Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype - 07/02/19
, Yu Nee Lee, PhD l, ⁎ Abstract |
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.
Le texte complet de cet article est disponible en PDF.Graphical abstract |
Key words : Recombination-activating gene 2, VDJ recombination, severe combined immunodeficiency, Omenn syndrome, autoimmunity, genotype-phenotype correlation
Abbreviations used : AS, CADD, CID-G/AI, GFP, gnomAD, IRES, MAF, OS, RAG, SCID, v-Abl, WT
Plan
| Supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH; grant 2R01AI100887 to L.D.N.); the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health; and the JSPS Research Fellowship for Japanese Biomedical and Behavioral Research at NIH (to Y.Y.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government. |
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| Disclosure of potential conflict of interest: The authors declare that they have relevant no conflicts of interest. |
Vol 143 - N° 2
P. 726-735 - février 2019 Retour au numéro
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