Table_3_Rapid Low-Cost Microarray-Based Genotyping for Genetic Screening in Primary Immunodeficiency.pdf

Background: Genetic tests for primary immunodeficiency disorders (PIDs) are expensive, time-consuming, and not easily accessible in developing countries. Therefore, we studied the feasibility of a customized single nucleotide variant (SNV) microarray that we developed to detect disease-causing variants and copy number variation (CNV) in patients with PIDs for only 40 Euros.Methods: Probes were custom-designed to genotype 9,415 variants of 277 PID-related genes, and were added to the genome-wide Illumina Global Screening Array (GSA). Data analysis of GSA was performed using Illumina GenomeStudio 2.0, Biodiscovery Nexus 10.0, and R-3.4.4 software. Validation of genotype calling was performed by comparing the GSA with whole-genome sequencing (WGS) data of 56 non-PID controls. DNA samples of 95 clinically diagnosed PID patients, of which 60 patients (63%) had a genetically established diagnosis (by Next-Generation Sequencing (NGS) PID panels or Sanger sequencing), were analyzed to test the performance of the GSA. The additional SNVs detected by GSA were validated by Sanger sequencing.Results: Genotype calling of the customized array had an accuracy rate of 99.7%. The sensitivity for detecting rare PID variants was high (87%). The single sample replication in two runs was high (94.9%). The customized GSA was able to generate a genetic diagnosis in 37 out of 95 patients (39%). These 37 patients included 29 patients in whom the genetic variants were confirmed by conventional methods (26 patients by SNV and 3 by CNV analysis), while in 8 patients a new genetic diagnosis was established (6 patients by SNV and 2 patients suspected for leukemia by CNV analysis). Twenty-eight patients could not be detected due to the limited coverage of the custom probes. However, the diagnostic yield can potentially be increased when newly updated variants are added.Conclusion: Our robust customized GSA seems to be a promising first-line rapid screening tool for PIDs at an affordable price, which opens opportunities for low-cost genetic testing in developing countries. The technique is scalable, allows numerous new genetic variants to be added, and offers the potential for genetic testing not only in PIDs, but also in many other genetic diseases.

背景：原发性免疫缺陷病（PIDs）的基因检测成本高昂、耗时且在发展中国家难以获得。因此，本研究探讨了我们所开发的定制单核苷酸多态性（SNV）微阵列的可行性，该微阵列旨在仅用40欧元检测PIDs患者的致病性变异和拷贝数变异（CNV）。方法：探针被定制设计以对277个与PIDs相关的基因的9,415个变异进行基因分型，并添加到全基因组Illumina全球筛查阵列（GSA）中。使用Illumina GenomeStudio 2.0、Biodiscovery Nexus 10.0和R-3.4.4软件对GSA进行数据分析。通过将GSA与56例非PIDs受试者的全基因组测序（WGS）数据进行比较，对基因分型进行验证。对95例临床诊断的PIDs患者的DNA样本进行分析，其中60例（63%）患者具有通过下一代测序（NGS）PID组或Sanger测序建立的遗传诊断（其中26例通过SNV分析，3例通过CNV分析），以测试GSA的性能。通过Sanger测序验证了GSA检测到的额外SNVs。结果：定制阵列的基因分型准确率达到99.7%。检测罕见PIDs变异的灵敏度较高（87%）。两次运行的单样本重复性较高（94.9%）。在95例患者中，定制GSA能够对37例（39%）进行遗传诊断。这37例患者中，29例患者的遗传变异通过传统方法得到证实（26例通过SNV分析，3例通过CNV分析），而在8例患者中建立了新的遗传诊断（6例通过SNV分析，2例因CNV分析怀疑为白血病）。由于定制探针的覆盖范围有限，28例患者无法检测。然而，当添加新的更新变异时，诊断产量有望增加。结论：我们开发的稳健的定制GSA似乎是一种有前景的、经济实惠的PIDs一线快速筛查工具，为发展中国家低成本遗传检测提供了机遇。该技术可扩展，允许添加大量新的遗传变异，并具有在PIDs以及许多其他遗传疾病中进行遗传检测的潜力。