Ning Zhang,1,* Yi-Lin Sang,1,* Wu Zhu,1 Yu-Rong Wang,2 Yan-Yan Yu,2,3 Ya-Hui Chen,1 Juan Du,2,4 Wen-Bin He,4,5 Yue-Qiu Tan,2,4 Fu-Yan Wang1 

1Department of Immunology, Xiangya School of Basic Medical Sciences, Central South University, Changsha, Hunan, 410013, People’s Republic of China; 2Institute of Reproductive and Stem Cell Engineering, Xiangya School of Basic Medical Sciences, Central South University, Changsha, Hunan, 410078, People’s Republic of China; 3Clinical Laboratory, Hunan Provincial Tuberculosis Prevention and Control Institute & Hunan Chest Hospital, Changsha, Hunan, 410013, People’s Republic of China; 4Reproductive and Genetic Hospital of CITIC-Xiangya & Clinical Research Center for Reproduction and Genetics in Hunan Province, Changsha, Hunan, 410078, People’s Republic of China; 5Hunan Guangxiu Hospital Affiliated with Hunan Normal University, Health Science Center, Hunan Normal University, Changsha, Hunan, 410017, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yue-Qiu Tan, Institute of Reproductive and Stem Cell Engineering, Xiangya School of Basic Medical Sciences, Central South University, Changsha, Hunan, 410078, People’s Republic of China, Email tanyueqiu@csu.edu.cn Fu-Yan Wang, Department of Immunology, Xiangya School of Basic Medical Sciences, Central South University, Changsha, Hunan, 410013, People’s Republic of China, Email wfy4010@csu.edu.cn

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.

Keywords: IL2RG, X-linked severe combined immunodeficiency, IL2RG localization, STAT5 phosphorylation, transcriptional activity