Development of a diagnostic protocol, mutation search, and genotype-phenotype correlation in haematological and immunological diseases by targeted resequencing using three different gene panels

Introduction: Next Generation Sequencing (NGS) has driven the rapid increase in the number of recognizable inborn errors of immune system development and/or function often hampered by the wide heterogeneity of the many genetically diverse but phenotypically overlapping diseases. NGS has also led to the discovery of new genes implicated in well-defined biological pathways, revisiting frequencies and broadening their phenotypic spectrum. Objectives: Identification of the genetic causes of already known and unknown immune-mediated diseases with immunedysregulation; improvement of our knowledge about clinically overlapping phenotypes through the genetic characterization of the corresponding patients; optimization of the diagnostic work-up in order to administer disease specific treatments to patients. Methods: A total of 150 patients, selected based on a clinical history highly evocative for immune-dysregulation (peripheral and/or central cytopenia and/or lymphoprolipheration and/or autoimmunity or autoinflammation) and/or immunodeficiency were submitted to 3 custom gene panels of 146, 315 and 58 genes and sequenced through the Ion Personal Genome Machine (PGM™) System. Based on the clinical phenotype, frequency and impact on the protein, variants were selected and validate by standard Sanger sequencing. Results: The mutation detection rates for these panels were 15/51 (29%), and 16/69 (23%) and 2/30 (6%), respectively, for a total of 35 pathogenic variants that correlate with the respective clinical phenotypes. The patients could be classified in: ALPS/ALPS-like (97, 26 of which diagnosed), cytopenia (33, 4 diagnosed), undefined autoinfiammatory (12, 2 diagnosed) and suspected immunodeficiency (8, 1 diagnosed). Moreover, variants of unknown significance (VUS) in potentially causative genes were also found in additional 14 patients (6/30; 4/51; 4/69). A number of variants, either pathogenic, likely pathogenic, or VUS have been detected in some cases at genes unexpected on the basis of the phenotypes, among which PRKCD, PIK3CD, IL7R, NCF1, TNFRSF13C, CASP8, thus confirming wide heterogeneity in the phenotypic spectrum associated with diseases sharing haemato-immune rheumatological features. Conversely, several clinically similar cases did not reveal any relevant mutation, thus reflecting a genetic heterogeneity that is far from being disclosed yet. Conclusion: The NGS approach has demonstrated excellent performances in the 1) evaluation of large genes and mutation detection, 2) overall timeliness of the gene panels, relying on continuous literature updates, and 3) identification of unexpected phenotypes for well-defined monogenic disease and definition of different disease clinical entities characterized by overlapping phenotypes. By contrast, due to the remarkable variability in clinical presentations, defining the appropriate list of genes for a given phenotype represents one key difficulty in the design of these panels. In the near future we will have to focus on the functional study of the many variants, especially VUS, that have emerged in a massive study like ours