The IL-2RG R328X nonsense mutation allows partial STAT-5 phosphorylation and defines a critical region involved in the leaky-SCID phenotype

Abstract

In addition to their detection in typical X-linked severe combined immunodeficiency, hypomorphic mutations in the interleukin (IL)-2 receptor common gamma chain gene (IL2RG) have been described in patients with atypical clinical and immunological phenotypes. In this leaky clinical phenotype the diagnosis is often delayed, limiting prompt therapy in these patients. Here, we report the biochemical and functional characterization of a nonsense mutation in exon 8 (p.R328X) of IL2RG in two siblings: a 4-year-old boy with lethal Epstein-Barr virus-related lymphoma and his asymptomatic 8-month-old brother with a T^low B⁺ natural killer (NK)⁺ immunophenotype, dysgammaglobulinemia, abnormal lymphocyte proliferation and reduced levels of T cell receptor excision circles. After confirming normal IL-2RG expression (CD132) on T lymphocytes, signal transducer and activator of transcription-1 (STAT-5) phosphorylation was examined to evaluate the functionality of the common gamma chain (γ_c ), which showed partially preserved function. Co-immunoprecipitation experiments were performed to assess the interaction capacity of the R328X mutant with Janus kinase (JAK)3, concluding that R328X impairs JAK3 binding to γ_c . Here, we describe how the R328X mutation in IL-2RG may allow partial phosphorylation of STAT-5 through a JAK3-independent pathway. We identified a region of three amino acids in the γ_c intracellular domain that may be critical for receptor stabilization and allow this alternative signaling. Identification of the functional consequences of pathogenic IL2RG variants at the cellular level is important to enable clearer understanding of partial defects leading to leaky phenotypes.

Keywords: IL-2RG; JAK1; JAK3; STAT-5; X-linked severe combined immunodeficiency; hypomorphic mutations; interleukin receptor common gamma subunit; leaky SCID; whole exome sequencing.

Figure 1

Sanger sequencing electropherogram of IL2RG gene in the family reported. Family pedigree chart with the c.C982T/p.R328X mutation showing an X‐linked recessive inheritance pattern. Both patients were hemizygous carriers of the mutation located in exon 8, which was inherited from their asymptomatic mother. Squares = males; circles= females; solid square with line across = deceased patient; bicolor circle = carrier.

Figure 2

Immunological studies from patient II.II. (a) Absence of thymic shadow in the chest X‐ray. (b) Lymphocyte proliferation assay after phytohemagglutinin (PHA), muromonab‐CD3 (OKT3) and OKT3 + interleukin (IL)‐2 stimulation compared with a healthy control and a typical severe combined immunodeficiency (SCID patient. (c) Distribution of the T cell receptor‐Vb (TCR‐Vβ) repertoire in the patient compared with reference values of healthy controls analyzed by flow cytometry in T cells. (d) Expression of CD132 in T lymphocytes in the patient showed mean fluorescence intensity (MFI) and percentage comparable to a healthy control analyzed by flow cytometry. (f) Intracellular staining of signal transducer and activator of transcription‐5 (STAT‐5) phosphorylation in CD3⁺ T lymphocytes using increasing concentrations of IL‐2 compared with a healthy control analyzed by flow cytometry.

Figure 3

Co‐precipitation of Janus kinase (JAK3) and interleukin (IL)‐2RG R328X. African green monkey kidney (COS‐7) cells were transiently transfected with Myc‐tagged IL‐2RG wild‐type (WT) or Y325X or R328X mutants in combination with JAK3. Cell lysates were immunoprecipitated with anti‐JAK3 in all cases. Western blots were conducted with the indicated antibodies. Whole cell lysates were included as controls.

Figure 4

Gene map of IL2RG gene showing leaky mutations and detail of the constructs used in the co‐precipitation experiments. (a) Previously reported leaky mutations are indicated with gray dots. R328X mutation is indicated with a black dot. (b) Schematic representation of the interleukin (IL)‐2 receptor showing the location and sequence of the mutants used in the co‐precipitation experiments (Y325X and R328X). c.‐13C>T 40, c.52delG 41, c.260T>C 42, c.890A>G 43. All other mutations are listed in 19.