基于长程定量 PCR 和长序列测序的血友病靶向分析的临床验证和应用：来自中国一家血友病治疗中心的经验。

IF 5.5 2区医学 Q1 HEMATOLOGY

Journal of Thrombosis and Haemostasis Pub Date : 2024-12-01 DOI:10.1016/j.jtha.2024.08.013

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

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引用次数: 0

摘要

背景靶向长读测序（LRS）有望全面分析分别由 F8 和 F9 基因引起的血友病 A（HA）和血友病 B（HB）的各种复杂变异。方法对检索对象的基因变异进行回顾性和前瞻性分析。为进一步分析未确诊病例，还进行了全基因组测序（WGS）。利用长程 PCR 和长程 PCR 结合测序技术绘制并验证了新型基因组重排的断点。在对 90 例 HA 病例的回顾性分析中，与常规方法相比，HA-LRS 检测法在 84 例病例中显示出一致的结果，并在 6 例病例中通过进一步验证确认了 6 个大缺失的特征及其确切断点（常规方法仅在扩增涉及的外显子时出现失败）。在对 21 名 HA 受试者的前瞻性分析中，在 20 个病例中发现了 20 个 F8 变异。其余一名 HA 患者没有发现重复/缺失或 SNV/InDel，但通过 LRS 观察到了涉及 F8 第 14 和 21 号外显子的潜在重组。WGS 分析和进一步验证确定了涉及 F8 第 14-21 号外显子的 30,478bp 串联重复。结论基于 LRS 的靶向分析能高效、全面地鉴定血友病基因的 SNVs/InDels 和基因组重排，尤其是我们首次扩大了包括 F9 基因在内的面板。然而，进一步研究复杂的总重排仍是必要的。

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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

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 repeat involving exons 14 to 21 of F8. Among the 11 HB patients, HB-LRS analysis detected 11 SNVs/InDels in F9, consistent with routine methods.

Conclusion

Targeted LRS-based analysis was efficient and comprehensive in identifying SNVs/InDels and genomic rearrangements of hemophilia genes, especially when we first expanded the panel to include F9. However, further investigation for complex gross rearrangement is still essential.

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来源期刊

Journal of Thrombosis and Haemostasis 医学-外周血管病

CiteScore

24.30

自引率

3.80%

发文量

321

审稿时长

1 months

期刊介绍： The Journal of Thrombosis and Haemostasis (JTH) serves as the official journal of the International Society on Thrombosis and Haemostasis. It is dedicated to advancing science related to thrombosis, bleeding disorders, and vascular biology through the dissemination and exchange of information and ideas within the global research community. Types of Publications: The journal publishes a variety of content, including: Original research reports State-of-the-art reviews Brief reports Case reports Invited commentaries on publications in the Journal Forum articles Correspondence Announcements Scope of Contributions: Editors invite contributions from both fundamental and clinical domains. These include: Basic manuscripts on blood coagulation and fibrinolysis Studies on proteins and reactions related to thrombosis and haemostasis Research on blood platelets and their interactions with other biological systems, such as the vessel wall, blood cells, and invading organisms Clinical manuscripts covering various topics including venous thrombosis, arterial disease, hemophilia, bleeding disorders, and platelet diseases Clinical manuscripts may encompass etiology, diagnostics, prognosis, prevention, and treatment strategies.