背景与目的: 人类白细胞抗原 (Human leukocyte antigen，HLA) 基因是人类免疫系统的重要组成部分，HLA参与抗原呈递和免疫应答并与多种自身免疫疾病和传染病的易感性相关。HLA基因的多态性与表达水平也影响着疾病的发生和发展，但目前对于HLA基因群体的表达水平及表达调控研究并不充分，仍有意义对HLA基因的表达调控进行进一步挖掘。本研究的目的是基于中国不同民族永生细胞库遗传多样性资源对HLA基因进行高分辨率分型，HLA基因的表达数量性状位点（Expression Quantitative Trait Loci, eQTL）分析以及HLA基因的表达水平及调控探究，从而深入理解影响HLA基因表达的分子机制。

方法：在本研究中，我们利用全基因组测序（Whole Genome Sequencing, WGS），RNA-seq（RNA sequencing）数据，对89个来自于中国不同民族永生细胞库中的永生化B淋巴细胞分别进行了HLA基因分型、HLA基因表达定量以及eQTL定位分析。其中采用生物信息学方法对89例样本的全基因组测序结果进行了HLA-Ⅰ，Ⅱ类经典基因的等位基因分型；结合89例样本的全基因测序数据和转录组测序数据进行了HLA的基因表达定量以及eQTL分析。根据不同HLA基因的eQTL分析结果，进行了功能注释，利用多种数据库对重要的顺式表达数量性状位点（cis-eQTLs）进行鉴定。对效应最强的区域进行进一步注释探究，对位于基因HLA-H启动子区域的阳性位点进行验证，采用双荧光素酶报告基因方法对包含不同单核苷酸多态性（Single Nucleotide Polymorphism, SNP）的启动子活性生物学功能探索，探究其调控HLA基因表达水平的作用机制。

结果：HLA 基因分型结果：89例少数民族样本HLA分型共检出26种HLA-A等位基因，51种HLA-B等位基因，20种HLA-C等位基因，4种HLA-DPA1等位基因，25种HLA-DPB1等位基因，15种HLA-DQA1等位基因，16种HLA-DQB1等位基因，26种HLA-DRB1等位基因。HLA基因表达定量结果：HLA-B是总体上表达量最高的基因，HLA-A和HLA-DRA次之。其中不同HLA等位基因的表达水平也存在差异。eQTL分析结果：eQTL分析和可视化的结果提示，大多数HLA基因的表达数量性状位点均位于距离其转录起始位点50kb以内，可能处于调控对应基因表达的调控元件上。其中，HLA-H基因的信号最显著，且显著区域包含了HLA-H基因启动子。HLA-H的表达水平在群体中存在明显的差异，基因分型为HLA-H*02:07的个体表达量明显高于其他非HLA-H*02:07的个体，两组不同的HLA-H基因的表达水平的个体在HLA-H基因启动子阳性区域存在两个不同的单倍型。双荧光素酶报告基因结果表明包含不同单倍型的启动子活性存在显著差异，提示该区域上的SNPs可能为调控HLA-H基因表达的因素之一。预测了HLA-H 基因启动子区域存在可与四种不同转录因子（ZNF354A，ZNF320，ZNF675 以及 PRDM9）潜在结合的位点。

结论：本研究进一步揭示了HLA等位基因的高度多样性，进一步证实了不同人群中HLA基因的表达水平有差异。HLA基因的表达调控研究结果显示大多数HLA基因的表达数量性状位点位于其转录起始位点50kb以内，HLA-H基因启动子区域的信号最为显著。此外，我们证实了HLA-H的不同SNP类型的启动子活性存在显著差异，这些差异可能是调控HLA-H基因表达的重要因素。同时预测到了相应的转录因子结合位点，转录因子也可能为调控 HLA-H 基因表达的因素之一。初步解析了HLA-H基因的表达调控机制，为进一步理解HLA基因在人类健康和疾病中的作用为未来的研究提供了重要的基础。

Background and Purpose: Human leukocyte antigen (HLA) genes are a crucial component of the human immune system, involved in antigen presentation and immune response, and are associated with susceptibility to various autoimmune diseases and infectious diseases. The polymorphism and expression levels of HLA genes also affect the occurrence and development of diseases. However, current research on the expression levels and regulatory mechanisms of HLA genes in populations is not sufficient, warranting further exploration. This study aims to perform high-resolution typing of HLA genes based on the genetic diversity resources of immortalized cell lines from different ethnic groups in China, analyze the Expression Quantitative Trait Loci (eQTL) of HLA genes, and investigate the expression levels and regulatory mechanisms of HLA genes to understand the molecular mechanisms affecting HLA gene expression more deeply.

Methods: In this study, we utilized Whole Genome Sequencing (WGS) and RNA sequencing (RNA-seq) data to perform HLA gene typing, quantitative expression analysis, and eQTL mapping on immortalized B lymphocytes from 89 individuals representing different ethnic groups in China. Bioinformatics methods were used to perform allele typing of classical HLA-I and II genes from whole-genome sequencing results of 89 samples; combined with transcriptome sequencing data for quantitative gene expression analysis and eQTL analysis of HLA. Functional annotations were conducted based on eQTL analysis results, and important cis-eQTLs were identified using various databases. The most significant regions were further annotated, and positive loci in the promoter region of the HLA-H gene were validated. The biological function of promoter activity containing different Single Nucleotide Polymorphisms (SNPs) was explored using a dual-luciferase reporter gene method to investigate the regulatory mechanisms affecting HLA gene expression levels.

Results: HLA gene typing results revealed 26 HLA-A alleles, 51 HLA-B alleles, 20 HLA-C alleles, 4 HLA-DPA1 alleles, 25 HLA-DPB1 alleles, 15 HLA-DQA1 alleles, 16 HLA-DQB1 alleles, and 26 HLA-DRB1 alleles among the 89 minority samples. HLA-B had the highest overall expression, followed by HLA-A and HLA-DRA, with varying expression levels among different HLA alleles. eQTL analysis indicated that most HLA gene expression quantitative trait loci are located within 50kb of their transcription start sites, likely on regulatory elements controlling gene expression. The HLA-H gene showed the most significant signal, including its promoter region. There were noticeable differences in HLA-H expression levels across the population, with individuals carrying the HLA-H*02:07 genotype showing significantly higher expression than those without. Two different haplotypes were identified in the positive promoter region of the HLA-H gene between groups with different expression levels. Dual-luciferase reporter gene results suggested significant differences in promoter activity between different haplotypes, indicating that SNPs in this region might be one of the factors regulating HLA-H gene expression. Potential binding sites for four different transcription factors (ZNF354A, ZNF320, ZNF675, and PRDM9) were predicted in the HLA-H gene promoter region.

Conclusion: This study further reveals the high diversity of HLA alleles and confirms the differential expression levels of HLA genes across different populations. The expression regulation study of HLA genes showed that most eQTLs are located within 50kb of the transcription start sites, with the HLA-H gene promoter region showing the most significant signal. Additionally, we confirmed significant differences in promoter activity among different SNP types of HLA-H, which may be crucial factors regulating HLA-H gene expression. The corresponding transcription factor binding sites were also predicted, which may also play a role in regulating HLA-H gene expression. This preliminary analysis of the expression regulatory mechanism of the HLA-H gene provides an important foundation for further understanding the role of HLA genes in human health and disease.