Functional characterization of MDR3 promoter mutations detected in intrahepatic cholestasis of pregnancy

Background Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with risk of intrauterine fetal death and prematurity. There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporter MDR3 (multidrug resistance protein 3) is a risk factor for ICP. The aim of this study was to functionally characterize newly detected MDR3 promoter variants in ICP patients. Methods Luciferase reporter constructs of the human MDR3 5'-regulatory regions were generated by PCR and ligated into the pGL3-Basic vector. DNA from three different ICP patients (covering the wildtype sequence and eight promoter mutations, three of which were ICP-specific: intron -4: C(-10)T; exon-3: A(-358)G; intron-3: C(-31)T) was used. The human hepatocyte derived cell lines Huh7 and HepG2 were transiently transfected with reporter constructs to assay for promoter activity. Results In both cell lines, no differences in promoter activity of the mutated constructs were observed with respect to the promoter containing the reference sequence. In HepG2 cells, activity of the two mutated constructs amounted to 96% and 103% of reference activity, respectively. In Huh-7 cells activity were 90% and 85% of reference activity, respectively. Conclusion Results indicate that the tested promoter mutations do not change the activity of the MDR3-promoter. They therefore do not seem to play a pathophysiological role in ICP development. Clinical Pharmacology & Therapeutics (2004) 75, P16–P16; doi: 10.1016/j.clpt.2003.11.061