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.

Keywords: Haloplex; Ion Torrent; Next Generation Sequencing; gene panels; primary immunodeficiencies.

Figure 1

Clinical diagnosis of patients at admission. (A) Percentage of patients for three main categories. Percentage of each clinical diagnosis in patients belonging to (B) T cell defects, (C) Humoral defects and (D) Other PIDs categories. For each category the total number of patients is indicated.

Figure 2

Flowchart indicating the strategy of the study. (1) Indicates the only patient in Humoral defect group who has been analyzed by Ion Torrent panel 1.

Figure 3

Comparison between different number of genetic diagnoses obtained by Ion Torrent and Haloplex. (A) Histogram showing the number of overall diagnosed (red), under investigation (orange) or undiagnosed (gray) patients. (B) Histogram showing the Ion Torrent diagnoses. (C) Histograms showing Haloplex diagnoses. (D) Diagnostic findings by Haloplex in negative Ion Torrent patients. The percentages refer only to diagnosed patients.

Figure 4

Type and zygosity of mutations and mutated genes distribution. Types and percentage of mutations found in diagnosed PID patients for Ion Torrent (A) and Haloplex (B). (C) Overall observed zygosity for diagnosed PID patients. (D) Total number of detected mutated genes.