Investigation of Genetic Defects in Severe Combined Immunodeficiency Patients from Turkey by Targeted Sequencing

Abstract

Primary immunodeficiencies (PIDs) represent a large group of disorders with an increased susceptibility to infections. Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiencies (PIDs) with marked Tcell lymphopenia. Investigation of the genetic aetiology using classical Sanger sequencing is associated with considerable diagnostic delay. We here established a custom-designed, next-generation sequencing (NGS)-based panel to efficiently identify disease-causing genetic defects in PID patients and applied this method in SCID patients of Turkish origin with previously undefined genetic aetiology. We used HaloPlex enrichment technology, a targeted, NGS-based method which was designed to diagnose patients with SCID and other PIDs. Our HaloPlex panel included a total of 356 PID-related genes, and we searched disease-causing mutations in 19 Turkish SCID patients without a genetic diagnosis. The coverage of targeted regions ranged from 97.47% to 99.62% with an average of 98.31% for all patients. All known SCID genes were covered with a percentage of at least 97.3%. We made a genetic diagnosis in six of 19 (33%) patients, including four novel disease-causing mutations identified in RAG1, JAK3 and IL2RG, respectively. We showed that this NGS-based method can provide rapid genetic diagnosis for patients suffering from SCID, potentially facilitating clinical treatment decisions.

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies

“…Phenotypic and genotypic heterogeneity of PIDs make genetic diagnosis often complex and delayed. Indeed, more than one genotype might cause similar clinical phenotypes, but identical genotypes will not often produce the same phenotype and finally clinical penetrance may be different (6)(7)(8)(9). The characterization of PIDassociated genes is expected to significantly contribute to define the molecular events governing immune system development and will provide new insights into the pathogenesis of PIDs.…”

Clinical efficacy of a next‐generation sequencing gene panel for primary immunodeficiency diagnostics

“…Exclusion criteria included prior knowledge of the genetic cause of PID and/or previous bone marrow transplant or gene therapy. A target cohort size of 20‐30 participants was chosen based on the results of previously published studies …”

Targeted mutation screening of 292 candidate genes in 38 children with inborn haematological cytopenias efficiently identifies novel disease‐causing mutations

“…Regions of interest included all exons, exon–intron boundaries and promoter regions. Sequencing was performed on a HiSeq3000 platform (Illumina, San Diego, CA, USA) as described (Salzer et al , ; Erman et al , ). 98·5% of enriched exonic bases were considered callable with a minimum read depth of 2.…”

“…Additional details and genetic results are outlined in the Supplementary Results. All identified variants which were deemed potentially disease‐causing were validated with capillary sequencing as described (Salzer et al , ; Erman et al , ) or multiplex ligation‐dependent probe amplification.…”

“…Additional details and genetic results are outlined in the Supplementary Results. All identified variants which were deemed potentially disease-causing were validated with capillary sequencing as described (Salzer et al, 2016;Erman et al, 2017) or multiplex ligation-dependent probe amplification. Collectively, we identified a causative genetic defect in 17/ 38 patients (44Á7%) including novel mutations in 13 of them ( Fig 1A).…”