cDNA and targeted RNA sequencing to unravel hidden genetic defects in PID patients with only one-disease related variant in genes with recessive inheritance

Background:The emerging of Next-Generation Sequencing (NGS) technologies has greatly facilitated genetic diagnosis of PID patients. Nevertheless the underlying genetic cause is only identified in 5-15% of PID patients. Currently genetic workup consists of targeted sequencing and/or whole exome sequencing (WES) at the gDNA level, which fails to detect large deletions, deep-intronic mutations, or changes in gene expression. Therefore, the aim of this project is to reveal genetic alterations missed at the gDNA level.Methods:cDNA and targeted RNA sequencing will be performed in patients with only one-disease related variant identified by WES or targeted analysis of known PID genes with recessive inheritance. For this study we selected patients with a clear PID phenotype based on symptoms and immunologic work-up in whom only 1 defect allele was detected in a relevant candidate gene based on pathway analysis. Results:In first instance we have optimised long-range PCRs to analyse the complete coding region of the relevant genes at the cDNA level. cDNA sequencing evaluating biallelic expression levels and/or aberrant transcripts in the patients is ongoing. In parallel we will evaluate an NGS-based approach capturing all genes of interest to evaluate their expression. Conclusions:NGS technologies enhanced the number of disease-related PID genes but final genetic confirmation of a PID diagnosis remains challenging. Identification of the underlying genetic defect is of utmost importance for risk stratification, improved therapeutic management strategies and genetic counselling. We believe that the implementation of RNA sequencing technologies in the workup of PID patients will improve patient diagnosis and management.Background:The emerging of Next-Generation Sequencing (NGS) technologies has greatly facilitated genetic diagnosis of PID patients. Nevertheless the underlying genetic cause is only identified in 5-15% of PID patients. Currently genetic workup consists of targeted sequencing and/or whole exome sequencing (WES) at the gDNA level, which fails to detect large deletions, deep-intronic mutations, or changes in gene expression. Therefore, the aim of this project is to reveal genetic alterations missed at the gDNA level.Methods:cDNA and targeted RNA sequencing will be performed in patients with only one-disease related variant identified by WES or targeted analysis of known PID genes with recessive inheritance. For this study we selected patients with a clear PID phenotype based on symptoms and immunologic work-up in whom only 1 defect allele was detected in a relevant candidate gene based on pathway analysis. Results:In first instance we have optimised long-range PCRs to analyse the complete coding region of the relevant genes at the cDNA level. cDNA sequencing evaluating biallelic expression levels and/or aberrant transcripts in the patients is ongoing. In parallel we will evaluate an NGS-based approach capturing all genes of interest to evaluate their expression. Conclusions:NGS technologies enhanced the number of disease-related PID genes but final genetic confirmation of a PID diagnosis remains challenging. Identification of the underlying genetic defect is of utmost importance for risk stratification, improved therapeutic management strategies and genetic counselling. We believe that the implementation of RNA sequencing technologies in the workup of PID patients will improve patient diagnosis and management.C