Radiosensitive SCID patients with Artemis gene mutations show a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint in bone marrow

Severe combined immunodeficiency disease (SCID) can be immunologically classified by the absence or presence of T, B, and natural killer (NK) cells. About 30% of T(-)B(-)NK(+) SCID patients carry mutations in the recombination activating genes (RAG). Some T(-)B(-)NK(+) SCID patients without RAG gene mutations are sensitive to ionizing radiation, and several of these radiosensitive (RS) SCID patients were recently shown to have large deletions or truncation mutations in the Artemis gene, implying a role for Artemis in DNA double-strand break (dsb) repair. We identified 5 RS-SCID patients without RAG gene mutations, 4 of them with Artemis gene mutations. One patient had a large genomic deletion, but the other 3 patients carried simple missense mutations in conserved amino acid residues in the SNM1 homology domain of the Artemis protein. Extrachromosomal V(D)J recombination assays showed normal and precise signal joint formation, but inefficient coding joint formation in fibroblasts of these patients, which could be complemented by the wild-type Artemis gene. The cells containing the missense mutations in the SNM1 homology domain had the same recombination phenotype as the cells with the large deletion, indicating that these amino acid residues are indispensable for Artemis function. Immunogenotyping and immunophenotyping of bone marrow samples of 2 RS-SCID patients showed the absence of complete V(H)-J(H) gene rearrangements and consequently a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint-that is, at the transition from CyIgmu(-) pre-B-I cells to CyIgmu(+) pre-B-II cells. The completeness of this arrest illustrates the importance of Artemis at this stage of lymphoid differentiation.     

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.     

Oligoclonal expansion of T lymphocytes with multiple second-site mutations leads to Omenn syndrome in a patient with RAG1-deficient severe combined immunodeficiency

Omenn syndrome (OS) is a rare primary immunodeficiency characterized by the presence of activated/oligoclonal T cells, eosinophilia, and the absence of circulating B cells. OS patients carry leaky mutations of recombination activating genes (RAG1 or RAG2) resulting in partial V(D)J recombination activity, whereas null mutations cause severe combined immunodeficiency with absence of mature T and B cells (T-B- SCID). Here we describe somatic mosaicism due to multiple second-site mutations in a patient with RAG1 deficiency. We found that he is homozygous for a single base deletion in the RAG1 gene, which results in frameshift and likely abrogates the protein function. However, the patient showed typical OS features. Molecular analysis revealed that several second-site mutations, all of which restored the RAG1 reading frame and resulted in missense mutations, were demonstrated in his T cells. These findings suggest that his revertant T-cell mosaicism is responsible for OS phenotype switched from T-B- SCID.     

Identification of anti-herpes simplex virus antibody-producing B cells in a patient with an atypical RAG1 immunodeficiency

Mutations of the RAG1 or RAG2 protein that eliminate their recombination activity result in T-B-severe combined immunodeficiency (SCID), whereas mutations retaining partial recombination activity lead to Omenn syndrome, a peculiar SCID characterized by increased host T cells and absence of circulating B cells. The prognosis of this disease is fatal, unless hematopoietic stem cell transplantation is performed. This study reports a case of atypical SCID, carrying RAG1 mutations. The patient survived for 6 years without hematopoietic stem cell transplantation. The missense mutation, tested by in vivo recombination assay, revealed residual recombination activity. By the age of 5 years, the patient developed host B cells, but not T cells, possibly due to engrafted maternal T cells. In addition, the host B cells were able to produce antibodies, including anti-herpes simplex virus-antibodies. The fact that host B cells could produce antibodies in this patient could explain not only the mild phenotype observed but also, at least in part, how patients with Omenn syndrome produce immunoglobulin E and sometimes immunoglobulin M, as the same missense mutation of RAG1 gene has been reported in a patient with Omenn syndrome.     

Lack of iNKT cells in patients with combined immune deficiency due to hypomorphic RAG mutations

Hypomorphic mutations of the RAG genes in humans are associated with a spectrum of clinical and immunologic presentations that range from T(-) B(-) severe combined immune deficiency (SCID) to Omenn syndrome. In most cases, residual V(D)J recombination activity allows for development of few T-cell clones, which expand in the periphery and infiltrate target organs, resulting in tissue damage. Invariant natural killer T (iNKT) cells play an important immunoregulatory role and have been associated with protection against autoimmunity. We now report on 5 unrelated cases of combined immune deficiency due to hypomorphic RAG mutations, and demonstrate the absence of iNKT cells in all 5 patients. These findings suggest that lack of this important immunoregulatory cell population may contribute to the pathophysiology of Omenn syndrome.     

Recombinase activating gene enzymes of lymphocytes

Expression of T-cell receptor and surface immunoglobulins on T and B lymphocytes, respectively, is strictly dependent on the variable, (diversity) joining exon (V(D)J) recombination process, which is initiated by the lymphoid-specific recombinase activating gene proteins 1 and 2 (RAG1 and RAG2). Recent advances have highlighted the functional organization of the RAG1 and RAG2 proteins and have provided important information on the regulation of RAG gene expression. Depending on the severity of their effects on the V(D)J recombination process, mutations of the RAG genes account for a spectrum of combined immune deficiencies in humans.     

N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements

The proteins encoded by RAG1 and RAG2 can initiate gene recombination by site-specific cleavage of DNA in immunoglobulin and T-cell receptor (TCR) loci. We identified a new homozygous RAG1 gene mutation (631delT) that leads to a premature stop codon in the 5' part of the RAG1 gene. The patient carrying this 631delT RAG1 gene mutation died at the age of 5 weeks from an Omenn syndrome-like T(+)/B(- )severe combined immunodeficiency disease. The high number of blood T-lymphocytes (55 x 10(6)/mL) showed an almost polyclonal TCR gene rearrangement repertoire not of maternal origin. In contrast, B-lymphocytes and immunoglobulin gene rearrangements were hardly detectable. We showed that the 631delT RAG1 gene can give rise to an N-terminal truncated RAG1 protein, using an internal AUG codon as the translation start site. Consistent with the V(D)J recombination in T cells, this N-terminal truncated RAG1 protein was active in a plasmid V(D)J recombination assay. Apparently, the N-terminal truncated RAG1 protein can recombine TCR genes but not immunoglobulin genes. We conclude that the N-terminus of the RAG1 protein is specifically involved in immunoglobulin gene rearrangements.     

Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes

The nature of Hodgkin and Reed-Sternberg (HRS) cells remains in question. Immunophenotypic studies favor a relation to the lymphoid lineage with the existence of B- and T-cell types. However, studies on the detection of antigen (Ag) receptor gene rearrangements provided inconsistent results. They concur in that rearranged Ig and T-cell receptor (TCR) genes are not demonstrable in most Hodgkin's disease (HD) cases. To clarify whether this is because of the insensitivity of the method of detection or a real absence of clonal Ig heavy chain (IgH) rearrangements, a polymerase chain reaction (PCR) method with high sensitivity was applied, allowing the detection of less than 50 cells with clonally rearranged IgH genes in a mixture of 100,000 germline or individually rearranged cells. In 67 cases of HD, most of those (67%) with B-Ag+ HRS cells express clonal VDJ rearrangements of the IgH gene. No cases with T-cell Ag+ HRS cells harbored detectable clonal VDJ rearrangements. Of 10 sequenced rearranged IgH genes, the VH segment of six contained considerable somatic mutations. These results suggest that the demonstrated VDJ rearrangements stem from the HRS cells themselves and that the HRS cells of cases with rearranged IgH genes are B-cell related and correspond in their differentiation stage either to naive pregerminal center B cells or (more commonly) to germinal center/postgerminal center-derived memory B cells.     

Omenn syndrome due to ARTEMIS mutations

Omenn syndrome (OS) is characterized by severe combined immunodeficiency (SCID) associated with erythrodermia, hepatosplenomegaly, lymphadenopathy, and alopecia. In patients with OS, B cells are mostly absent, T-cell counts are normal to elevated, and T cells are frequently activated and express a restricted T-cell receptor (TCR) repertoire. Thus far, inherited hypomorphic mutations of the recombination activating genes 1 and 2 (RAG1/2) have been described in OS. We report on a first patient with clinical and immunologic features of OS caused by hypomorphic ARTEMIS mutations. The patient's T cells expressed alpha/beta receptors with an oligoclonal repertoire but normal V(D)J recombination coding joints. Sequencing of the ARTEMIS gene revealed a compound heterozygosity in this nonhomologous end-joining (NHEJ) factor, explaining the enhanced radiosensitivity of the patient's primary dermal fibroblasts. The maternal allele contained a null mutation within the active center, whereas the expression of the paternal allele with a start codon (AUG to ACG) mutation partially restored V(D)J recombination and ARTEMIS function in vivo and in vitro.     

Omenn syndrome--review of several phenotypes of Omenn syndrome and RAG1/RAG2 mutations in Japan.

Omenn syndrome (OS) is a form of severe combined immunodeficiency (SCID) characterized by erythrodermia, hepatosplenomegaly, lymphadenopathy, and alopecia. In patients with OS, B cells are mostly absent, T-cell counts are normal to elevated, and T cells are frequently activated and express a restricted T-cell receptor (TCR) repertoire. Thus far, inherited hypomorphic mutations of the recombination activating genes either 1 or 2 (RAG1/2) have been detected in most OS patients. We have recently experienced a rare case of OS showing the revertant mosaicism due to multiple second-site mutations leading to typical OS clinical features with RAG1-deficient SCID. In this review, we will focus on the variation of several phenotypes of OS.     

Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus

Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a "single-cell" model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Igkappa gene (IGK) rearrangements, followed by IGK deletions and Iglambda gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified. This might be explained by the fact that regulation of the ordered recombination process is not fully strict, particularly when the IGL locus is involved. Unfavorable somatic mutations followed by receptor editing might have contributed to this finding. Eight of these 10 cases indeed contained somatic mutations. In cases with 2 functional Ig light chain rearrangements, both alleles were transcribed, but monospecific Ig expression was still maintained. This suggests that in these cases allelelic exclusion is not regulated at the messenger RNA level but either at the level of translation or protein stability or via preferential pairing of Ig light and Ig heavy chains. Nevertheless, ordered rearrangement processes are the main determinant for monospecific Ig light chain expression.     

Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome

Omenn syndrome (OS) is an inherited disorder characterized by an absence of circulating B cells and an infiltration of the skin and the intestine by activated oligoclonal T lymphocytes, indicating that a profound defect in the lymphoid developmental program could be accountable for this condition. Inherited mutations in either the recombination activating genes RAG1 or RAG2, resulting in partial V(D)J recombinase activity, were shown to be responsible for OS. This study reports on the characterization of new RAG1/2 gene mutations in a series of 9 patients with OS. Given the occurrence of the same mutations in patients with T-B-severe combined immune deficiency or OS on 3 separate occasions, the proposal is made that an additional factor may be required in certain circumstances for the development of the Omenn phenotype. The nature of this factor is discussed.     

The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatin-dependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.     

Monocytoid B cells are distinct from splenic marginal zone cells and commonly derive from unmutated naive B cells and less frequently from postgerminal center B cells by polyclonal transformation.

Monocytoid B cells represent a morphologically conspicuous B-cell population that constantly occurs in Toxoplasma gondii-induced Piringer's lymphadenopathy. Although widely believed to be closely related to splenic marginal zone B cells, neither this relationship, nor the B-cell differentiation stage of monocytoid B cells, nor their cellular precursors have been established. We have therefore examined monocytoid B cells for their expression of B-cell differentiation markers and the Ig isotypes at the RNA and protein level as well as for rearranged Ig heavy chain (H) genes and somatic mutations within the variable (V) region. The results obtained were compared with the corresponding features of other B-cell populations. The monocytoid B cells displayed immunophenotypical differences to all other B-cell populations. IgM and IgD expression was absent from most monocytoid B cells at the RNA and protein levels. Unrelated (polyclonal) Ig rearrangements were found in 85 of the 95 cells studied. Seventy-four percent of the rearranged VH genes were devoid of somatic mutations, whereas the remaining 26% carried a low number of somatic mutations. The majority of these showed no significant signs of antigen selection. This finding in conjunction with the predominantly unrelated Ig gene rearrangements indicates that most monocytoid B cells arise not by clonal proliferation but by transformation of polyclonal B cells. The B cells undergoing a monocytoid B-cell transformation are in the majority (74%) naive B cells, and only a minority are (26%) non-antigen-selected postgerminal center B cells. Thus, our data show that monocytoid B cells represent a distinct B-cell subpopulation.