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Human RAG mutations: biochemistry and clinical implications

Key Points

  • Recombination-activating gene (RAG) mutations in humans are associated with a broad spectrum of clinical phenotypes, ranging from severe, early-onset infections to inflammation and autoimmunity.

  • There is a correlation between the severity of the clinical and immunological phenotypes and the recombination activity of the mutant RAG protein, and hypomorphic mutations that severely affect recombination activity are associated with restriction of the T cell and B cell repertoires. However, environmental factors may also contribute to determining the disease phenotype.

  • Crystal structure and cryo-electron microscopy studies have revealed the structure of the heterotetrameric RAG complex bound to DNA. Fine definition of this structure has also offered important insights into the disease-causing effects of naturally occurring RAG mutations.

  • Studies in patients and in mice have demonstrated that RAG mutations affect central and peripheral T cell and B cell tolerance, including defective expression of autoimmune regulator (AIRE), reduced number and function of regulatory T cells, impaired receptor editing and increased levels of B cell-activating factor (BAFF), allowing the rescue of self-reactive B cells.

  • A broad range of autoantibodies has been demonstrated in patients with RAG mutations presenting with inflammation and autoimmunity. Neutralizing antibodies specific for interferon-α (IFNα) and IFNω have been documented particularly in patients with a history of severe viral infections.

  • Recent data indicate that RAG expression during the early stages of lymphoid development selects cells with improved fitness. NK cells from Rag−/− mice have an activated phenotype and increased cytotoxicity. If confirmed in humans, these data may account for the high rate of graft rejection observed after unconditioned haematopoietic stem cell transplantation in patients with RAG deficiency.


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.

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Figure 1: Characterization and distribution of human RAG mutations.
Figure 2: Effects of mutations associated with CID–G/AI on the structure of the RAG complex.
Figure 3: RAG deficiency results in impairment of several tolerance checkpoints.
Figure 4: The interaction of genetic, immunological and environmental factors in determining the phenotype of human RAG deficiency.


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L.D.N. was supported by grants from the National Institute of Allergy and Infectious Diseases, US National Institutes of Health (NIH; 5R01AI100887) and the March of Dimes (1-FY13-500). J.E.W. was supported by the NIH grant 5K08AI103035.

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Correspondence to Luigi D. Notarangelo.

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Missense RAG1 and RAG2 mutations: protein expression, recombination activity, structural effects and in vivo clinical phenotype (PDF 364 kb)


Non-homologous end joining pathway

(NHEJ pathway). An error-prone pathway that mediates joining of DNA double-strand breaks without requiring a homologous template. In mammals, the NHEJ pathway involves several proteins — Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, Cernunnos (also known as XLF and NHEJ1), X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV. Genetic defects of Artemis are the most common cause of radiosensitive severe combined immunodeficiency (SCID) in humans.

Haematopoietic stem cell transplantation

(HSCT). A therapeutic procedure that involves transfusion of donor HSCs into a recipient. HLA matching between the recipient and the donor determines compatibility. HSCT from a haploidentical donor (such as a parent) is associated with a high risk of graft-versus-host disease, unless T cells are depleted. Chemotherapy is often used before HSCT to eliminate the recipient's blood cells and favour engraftment of donor-derived HSCs but is not strictly necessary in infants with severe combined immunodeficiency (SCID).

Cellular radiosensitivity

Susceptibility of cells to the damaging effects of ionizing radiation, resulting in genomic instability, tumour development or cell death. Genetic defects that affect mechanisms of repair of DNA double-strand breaks are associated with increased cellular radiosensitivity.

SCID mouse

An animal model of severe combined immunodeficiency (SCID) that is characterized by lack of T cells and B cells, and increased cellular radiosensitivity. The SCID mouse carries mutations of the Prkdc gene, encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

Purifying selection

In population genetics, purifying selection refers to the selective removal of deleterious alleles from a given population. Purging of these genetic variants occurs when they cause early death or affect the reproductive fitness of affected individuals.

CDR3 spectratyping

(Complementarity-determining region 3 spectratyping). A PCR-based method that measures the diversity of T cell and B cell repertoires, based on the length of the CDR3 of immunoglobulin and T cell receptor transcripts. A Gaussian distribution of CDR3 length is detected in polyclonal T cells and B cells, whereas a single peak is observed in patients with leukaemia or lymphoma, and an altered distribution may be detected in patients with infections, autoimmune diseases or severe impairment of T cell and/or B cell development.

Autoimmune polyendocrinopathy candidiasis and ectodermal dystrophy

(APECED). A monogenic autoimmune disease caused by mutations of the autoimmune regulator (AIRE) gene, affecting central T cell tolerance. Common manifestations of this disease include autoimmune hypoparathyroidism, Addison disease, type 1 diabetes, candidiasis, alopecia and nail dystrophy.


Transgenic products that are generated through a process replacing the original codons with synonymous codons for which a more abundant tRNA is available. This facilitates the rate of translation and ultimately results in the production of higher amounts of the protein.

Whole-exome sequencing

A process by which all exons contained in the genome (collectively comprising the exome) are amplified and subjected to high-throughput sequencing. DNA genetic variants that are present in a given individual are identified by comparing the exome of that subject to the normal reference sequence.

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Notarangelo, L., Kim, MS., Walter, J. et al. Human RAG mutations: biochemistry and clinical implications. Nat Rev Immunol 16, 234–246 (2016).

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