Human DNA-dependent protein kinase catalytic subunit deficiency: a comprehensive review and update.

BACKGROUND: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has an essential role in the non-homologous end-joining pathway that repairs DNA double-strand breaks in V(D)J recombination involved in the expression of T- and B-cell receptors. Whereas homozygous mutations in PRKDC define the scid mouse, a model that has been widely used in biology, human mutations in PRKDC are extremely rare and the disease spectrum has not been described so far. OBJECTIVE: To provide an update on the genetics, clinical spectrum, immunological profile, and therapy of DNA-PKcs deficiency in human. METHODS: The clinical, biological, and treatment data from the 6 cases published to date and from 1 new patient were obtained and analyzed. Rubella PCR was performed on available granuloma material. RESULTS: We report on 7 patients; Six patients displayed the autosomal recessive p.L3062R mutation in PRKDC gene encoding DNA-PKcs. Atypical severe combined immunodeficiency with inflammatory lesions, granulomas, and autoimmunity was the predominant clinical manifestation (n=5/7). Rubella viral strain was detected in the granuloma of 1 patient over the 2 tested. T-cell counts, including naïve CD4+CD45RA+ T cells and T-cell function were low at diagnosis for 6 patients. For most patients with available values naïve CD4+CD45RA+ T cells decreased over time (n=5/6). Hematopoietic stem cell transplantation (HSCT) was performed in 5 patients, of whom 4 are still alive without transplant-related morbidity. Sustained T- and B-cell reconstitution was respectively observed for 4 and 3 patients, after a median follow-up of 8 years (range 3-16 y). CONCLUSION: DNA-PKcs deficiency mainly manifests as an inflammatory disease with granuloma and autoimmune features, along with severe infections.

Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells.

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

V(D)J and immunoglobulin class switch recombinations: a paradigm to study the regulation of DNA end-joining.

The immune system is the site of intense DNA damage/modification, which occur during the development and maturation of B and T lymphocytes. V(D)J recombination is initiated by the Rag1 and Rag2 proteins and the formation of a DNA double-strand break (DNA dsb). This DNA lesion is repaired through the use of the non-homologous end-joining (NHEJ) pathway, several factors of which have been identified through the survey of immunodeficient conditions in humans and mice. Upon antigenic recognition in secondary lymphoid organs, mature B cells further diversify their repertoire through class switch recombination (CSR). CSR is a region-specific rearrangement process triggered by the activation-induced cytidine deaminase factor and also proceeds through the introduction of DNA dsb. However, unlike V(D)J recombination, CSR does not rely strictly on NHEJ for the repair of the DNA lesion. Instead, CSR, but not V(D)J recombination, requires the major factors of the DNA damage response. V(D)J recombination and CSR thus represent an interesting paradigm to study the regulation among the various DNA repair pathways.

A case report of a patient with microcephaly, facial dysmorphism, chromosomal radiosensitivity and telomere length alterations closely resembling "Nijmegen breakage syndrome" phenotype.

Genetic heterogeneity in Nijmegen breakage syndrome (NBS) is highlighted by patients showing clinical and cellular features of NBS but with no mutations in NBS1 and normal levels of nibrin. NBS is an autosomal recessive disorder, whose clinical cellular signs include growth and developmental defects, dysmorphic facies, immunodeficiency, cancer predisposition, chromosomal instability and radiosensitivity. NBS is caused by mutations in the NBS1 gene, whose product is part of the MRE11/RAD50/NBS1 complex involved in the DNA double-strand break (DSB) response pathway. Since the identification of the NBS1 gene, patients with NBS clinical signs, particularly severe congenital microcephaly, are screened for mutations in the NBS1 gene. Further analyses include X-ray-induced chromosome aberrations, telomere analysis, kinetics of DSBs repair, levels of a panel of proteins involved in the maintenance of genetic stability, radiation-induced phosphorylation of various substrates and cell cycle analysis. We describe a patient with a NBS clinical phenotype, chromosomal sensitivity to X-rays but without mutations in the whole NBS1 or in the Cernunnos gene. Enhanced response to irradiation was mediated neither by DSBs rejoining defects nor by the NBS/AT-dependent DNA-damage response pathway. Notably, we found that primary fibroblasts from this patient displayed telomere length alterations. Cross-talk between pathways controlling response to DSBs and those involved in maintaining telomeres has been shown in the present patient. Dissecting the cellular phenotype of radiosensitive NBS-like patients represents a useful tool for the research of new genes involved in the cellular response to DSBs.

T early alpha (TEA) regulates initial TCRVAJA rearrangements and leads to TCRJA coincidence.

Both TCRA alleles are rearranged in mature T lymphocytes, as a result of a lack of allelic exclusion at the TRCA locus. We show in a series of T cell clones that the two TCRJA segments are not randomly, but rather coincidentally, rearranged in a given T cell. The TCRJA coincidence relies, in part, on the presence of "T early alpha" (TEA), a cis-regulatory genetic element located upstream of the TCRJA cluster. TEA promotes specific recombinational accessibility that targets primary TCRVAJA rearrangements on the 5' side of the TCRA locus. In a model of multiple waves of TCRVAJA recombination, this cis-regulatory effect of TEA allows for the scanning of the entire TCRJA cluster, thereby increasing the TCR alpha/beta diversity potential.

Gene therapy of severe combined immunodeficiencies.

Recent advances in gene transfer in human hematopoietic cells, combined with a better understanding of the genetic aspects of several immunodeficiencies, has offered new opportunities in the domain of gene therapy. Severe combined immunodeficiency (SCID) appear to represent a good model for the application of gene therapy, combining an expected selective advantage for transduced cells, an absence of immunological response to the vector and/or the therapeutic transgene, together with accessibility to hematopoietic stem cells (HSC). Ex vivo retroviral transduction of a therapeutic transgene in HSC prior to transplantation appears to be a particularly effective and long-lasting means of restoring the expression of a mutated gene in the lymphoid lineage. Furthermore, encouraging therapeutic benefits as a result of a gene therapy protocol for the treatment of X-linked severe combined immunodeficiencies (SCID-X1) invites many questions as to the reasons for this therapeutic benefit. This review outlines the results that have been achieved in gene therapy for SCID-X1, ADA-SCID as well as other types of SCID, and discusses the possible relationship between the physiopathology of each disease and the success of relevant trials.

Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency.

The V(D)J recombination process insures the somatic diversification of immunoglobulin and antigen T cell receptor encoding genes. This reaction is initiated by a DNA double-strand break (dsb), which is resolved by the ubiquitously expressed DNA repair machinery. Human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID) is characterized by a defect in the V(D)J recombination leading to an early arrest of both B and T cell maturation. We previously mapped the disease-related locus to the short arm of chromosome 10. We herein describe the cloning of the gene encoding a novel protein involved in V(D)J recombination/DNA repair, Artemis, whose mutations cause human RS-SCID. Protein sequence analysis strongly suggests that Artemis belongs to the metallo-beta-lactamase superfamily.

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.

Three-dimensional clustering of human RAG2 gene mutations in severe combined immune deficiency.

The V(D)J recombination, which leads to the somatic rearrangement of variable, diversity, and joining segments, is the mechanism accountable for the diversity of T cell receptor- and Ig-encoding genes. The products of the RAG1 and RAG2 genes are the lymphoid-specific factors responsible for the initiation of the V(D)J recombination through the generation of a DNA double strand break. RAG1 or RAG2 gene inactivation in the mouse leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and, ultimately, to severe combined immune deficiency (SCID). A human SCID condition is also characterized by an absence of mature T and B lymphocytes and is associated with mutations in either RAG1- or RAG2-encoding genes. Based on the predicted beta-propeller three-dimensional structure model for RAG2, we found that six out of the seven mutations described to date in T-B-SCID patients are clustered on one side of the propeller, in regions exposed to solvent. This finding reinforces the biological significance of this predicted model and suggests that RAG1 interacts with RAG2 on one of the side of the scaffold formed by the beta-propeller.

A new gene involved in DNA double-strand break repair and V(D)J recombination is located on human chromosome 10p.

V(D)J recombination, accountable for the diversity of T cell receptor- and immunoglobulin-encoding genes, is initiated by a lymphoid-specific DNA double-strand break. The general DNA repair machinery is responsible for the resolution of this break. Any defect in one of the known components of the DNA repair/V(D)J recombination machinery (Ku70, Ku80, DNA-PKcs, XRCC4 and DNA ligase IV) leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and ultimately to severe combined immune deficiency (SCID) in several animal models. A human SCID condition is also characterized by an absence of mature T and B lymphocytes, and is associated with an increase in sensitivity to DNA-damaging agents (RS-SCID). None of the above-mentioned genes are defective in these patients, arguing for the likelihood of the existence of yet another unknown component of the V(D)J recombination/DNA repair apparatus. Athabascan-speaking (SCIDA) Navajo and Apache Native Americans have a very high incidence of T(-)B(-)SCID. The SCIDA locus is highly linked with markers on chromosome 10p, although the exact molecular defect has not been recognized in these patients. We show here that cells with the SCIDA defect are impaired in the DNA repair phase of V(D)J recombination similarly to RS-SCID, precisely an absence of V(D)J coding joint formation. Moreover, genotyping analysis in several RS-SCID families corroborates a linkage of the RS-SCID locus to the SCIDA region on chromosome 10p. These results demonstrate the presence of a new essential DNA repair/V(D)J recombination gene in this region, the mutation of which causes RS-SCID in humans.

[Gene therapy for immune deficiencies]. / Thérapie génique des déficits immunitaires.

Gene therapy offers an attractive option to the most severe forms of primary immunodeficiency diseases. Identification of disease associated genes as well as advances in the technology of gene transfer into hematopoietic progenitor cells have set the basis for the first clinical trials. Settings characterized by the potential for a selective advantage provided to transduced cells are the first diseases to target. The recent example of successful treatment of Severe Combined Immunodeficiency-X1 (gamma c deficiency) illustrates this potential.

RORgammaT, a thymus-specific isoform of the orphan nuclear receptor RORgamma / TOR, is up-regulated by signaling through the pre-T cell receptor and binds to the TEA promoter.

TEA (T early alpha) is a genetic element located upstream of the TCR-Jalpha cluster. Thymocytes from mice carrying a targeted deletion of TEA do not rearrange their TCRalpha locus on a window spanning the first nine Jalpha segments. This led us to the hypothesis of TEA having a "rearrangement focusing" activity on the 5' side of the TCR-Jalpha region. We analyzed DNAseI and "phylogenetic" footprints within the TEA promoter in an attempt to identify trans-acting factors that could account for its regulatory function on DNA accessibility. One of these footprints corresponded to a putative DNA-binding site for an orphan nuclear receptor of the ROR / RZR family. The RORgammaT cDNA clone was isolated from a thymus library using a probe corresponding to the DNA-binding domain of RORgamma / TOR. RORgammaT is a thymus-specific isoform of RORgamma, expressed almost exclusively in immature double-positive thymocytes. RORgammaT binds, to the TEA promoter in vitro. Lastly, the expression of RORgammaT is stimulated in two situations that mimic activation through the pre-TCR and in which the thymocytes have their TCR-alpha locus in an "open", yet unrearranged DNA configuration. We propose that the expression of RORgammaT may be part of the pre-TCR activation cascade leading to the maturation of alpha / beta T cells and may participate in the regulation of DNA accessibility in the TCR-Jalpha locus.

Lymphoproliferative syndrome with autoimmunity: A possible genetic basis for dominant expression of the clinical manifestations.

Fas (CD95/Apo-1) mutations were previously reported as the genetic defect responsible for human lymphoproliferative syndrome associated with autoimmune manifestations (also known as autoimmune lymphoproliferative syndrome or Canale-Smith syndrome). We have identified 14 new heterozygous Fas mutations. Analysis of patients and families allow us to further dissect this syndrome with regards to the relationship between Fas mutations, inheritance pattern, and phenotype as observed on long-term follow-up. In vitro studies show that lymphocytes from all Fas mutant carriers exhibit a Fas-antibody-induced apoptosis defect. However, among the 8 inherited mutations, 4 of 4 Fas missense mutations were associated with high clinical penetrance, whereas 3 of 4 mutations leading to a truncated Fas product were associated with variable clinical penetrance. This suggests that a second defect, in another yet undefined factor involved in apoptosis and/or lymphoproliferation control, is necessary to induce full clinical expression of the disease. These results also indicate that the currently available antibody-mediated in vitro apoptosis assay does not necessarily reflect the in vivo ability of abnormal Fas molecules to trigger lymphocyte death. In addition, we found that lymphoproliferative manifestations resolved with age, whereas immunological disorders [ie, hypergammaglobulinemia and detection of TcR alphabeta(+) CD4(-) CD8(-) lymphocytes] persisted. This observation suggests that Fas-mediated apoptosis plays a more important role in lymphocyte homeostasis in early childhood than later on in life.

Combined immunodeficiency associated with increased apoptosis of lymphocytes and radiosensitivity fibroblasts.

Severe immunodeficiency characterized by lymphopenia was found in two siblings, one of whom was examined in detail. The calcium flux, pattern of tyrosine phosphorylation of proteins, and interleukin 2 (IL-2) production and proliferation in response to mitogens suggested that the peripheral blood T cells activated normally. The peripheral blood T cells were shown to have an activated phenotype with increased expression of CD45RO+ and CD95/Fas. Increased spontaneous apoptosis occurred in unstimulated lymphocyte cultures. The elevated apoptosis was not due to alterations in expression or to mutations in Bcl-2, Bcl-X(L), or Flip, nor could the spontaneous apoptosis be prevented by blocking Fas, suggesting that it was independent of Fas signaling. This is the first inherited combined immunodeficiency associated with impaired lymphocyte survival. Fibroblasts derived from the patient showed appreciable radiosensitivity in clonal assays, but apoptosis was not elevated. Our results show that the fibroblasts represent a new radiosensitive phenotype not associated with cell cycle checkpoint defects, V(D)J recombination defects, or elevated chromosome breakage. We suggest that the affected gene plays a role in an undetermined damage response mechanism that results in elevated spontaneous apoptosis in lymphoid cells and radiosensitivity in fibroblasts.

A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency.

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku-DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination.

Highly restricted human T cell repertoire in peripheral blood and tissue-infiltrating lymphocytes in Omenn's syndrome.

Omenn's syndrome is an inherited human combined immunodeficiency condition characterized by the presence of a large population of activated and tissue-infiltrating T cells. Analysis of the TCRB repertoire revealed a highly restricted TCRBV usage in three patients. More strikingly, T cell clones from the three patients expressed TCRB chains with VDJ junction similarities, suggesting a common antigenic specificity. Analysis of the TCRA repertoire in one patient also revealed a restricted TCRAV usage. Finally, analysis of the TCRBV repertoire of tissue-infiltrating T cells in one patient suggested nonrandom tissue migration. These results suggest that the oligoclonal expansion of T cells observed in Omenn's syndrome could be the consequence of autoimmune proliferation generated by a profound defect in lymphocyte development.

Activation induces apoptosis in Herpesvirus saimiri-transformed T cells independent of CD95 (Fas, APO-1).

Signaling via the T cell receptor (TCR)/CD3 complex of pre-activated T cells induces apoptosis. Such an activation-induced cell death (AICD) is thought to play an important role in the regulation of cellular immune responses. In this study we analyzed pathways of AICD by using human T cells transformed by Herpesvirus saimiri. These growth-transformed T cells show the phenotype of activated mature T cells and continue to express a functionally intact TCR. We show that human H. saimiri-transformed T cell clones readily undergo cell death upon signaling via the TCR/CD3 complex or via phorbol 12-myristate 13-acetate (PMA) + ionomycin. The AICD in H. saimiri-transformed T cells was detectable a few hours after activation and it was not affected by the presence of interleukin (IL)-2 or by anti-CD4 cross-linking. However, AICD required tyrosine phosphorylation, since it could be blocked by herbimycin A. Cyclosporin A (CsA) did not block the development of AICD, but other consequences of activation in H. saimiri-transformed T cells like the production of interferon-gamma. Surprisingly, the development of AICD was not reduced by neutralizing antibodies to tumor necrosis factor (TNF)-alpha or blocking antibodies directed to CD95 (Fas, APO-1), although H. saimiri-transformed T cells were sensitive to CD95 ligation. To confirm that this form of AICD is really independent of CD95, we have established an H. saimiri-transformed T cell line from a patient with a homozygous deletion in the CD95 gene. This CD95-deficient T cell line was as sensitive to AICD as other CD95-expressing H. saimiri-transformed T cells. In conclusion, we describe here a type of AICD in H. saimiri-transformed T cells that is independent of CD95 and TNF-alpha, not sensitive to CsA, but requires tyrosine phosphorylation. This system should be useful for the investigation of CD95-independent forms of AICD.