Abstract:

Objective To investigate the methylation status of ITGAL gene promoter and the effects of mRNA expression in T cells of patients with biliary atresia (BA). Methods The BA infants were recruited from the patients diagnosed as BA by surgery at the Children's Hospital of Fudan University. At the same time, the control group was collected from the patients diagnosed as inguinal hernia by surgery from the same hospital. CD4+ and CD8+T cells were isolated from the peripheral venous blood of infants in BA and control groups. Of them the CD4+ and CD8+T cells of 5 infants with BA were cultured, and then treated with 5-azaC. Genomic DNA and mRNA were extracted from CD4+ and CD8+T cells. Bisulfite DNA sequencing was performed to determine the methylation status of ITGAL gene promoter. The mRNA expression of ITGAL was detected using RT-PCR. Results Twenty-five infants with BA (20 were divided into BA group, 5 were treated with 5-azaC) and 20 controls were analyzed. ①The promoter region (-250 to 250 bp) of the ITGAL gene was unmethylated in BA and control groups. The average methylation level of ITGAL gene promoter region (-1450 to -950 bp) in BA group was higher than that in control group (P<0.001 for CD4+T cells and P=0.02 for CD8+T cells). The average methylation levels from CD8+T cells in BA compared with that in control group and the levels from CD4+T cells compared with that from CD8+T cells in control group were not significantly different (P>0.05). The average methylation levels of ITGAL gene promoter were lower in CD4+ and CD8+T cells in 5-azaC treated group than those in 5-azaC untreated group (P<0.001). ②The mRNA expression of ITGAL in CD4+ T cells in BA group was lower than that in control group (P=0.007) and CD8+T cells in BA group (P=0.013). The mRNA expression of ITGAL in CD8+T cells between BA and control groups and the difference between CD4+ and CD8+T cells in control group were not significantly different (P=0.266 and 0.227). The mRNA expressions of ITGAL in CD4+ and CD8+T cells in 5-azaC treated group were higher than those in the 5-azaC untreated group (P=0.013 and 0.025). Conclusions ITGAL gene promoter in peripheral blood CD4+ T cells from infants with BA is significantly hypermethylated, which impacts the mRNA expression of ITGAL, and may cause the abnormal T cell function.

Key words: 5-azaC, Biliary atresia, DNA methylation, ITGAL