4 enzyme in hepatocytes: a potential mechanism of negative feedback control of FXR activity.Gastroenterology. 2003; 124: 1926-1940Abstract Full Text Full Text PDF PubMed Scopus (164) Google ScholarP-labeled double-stranded human BSEP oligonucleotide probe (0.2 pmol). Where indicated, α-FXR antibody or cold oligonucleotides (specific (BSEP) or nonspecific (hPPRE) were added at a 50-fold molar excess (Supplementary Table 3; see supplementary material on line at www.gastrojournal.org). After 30 minutes at room temperature, the protein–DNA complexes were resolved on a 6% polyacrylamide gel.ResultsFXR Mutation Analysis in ICP PatientsIn an initial screen, sequencing of the entire FXR coding region including intron/exon boundaries in 92 British ICP cases of mixed ethnicity revealed 4 novel heterozygous variants in FXR (NR1H4;Figure 1). Subsequently, these variants were genotyped in a collection of cases and controls of British and Swedish Caucasian origin (Table 1). M1V was detected uniquely in 1 British case, and the W80R variant was not detected at all in Caucasians. Carriers of the −1g>t variant are more common in British controls than cases (OR, 0.79; 95% CI, 0.27–2.25), but this difference is not statistically significant (P = .79). In contrast with the UK results, the −1g>t variant is more common in cases than controls in the Swedish collection, although numbers are very low (2 in cases and 1 in controls; Table 1). Combined analysis of the 2 studies gives no evidence for association at the −1g>t variant (OR, 0.92; 95% CI, 0.35–2.44; P = .96). Carriers of M173T are more common among British cases than controls (OR, 2.22; 95% CI, 0.70–8.3), but this difference does not reach statistical significance (P = .20). In the Swedish case-control study, M173T carriers are again more common in cases, with 4 copies in cases and none in controls (P = .04) and combined analysis of both collections gives significant evidence for an association of ICP with M173T (OR, 3.2; 95% CI, 1.1–11.2; P = .02).Table 1Allele Frequencies of the FXR Variants in ICP Cases and Controls for a British and Swedish Caucasian CollectionExonNucleotide AlterationPredicted EffectCohortaThe British cohort consists of 293 cases and 290 controls, the Swedish cohort of 49 cases and 59 controls.Case CarriersControl CarriersAllele Frequency Cases (95% CI)Allele Frequency Controls (95% CI)3−1g>tTranslation defectBritish8141.4% (0.6–2.7)2.4% (1.5–4.0)Swedish212.0% (0.2–7.2)0.8% (0.0–4.6)31a>gM1VBritish100.1% (0.0–0.9)0.0% (0.0–0.6)Swedish000.0% (0.0–3.7)0.0% (0.0–3.1)4238t>cW80RbThis variant was only present in 1 UK case of Asian origin.British000.0% (0.0–0.6)0.0% (0.0–0.6)Swedish000.0% (0.0–3.7)0.0% (0.0–3.1)5518t>cM173TBritish1151.9% (0.9–3.3)0.8% (0.2–2.0)Swedish404.1% (1.1–10.1)0.0% (0.0–3.1)a The British cohort consists of 293 cases and 290 controls, the Swedish cohort of 49 cases and 59 controls.b This variant was only present in 1 UK case of Asian origin. Open table in a new tab The frequency of each variant was established in the 77 UK cases of mixed ethnic origin and in a cohort of 47 German cases. There were no further copies of M173T or M1V. There were 15 cases of −1g>t (1 of these cases also had the W80R variant; data not shown). Allele frequencies were not analyzed in these groups because there were no matched controls available.Clinical Characteristics of ICP Patients With FXR MutationsClinical and biochemical characteristics of the British ICP patients with mutations are presented in Supplementary Table 4 (see supplementary material on line at www.gastrojournal.org). Serum bile acids were raised in all patients in whom they were measured. All had raised liver transaminase concentrations in serum. The γ-glutamyl transpeptidase (GGT) activity was measured in 18 cases and it was elevated in 5. Other clinical features included cyclical itch (ie, pruritus at specific stages of the menstrual cycle) which affected 16% (5/32) of cases (Supplementary Table 4; see supplementary material on line at www.gastrojournal.org). A personal or family history of gallstones was present in 31% (10/32) of cases. Intrauterine death, fetal distress, or spontaneous prematurity occurred in 41% (13/32) of cases. The controls all had healthy pregnancies and displayed none of the mentioned clinical characteristics.Functional Analysis of the −1g>t and M1V VariantsTwo ATG codons are present at the amino-terminus of FXRα1 and α2 sep
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