运用二代测序技术确认PCR-SSOP法检出的HLA罕见等位基因

Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP

目的:确认PCR-序列特异性寡核苷酸探针(SSOP)法检出的4例磁珠探针HLA基因分型结果格局异常和3例模棱两可结果样本的HLA真实基因型。方法:对HLA高分辨组织配型血样采用PCR-SSOP法检出的4例磁珠探针HLA基因分型结果格局异常和3例模棱两可结果进一步采用PCR-直接测序分型(SBT)技术和二代测序(NGS)技术复检确认。结果:采用PCR-SSOP方法检出4例磁珠探针HLA基因分型结果格局异常的样本,加做SBT及NGS两种方法复检后结果显示,样本1的HLA-A分型结果与已知基因型不完全匹配,NGS分析显示,该等位基因与同源性最接近的等位基因A*31:01:02:01在第2外显子154位G>A变异,导致28位编码氨基酸由缬氨酸变为蛋氨酸(p.Val28Met),样本2的HLA-C结果为C*03:119, 06:02,样本3的HLA-C结果为C*03:03, 07:137,样本4的HLA-B结果为B*55:02,55:12。采用PCR-SSOP方法检出3例模棱两可结果样本,加做SBT及NGS两种方法复检后结果显示,样本5的HLA-B结果为B*15:58, 38:02,样本6的HLA-DRB1结果为DRB1*04:05, 14:101,样本7的HLA-DQB1结果为DQB1*03:34,05:02。其中等位基因C*03:119、C*07:137和DRB1*14:101均未收录于中国造血干细胞捐献者资料库的常见及确认等位基因(CWD)表2.4版本。结论:PCR-SSOP法磁珠探针HLA基因分型结果格局异常提示可能为HLA罕见等位基因或新等位基因,需要测序验证。

Objective: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.Methods: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.Results: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 HLA-A genotype of sample 1 did not match any known genotypes.NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01 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 HLA-C genotype of sample 2 was C*03:119,06:02,sample3 was C*03:03,07:137,and sample 4 was B*55:02,55:12.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 HLA-B genotype of sample5 was B*15:58,38:02,sample 6 was DRB1*04:05,14:101,and sample 7 was DQB1*03:34,05:02.Among them,alleles C*03:119,C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database.Conclusion: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.