Objective: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system.
Methods: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods.
Results: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System.
Conclusion: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.
题目: 基于二代测序的HLA-II类等位基因多态性研究及等位基因丢失防范策略.
目的: 研究二代测序技术（NGS）检测深圳地区随机健康无关汉族人群HLA-DRB1、DQB1、DQA1、DRB3、DRB4、DRB5、DPA1、DPB1等位基因多态性的精确性，探讨HLA-DRB1等位基因丢失的原因及室内关键质控体系建立策略。.
方法: 采用MiSeqDxTM NGS平台对1 012例样本完成HLA-II类等位基因分型。对质控体系软件提示的疑难样本和HLA-DRB1纯合子样本采用PCR-SSOP法或PCR-SBT法进行确认。.
结果: 检出HLA-DRB1、DRB3、DRB4、DRB5、DQA1、DQB1、DPA1、DPB1等位基因分别有45、7、5、7、17、21、10、27种。常见等位基因（频率>10%）有HLA-DRB1*09:01（17.09%）、15:01（10.72%）；DRB3*02:02（25.99%）、03:01（10.18%）；DRB4*01:03（36.46%）；DRB5*01:01（15.42%）；DQA1*01:02（20.01%）、03:02（17.19%）；DQB1*03:01（19.47%）、03:03（17.98%）、05:02（11.66%）、06:01（10.67%）；DPA1*02:02（54.45%）、01:03（31.18%）；DPB1*05:01（39.13%）、02:01（16.90%）。HLA-DRB1和DQB1位点基因频率与中国常见及确认的HLA等位基因表（CWD2.4）进行统计学比较,差异无统计学意义（χ2=12.68，P >0.05)。对NGS检出的94例HLA-DRB1纯合子样本采用PCR-SSOP法进行复检，检出HLA-DRB1位点漏检等位基因1例，通过SBT法确认为漏检DRB1*04:03等位基因，为此建立了本实验室室内质控体系。检出新等位基因2例，获WHO HLA系统因素命名委员会命名。.
结论: 基于NGS-HLA分型方案的HLA分型结果，模棱两可结果比率更低。HLA-II 类等位基因在深圳地区无关健康供者汉族人群中存在遗传多态性。在临床组织相容性试验中独立使用NGS方法存在局限性，需要内部质量控制策略来防范偶发的等位基因丢失事件。.
Keywords: human leukocyte antigen; allele frequency; next-generation sequencing; allele dropout.