Background: Exposure to non-matching human platelet alloantigens (HPA) may result in alloimmunization. Antibodies to HPA can be responsible for post-transfusion purpura, refractoriness to donor platelets, and fetal and neonatal alloimmune thrombocytopenia. For the supply of compatible apheresis platelet concentrates, the HPA genotypes are determined in a routine manner. Methods: Here, we describe a novel method for genotyping twelve different HPA systems simultaneously, including HPA-1 to HPA-5, HPA-9w, HPA-10w, HPA-16w, HPA-19w, HPA-27w, and the novel HPA-34w by means of amplicon-based next-generation sequencing (NGS). Blood donor samples of 757 individuals with a migration background and 547 of Western European ancestry were genotyped in a mass-screening setup. An in-house software was developed for fast and automatic analysis. TaqMan assay and Sanger sequencing results served for validation of the NGS workflow. Finally, blood donors were divided in several groups based on their country of origin and the allele frequencies were compared. Results: For 1,299 of 1,304 samples (99.6%) NGS was successfully performed. The concordance with TaqMan assay and Sanger sequencing results was 99.8%. Allele-calling dropouts that were observed for two samples with the TaqMan assay caused by rare single nucleotide polymorphisms were resolved by NGS. Additionally, twenty rare and two novel variants in the coding regions of the genes ITGB3, GPB1A, ITGBA2, and CD109 were detected. The determined allele frequencies were similar to those published in the gnomAD database. Conclusions: No significant differences were observed in the distribution of allele frequencies of HPA-1 through HPA-5 and HPA-15 throughout the analyzed groups except for a lower allele frequency for the HPA-1b allele in the group of donors with Southern Asian ancestry. In contrast, other nucleotide variants that have not yet been phenotypically characterized occurred three times more often in blood donors with a migration background. High-throughput amplicon-based NGS is a reliable method for screening HPA genotypes in a large sample cohort simultaneously. It is easily upgradeable for genotyping additional targets without changing the setup or the analysis pipeline. Mass-screening methods will help building up blood donor registries to provide matched blood products.

中文翻译：

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使用下一代测序对 12 种人类血小板抗原系统的献血者进行高通量筛选，揭示了罕见多态性和两种新型蛋白改变变异体的检测

背景：暴露于不匹配的人类血小板同种异体抗原 (HPA) 可能会导致同种异体免疫。HPA 抗体可导致输血后紫癜、供体血小板不耐受以及胎儿和新生儿同种免疫性血小板减少症。对于相容的单采血小板浓缩物的供应，HPA 基因型以常规方式确定。方法：在这里，我们描述了一种同时对 12 种不同 HPA 系统进行基因分型的新方法，包括 HPA-1 至 HPA-5、HPA-9w、HPA-10w、HPA-16w、HPA-19w、HPA-27w 和新型 HPA -34w 通过基于扩增子的下一代测序 (NGS)。在大规模筛选设置中对 757 名具有迁移背景和 547 名西欧血统的个体的献血者样本进行了基因分型。开发了一种内部软件，用于快速和自动分析。TaqMan 检测和 Sanger 测序结果用于验证 NGS 工作流程。最后，献血者根据其原籍国分为几组，并比较等位基因频率。结果：1,304 个样本中的 1,299 个 (99.6%) 成功进行了 NGS。与 TaqMan 测定和 Sanger 测序结果的一致性为 99.8%。由罕见的单核苷酸多态性引起的 TaqMan 分析中观察到的两个样本的等位基因调用丢失由 NGS 解决。此外，在基因 ITGB3、GPB1A、ITGBA2 和 CD109 的编码区中检测到 20 个罕见变异和 2 个新变异。确定的等位基因频率与 gnomAD 数据库中公布的相似。结论：除了具有南亚血统的供体组中 HPA-1b 等位基因的等位基因频率较低外，在整个分析组中，HPA-1 到 HPA-5 和 HPA-15 的等位基因频率分布没有观察到显着差异。相比之下，其他尚未进行表型表征的核苷酸变体在具有迁移背景的献血者中发生的频率要高出三倍。基于高通量扩增子的 NGS 是同时在大样本队列中筛选 HPA 基因型的可靠方法。它可以轻松升级以对其他目标进行基因分型，而无需更改设置或分析流程。大规模筛查方法将有助于建立献血者登记处以提供匹配的血液制品。