Diagnostics of Primary Immunodeficiency Diseases: A Sequencing Capture Approach

Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of “next generation” sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.

原发性免疫缺陷病（Primary Immunodeficiencies, PID）是一类遗传性免疫系统缺陷疾病，患者感染风险显著升高。由于其临床表型多样且现有诊断流程复杂，日常临床实践中对原发性免疫缺陷病作出精准诊断往往颇具挑战。得益于“下一代”测序技术与靶标富集技术的问世，多重诊断检测方法的开发现已具备可行性。本研究采用基于筛选富集的靶向检测方法，对179个已知原发性免疫缺陷病相关致病基因中的致病变异进行检测。我们通过对33名患者的DNA进行测序，评估了该检测方法用于原发性免疫缺陷病分子诊断的应用价值；其中18名患者在实验启动前已被确认携带至少一种已知致病突变。本研究成功在60%的受试患者样本中明确了致病突变，这表明靶向测序策略可解决绝大多数原发性免疫缺陷病的诊断难题。在未明确病因的患者样本中检出的致病突变，分别位于STAT3、IGLL1、RNF168及PGM3基因中。基于本研究结果，我们提出一套原发性免疫缺陷病诊断的分步方案：先开展靶向重测序，若靶向外显子区域未检出致病变异，则进一步辅以全转录组测序或全基因组测序。