Zhong Xian-Xin, Wu Wang-Da, Quan Zhan-Rou, Gao Su-Qing
Department of Clinical Laboratory Examination, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, Guangdong Province, China.
Institute of Transfusion Medicine of Shenzhen Blood Center, Shenzhen 518020, Guangdong Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2023 Feb;31(1):203-208. doi: 10.19746/j.cnki.issn.1009-2137.2023.01.032.
To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method.
All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology.
A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The genotype of sample 2 was , sample 3 was , and sample 4 was . A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the genotype of sample 5 was , sample 6 was , and sample 7 was . Among them, alleles and were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database.
The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.
对包括4例磁珠探针HLA基因分型结果模式异常及3例采用聚合酶链反应-序列特异性寡核苷酸探针(SSOP)法检测结果不明确的样本进行HLA基因分型确认。
所有来源于HLA高分辨率分型实验室的样本均采用PCR-SSOP法进行检测。对4例磁珠探针HLA基因分型结果模式异常样本及3例结果不明确样本进一步采用基于聚合酶链反应的序列分型(SBT)技术和二代测序(NGS)技术进行确认。
采用PCR-SSOP法检测出4例磁珠探针HLA基因分型结果模式异常样本。SBT和NGS结果显示,样本1的基因型与任何已知基因型均不匹配。NGS分析显示,该新等位基因与最接近匹配的等位基因在第2外显子154位存在G>A差异,导致第28密码子处一个氨基酸由缬氨酸替换为甲硫氨酸(p.Val28Met)。样本2的基因型为 ,样本3的基因型为 ,样本4的基因型为 。采用PCR-SSOP法初步检测出3例结果不明确样本。SBT和NGS复检结果显示,样本5的基因型为 ,样本6的基因型为 ,样本7的基因型为 。其中,等位基因 和 未包含在中国造血干细胞捐献者数据库的常见及充分记录等位基因(CWD)v2.4中。
PCR-SSOP法检测磁珠探针HLA基因分型结果出现异常模式,提示可能为罕见等位基因或新等位基因,需通过测序进行验证。