Human RAG mutations: biochemistry and clinical implications

Abstract

The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate the V(D)J recombination process, which ultimately enables the generation of T cells and B cells with a diversified repertoire of antigen-specific receptors. Mutations of the RAG genes in humans are associated with a broad spectrum of clinical phenotypes, ranging from severe combined immunodeficiency to autoimmunity. Recently, novel insights into the phenotypic diversity of this disease have been provided by resolving the crystal structure of the RAG complex, by developing novel assays to test recombination activity of the mutant RAG proteins and by characterizing the molecular and cellular basis of immune dysregulation in patients with RAG deficiency.

Figure 1. Characterization and distribution of human RAG mutations

a |Schematic representation of the recombination-activating gene 1 (RAG1) protein with the various mutations colour-coded according to the clinical presentation. See Supplementary information S1 (table) for references. RAG1 catalytic and zinc-binding residues are indicated by red and black stars, respectively. b | Number of RAG1 missense mutations in the various domains. c | Frequency of RAG1 mutations calculated by dividing the number of mutations in a given region by the number of amino acids in that region. d | Schematic representation of the RAG2 protein with the various mutations colour-coded according to the clinical presentation. The trimethylated histone H3 lysine 4 (H3K4me3)-binding residue and T490 phosphorylation site of RAG2 are indicated by green and blue stars, respectively. e | Number of RAG2 missense mutations in the various domains. f | Frequency of RAG2 mutations calculated by dividing the number of mutations in a given region by the number of amino acids in that region. BI, basic I domain; BIIa/b, basic IIa/b domain; BIII, basic III domain; CTD, carboxy-terminal domain; DDBD, dimerization and DNA-binding domain; NBD, nonamer-binding domain; PHD, plant homeodomain; preR, pre-RNase H; RNH, catalytic RNase H.

Figure 2. Effects of mutations associated with CID–G/AI on the structure of the RAG complex

a| Structure of the recombination-activating gene 1 (RAG1)–RAG2 heterotetramer. Two RAG1 core subunits are shown in blue and grey, and two RAG2 core subunits are shown in purple and pink. Side chains of mutations found only in patients with combined immunodeficiency associated with granulomas and/or autoimmunity (CID–G/AI) are shown as yellow spheres, and those found in both patients with CID–G/AI and patients with severe combined immunodeficiency (SCID) or Omenn syndrome are shown in orange. For clarity, mutations are shown in one subunit of RAG1 and RAG2 only. All residues are numbered according to human RAG proteins. b,c | Zoom-in views of mutations that are unique to patients with CID–G/AI. Molecular surfaces are shown together with the ribbon diagram. L514R and Y728H from RAG1 and M322T from RAG2 are partially exposed (shown in yellow), whereas V8I, F62L and T77N from RAG2 are buried inside the protein (shown in purple). These mutations seem to lead to mild structural destabilization of the RAG proteins. The R841W mutation is at the interface of the closed conformation of the RAG complex.

Figure 3. RAG deficiency results in impairment of several tolerance checkpoints

a| Central T cell tolerance. Impaired V(D)J recombination as a result of recombination-activating gene (RAG) deficiency leads to a restricted T cell receptor (TCR) repertoire, T cell lymphopaenia and aberrant thymus architecture. Altered T cell development hinders lymphostromal crosstalk in the thymus and the maturation of medullary thymic epithelial cells (mTECs); the resulting lack of expression of autoimmune regulator (AIRE) and of AIRE-dependent tissue-restricted antigens (TRAs) impairs the negative selection of self-reactive T cells or their conversion to regulatory T (T_Reg) cells. Autoreactive T cells are exported to the periphery and expand in number. b | Peripheral T cell tolerance. The generation of T_Reg cells is limited in the thymus, which results in decreased T_Reg cell count and decreased suppressive function in the periphery. c | Central B cell tolerance. In the bone marrow, decreased V(D)J recombination results in a restricted B cell receptor (BCR) repertoire enriched in autoreactive B cells secondary to impaired receptor editing. d | Peripheral B cell tolerance. In the periphery, B cell lymphopaenia and an inflammatory state induce increased levels of B cell-activating factor (BAFF). In a BAFF-rich environment, the survival of autoreactive cells expressing low levels of BAFF receptor (BAFFR) is favoured. Autoreactive cells are shown in red, non-self-reactive cells in purple. FOXP3, forkhead box P3.

Figure 4. The interaction of genetic, immunological and environmental factors in determining the phenotype of human RAG deficiency

The recombination-activating gene (RAG) genotype determines the levels of recombination activity of the mutant RAG proteins. Mechanisms contributing to immune deficiency are shown on the left (blue) and those associated with autoimmunity are shown on the right (red), for both T cell- and B cell-dependent immune responses. RAG mutations with higher residual recombination activity are more likely to result in immune dysregulation. Ultimately, exposure to environmental triggers affects the immune deficiency and immune dysregulation status of the patient, thereby determining the clinical phenotype. AIRE, autoimmune regulator; BAFF, B cell-activating factor; D, diversity; EBV, Epstein–Barr virus; J, joining; T_Reg cell, regulatory T cell; V, variable.

Immunoglobulin or TCR genes