Role Of MDR3, BSEP And FIC1 Gene In The Pathogenesis Of Intrahepatic Cholestasis Of Pregnancy And In The Alterations Induced By Cholestasis In Bile Acid Transport Across The Human Placenta

Part I Study on MDR3 gene mutation in patients with intrahepatic cholestasis of pregnancyBackground Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that causes maternal pruitus and hepatic impairment and can cause fetal death, spontaneous prematurity and sudden intrauterine death. The genetic etiology of most cases of ICP is unknown. There is increasing evidence that genetically determined dysfunction in mutidrug resistance gene 3 (MDR3, ABCB4) might be risk factors for ICP development. Heterozygous carriers of mutations causing progressive familial intrahepatic cholestasis (PFIC) diseases may experience ICP in some of European countries such as France, England and Finland. But we have no idea of the distribution of MDR3 gene mutation in patients complicated with ICP in China.Objective This study aimed to describe the extent of genetic variability in MDR3 and identify new disease-causing mutation in the development of ICP.Methods 30 women with ICP and 60 women with uneventful pregnancies were recruited between November 2003 and April 2005. Liver function tests including γ-glutamyltranspeptidase (γ-GT) and serum total bile acids (TBA) were evaluated at entry. Using PCR-SSCP assay, we screened the mutation of five exons of MDR3 gene (exon6, exon9, exon12, exon14 and exon23).Result Important ICP-induced changes in serum TBA level were observed. 4 had a raised γ-GT, 16 had a normal γ-GT, and the γ-GT was not measured in 10 cases. All samples were amplified by PCR assay, but there are no abnormal moving bands, which were found by SSCP electrophoresis.Conclusion No new mutations were observed in the five exons of MDR3. The MDR3 gene mutation is not likely to be implicated in these Chinese patients with ICP in central China.Part II Expression of MDR3(ABCB4), BSEP(ABCB11) and FIC1(ATP8B1) Gene Product in 1st and 3rd Trimester Human PlacentaBackground Since the excretion of potentially toxic cholephilic organic anions produced by the fetus, such as bile acids and biliary pigments, cannot be performed by the fetal liver alone, the placenta and the maternal liver must play a key role collaborating in this function. Simple diffusion is not the major route for transplacental transfer of fetal bile acids. The uptake of fetal bile acids across the basal plasma membrane is carried out by sodium independent anion exchange. The export of BAs from the trophoblast toward the maternal blood is probably mainly carried out by ATP-dependent transport systems and ATP-independent mechanisms. As the major mechanism of bile acids transfer across the placenta, the existence of transport proteins in the trophoblast, some of which have unidirectional transport characteristics, accounts for the vectorial properties of the overall process. But the character of these proteins in the trophoblasts may be ascertained. FIC1(ATP8B1), BSEP(ABCB11) and MDR3(ABCB4) genes are relevant to bile formation in the human liver. Mutations in FIC1 (ATP8B1), in BSEP (ABCB11) as well as in MDR3 (ABCB4) can cause different progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis (BRIC) and Greenland familial cholestasis (GFC). The expression of FIC1(ATP8B1), BSEP(ABCBll) and MDR3(ABCB4) genes in placenta and their relevance in overall bile acids transport across this organ has not been evaluated yet.Objective To determine the expression of three genes MDR3, BSEP and FIC1, which influence bile acid transport, at the level of transcription and protein in normal human placenta.Method Fifteen 1st trimester placental specimens between 9 and 12 weeks' gestation and twenty 3rd trimester placentae between 38 and 40 weeks' gestation were collected following termination of pregnancy. MDR3, BSEP and FIC1 mRNA were detected in all placentae by using real time reverse transcript polymerase chain reaction (real time RT-PCR). Immunohistochemistry was employed to locate the expression of MDR3 P-Glycoprotein and BSEP protein at the level of protein in two group placentae. The quantity of the expression of MDR3 P-Glycoprotein and BSEP protein was determined by Western blot analysis in all placentae.Result All of three genes MDR3, BSEP and FIC1 at the level of transcription were found in 1st and 3rd trimester human placenta and to be differentially expressed. FIC1 mRNA was found to be down regulated significantly in 3rd trimester placenta compared with 1st trimester(P < 0.01), while MDR3 mRNA was up regulated significantly(P < 0.05). Low levels of BSEP transcripts were detected in both 1st and 3rd placenta but the expression was found no variation (P>0.05).Both MDR3 P-glycoprotein and BSEP protein were found to express in placenta. MDR3 is found in apical membrane of villous syncytiotrophoblast. The distribution of BSEP in placenta is similar to that of MDR3. Western blot analysis showed that compared with 1st trimester placenta, the expression of MDR3 P-glycoprotein was significantly increased (P<0.05) while the expression of BSEP protein was maintained in 3rd trimester placenta (P>0.05).Conclusion The product of three genes that influence bile acid transport were all found to express in placenta and varies according to the needs of growth of fetuses during pregnancy. These genes may be involved in bile acid transport across the human placenta.Part III Role of MDR3, BSEP and FIC1 gene in the alterations induced by intrahepatic cholestasis of pregnancy in bile acid transport across the human placentaBackground Intrahepatic cholestasis of pregnancy (ICP) poses little medical risk to the mother, but poses significant risk to the fetus of perinatal mortality, preterm delivery, fetal distress, and meconium staining. Current evidence suggests that transport of bile acids across the placenta from the fetal to the maternal circulation is impaired and the level of fetal serum bile acids rose. Exactly how this leads to fetal compromise is unknown and raise bile acids of fetus may play a role in this course. We have found three genes MDR3, BSEP and FIC1, which influence bile acid transport, expressing in normal 1st and 3rd trimester human placenta. How these genes express in ICP placenta and its relations with the alterations induced by ICP in bile acid transport across the human placenta are worth a try. Objective To investigate the expression of three genes MDR3, BSEP and FIC1, which influence bile acid transport, in intrahepatic cholestasis of pregnancy (ICP) placenta and discusses their roles in alterations induced by ICP in bile acid transport across the human placenta.Method Twenty trimester placentae from normal mature pregnancy between 38 and 40 weeks' gestation and thirty-six placentae from pregnancy complicated with ICP were collected following termination of pregnancy. Real time RT-PCR was applied to detect MDR3, FIC1 and BSEP mRNA in normal placenta and ICP placenta. Immunohistochemistry was employed to detect the expression of MDR3 P-Glycoprotein and BSEP protein in two group placentae and the quantity of the two proteins was determined by Western blot analysis in ICP placenta.Result The three gene transcripts were found in ICP placenta, and MDR3 P-glycoprotein and BSEP also existed in ICP placenta. The distribution of MDR3 and BSEP in ICP group is similar to that of placenta of normal group. They are all found in villous syncytiotrophoblast. Compared with normal placenta, FIC1 gene and MDR3 gene transcripts was found to be down regulated significantly in placenta of ICP group(P < 0.05 and P < 0.01 respectively); Western blot analysis showed that the level of MDR3 P-glycoprotein was significantly lower in ICP placenta (P < 0.01). Although the mRNA and protein express of BSEP were detected in ICP placenta, the expression was found no variation (P>0.05).Conclusion The three genes that influence bile acid transport were all found to express in ICP placenta. The abnormal expression of FIC1 gene and MDR3 gene in ICP placenta may be related to the alterations induced by ICP in bile acid transport across human placentae and BSEP gene had no correlation with the functional changes of bile acid transport across the placenta. These genes may play a role in the bad outcomes of fetuses complicated with ICP.