应用二代测序技术排除PCR-SBT零错配的HLA-C基因型  

Exclusion of HLA-C Genotype with Zero Mismatched PCR-SBT Results by Next Generation Sequencing

作　　者：钟艳平[1] 陈浩 周丹[1] 邹红岩[1] ZHONG Yan-Ping;CHEN Hao;ZHOU Dan;ZOU Hong-Yan(Shenzhen Institute of Transfusion Medicine,Shenzhen Blood Center,Shenzhen 518020,Guangdong Province,China)

机构地区：[1]深圳市血液中心输血医学研究所,广东深圳518020

出　　处：《中国实验血液学杂志》2022年第4期1213-1218,共6页Journal of Experimental Hematology

基　　金：深圳市医学重点学科建设经费资助(SZXK070);深圳市科技计划项目(JCYJ20190806152001762);深圳市医疗卫生三名工程项目(SZSM201811092)。

摘　　要：目的:对3例PCR-SBT结果零错配的HLA-C罕见等位基因进行全长测序分析,以确定其真实基因型。方法:从临床移植配型样本中筛查出3例PCR-SBT结果零错配的HLA-C罕见等位基因,应用下一代测序技术检测序列全长以准确分型。结果:3例样本PCR-SBT分型结果分别为HLA-C*03:04,HLA-C*12:167;HLA-C*07:291,HLA-C*15:02;HLA-C*01:43,HLA-C*08:16;除HLA-C*03:04、HLA-C*15:02为HLA常见等位基因,其他等位基因均不在中国常见及确认HLA等位基因CWD表(2.3版本)中。NGS全长测序发现3例样本HLA-C基因型均为常见等位基因和新等位基因的组合,3个新等位基因分别在第6、2、4外显子存在一个碱基突变。新鉴定的等位基因序列已提交给Genbank数据库(MK629722、MK335474、MK641803),被WHO HLA命名委员会正式命名为HLA-C*03:04:74、HLA-C*15:192、HLA-C*08:01:25。3例样本的HLA高分辨分型结果应该为HLA-C*03:04:74,12:03;HLA-C*07:02,15:192;HLA-C*01:02,08:01:25。结论:HLA分型结果中含有罕见等位基因的零错配结果应谨慎对待,须通过NGS或克隆等方法对全长序列加以复核确认。本实验室通过加做NGS最终确认3例PCR-SBT零错配的HLA-C基因型均为常见等位基因和新等位基因的组合,为临床移植配型提供了准确依据,丰富了人类HLA遗传数据库。Objective:Three cases of rare alleles of HLA-C with zero mismatched PCR-SBT results were analyzed by full-length sequencing to determine the true genotypes.Methods:Three rare HLA-C alleles with zero mismatched PCRSBT results were screened from clinical transplant matching samples,and the full-length sequence was detected by nextgeneration sequencing technology.Results:The results of PCR-SBT typing of 3 samples were:HLA-C*03:04,12:167;HLA-C*07:291,15:02;HLA-C*01:43,08:16.Other alleles were not in the CWD table of common and confirmed HLA alleles in China(version 2.3)except common allele HLA-C*03:04,HLA-C*15:02.NGS full-length sequencing revealed that the HLA-C genotypes of the three samples were a combination of common alleles and novel alleles,and the three novel alleles had a base mutation in exons 6,2,and 4,respectively.The novel allele sequences have been submitted to the Genbank database(MK629722,MK335474,MK641803),which were officially named HLA-C*03:04:74,HLA-C*15:192,HLA-C*08:01:25 by the WHO HLA Nomenclature Committee.The HLA high-resolution typing results of 3 samples were:HLA-C*03:04:74,HLA-C*12:03;HLA-C*07:02,HLA-C*15:192;HLA-C*01:02,HLA-C*08:01:25.Conclusion:HLA typing results containing rare alleles should be treated cautiously,and the full-length sequence should be verified by NGS or cloning.The laboratory finally confirmed that the 3 cases of PCR-SBT zero mismatch HLA-C genotypes are the combination of common alleles and novel alleles by NGS sequencing,which provides an accurate basis for clinical transplantation matching and enriches the human HLA genetic database.

关 键 词：人类白细胞抗原 罕见等位基因 新等位基因 聚合酶链反应-测序分型法 二代测序技术 

分 类 号：R394[医药卫生—医学遗传学]