Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis. Targeted NGS panel for autoinflammation and vasculitis

Background: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the “Vasculitis and Inflammation Panel” (VIP) for AID and vasculitis. Methods: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. Results: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%).Conclusions: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID.

背景：单基因自身炎症性疾病（Monogenic autoinflammatory diseases, AID）是一类快速增长、遗传异质性显著但表型相互重叠的全身性炎症性疾病，与先天免疫失调密切相关。该类疾病可造成严重的发病负担、死亡风险及经济损失。本研究旨在开发并评估一款针对AID与血管炎的二代测序（Next Generation Sequencing, NGS）靶向基因panel——“血管炎与炎症检测panel（Vasculitis and Inflammation Panel, VIP）”的临床应用价值。
方法：采用安捷伦（Agilent）SureDesign工具设计了两款VIP检测panel：VIP1靶向113个基因，后续升级版VIP2靶向166个基因。使用QXT靶向富集系统（QXT Target Enrichment System）构建捕获并完成索引的文库，对72例患者的样本完成文库制备后，以16个样本为一个多重测序组，在Illumina MiSeq测序仪上以150bp双端测序模式进行测序。本研究队列包含22例携带已验证致病突变的阳性对照DNA样本，以及50例疑似AID患者的前瞻性样本——这些患者此前基于Sanger测序（Sanger sequencing）的基因筛查未获得明确诊断结果。
结果：VIP对22例阳性对照样本中各类已知突变的检测均具备良好的灵敏度与特异性，涵盖基因缺失、小片段插入缺失（insertions-deletions, INDELs）以及等位基因分数低至3%的体细胞嵌合突变。在50例未分类AID患者中，6例（12%）携带至少1个5类（明确致病性）变异，11例（22%）携带至少1个可能致病性变异（4类）。总体而言，VIP检测使50例患者中的16例（32%）获得了明确或高度疑似的分子诊断。
结论：这款全面的靶向基因检测panel具备较高的诊断效能与准确性，证实了针对疑似AID患者采用广谱NGS检测的临床应用价值。