Abstract
PURPOSE: X-linked severe combined immunodeficiency (X-SCID) is an inherited immune disorder caused by pathogenic variants in the IL2RG gene, leading to recurrent infections. Identifying these variants and elucidating their pathogenic mechanisms are crucial for precise diagnosis and treatment, prenatal diagnosis, and preimplantation genetic testing (PGT). This study aimed to identify candidate variants in four families with suspected immunodeficiency, assess their pathogenicity, elucidate their pathogenic mechanisms, and provide a basis for precise treatment, prenatal diagnosis, and PGT. PATIENTS AND METHODS: Four families with suspected immunodeficiency were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Whole exome sequencing (WES) was used to identify the genetic etiology. Functional experiments were performed to assess the pathogenicity of the identified IL2RG variants, and to elucidate their pathogenic mechanisms. RESULTS: WES identified four IL2RG variants: three hemizygous (c.569G>C:p.R190P, c.515T>C:p.L172P, c.217A>C:p.T73P) and one heterozygous (c.1091C>T:p.T364I) variants. Three of these variants were novel. Initially three variants (p.R190P, p.T73P, and p.T364I) were classified as variants of uncertain significance (VUS) and one (p.L172P) was likely pathogenic (LP) according to ACMG/AMP guidelines. Functional analyses revealed reduced STAT5 phosphorylation and transcriptional activity across all variants, supporting the reclassification of three variants (p.R190P, p.L172P, and p.T73P) as likely pathogenic (LP), and one variant (p.T364I) as VUS with a Bayesian score of 5. Furthermore, IP-MS analysis revealed that the mutant IL2RG resulted in reduced cell-surface expression and abnormal nuclear localization. Therefore, the identified IL2RG variants impair IL-2-induced STAT5 phosphorylation and transcriptional activity to cause X-linked severe combined immunodeficiency in these families. CONCLUSION: This study highlights the critical role of functional analysis in clarifying variant pathogenicity and provides a clear example of pathogenicity assessment for IL2RG variants. Integrating genomic and functional data enhance diagnostic precision and informs precise treatment strategies, genetic counseling, prenatal diagnosis, and PGT for X-SCID.