AUTHOR=Suratannon Narissara , van Wijck Rogier T. A. , Broer Linda , Xue Laixi , van Meurs Joyce B. J. , Barendregt Barbara H. , van der Burg Mirjam , Dik Willem A. , Chatchatee Pantipa , Langerak Anton W. , Swagemakers Sigrid M. A. , Goos Jacqueline A. C. , Mathijssen Irene M. J. , Dalm Virgil A. S. H. , Suphapeetiporn Kanya , Heezen Kim C. , Drabwell Jose , Uitterlinden André G. , van der Spek Peter J. , van Hagen P. Martin ,  The South East Asia Primary Immunodeficiencies (SEAPID) Consortium 

TITLE=Rapid Low-Cost Microarray-Based Genotyping for Genetic Screening in Primary Immunodeficiency

JOURNAL=Frontiers in Immunology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.00614

DOI=10.3389/fimmu.2020.00614

ISSN=1664-3224

ABSTRACT=<p><bold>Background:</bold> 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.</p><p><bold>Methods:</bold> 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.</p><p><bold>Results:</bold> 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.</p><p><bold>Conclusion:</bold> 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.</p>