Wei Ling, Lopez Genghis H, Zhang Yang, Wen Jizhi, Wang Zhen, Fu Yongshui, Hyland Catherine A, Flower Robert L, Luo Guangping, Ji Yanli
Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, P.R. China.
Clinical Services and Research, Australian Red Cross Blood Service, Queensland, Australia.
Transfusion. 2018 Jul;58(7):1763-1771. doi: 10.1111/trf.14641. Epub 2018 Jun 13.
MNS hybrid GP(B-A-B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B-A-B) hybrid glycophorins based on high-resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population.
DNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B-A-B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation-dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles.
A total of five GYP(B-A-B) genotypes (201/3104, 6.5%) were identified, which were GYPMur heterozygote (n = 194), GYPMur homozygote (n = 3), GYPBun heterozygote (n = 2), GYPHF heterozygote (n = 1), and a novel GYP(B-A-B) hybrid allele (n = 1). Genotyping results for GYPMur and wild-type GYPB samples obtained by HRM were consistent with MLPA, while GYPBun and GYPHF heterozygote identified by HRM could only be identified to have one copy of 5' inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single-nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYPMur and GYP*Sch alleles.
The HRM assay could distinguish the GYP(B-A-B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B-A-B) genotyping kits for the Chinese population.
MNS杂合GP（B-A-B）血型糖蛋白在东南亚人群中更为常见，针对其携带抗原的同种抗体具有临床意义。由于缺乏商业试剂，通过血清学技术检测杂合糖蛋白受到限制。在本研究中，基于高分辨率熔解曲线分析（HRM）的GP（B-A-B）杂合糖蛋白基因分型方法被应用于中国南方汉族人群的基因分型分析。
收集了3104名中国南方汉族献血者的DNA样本。通过HRM分析对GYP（B-A-B）基因型进行分析。部分样本（n = 106）也通过多重连接依赖探针扩增（MLPA）分析进行检测。对熔解曲线图谱有变异的样本进行直接测序。
共鉴定出5种GYP（B-A-B）基因型（201/3104，6.5%），分别为GYPMur杂合子（n = 194）、GYPMur纯合子（n = 3）、GYPBun杂合子（n = 2）、GYPHF杂合子（n = 1）和一个新的GYP（B-A-B）杂合等位基因（n = 1）。通过HRM获得的GYPMur和野生型GYPB样本的基因分型结果与MLPA一致，而通过HRM鉴定出的GYPBun和GYPHF杂合子在MLPA中仅能鉴定出GYPB假外显子3的5'非活性剪接位点有一个拷贝。此外，在31个样本中鉴定出10个单核苷酸多态性（SNP），包括4个已知SNP和6个新SNP。一个样本被鉴定同时携带GYPMur和GYP*Sch等位基因。
HRM分析能够成功区分GYP（B-A-B）杂合等位基因。在为中国人群开发GYP（B-A-B）基因分型试剂盒时，应考虑GYPB基因内鉴定出的多态性。
