Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Instituto Murciano de Investigación Biosanitaria (IMIB-Arrixaca), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Universidad de Murcia, Murcia, Spain.
Department of Haematology, NHS Blood and Transplant Centre, University of Cambridge, Cambridge, United Kingdom.
Thromb Haemost. 2022 Aug;122(8):1369-1378. doi: 10.1055/s-0042-1749345. Epub 2022 Jun 28.
The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in are identified for up to 70% of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in . Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases with type I ATD proven by functional and antigen assays, who were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all eight SVs detected by multiple ligation-dependent probe amplification, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize, and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions.
遗传性抗凝血酶缺陷症（ATD）的鉴定对于预防潜在的致命性血栓事件至关重要。 已鉴定出 的致病变异占病例的 70%左右，其中大多数为单核苷酸变异和插入缺失。由于 中重复元件数量较多，因此检测和表征 ATD 的结构变异（SVs）仍然具有挑战性。在这里，我们对 10 个家族性和 9 个单发性 I 型 ATD 病例进行了长读长全基因组测序，这些病例通过功能和抗原测定得到证实，他们是从 340 名患有这种罕见疾病的患者队列中选择的，因为基因分析要么是阴性的，要么是模棱两可的，要么是不完全特征的。我们开发了一种分析工作流程来识别与疾病相关的 SVs。这种方法独立于其大小或类型，解决了通过多重连接依赖性探针扩增检测到的所有 8 个 SVs，并首次鉴定了以前被错误分类为缺失的复杂重排。值得注意的是，我们在 11 例以前未知缺陷的病例中的 2 例中确定了导致 ATD 的机制：插入了一个新的 2.4kb SINE-VNTR-Alu 反转录元件，该元件通过从头组装进行了表征，并在先证者和受影响的亲属中通过特异性聚合酶链反应扩增和测序进行了验证。所有 SVs 的核苷酸水平分辨率允许进行断点分析，这揭示了重复元件和微同源性，支持所有 SVs 基于共同复制的机制。我们的研究强调了长读测序技术作为一种补充方法的实用性，用于鉴定、表征和揭示涉及 ATD 的致病 SVs 的分子机制，并扩大了由反转录转座子插入引起的遗传疾病目录。
