Promoter hypermethylation of the AE2/SLC4A2 gene in PBC

Abstract

Background & Aims: Patients with primary biliary cholangitis (PBC) exhibit reduced AE2/SLC4A2 gene expression in the liver and peripheral blood mononuclear cells (PBMCs). AE2 encodes a Cl–/HCO3– exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Reduced AE2 expression in PBC may be pathogenic, as Ae2-knockout mice reproduce characteristic PBC features. Herein, we aimed to identify CpG-methylation abnormalities in AE2 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 AE2 promoter regions through pyrosequencing of bisulphite-modified genomic DNA from liver specimens and PBMCs. AE2a and alternative AE2b1 and AE2b2 mRNA levels were measured by real-time PCR. Human lymphoblastoid-T2 cells were treated with 5-aza-2´-deoxycytidine for demethylation assays. Results: AE2 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 AE2a-CpG sites and 4 alternate-AE2b2-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 AE2a-CpG sites distinguished PBC from controls, and mean methylation rates correlated negatively with AE2a mRNA levels in these immune cells. Alternate AE2b2/AE2b1 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 AE2b2/AE2b1 expression and upregulated AE2a-promoter expression. Conclusions: Disease-specific hypermethylation of AE2 promoter regions and subsequent downregulation of AE2-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 AE2/SLC4A2 gene. Herein, we found that AE2 promoter regions are hypermethylated in the liver and peripheral blood mononuclear cells of patients with PBC. This increased methylation is associated with downregulated AE2-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. Keywords: AE2 chloride/bicarbonate exchanger, cholestasis, DNA methylation, epigenetic repression, primary biliary cholangitis/cirrhosis