Promoter hypermethylation of the gene in PBC.

BACKGROUND & AIMS: Patients with primary biliary cholangitis (PBC) exhibit reduced gene expression in the liver and peripheral blood mononuclear cells (PBMCs). encodes a Cl/HCO exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Reduced expression in PBC may be pathogenic, as -knockout mice reproduce characteristic PBC features. Herein, we aimed to identify CpG-methylation abnormalities in promoter regions that might contribute to the reduced gene transcription in PBC livers and PBMCs.

METHODS

CpG-cytosine methylation rates were interrogated at 1-base pair resolution in upstream and alternate promoter regions through pyrosequencing of bisulphite-modified genomic DNA from liver specimens and PBMCs. and alternative and mRNA levels were measured by real-time PCR. Human lymphoblastoid-T2 cells were treated with 5-aza-2´-deoxycytidine for demethylation assays.

RESULTS

promoters were found to be hypermethylated in PBC livers compared to normal and diseased liver specimens. Receiver operating characteristic (ROC) curve analysis showed that minimal CpG-hypermethylation clusters of 3 -CpG sites and 4 alternate--CpG sites specifically differentiated PBC from normal and diseased controls, with mean methylation rates inversely correlating with respective transcript levels. Additionally, in PBMCs a minimal cluster of 3 hypermethylated -CpG sites distinguished PBC from controls, and mean methylation rates correlated negatively with mRNA levels in these immune cells. Alternate promoters in PBMCs were constitutively hypermethylated, in line with absent alternative mRNA expression in diseased and healthy PBMCs. Demethylation assays treating lymphoblastoid-T2 cells with 5-aza-2´-deoxycytidine triggered expression and upregulated -promoter expression.

CONCLUSIONS

Disease-specific hypermethylation of promoter regions and subsequent downregulation of -gene expression in the liver and PBMCs of patients with PBC might be critically involved in the pathogenesis of this complex disease.

LAY SUMMARY

Primary biliary cholangitis (PBC) is a chronic immune-associated cholestatic liver disease with unclear complex/multifactorial etiopathogenesis affecting mostly middle-aged women. Patients with PBC exhibit reduced expression of the gene. Herein, we found that promoter regions are hypermethylated in the liver and peripheral blood mononuclear cells of patients with PBC. This increased methylation is associated with downregulated -gene expression, which might contribute to the pathogenesis of PBC. Therefore, novel epigenetic targets may improve treatment in patients with PBC who respond poorly to current pharmacological therapies.