Immunohematology and Genetic Testing (red cells, leukocytes and platelets)
Xuan You, n/a
Blood Center of Zhejiang Province, Zhejiang, China (People's Republic)
The human leukocyte antigen (HLA) expression levels influence the outcome of immune responses against both pathogens and tumors. HLA-A mRNA expression levels vary significantly in different HLA-A locus. In this study, we have examined the expression levels of HLA-A allele in the Zhejiang Han population, and the DNA methylation in the HLA-A promoter region was sequenced by target bisulfite sequencing, to evaluate the DNA methylation on HLA-A expression.
Study
Design/Methods:
HLA-A homozygous individuals (n=45) were recruited from the voluntary blood donors in Blood Center of Zhejiang Province after information consents. HLA-A was amplified using SYBR® Green qPCR method and the housekeeping gene B2M was used as internal control. The HLA-A expression level was calculated relative to B2M using the 2-ΔΔCt method. Bisulfite-treated DNA was amplified using primers HLA-F (GAGTTAGGGAGTTTAGTTTAGGGA) and HLA-R (ACACTAATTAACTTCTCTAAAAACC). The library for the amplicon was prepared with KAPA HTP Library Preparation Kit and was sequenced with Illumina Miseq platform. All the sequencing datas in FASTQ format were analyzed by CLC Genomics Workbench 20.0 (QIAGEN). Peripheral blood mononuclear cells (PBMCs) from samples containing HLA-A*02 or HLA-A*11 with different expression levels were treated with 10 μM 5-azacytidine(5azaCR). RNA was extracted after 24 h of culture, and the HLA-A mRNA expression levels were detected by qPCR.
Results/Findings:
The results of qPCR analyses exhibit a differential expression of HLA-A alleles. The average expression level of HLA-A*02 (n=15) was much higher than that of HLA-A*11 (n=18) (P < 0.0001). The average expression of HLA-A*24 (n=9) and HLA-A*33 (n=3) were between the above two. A 500bp bisulfite converted DNA fragment upstream of the transcription start site was subjected to deep sequencing. The results showed that 12-14 CpG sites in these four HLA-A alleles. Two CpG sites (position-155 and -304) were only found to be methylated in HLA-A*11, in which, -155 is predicted to contain a binding site for tumor suppressor p53 (TP53), and -304 is predicted to contain a binding site for differentiated embryo chondrocyte expressed gene 1 (DEC1) by AnimalTFDB. The mRNA expression level of HLA-A*11 (low expression) increased after treated by 5azaCR (P< 0.05). However, no effect of 5azaCR treatment was identified on expression of HLA-A*02 (high expression).
Conclusions:
Differentially methylated regions on HLA-A allele of low expression could cause a decrease in transcription factor binding sites and affect the expression sites which needs further research. This work was sponsored by Zhejiang Provincial Natural Science Foundation of China under Grant No.LGF22H080004 and Science Research Foundation of Zhejiang Healthy Bureau under Grant No. 2024KY941.