Targeted Next-Generation Sequencing in the Molecular Diagnosis of Severe Combined Immunodeficiency

Background and Objectives: Severe combined immunodeficiency (SCID) represents a group of rare and potentially fatal monogenic disorders arising from pathogenic variants in a broad spectrum of genes. Diagnostic delays beyond the first few months of life have been associated with poor overall survival and hematopoietic stem cell transplantation (HSCT) outcomes. Therefore, the aim of our study was to apply an NGS assay enabling the rapid and reliable diagnosis of SCID. Materials and Methods: We developed a targeted NGS panel of 30 genes implicated in the pathogenesis of most SCID cases and we applied it to three Greek infants with suspected SCID. Results: Each patient displayed a distinct immunophenotype-T^-B^-NK^-, T^-B^-NK⁺ and T^-B⁺NK^-, respectively-and was found to harbor pathogenic or likely pathogenic variants in the analyzed SCID-related genes. In particular, patient 1 carried two heterozygous ADA variants (c.58G>A, p.Gly20Arg and c.956_960del, p.Glu319Glyfs); patient 2 harbored two discrete pathogenic variants in the DCLRE1C gene (a large deletion of exons 1-3 and the nonsense mutation c.241C>T, p.Arg81*), causing Artemis deficiency; and patient 3 carried a hemizygous IL2RG missense variant (c.437T>C, p.Leu146Pro), associated with X-linked SCID. All variants were confirmed by Sanger sequencing. Conclusions: Our method successfully identified the underlying genetic defects in all patients, thereby establishing a molecular diagnosis of SCID. These findings highlight the potential of targeted NGS assays for achieving rapid and accurate molecular diagnosis of SCID, which is crucial for the timely treatment of life-threatening conditions in affected children.