Clinical validation and application of targeted long-range polymerase chain reaction and long-read sequencing–based analysis for hemophilia: experience from a hemophilia treatment center in China

作者：Meizhen Shi, Yunting Ma, Xianwei Peng, Xu Zhou, Zifeng Cheng, Bobo Xie, Xianda Wei, Chunrong Gui, Aiping Mao, Wenting Lin, Jiefeng Luo, Yinghui Lai, Baoheng Gui · 发表于：Journal of Thrombosis and Haemostasis · 年份：2024 · DOI：10.1016/j.jtha.2024.08.013 · 被引用次数：6 · 研究领域：Hemophilia Treatment and Research、Platelet Disorders and Treatments、Coagulation, Bradykinin, Polyphosphates, and Angioedema

BACKGROUND: Targeted long-read sequencing (LRS) is expected to comprehensively analyze diverse complex variants in hemophilia A (HA) and hemophilia B (HB) caused by the F8 and F9 genes, respectively. However, its clinical applicability still requires extensive validation. OBJECTIVES: To evaluate the clinical applicability of targeted LRS-based analysis compared with routine polymerase chain reaction (PCR)-based methods. METHODS: Gene variants of retrieved subjects were retrospectively and prospectively analyzed. Whole-genome sequencing was performed to further analyze undiagnosed cases. Breakpoints of novel genomic rearrangements were mapped and validated using long-distance PCR and long-range PCR combined with sequencing. RESULTS: In total, 122 subjects were retrieved. In retrospective analysis of the 90 HA cases, HA-LRS assay showed consistent results in 84 cases compared with routine methods and characterized 6 large deletions with their exact breakpoints confirmed by further validation in 6 cases (routine methods only presented failure in amplifying the involved exons). In prospective analysis of the 21 HA subjects, 20 variants of F8 were identified in 20 cases. For the remaining HA patient, no duplication/deletion or single-nucleotide variant (SNV)/insertion and deletion (InDel) was found, but a potential recombination involving exons 14 and 21 of F8 was observed by LRS. Whole-genome sequencing analysis and further verification defined a 30 478 base pairs (bp) tandem rep...