Background Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. Methods Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. Results We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). Conclusions A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

中文翻译：

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通过靶向捕获下一代测序检测慢性淋巴细胞白血病 IGHV 体细胞高突变

背景免疫球蛋白重变（IGHV）基因的体细胞高突变（SHM）状态在确定慢性淋巴细胞白血病（CLL）患者的预后和治疗中起着至关重要的作用。确定 SHM 状态的常用方法是多重聚合酶链反应和免疫球蛋白重基因座的桑格测序；然而，该技术通量低，容易失败，并且不允许与其他诊断测定进行多重分析。方法在这里，我们设计并验证了一种 DNA 靶向捕获方法，用于检测免疫球蛋白重变异体细胞超突变 (IGHV SHM) 状态，作为更大的下一代测序 (NGS) 面板的子模块，该面板还包括 ATM、BIRC3、CHD2、KLHL6、 MYD88、NOTCH1、NOTCH2、POT1、SF3B1、TP53 和 XPO1。该检测以 FASTQ 文件作为输入，并按照欧洲 CLL 指南研究计划输出包含 IGHV SHM 状态和 V 等位基因使用情况的报告。结果 我们在 35 个 CLL 患者样本上验证了该方法，其中 34 个样本使用 Sanger 测序进行了表征。NGS 小组确定了 35 名 CLL 患者中 34 名的 IGHV SHM 状态。我们通过 NGS 和 Sanger 测序调用显示了 33 个 CLL 样本的 100% 敏感性和特异性。此外，我们证明该面板可以与其他靶向捕获面板相结合，以检测预后重要的 CLL 单核苷酸变异、插入/缺失和拷贝数变异（TP53 拷贝数丢失）。结论 IGHV SHM 检测的靶向捕获方法可以集成到更广泛的测序面板中，从而可以在单分子检测中进行广泛的 CLL 预测。