Methylation levels and mRNA expression of ABCB1 in relation to tacrolimus blood concentration
Through methylation chip screening, the methylation levels of three ABCB1 sites (cg12501229, cg00634941, and cg05496710) located on chromosome 7 were found to be significantly different between the high and low tacrolimus blood concentration groups (Figure 2A ). Using the pyrophosphorylation assay, the methylation levels of these three sites were further evaluated in the 23 donor liver specimens carrying the CYP3A5*3/*3 genotype. We found that the methylation levels of these sites in the high C0/D group were all significantly lower than those in the low C0/D group (Figure 2B-D ).
Furthermore, ABCB1 mRNA expression levels were determined by RT-qPCR, and correlation analyses revealed that ABCB1 mRNA expression in donor livers was positively correlated with the tacrolimus C0/D ratio (r = 0.458, P < 0.05) (Figure 3A ). An ABCB1 mRNA expression level of 0.15 was used as the cut-off value to analyse the tacrolimus C0/D ratio and differences between the low-and high-expression groups (Figure 3B and C ). Our results showed that the greater proportion of patients with high blood tacrolimus concentrations were found in the high ABCB1 expression group.