Analysis of genetic variation of the pregnane x receptor in putative cholestatic adverse drug reactions using archival liver biopsies : identification of a novel dysfuctional protein varient

A central role in drug clearance makes PXR a strong candidate for altered functionality in adverse drug reactions (ADR), particularly drug-induced cholestasis. By screening in archive of clinical liver biopsies, 53 patients displaying evidence of cholestatic ADRs have been identified. These liver specimens were formalin-fixed, therefore nucleic acid yield and integrity is compromised. To overcome this, DNA extraction and amplification methods were optimised for sequencing of the open reading frames of the PXR gene allowing as little as lng of genomic DNA to be analysed. Simultaneous mRNA extraction and RT-PCR analysis was also developed. In the majority of cases sequence analysis revealed no significant variants. However, in two cases (3.6%) a non-synonymous T/C base substitution encoding a novel PXR protein variant, C301R, was identified as a heterozygote. In a normal control cohort of 308 no incidence of C301R was observed (p=0,023). Functional analysis by reporter gene co-transfection assay of this variant discovered an apparent attenuation of ligand (hyperforin and rifampicin)-mediated transcriptional activation, of both DR3 and ER6 CYP3A response elements compared to the wild-type human PXR. The C301R variant displayed no loss of DNA response element binding compared to wild-type PXR. The identification of this variant protein at a significantly increased frequency compared to a normal population, in a hepatic ADR cohort, would suggest that this apparently functional PXR variant may be one single predisposing factor to ADR. Similar utilisation of archival biopsies could be used to identify further candidate genes associated to ADR pre-disposition.