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

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.

Keywords: Omenn syndrome; Recombination-activating gene 2; VDJ recombination; autoimmunity; genotype-phenotype correlation; severe combined immunodeficiency.

FIG 1.

Distribution of RAG2 mutations. Graphic presentation of different RAG2 mutations according to their position in the RAG2 protein and abundance of the patients according to various clinical presentations. For 5 RAG2 mutations (M1T, M110L, Y195D, C446W, and H481P), complete genetic and clinical information for the patients is not available. PHD, Plant homology domain.

FIG 2.

Assay to measure recombination activity of human RAG2 variants. A, Schematic representation of the recombination assay. Rag2^−/− v-Abl–transformed pro-B cells in which an inverted GFP cassette flanked by a recombination signal sequence (RSS) had been integrated stably in the genome (top) were transduced with a retroviral vector encoding either WT or mutant human RAG2 (hRAG2) and hCD2 as a reporter (middle). In this system GFP expression is measured by using fluorescence-activated cell sorting as a readout of successful recombination induced by the RAG2 protein in the presence of WT-mRag1 and the intact nonhomologous end-joining (NHEJ) pathway (bottom). LTR, Long terminal repeat. B, Representative fluorescence-activated cell-sorting dot plot of Rag2^−/− v-Abl pro-B cells transduced with an empty vector (pBMN[mock]) or with retroviral vectors encoding either WT human RAG (WT-hRAG), WT mouse Rag2 (WT-mRAG2), or RAG2 mutants identified in patients with various clinical presentations (T⁻B⁻SCID, OS, and CID-G/AI).

FIG 3.

Recombination activity of mutant RAG2 proteins. A, Graphic representation of the recombination activity of various RAG2 mutations according to their position in different domains of the RAG2 protein (core domain: amino acids 1–383; plant homology domain [PHD]: amino acids 414–487). Solid circles identify nonsense mutations, and triangles represent missense mutations. Means ± SEMs are shown to illustrate recombination activity. For each RAG2 variant, the assay was performed in triplicates. B, Frequency of pathogenic mutations (defined as recombination activity <80% WT-hRAG2) per amino acid length of various domains of the hRAG2 protein. C, Recombination activity (expressed as percentage of the activity of WT-hRAG2) of nonsense and missense hRAG2 mutations. Bars represent means ± SEMs. Statistical analysis was performed with the Mann-Whitney test. D, Recombination activity (expressed as percentage of the activity of WT-hRAG2) of missense mutations affecting the PHD and non-PHD domains of the hRAG2 protein. Bars represent means 6 SEMs. Statistical analysis was performed with the Mann-Whitney test. n.s., Not significant.

FIG 4.

Correlation between RAG2 recombination activity and severity of clinical presentation. Representation of recombination activity of mutant RAG2 alleles according to the clinical phenotype in 55 patients with RAG2 deficiency. Patients with variants resulting in values of 100% were omitted from this analysis. In the case of patients with compound heterozygous mutations, recombination activity corresponding to the allele with the higher activity is shown. Bars represent means ± SEMs.

FIG 5.

Recombination activity of compound heterozygous RAG2 variants. A, Analysis of the recombination activity supported by bicistronic vectors simultaneously expressing 2 RAG2 variants and comparison with recombination activity of single variants. Experiments were done in triplicates. B, Pearson correlation analysis between recombination activity supported by bicistronic vectors versus number of unique sequences. C, Graphic representation of Igh repertoire diversity. D, Quantitative measurement of diversity and unevenness of the Igh repertoire.