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

Narissara Suratannon,Kim C Heezen,Peter J Van Der Spek,Anton W Langerak,Pantipa Chatchatee,Virgil A S H Dalm,Jose Drabwell,Joyce B J Van Meurs,Linda Broer,André G Uitterlinden,Laixi Xue,Kanya Suphapeetiporn,Barbara H Barendregt,Mirjam Van Der Burg,P Martin Van Hagen,Rogier T A Van Wijck,Willem A Dik,Irene M J Mathijssen ,Sigrid Swagemakers ,Jacqueline A.c Goos

doi:10.3389/fimmu.2020.00614

Abstract

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.

Highlights

Primary immunodeficiency disorders (PIDs) are a heterogeneous group of diseases, including more than 400 distinct monogenic inherited disorders, that affect the development and function of the immune system (1–3)
Assessment of the Reproducibility To assess the reproducibility of the array, we focused only on the custom content of the Global Screening Array (GSA) and compared the post-quality control (QC) data for the 41 overlapping PID samples in the two array runs
Of the 9,415 custom variants added to the standard GSA v1 content, 8,883 and 8,852 variants were included in the post-QC data of the custom content for the first and second runs, respectively

Summary

Introduction

Primary immunodeficiency disorders (PIDs) are a heterogeneous group of diseases, including more than 400 distinct monogenic inherited disorders, that affect the development and function of the immune system (1–3). Current genetic diagnostic approaches for PIDs are based on Sanger sequencing, next-generation sequencing (NGS), and copy number variant (CNV) analysis. These techniques are time-consuming, costly and involve complicated data interpretation. The cost of SNP arrays has decreased substantially (from 300 Euro to 40 Euros per sample), driven by the very large sample sizes needed to perform genomewide association studies (GWAS). Both Affymetrix/Thermofisher and Illumina have designed cost-effective arrays that contain ∼800,000 variants, allowing a wide range of genetic variants to be assessed. 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

Results

Discussion

Conclusion