A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency

Abstract

Background: The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T(-)B(-) severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes.

Objective: We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations.

Methods: We have developed a flow cytometry-based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1(-/-) pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology.

Results: Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity.

Conclusions: Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process.

Keywords: Omenn syndrome; Recombination-activating gene 1; V(D)J recombination; autoimmunity; genotype-phenotype correlation; immune repertoire; severe combined immune deficiency.

FIG 1

Schematic representation of the experimental outline and readout. A, A mouse Abelson (Abl) virus–transformed pro–B-cell line deficient for mRag1 was infected with a retrovirus containing an inverted GFP cassette. Subclones with single-copy stable integrants were transduced with vectors expressing wild-type RAG1 or various hRAG1 mutations (see Table E1) and then treated with imatinib to promote cell differentiation and induction of RAG1 activity. B and C, The level of GFP expression indicated the recombinase activity level on imatinib stimulation of Rag1−/− Abl pro-B cells transduced with an empty vector, or with vectors encoding either for wild-type hRAG1 (hWT) or wild-type mRag1 (mWT; Fig 1, B) or for one representative hRAG1 mutation for each of the 5 different phenotypic subgroups of the disease (Fig 1, C). LTR, Long terminal repeats.

FIG 2

Activity levels of 79 genetic variants of hRAG1. A, Schematic representation of 79 genetic variants of hRAG1 affecting the various domains: RING, zinc finger RING type domain (amino acids 168–283); ZFA, zinc finger A; NBR (amino acids 387–461); HBR (amino acids 531–763); ZFB, zinc finger B; and the core domain from amino acids 385 to 1011. The conserved cysteine (C) and histidine (H) residues are marked with black lines, and the basic domains are marked with bars. They are as follows: C1 (103/115); C2 (169/181); C3 (204/214) CH (269/275); CC (905/910); HH (940/945); BI (142–147); BIIa (219–225); BIIb (234–237); and BIII (244–257). The RAG1 variants are color coded, corresponding to the clinical phenotype of patients in which they were identified (red = T⁻/B⁻ SCID, orange = OS, green = γδ-T, blue = atypical/leaky SCID, and purple = CID-G/A). The asterisk marks mutations associated with other phenotypes. Mutations in black correspond to alleles with the lower recombination activity that had been identified in patients who were compound heterozygous for RAG1 mutations. Known polymorphisms are indicated in pink, and gray is used to identify variants detected in patients for whom incomplete clinical and immunologic information was available. B, Bar diagram representing the activity level of various hRAG1 mutants relative to wild-type hRAG1. Values are expressed as percentages ± SEMs. For each mutant, 3 to 5 independent experiments were performed. Mutations falling in the NBR and HBR are contained in shaded areas. C, Recombination activity of missense mutations falling in the NBR/HBR versus other regions of hRAG1. D, Recombination activity of hRAG1 mutants identified in patients with a distinct clinical and immunologic phenotype. E, Recombination activity of hRAG1 mutants identified in patients with virtual lack of circulating B cells (<30 cells/µL) and in those with residual B cells (≥30 cells/µL). The Mann-Whitney U test was performed to demonstrate statistical significance for all the 1-tailed P values in the graphs: **P < .01, ***P < .001, and ****P < .0001.

FIG 3

Protein expression of hRAG1 mutants. A–C, Protein expression of hRAG1 mutants affecting NBR (Fig 3, A), HBR (Fig 3, B), and non-NBR/HBR (Fig 3, C) domains. Expression of β-actin was used to normalize the density for each of the hRAG1 mutants. Results are shown as adjusted density (ImageJ). One representative of 2 immunoblots is shown. D, Adjusted density of hRAG1 protein expression of mutants affecting the NBR/HBR or non-NBR/HBR domains of the molecule. ns, Not significant. Statistical analysis was performed with the Mann-Whitney U test. E, Correlation between adjusted density of protein expression and recombination activity of RAG1 mutants (Spearman r_s = 0.351, P = .023).

FIG 4

Diversity and CDR-H3 characteristics and composition of the rearranged Ighc repertoire of hRAG1 mutations. A–F, Tree maps (iRepertoire) were generated to depict graphically the diversity and frequency of different V–J pairings induced by various hRAG1 mutants: wild-type hRAG1 (Fig 4, A); R699Q (Fig 4, B); G516A (Fig 4, C); M435V (Fig 4, D); R314W (Fig 4, E); and K992E (Fig 4, F). Each dot represents a unique V_H-J_H recombination, and the size of the dot indicates the relative frequency of that specific V-J_H rearrangement. G–I, Bar diagrams representing the uses of V_H (Fig 4, G), D_H (Fig 4, H), and J_H (Fig 4, I) genes for wild-type hRAG1 and various hRAG1 mutants. For the V_H genes, only the genes that had positive values are included. J–M, Characterization of CDR-H3 sequences: average CDR-H3 length (± SEM; Fig 4, J); average index of hydrophobicity (± SEM) according to a normalized Kyte-Doolittle scale (Fig 4, K); differential use of 3 RFs (Fig 4, L); and average Shannon entropy index (± SEM) of the CDR-H3 loop (Fig 4, M). *P < .05, **P < .01, ***P < .001, and ****P < .0001 unpaired 2-tailed t test.