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Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination
Chrystelle Couëdel, … , Anna Villa, Patricia Cortes
Chrystelle Couëdel, … , Anna Villa, Patricia Cortes
Published March 15, 2010
Citation Information: J Clin Invest. 2010;120(4):1337-1344. https://doi.org/10.1172/JCI41305.
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Research Article Immunology

Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination

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Abstract

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.

Authors

Chrystelle Couëdel, Christopher Roman, Alison Jones, Paolo Vezzoni, Anna Villa, Patricia Cortes

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Figure 2

T-B-SCID/OS Rag2PHD mutations affect Rag2 stability and cellular localization in pro-B cells.

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T-B-SCID/OS Rag2PHD mutations affect Rag2 stability and cellular localiz...
A mouse Rag2–/– pro-B cell line was retrovirally transduced to express FNT-tagged full-length wild-type Rag2 or T-B-SCID/OS mutants. (A) Western blot analysis was performed on whole cell extracts from retrovirally transduced pro-B cell lines for Flag, Rag2, and β-actin. Samples were loaded on the same gel, and the length of exposure was identical. The vertical line between FNT–mouse Rag2–C446W and –W453R mutants indicates a sample that was excluded from our study. (B) Total mRNA was isolated from retrovirally transduced pro-B cell lines, and Rag2 transcription levels were determined by Q-PCR. The average of 2 independent experiments are graphed as fold increase compared with mRNA expression of FNT–mouse Rag2. (C) Retrovirally transduced pro-B cell lines were pulse labeled with [35S] methionine/cysteine to determine degradation of wild-type Rag2 and T-B-SCID/OS mutants. Radiolabeled proteins were IP at various times after pulse with anti-Rag2 antibody, fractionated by SDS PAGE, and quantified on a PhosphorImager. Data were normalized to the radioactivity levels at the end of pulse. The graph presents the average from 2 independent experiments. (D) Cellular localization of Rag2 in retrovirally transduced pro-B cell lines was determined by Western blotting analysis of fractionated cytoplasm (C) and nuclear (N) extracts (top). Purity and loading of the fractions were analyzed by Western blotting for α-tubulin (middle) and Lamin B1 (bottom). The vertical line between FNT–mouse Rag2–C446W and –W453R is as in A.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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