背景:由于涉及大量基因及其分子特征,先天性免疫错误(IEI)的遗传诊断很复杂。据报道,错义变体是IEI的最常见原因。然而,拷贝数变异(CNV)的频率可能被低估,因为它们的检测需要特定的定量技术.在这一点上,下一代测序(NGS)的使用正在获得相关性。
方法:在本文中,我们基于允许检测单核苷酸变体(SNV)和CNV的三种诊断算法,介绍了我们在IEI基因诊断方面的经验.按照这个近似,在2014年至2021年之间评估了703个指标案例。桑格测序,MLPA,CGH阵列,进行了跨断点PCR或定制的基于NGS的多基因靶向组.
结果:在703例指标病例中,142例(20%)达到了基因诊断,其中19个将缺失呈现为因果变体。在家庭研究中,还在5名受影响的亲属和16名健康携带者中检测到缺失。此外,我们编译,表征并呈现我们的诊断算法检测到的所有CNV,代表迄今为止与IEI相关的最大删除队列。此外,三个生物信息学工具(LACONv,XHMM,VarSeq™)基于NGS数据进行评估。VarSeq™是最敏感和特异性的生物信息学工具;检测位于捕获区域中的21/23(91%)缺失。
结论:根据我们的结果,我们提出了一种指导分子诊断的策略,可以由IEI领域的专家和非专家中心遵循。
BACKGROUND: Genetic diagnosis of inborn errors of immunity (IEI) is complex due to the large number of genes involved and their molecular features. Missense variants have been reported as the most common cause of IEI. However, the frequency of copy number variants (CNVs) may be underestimated since their detection requires specific quantitative techniques. At this point, the use of Next Generation Sequencing (NGS) is acquiring relevance.
METHODS: In this article, we present our experience in the genetic diagnosis of IEI based on three diagnostic algorithms that allowed the detection of single nucleotide variants (SNVs) and CNVs. Following this approximation, 703 index cases were evaluated between 2014 and 2021. Sanger sequencing, MLPA, CGH array, breakpoint spanning PCR or a customized NGS-based multigene-targeted panel were performed.
RESULTS: A genetic diagnosis was reached in 142 of the 703 index cases (20%), 19 of them presented deletions as causal variants. Deletions were also detected in 5 affected relatives and 16 healthy carriers during the family studies. Additionally, we compile, characterize and present all the CNVs detected by our diagnostic algorithms, representing the largest cohort of deletions related to IEI to date. Furthermore, three bioinformatic tools (LACONv, XHMM, VarSeq™) based on NGS data were evaluated. VarSeq™ was the most sensitive and specific bioinformatic tool; detecting 21/23 (91%) deletions located in captured regions.
CONCLUSIONS: Based on our results, we propose a strategy to guide the molecular diagnosis that can be followed by expert and non-expert centres in the field of IEI.