case_images: PMC6460992_01
case_images: PMC6460992_02
case_images: Gene Map of IL2RG gene showing novel variants for patients A and B. (A)
  IL2RG cDNA map with sites of variants (indicated by arrows) found in patients A and B with X-linked SCID. Patient A has a novel hemizygous missense variant: c.G175C in exon 2 (p.E59Q), which was designated a variant of unknown significance. Patient B has a novel variant in a non-coding region of IL2RG: a deletion within the 3' UTR (AA *307 *308). (B) Chromatogram of Sanger sequencing of the 3' UTR of the IL2RG gene for patient B, his mother and a healthy control (HC). Sequencing reveals patient B and his mother (a carrier) have a 2 base pair deletion within the polyA signal sequence: AATAAA is changed to AATA, as indicated by the blue rectangles.
case_images: fped-07-00055-g0001.jpg
case_images: PMC6460992_06_fped-07-00055-g0001.jpg
case_images: F1
case_images: Further genetic testing of the noncoding regions of IL2RG, revealed a 3 prime untranslated region (3' UTR) deletion NM_000206.2 c.*307_*308del within the polyA site (AATAAA was changed to AATA) (Figures 1A,B).
case_images: PMC6460992_03
case_images: Immune functional assays for interleukin receptor signaling in subsets of lymphocytes. (A) X chromosome inactivation (XCI) with (+) or without (-) Hpall. Hpall digests unmethylated alleles. The X chromosome that is active is under methylated, thus, once digested by Hpall, PCR amplification does not occur and band disappears. XCI in CD3+ T cells is skewed toward. (B) Histogram of T cell and monocyte response with STAT phosphorylation to several gammac dependent (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) and independent (IL-6, IFN-alpha, IFN-gamma) cytokines in PBMCs of patient A. (C) Total lymphocyte response with STAT5 phosphorylation to IL-21 and IL-2 stimulation in patient A, patient B and a healthy control (unstimulated in red, stimulated in blue). (D) Level of CD132 (gammac) expression on CD3+ T cells of patient B and a healthy control (patient unstimulated in green, patient stimulated in red, and healthy control in blue). Eighty-four percent of patient's cells expressed CD 132 when stimulated, however, mean fluorescence intensity was lower in patient than control. X inactivation and STAT phosphorylation studies in Figures 2A,B were performed by the Department of Laboratory Medicine, Clinical Center, National Institutes of Health (Bethesda, MD).
case_images: fped-07-00055-g0002.jpg
case_images: PMC6460992_05_fped-07-00055-g0002.jpg
case_images: F2
case_images: Maternal XCI studies by two independent laboratories using PBMCs demonstrated mild skewing, while subsequent testing of maternal T cells indicated complete skewing toward inactivation of the X-chromosome with the allele containing the pathogenic variant carried by the patient (Table 1 and Figure 2A lower panel). IL-21 and IL-2 signaling in the patient's total lymphocytes demonstrated impaired signaling (Figure 2C).
case_images: PMC6460992_04
case_images: Immune functional assays for interleukin receptor signaling in subsets of lymphocytes. (A) X chromosome inactivation (XCI) with (+) or without (-) Hpall. Hpall digests unmethylated alleles. The X chromosome that is active is under methylated, thus, once digested by Hpall, PCR amplification does not occur and band disappears. XCI in CD3+ T cells is skewed toward. (B) Histogram of T cell and monocyte response with STAT phosphorylation to several gammac dependent (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) and independent (IL-6, IFN-alpha, IFN-gamma) cytokines in PBMCs of patient A. (C) Total lymphocyte response with STAT5 phosphorylation to IL-21 and IL-2 stimulation in patient A, patient B and a healthy control (unstimulated in red, stimulated in blue). (D) Level of CD132 (gammac) expression on CD3+ T cells of patient B and a healthy control (patient unstimulated in green, patient stimulated in red, and healthy control in blue). Eighty-four percent of patient's cells expressed CD 132 when stimulated, however, mean fluorescence intensity was lower in patient than control. X inactivation and STAT phosphorylation studies in Figures 2A,B were performed by the Department of Laboratory Medicine, Clinical Center, National Institutes of Health (Bethesda, MD).
case_images: fped-07-00055-g0002.jpg
case_images: PMC6460992_05_fped-07-00055-g0002.jpg
case_images: F2
case_images: Maternal XCI studies by two independent laboratories using PBMCs demonstrated mild skewing, while subsequent testing of maternal T cells indicated complete skewing toward inactivation of the X-chromosome with the allele containing the pathogenic variant carried by the patient (Table 1 and Figure 2A lower panel). IL-21 and IL-2 signaling in the patient's total lymphocytes demonstrated impaired signaling (Figure 2C).
case_images: T cell repertoire study. Spectratyping of CD3+ T cells for TCRbeta was performed for patient B. The patient has essentially absent TCRVbeta repertoire diversity (Cellular and Molecular Immunology Laboratory, Mayo Clinic). The majority of the 23 Vbeta families assessed are completely absent and those that show the presence of T cell receptor Vbeta families are profoundly oligoclonal (<=5 independent peaks). Patient in red, house-keeping gene (beta-actin) in blue, and size marker in orange.
case_images: fped-07-00055-g0003.jpg
case_images: PMC6460992_04_fped-07-00055-g0003.jpg
case_images: F3
case_images: Spectratyping demonstrated that most of the 23 Vbeta families assessed were completely absent and the families present were profoundly oligoclonal (<=5 independent peaks) in CD3+ T cells, which was consistent with a Tlow leaky SCID phenotype (Figure 3).
case_images: PMC6460992_05
case_images: Immune functional assays for interleukin receptor signaling in subsets of lymphocytes. (A) X chromosome inactivation (XCI) with (+) or without (-) Hpall. Hpall digests unmethylated alleles. The X chromosome that is active is under methylated, thus, once digested by Hpall, PCR amplification does not occur and band disappears. XCI in CD3+ T cells is skewed toward. (B) Histogram of T cell and monocyte response with STAT phosphorylation to several gammac dependent (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) and independent (IL-6, IFN-alpha, IFN-gamma) cytokines in PBMCs of patient A. (C) Total lymphocyte response with STAT5 phosphorylation to IL-21 and IL-2 stimulation in patient A, patient B and a healthy control (unstimulated in red, stimulated in blue). (D) Level of CD132 (gammac) expression on CD3+ T cells of patient B and a healthy control (patient unstimulated in green, patient stimulated in red, and healthy control in blue). Eighty-four percent of patient's cells expressed CD 132 when stimulated, however, mean fluorescence intensity was lower in patient than control. X inactivation and STAT phosphorylation studies in Figures 2A,B were performed by the Department of Laboratory Medicine, Clinical Center, National Institutes of Health (Bethesda, MD).
case_images: fped-07-00055-g0002.jpg
case_images: PMC6460992_05_fped-07-00055-g0002.jpg
case_images: F2
case_images: Maternal XCI studies by two independent laboratories using PBMCs demonstrated mild skewing, while subsequent testing of maternal T cells indicated complete skewing toward inactivation of the X-chromosome with the allele containing the pathogenic variant carried by the patient (Table 1 and Figure 2A lower panel). IL-21 and IL-2 signaling in the patient's total lymphocytes demonstrated impaired signaling (Figure 2C).
case_images: PMC6460992_06
case_images: Gene Map of IL2RG gene showing novel variants for patients A and B. (A)
  IL2RG cDNA map with sites of variants (indicated by arrows) found in patients A and B with X-linked SCID. Patient A has a novel hemizygous missense variant: c.G175C in exon 2 (p.E59Q), which was designated a variant of unknown significance. Patient B has a novel variant in a non-coding region of IL2RG: a deletion within the 3' UTR (AA *307 *308). (B) Chromatogram of Sanger sequencing of the 3' UTR of the IL2RG gene for patient B, his mother and a healthy control (HC). Sequencing reveals patient B and his mother (a carrier) have a 2 base pair deletion within the polyA signal sequence: AATAAA is changed to AATA, as indicated by the blue rectangles.
case_images: fped-07-00055-g0001.jpg
case_images: PMC6460992_06_fped-07-00055-g0001.jpg
case_images: F1
case_images: Further genetic testing of the noncoding regions of IL2RG, revealed a 3 prime untranslated region (3' UTR) deletion NM_000206.2 c.*307_*308del within the polyA site (AATAAA was changed to AATA) (Figures 1A,B).