Characterization of inherited IL2RG gene mutation, second-site suppressor mutation, and skin-infiltrating lymphocytes. (A) The IL2RG gene was amplified from DNA extracted from normal, the patient's, and the mother's peripheral blood mononuclear cells (PBMCs). Direct sequence was performed using an automated sequencer. Bars show the locations of the mutation. (B) RT-PCR analysis for IL2RG mRNA using PBMCs obtained from normal control (lane 1) and the patient (lane 2). Lane M contains a 100-bp molecular size marker. (C) Direct sequence analysis of IL2RG cDNA using PBMCs from normal control and the patient. (D) Schematic representation of effect of the inherited splice-site mutation. (E) Analysis of γc expression. Shown are the results of γc expression on B-cell and T-cell lines, and cloned T cells #3-4 and #3-1 established from the patient. (F) Direct sequence analysis of IL2RG gene using DNA obtained from the clone #3-1. A bar shows the locations of the inherited mutation and an arrow highlights the second-site mutation. (G) Direct sequence analysis of IL2RG cDNA from the clone #3-1. (H) Direct sequencing analysis of IL2RG gene using DNA obtained from the patient's primary CD4⁺ and CD8⁺ T cells and CD19⁺ B cells, and his skin. (I) GeneScan analysis of IL2RG cDNA amplified from the clone #3-1, the primary lymphocytes including CD4⁺ and CD8⁺ T cells and CD19⁺ B cells, and the skin of the patient. A peak of the size of approximately 454 nucleotides represents wild-type mRNA and a second peak of approximately 482 nucleotides is generated by aberrant splicing. (J) FACS analysis of skin-infiltrating lymphocytes. The percentage of cells gated in each region is shown. (K) Immunohistochemical staining of the skin biopsy specimen. The samples were stained with anti-CD8 mAb.