Analysis of mutations and recombination activity in RAG-deficient patients

Mutations in the recombination activating genes (RAG1 or RAG2) can lead to a variety of immunodeficiencies. Herein, we report 5 cases of RAG deficiency from 5 families: 3 of Omenn syndrome, 1 of severe combined immunodeficiency, and 1 of combined immunodeficiency with oligoclonal TCRγδ(+) T cells, autoimmunity and cytomegalovirus infection. The genetic defects were heterogeneous and included 6 novel RAG mutations. All missense mutations except for Met443Ile in RAG2 were located in active core regions of RAG1 or RAG2. V(D)J recombination activity of each mutant was variable, ranging from half of the wild type activity to none, however, a significant decrease in average recombination activity was demonstrated in each patient. The reduced recombination activity of Met443Ile in RAG2 may suggest a crucial role of the non-core region of RAG2 in V(D)J recombination. These findings suggest that functional evaluation together with molecular analysis contributes to our broader understanding of RAG deficiency.     

Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1

Summary: V(D)J recombination generates functional immunoglobulin and T‐cell receptor genes in developing lymphocytes. The recombination‐activating gene 1 (RAG1) and RAG2 proteins catalyze site‐specific DNA cleavage in this recombination process. Biochemical studies have identified catalytically active regions of each protein, referred to as the core regions. Here, we review our progress in the identification and characterization, in biophysical and biochemical terms, of topologically independent domains within both the non‐core and core regions of RAG1. Previous characterizations of a structural domain identified in the non‐core region of RAG1 from residues 265–380, referred to as the zinc‐binding dimerization domain, are discussed. This domain contains two zinc‐binding motifs, a RING finger and a C₂H₂ zinc finger. Core RAG1 also consists of multiple domains, each of which functions individually in one or more of the essential macromolecular interactions formed by the intact core protein. Two structural domains referred to as the central and the C‐terminal domains that include residues 528–760 and 761–979 of RAG1, respectively, have been identified. The interactions of the central and C‐terminal domains in core RAG1 with the recombination signal sequence (RSS) have contributed additional insight to a developing model for the RAG1–RSS complex.     

A plant homeodomain in RAG-2 that binds Hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement

V(D)J recombination is initiated by the recombination activating gene (RAG) proteins RAG-1 and RAG-2. The ability of antigen-receptor-gene segments to undergo V(D)J recombination is correlated with spatially- and temporally-restricted chromatin modifications. We have found that RAG-2 bound specifically to histone H3 and that this binding was absolutely dependent on dimethylation or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction required a noncanonical plant homeodomain (PHD) that had previously been described within the noncore region of RAG-2. Binding of the RAG-2 PHD finger to chromatin across the IgH D-J(H)-C locus showed a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the RAG-2 PHD finger abolished binding to H3K4me3 and greatly impaired recombination of extrachromosomal and endogenous immunoglobulin gene segments. Together, these findings are consistent with the interpretation that recognition of hypermethylated histone H3 K4 promotes efficient V(D)J recombination in vivo.     

Severe combined immunodeficiency in Frisian Water Dogs caused by a RAG1 mutation

Mortality of pups at 8-12 weeks of age was frequently observed in Frisian Water Dogs. Blood parameters and clinical signs of newborns from three litters were monitored. Three pups from two litters showed strongly reduced levels of immunoglobulins and lymphocytes. These dogs were euthanized after first display of disease. Concurrent clinical and pathological features were consistent with a diagnosis of severe combined immunodeficiency (SCID). Defective V(D)J recombination is one of the causes of SCID in humans and animals. Eight genes involved in V(D)J recombination were investigated by segregation analysis of closely located microsatellite markers and by DNA sequence analysis. A nonsense mutation in the gene coding for V(D)J recombination factor RAG1 was identified in DNA from the cases at a position similar to that of nonsense mutations found in human SCID. It was concluded that SCID due to a mutation of RAG1 led to the high mortality.     

Developmental anomalies and neoplasia in animals and cells deficient in the large zinc finger protein KRC

The large zinc finger protein KRC binds to the signal sequences of V(D)J recombination and the kappaB motif. Disruption of KRC expression in cell lines resulted in increased cell proliferation, anchorage independence of growth, and uncoupling of nuclear division and cell division. In this report, the function of KRC was studied in a RAG2-deficient blastocyst complementation animal model. KRC-deficient embryonic stem cells were generated by homologous recombination and were introduced into RAG2(-/-) blastocysts to generate KRC(-/-);RAG2(-/-) chimeric mice. The lymphoid compartments of chimeras examined at 5 weeks of age were developed, suggesting that KRC is not essential for V(D)J recombination development. However, by 6 months of age, there was a marked deficit in CD4(+)CD8(+) thymocytes in the chimeras, suggesting that KRC may be involved in T-lymphocyte survival. Additionally, one chimera developed anomalies, including postaxial polydactyly, hydronephrosis, and an extragonadal malignant teratoma. DNA analysis showed that the teratoma was derived from KRC(-/-) embryonic stem cells. The teratoma had compound tissue organization and was infiltrated with B lymphocytes. Subsequently, several immortalized KRC-deficient cell lines were established from the teratoma. In this study, growth anomalies and neoplasia were observed in animals and cells deficient in KRC, and other studies have shown allelic loss occurring at the chromosomal region of the human KRC counterpart in various tumors. We propose that KRC may be a previously unidentified tumor-suppresser gene.     

A direct interaction between the RAG2 C terminus and the core histones is required for efficient V(D)J recombination

V(D)J recombination is a tightly controlled process of somatic recombination whose regulation is mediated in part by chromatin structure. Here, we report that RAG2 binds directly to the core histone proteins. The interaction with histones is observed in developing lymphocytes and within the RAG1/RAG2 recombinase complex in a manner that is dependent on the RAG2 C terminus. Amino acids within the plant homeo domain (PHD)-like domain as well as a conserved acidic stretch of the RAG2 C terminus that is considered to be a linker region are important for this interaction. Point mutations that disrupt the RAG2-histone association inhibit the efficiency of the V(D)J recombination reaction at the endogenous immunoglobulin locus, with the most dramatic effect in the V to DJ(H) rearrangement.     

Regions of RAG1 protein critical for V(D)J recombination

The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutations for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conserved sequence. We show here that this “dispensable” region of RAG1 is not necessary for coding joint formation or for recombination of an integrated substrate, and that this portion is not functionally redundant with the “dispensable” region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.     

Characterization of immune function and analysis of RAG gene mutations in Omenn syndrome and related disorders

Omenn syndrome was recently found to be caused by missense mutations in RAG1 or RAG2 gene that result in partial V(D)J recombination activity. Although the clinical hallmarks of the disease are well defined, there have been several cases with clinical findings similar to, but distinct from Omenn syndrome. The data on immune functions and RAG gene mutations of such cases are limited. We described five Japanese infants from four unrelated families, including two cases of Omenn syndrome and three cases of related disorders. Sibling cases with typical Omenn phenotype were found to be compound heterozygotes of R396C and L885R mutations in RAG1. The former has been reported in European cases and may constitute a hot spot. The latter is a novel missense mutation. Infants with related disorders exhibited erythroderma, eosinophilia, hypogammaglobulinaemia, decreased number of B cells and skewing to Th2, and their lymph node specimens showed architectural effacement, lymphocyte depletion and histiocytic hyperplasia, each of which is seen characteristically in Omenn syndrome. However, in these cases serum IgE levels were low or undetectable. We found no mutation in RAG genes except for a K820R substitution in RAG1, which was regarded to be a functional polymorphism, in two of these cases. Our study suggests that RAG missense mutation may be a genetic abnormality unique to Omenn syndrome with characteristic clinical and laboratory findings. Variations of Omenn syndrome, or related disorders, may represent a different type of immunodeficiency, distinct from abnormalities in lymphoid-specific recombinase activity.     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase

The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking and hairpin formation) is severely impaired. Because both chemical steps are affected by the same mutations, a single active site appears responsible for both reactions. Two independent lines of evidence demonstrate that at least two of these acidic residues are directly involved in coordinating a divalent metal ion: The substitution of Cys for Asp allows rescue of some catalytic function, whereas an alanine substitution is no longer subject to iron-induced hydroxyl radical cleavage. Our results support a model in which the RAG1 protein contains the active site of the V(D)J recombinase and are interpreted in light of predictions about the structure of RAG1.     

New concepts in the regulation of an ancient reaction: transposition by RAG1/RAG2

Summary: The lymphoid‐specific factors, recombination‐activating gene 1 (RAG1) and RAG2, initiate V(D)J recombination by introducing DNA double‐stand breaks at specific sites in the genome. In addition to this critical endonuclease activity, the RAG proteins catalyze other chemical reactions that can affect the outcome of V(D)J recombination, one of which is transposition. While the transposition activity of the RAG proteins is thought to have been critical for the evolution of modern antigen‐receptor loci, it has also been proposed to contribute to chromosomal translocations and lymphoid malignancy. A major challenge has been to determine how the transposition activity of the RAG proteins is regulated in vivo. Although a variety of mechanisms have been suggested by recent studies, a clear resolution of this issue remains elusive.     

RAG1 and RAG2 in V(D)J recombination and transposition

RAG1 and RAG2 are the key components of the V(D)J recombinase machinery that catalyses the somatic gene rearrangements of antigen receptor genes during lymphocyte development. In the first step of V(D)J recombination--DNA cleavage--the RAG proteins act together as an endonuclease to excise the DNA between two individual gene segments. They are also thought to be involved in the subsequent DNA joining step. In vitro, the RAG proteins catalyze the integration of the excised DNA element into target DNA completing a process similar to bacterial transposition. In vivo, this reaction is suppressed by an unknown mechanism. The individual roles of RAG1 and RAG2 in V(D)J recombination and transposition reactions are discussed based on mutation analyses and structure predictions.     

Phenotypical heterogeneity in RAG-deficient patients from a highly consanguineous population

Mutations affecting recombination activation genes RAG1 and RAG2 are associated with variable phenotypes, depending on the residual recombinase activity. The aim of this study is to describe a variety of clinical phenotypes in RAG-deficient patients from the highly consanguineous Egyptian population. Thirty-one patients with RAG mutations (from 28 families) were included from 2013 to 2017. On the basis of clinical, immunological and genetic data, patients were subdivided into three groups; classical T^–B^– severe combined immunodeficiency (SCID), Omenn syndrome (OS) and atypical SCID. Nineteen patients presented with typical T^–B^–SCID; among these, five patients carried a homozygous RAG2 mutation G35V and five others carried two homozygous RAG2 mutations (T215I and R229Q) that were detected together. Four novel mutations were reported in the T^–B^–SCID group; three in RAG1 (A565P, N591Pfs*14 and K621E) and one in RAG2 (F29S). Seven patients presented with OS and a novel RAG2 mutation (C419W) was documented in one patient. The atypical SCID group comprised five patients. Two had normal B cell counts; one had a previously undescribed RAG2 mutation (V327D). The other three patients presented with autoimmune cytopaenias and features of combined immunodeficiency and were diagnosed at a relatively late age and with a substantial diagnostic delay; one patient had a novel RAG1 mutation (C335R). PID disorders are frequent among Egyptian children because of the high consanguinity. RAG mutations stand behind several variable phenotypes, including classical SCID, OS, atypical SCID with autoimmunity and T^–B⁺ CID.     

The bounty of RAGs: recombination signal complexes and reaction outcomes

Summary: V(D)J recombination is a form of site‐specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell‐specific proteins, recombination activating genes RAG‐1 and RAG‐2, and ubiquitously expressed architectural DNA‐binding proteins and DNA‐repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein‐DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence‐specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein‐DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post‐cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein‐DNA complexes that support them, and factors and forces that control them.     

Asymmetric DDE (D35E)-like sequences in the RAG proteins: implications for V(D)J recombination and retroviral pathogenesis

Experimental evidence suggests that the mechanism of vertebrate V(D)J recombination catalyzed by the vertebrate RAG proteins is similar to both retroviral integration and the transposition of IS630/Tc1-family transposons. The mechanism of both retroviral integration and IS630/Tc1 element transposition is well characterized and utilizes a functional metal ion binding site termed the DDE (or D35E) motif. We have previously identified a DDE-like region in the RAG-2 protein and a similar region within the RAG-1 protein. In this work, we propose that interference between DDE-like regions in the RAG proteins and the DDE-site of the HIV integrase may be a mechanism of retroviral pathogenesis in cells in which both the RAG proteins and retroviral integrase are co-expressed.     

Role of recombination activating genes in the generation of antigen receptor diversity and beyond

V(D)J recombination is the process by which antibody and T‐cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non‐homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence‐specific nuclease and its pathological role as a structure‐specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence‐specific and structure‐specific nuclease. It also deals with the off‐target cleavage of RAGs and its implications in genomic instability.     

The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

The enormous repertoire of the vertebrate specific immune system relies on the rearrangement of discrete gene segments into intact antigen receptor genes during the early stages of B-and T-cell development. This V(D)J recombination is initiated by a lymphoid-specific recombinase comprising the RAG1 and RAG2 proteins, which introduces double-strand breaks in the DNA adjacent to the coding segments. Much of the biochemical research into V(D)J recombination has focused on truncated or “core” fragments of RAG1 and RAG2, which lack approximately one third of the amino acids from each. However, genetic analyses of SCID and Omenn syndrome patients indicate that residues outside the cores are essential to normal immune development. This is in agreement with the striking degree of conservation across all vertebrate classes in certain non-core domains. Work from multiple laboratories has shed light on activities resident within these domains, including ubiquitin ligase activity and KPNA1 binding by the RING finger domain of RAG1 and the recognition of specific chromatin modifications as well as phosphoinositide binding by the PHD module of RAG2. In addition, elements outside of the cores are necessary for regulated protein expression and turnover. Here the current state of knowledge is reviewed regarding the non-core regions of RAG1 and RAG2 and how these findings contribute to our broader understanding of recombination.     

An immunodeficiency disease with RAG mutations and granulomas

We describe three unrelated girls who had an immunodeficiency disease with granulomas in the skin, mucous membranes, and internal organs. All three girls had severe complications after viral infections, including B-cell lymphoma associated with Epstein-Barr virus (EBV). Other findings were hypogammaglobulinemia, a diminished number of T and B cells, and sparse thymic tissue on ultrasonography. Molecular analysis revealed that the patients were compound heterozygotes for mutations in recombination activating gene 1 or 2 (RAG1 or RAG2). In each case, both parents were heterozygous carriers of a RAG mutation. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal activity). The parents and one sibling in the three families were healthy.