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Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype

Lookup NU author(s): Professor Andrew GenneryORCiD


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© 2018 American Academy of Allergy, Asthma & Immunology Background: Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. Objective: Using a flow cytometry–based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. Methods: Abelson virus–transformed Rag2−/− pro-B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild-type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP-expressing cells was evaluated by using flow cytometry, and high-throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. Results: The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild-type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. Conclusions: Our data support genotype-phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease-causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype.

Publication metadata

Author(s): Tirosh I, Yamazaki Y, Frugoni F, Ververs FA, Allenspach EJ, Zhang Y, Burns S, Al-Herz W, Noroski L, Walter JE, Gennery AR, van der Burg M, Notarangelo LD, Lee YN

Publication type: Article

Publication status: Published

Journal: Journal of Allergy and Clinical Immunology

Year: 2019

Volume: 143

Issue: 2

Pages: 726-735

Print publication date: 01/02/2019

Online publication date: 18/06/2018

Acceptance date: 27/04/2018

ISSN (print): 0091-6749

ISSN (electronic): 1097-6825

Publisher: Mosby Inc.


DOI: 10.1016/j.jaci.2018.04.027


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