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

Abstract

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs . The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% ( n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.

Mutational landscape of severe combined immunodeficiency patients from Turkey

“…However, many patients do not receive a molecular diagnosis despite extensive clinical and immunologic studies, mainly due to the large number of genes that can cause similar phenotypes. The recent advent of next‐generation sequencing technologies and its applications in PID research has accelerated the discovery of novel genes that are associated with PIDs (Cifaldi et al., 2019; Gallo et al., 2016). Targeted NGS panels have been commonly engaged in the clinical laboratories as powerful tools that aid molecular diagnosis by virtue of their capacity to investigate multiple genes simultaneously (Raje et al., 2014), with a reported success rate of 25%–40% among studies (Al‐Mousa et al., 2016; Erman et al., 2017; Fang et al., 2016; Nijman et al., 2014; Stoddard et al., 2014; Yu et al., 2016).…”

Novel Compound Heterozygous Mutations in IL-7 Receptor α Gene in a 15-Month-Old Girl Presenting With Thrombocytopenia, Normal T Cell Count and Maternal Engraftment

“…In the present case, a combination of self-tolerance perturbation, expressed as maternal engraftment and autoimmune manifestations, delayed the SCID diagnosis. Such a clinical scenario, characterized by early onset autoimmune diseases, isolated CD4-penia despite normal total T cell count and hypergammaglobulinemia, should guide physicians toward proceeding to an NGS approach in order to identify pathogenic variants of atypical PID (19).…”

How to evaluate for immunodeficiency in patients with autoimmune cytopenias: laboratory evaluation for the diagnosis of inborn errors of immunity associated with immune dysregulation

“…Targeted panels are most effective when there is a clear phenotype and/or family history, which supports the likelihood of a monogenic disease, and the panel under consideration is well represented for the genes of interest. 29 The most recent International Union of Immunological Societies classification 1 includes >400 genes, and most commercially available panels include most of these, which makes these panels practical and relatively rapid screens with a turnaround time averaging 2 or rarely 3 weeks. However, if the phenotype is more amorphous and ambiguous and there is either no family history or no samples for trio analysis (proband + parents) are available or there has been a previously negative targeted panel, WES is the most logical option.…”