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Open Access

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms

Thomas N. Burn, Kyutae D. Lee, Noor Dawany, Tanner F. Robertson, Megan R. Fisher, Craig H. Bassing and Edward M. Behrens
ImmunoHorizons March 1, 2020, 4 (3) 119-128; DOI: https://doi.org/10.4049/immunohorizons.2000001
Thomas N. Burn
*Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
†Division of Rheumatology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104;
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Kyutae D. Lee
‡Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and
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Noor Dawany
§Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104
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Tanner F. Robertson
*Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
‡Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and
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Megan R. Fisher
*Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
‡Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and
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Craig H. Bassing
*Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
‡Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and
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Edward M. Behrens
*Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
†Division of Rheumatology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104;
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  • FIGURE 1.
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    FIGURE 1.

    Novel nonsense mutation identified in 5′ region of Rag1 gene.

    (A) Diagram of RAG1 protein showing relative position of novel nonsense mutation (Q60X). (B) Sanger sequencing tracks aligning sequences from RAG1WT and RAG1NX mice in our colony to that of the reference mm10 genome (National Center for Biotechnology Information). (C) DN3 thymocytes (live singlets, CD4−, CD8−, CD25+, CD44−) and preselected DP thymocytes (live singlets, CD4+, CD8+, CD69−) were sorted from RAG1WT and RAG1NX mice. RNA was isolated and converted to cDNA. Relative RAG1 expression was measured by the ΔΔCt method using the housekeeping gene HPRT and a WT sample as a calibrator. Analyzed by two-way ANOVA with Tukey honest significant difference posttest. (D) RAG1 protein expressed in bulk thymocytes was analyzed by SDS-PAGE and Western blot. **p < 0.01, ***p < 0.0001.

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    FIGURE 2.

    RAG1NX mice have lymphocyte developmental blocks at Ag receptor rearrangement steps.

    Thymi from 4- to 5-wk-old WT, NX, and Het littermates were analyzed by flow cytometry. (A) CD4 versus CD8 frequencies and (B) total cell counts per thymus. Gating: live, singlets, dump− (B220, CD11b, CD11c, Gr1, NK1.1, Ter119), TCRγδ−. (C) DN1-4 frequencies and (D) total cell counts per thymus. Gating: CD4−, CD8−, TCRβlo. Early B cell development was analyzed in the bone marrow. (E and F) Frequencies of early B cell progenitors of B220+CD93+IgM− cells were quantified. Pregating: live, singlets, dump− (TCRβ, NK1.1, Ter119, CD11c, Gr1). Data combined from at least three independent experiments. Bars indicate mean ± SEM. Statistics: one-way ANOVA with Tukey honest significant difference posttest. **p < 0.01, ****p < 0.0001. n.s., p > 0.05.

  • FIGURE 3.
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    FIGURE 3.

    RAG1NX thymocytes have reduced TCRVβ recombination and an altered Vβ TCR repertoire.

    Sorted DN3 thymocytes from WT and NX mice were assayed by qPCR for frequencies of respective Vβ to DβJβ1 rearrangements (A) and Vβ to DβJβ2 rearrangements (B), four mice per genotype. A number sign (#) denotes not detected. Gating: live, singlets, dump− (B220, CD8, CD4, CD11b, CD11c, Gr1, NK1.1, Ter119), CD4−, CD8−, CD25+, CD44−. Vβ repertoire was assayed by flow cytometry of (C) DP thymocytes and (D) SP thymocytes. Representative gating in Supplemental Fig. 1A, 1B. (E) The ratio of Vβ usage by DP versus SP thymocytes was calculated. Data combined from two independent experiments. Error bars indicate SEM. Statistics: multiple t tests with Holm-Sidak correction. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001. n.s., p > 0.05.

  • FIGURE 4.
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    FIGURE 4.

    RAG1NX mice have reduced numbers of mature B and T cells and increased frequencies of Tregs and T memory cells.

    (A) Numbers of leukocytes were enumerated from spleens of 4- to 5-wk-old RAG1WT (WT), RAG1NX (NX), or heterozygous (Het) littermates by flow cytometry. Relative frequencies and numbers of peripheral Tregs (B), memory CD4+ T cells (C), and memory CD8+ T cells (D) in the spleen were quantified. Data combined from at least three independent experiments. Bars indicate mean ± SEM. Statistics: one-way ANOVA with Tukey honest significant difference posttest. Gating: live, singlets, B cells (CD19+TCRβ−), T cells (TCRβ+, CD19−, CD4+, or CD8+), NK cells (TCRβ−, CD19−, NK1.1+), dendritic cells (TCRβ−, CD19−, NK1.1−, CD11c+), neutrophils (TCRβ−, CD19−, NK1.1−, CD11c−, CD11b+, Ly6G+), inflammatory monocytes (TCRβ−, CD19−, NK1.1−, CD11c−, CD11b+, Ly6G−, Ly6Chi). TCM, central memory; TEM, effector memory. **p < 0.01, ****p < 0.0001. n.s., p > 0.05.

  • FIGURE 5.
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    FIGURE 5.

    RAG1NX mice develop increased age-associated B cells and ANA.

    (A–D) T cells from 4- to 5-wk-old WT and NX mice were stimulated ex vivo with PMA and ionomycin, and cytokine expression was measured by flow cytometry. Statistics: one-way ANOVA with Tukey honest significant difference posttest. Combined data from two independent experiments. (E) Twenty- to twenty-two–month-old WT and NX mice were compared for peripheral leukocyte populations. Gating: neutrophils (CD3−, Ly6G+), B cells (CD3−, Ly6G−, CD19+), T cells (CD3+, CD19−, Ly6G−, CD4+, or CD8+), eosinophils (CD3−, CD19−, Ly6G−, Siglec-F+), macrophages (CD3−, CD19−, Ly6G−, Siglec-F−, F4/80+), monocytes (CD3−, CD19−, Ly6G−, Siglec-F−, F4/80−, CD11b+). (F) Frequency of ABCs of total B cells was quantified in 20- to 22-mo-old female WT and NX mice. Statistics: Mann–Whitney U test. (G) ANA staining intensity on Hep-2a cells was assayed for in young (4- to 5-wk-old) and old (20- to 22-mo-old) WT and NX mice. Bars indicate mean ± SEM. Statistics: one-way nonparametric ANOVA with Tukey honest significant difference posttest. See representative images in Supplemental Fig. 2. *p < 0.05, ***p < 0.0001, ****p < 0.0001.

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    FIGURE 6.

    Smaller RAG1 isoforms arise via internal translation initiation.

    (A) RAG1 schematic highlighting positions of FLAG and HA epitope tags, Q60X mutation, and internal methionines that serve as potential TIS. (B and C) Expression of RAG1-WT, Q60X, and iM constructs in 293T cells assayed for FLAG and HA expression by flow cytometry. Pregated on GFP+ cells. (D and E) Expression of RAG1-WT, Q60X, and iM mutants in 293T cells analyzed by SDS-PAGE and Western blot for FLAG (D) and HA (E) expression. (F and G) RAG1 WT versus internal methionine mutants (M1–M5) expressed in 293T cells were analyzed by SDS-PAGE and Western blot for FLAG (F) and HA (G) expression. All blots are representative of at least three independent experiments. (H) Analysis of strength of internal putative TIS Kozak sequences compared with canonical Kozak sequence and Noderer consensus sequence (21). (I) Noderer scores of putative TIS compared with Noderer scores surrounding random, out-of-frame AUG sequences within murine RAG1 transcript.

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ImmunoHorizons: 4 (3)
ImmunoHorizons
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1 Mar 2020
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A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms
Thomas N. Burn, Kyutae D. Lee, Noor Dawany, Tanner F. Robertson, Megan R. Fisher, Craig H. Bassing, Edward M. Behrens
ImmunoHorizons March 1, 2020, 4 (3) 119-128; DOI: 10.4049/immunohorizons.2000001

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A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms
Thomas N. Burn, Kyutae D. Lee, Noor Dawany, Tanner F. Robertson, Megan R. Fisher, Craig H. Bassing, Edward M. Behrens
ImmunoHorizons March 1, 2020, 4 (3) 119-128; DOI: 10.4049/immunohorizons.2000001
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