AUTHOR=Cifaldi Cristina , Brigida Immacolata , Barzaghi Federica , Zoccolillo Matteo , Ferradini Valentina , Petricone Davide , Cicalese Maria Pia , Lazarevic Dejan , Cittaro Davide , Omrani Maryam , Attardi Enrico , Conti Francesca , Scarselli Alessia , Chiriaco Maria , Di Cesare Silvia , Licciardi Francesco , Davide Montin , Ferrua Francesca , Canessa Clementina , Pignata Claudio , Giliani Silvia , Ferrari Simona , Fousteri Georgia , Barera Graziano , Merli Pietro , Palma Paolo , Cesaro Simone , Gattorno Marco , Trizzino Antonio , Moschese Viviana , Chini Loredana , Villa Anna , Azzari Chiara , Finocchi Andrea , Locatelli Franco , Rossi Paolo , Sangiuolo Federica , Aiuti Alessandro , Cancrini Caterina , Di Matteo Gigliola 

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

JOURNAL=Frontiers in Immunology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.00316

DOI=10.3389/fimmu.2019.00316

ISSN=1664-3224

ABSTRACT=<p><bold>Background:</bold> 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.</p><p><bold>Objective:</bold> To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes.</p><p><bold>Methods:</bold> Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: <italic>T cell defects, Humoral defects</italic> and <italic>Other PIDs</italic>. 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.</p><p><bold>Results:</bold> The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (<italic>n</italic> = 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.</p><p><bold>Conclusions:</bold> 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.</p>