Hemizygous IL2RG Variants Impair IL-2-Induced STAT5 Phosphorylation and Transcriptional Activity Causing X-Linked Severe Combined Immunodeficiency

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.

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

Figure 1

Family pedigree and conservation analysis of IL2RG variant sites. (A) Pedigrees of the four X-SCID families. The Sanger sequencing chromatogram of Family I from the proband (II-1), Families II and III from the proband’s mother (I-2), and Family IV from the proband’s aunt (III-1). Arrows indicate probands, black symbols indicate patients, white symbols represent unaffected individuals, and black spots indicate carriers. (B) Conservation analysis of IL2RG variants. Human IL2RG encodes a 369-amino acid protein. The protein contains two extracellular FN-III structural domains, FN-III (1) and FN-III (2), that facilitate its binding to cytokines. Cross-species alignment revealed that the aspartic acid residues at positions 73 and 172 in IL2RG are highly conserved across species, and residues at positions 190 and 364 are relatively conserved.

Figure 2

Reduced IL2RG expression on the cell surface affects IL-2-mediated signal transduction. (A) IL2RG mutant protein expression in HEK293 cells, as determined by Western blotting. (B) Co-IP analysis showed significantly reduced binding between JAK3 and the three IL2RG mutant proteins (p.R190P, p.L172P, and p.T364I), while the p.T73P mutant protein exhibited partial interaction with JAK3. (C) Flow cytometry analysis demonstrated that the surface expression of mutant IL2RG proteins was significantly reduced in HEK293 cells compared to that of wild-type IL2RG. (D) After stimulation with 1000 U/mL IL-2, STAT5 phosphorylation was substantially decreased in cells expressing mutant IL2RG proteins compared to wild-type IL2RG. (E) Luciferase reporter assays showed a significant reduction in STAT5 transcriptional activity in cells expressing the mutant IL2RG proteins compared to those expressing wild-type IL2RG. Cells in the blank control group were untransfected with any plasmid. Data are representative of three or four independent experiments, results are represented as mean ± SD, and statistical analyses were carried out via t test. (**p<0.01; ***p<0.001; ****p<0.0001).

Figure 3

IL2RG mutants display increased interactions with nuclear proteins. (A–D) GO analysis of WT and four mutant IL2RG proteins. The left panels show that all four IL2RG mutant proteins exhibited enhanced interactions with nuclear proteins and reduced interactions with plasma membrane proteins compared to wild-type IL2RG. The red dashed line indicates a more than 1.3-fold change in the abundance of interacting proteins in the mutant proteins compared to wild-type IL2RG. The right panels show the GO analysis of these differentially expressed proteins related to biological processes. The size of the dot represents the protein count, and the color of each dot corresponds to the p-value.