Yu Nee Lee

Hypomorphic Rag mutations can cause destructive midline granulomatous disease

Destructive midline granulomatous disease characterized by necrotizing granulomas of the head and neck is most commonly caused by Wegener granulomatosis, natural killer/T-cell lymphomas, cocaine abuse, or infections. An adolescent patient with myasthenia gravis treated with thymectomy subsequently developed extensive granulomatous destruction of midface structures, palate, nasal septum, airways, and epiglottis. His lymphocyte numbers, total immunoglobulin G level, and T-cell receptor (TCR) repertoire appeared normal. Sequencing of Recombination activating gene-1 (Rag1) showed compound heterozygous Rag1 mutations; a novel deletion with no recombinase activity and a missense mutation resulting in 50% Rag activity. His thymus was dysplastic and, although not depleted of T cells, showed a notable absence of autoimmune regulator (AIRE) and Foxp3(+) regulatory T cells. This distinct Rag-deficient phenotype characterized by immune dysregulation with granulomatous hyperinflammation and autoimmunity, with relatively normal T and B lymphocyte numbers and a diverse TCR repertoire expands the spectrum of presentation in Rag deficiency. This study was registered at www.clinicaltrials.gov as #NCT00128973.

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Timely and spatially regulated maturation of B and T cell repertoire during human fetal development

Immunocompetence in the developing fetus is temporally and spatially regulated. Developing Immunity The adaptive immune response plays a critical role in protecting the body from both foreign pathogens and internal dangers such as cancer. However, little is known about how the immune system develops during human gestation. Rechavi et al. analyzed differences in B and T lymphocyte ontogeny from 12 to 26 weeks of gestational age. They found that B cell development precedes T cell development and that repertoire maturation is both temporally and spatially regulated. These data can be used as a baseline to improve immune function in developing fetuses and to assess the effects of therapeutic interventions. Insights into the ontogeny of the human fetal adaptive immune system are of great value for understanding immunocompetence of the developing fetus. However, to date, this has remained largely uncharted territory, in large part because blood samples from healthy, early gestation fetuses have been hard to come by. In a comprehensive study, we analyzed levels of T cell receptor excision circles (TRECs), signal-joint κ receptor excision circles (sjKRECs), and intron recombination signal sequence–K-deleting element (iRSS-Kde) rearrangement, and T and B lymphocyte repertoire clonality in human fetuses from 12 to 26 weeks of gestational age. Using next-generation sequencing, we analyzed the diversity and complexity of T cell receptor β (TRB) and immunoglobulin heavy chain (IGH) repertoires in four fetuses at 12, 13, 22, and 26 weeks of gestation and in healthy full-term infants. We report the progressive increase of TREC, sjKREC, and iRSS-Kde levels over time and confirm that B cell development precedes T cell development in the human fetus. Temporally and spatially regulated maturation of B and T cell repertoire diversity and complexity during human fetal development was observed, including evidence that immunoglobulin somatic hypermutation and class switch recombination occur already during intrauterine life. Our results help define physiological levels of immunodeficiency in premature infants and may serve as a reference for future studies aimed at investigating the impact of intrauterine pathologies on fetal immune development and function.

Human RAG mutations: biochemistry and clinical implications

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.

Identification of Patients with RAG Mutations Previously Diagnosed with Common Variable Immunodeficiency Disorders

PurposeCombined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients.MethodsTwo different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients.ResultsTwo young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection.ConclusionAstute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.

Next Generation Sequencing Reveals Skewing of the T and B Cell Receptor Repertoires in Patients with Wiskott–Aldrich Syndrome

The Wiskott–Aldrich syndrome (WAS) is due to mutations of the WAS gene encoding for the cytoskeletal WAS protein, leading to abnormal downstream signaling from the T cell and B cell antigen receptors (TCR and BCR). We hypothesized that the impaired signaling through the TCR and BCR in WAS would subsequently lead to aberrations in the immune repertoire of WAS patients. Using next generation sequencing (NGS), the T cell receptor β and B cell immunoglobulin heavy chain (IGH) repertoires of eight patients with WAS and six controls were sequenced. Clonal expansions were identified within memory CD4+ cells as well as in total, naïve and memory CD8+ cells from WAS patients. In the B cell compartment, WAS patient IGH repertoires were also clonally expanded and showed skewed usage of IGHV and IGHJ genes, and increased usage of IGHG constant genes, compared with controls. To our knowledge, this is the first study that demonstrates significant abnormalities of the immune repertoire in WAS patients using NGS.

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies.

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-β (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency

BACKGROUND The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation

To the Editor: We would like to share a relevant and interesting immunodeficiency case that would be of interest to the readers of the Journal of Allergy and Clinical Immunology. This report describes an adult-onset idiopathic T-cell lymphopenia due to recombinase activating gene 1 (RAG1) deficiency in an HIV-negative male patient with no recurrent or opportunistic infections presenting at the age of 38 years with chronic dermatitis, pruritus, and hyperkeratosis. Clinical and immunologic examination revealed eosinophilia with modestly elevated IgE (747 kU/L), normal IgG, IgA, and IgM, profound pan–T-cell lymphopenia, high normal total B cells, and slightly reduced natural killer cells. Genetic analysis revealed a heterozygous frameshift mutation in the RAG1 gene, resulting in a truncated RAG1 protein. This is a late clinical presentation of an idiopathic T-cell lymphopenia secondary to a heterozygous hypomorphic RAG1 mutation and has important implications for the considerable phenotypic variability related to molecular defects in genes typically associated with severe combined immunodeficiency or Omenn syndrome (OS). A 38-year-old man presented with significant pruritic skin rash on his legs and eosinophilia that had been present for 2 years with poor resolution using over-the-counter topical treatments. Medical history revealed a healthy childhood and adulthood until 2 years earlier when the skin rash first appeared on the lower extremities. Gross examination of the skin revealed a generalized dermatitis with hyperkeratosis without histologic evidence of granulomas (Fig 1, A). There was also keratoderma of the feet and onychodystrophy. Skin biopsy of the lesions on the leg revealed a chronic dermatitis with eosinophilia, which was negative for IgG, IgM, or IgA with weak, discontinuous granular deposition of C3 along the basement membrane zone. Complete blood cell count with differential revealed significant lymphopenia, which on detailed lymphocyte subset evaluation by flow cytometry revealed profound pan–T-cell lymphopenia (Table I). There was a relative increase in the frequency of activated T cells (CD4+CD25+ and HLA-DR+ CD4 and CD8 T cells). Thymic function was significantly impaired for age, with almost absent CD4 recent thymic emigrants and TREC. Genetic analysis revealed the following heterozygous frameshift mutation (c.256_257delAA, K86VfsX33) in exon 2 of the gene encoding RAG1 that has been previously reported to be associated with SCID and OS, when present as a homozygous or compound heterozygous mutation.1 No other pathogenic genetic variations were found in either the known coding or promoter regions of the RAG1 gene or in any of the following SCID-associated genes: RAG2, JAK3, IL7R, ADA, CD3D, CD3E, DCLRE1C, and IL2RG. Genetic analysis for the above RAG1 mutation was performed not only on DNA extracted from whole blood but also from PBMCs and buccal brushing. In all 3 sample types, the same heterozygous mutation was identified, indicating that this was not a somatic mutation but a germline variation in the RAG1 gene. Genetic testing for this specific RAG1 mutation was performed on PBMCs isolated from whole blood of both parents of this patient, and the father was shown to have the same heterozygous mutation as the patient; however, the father was clinically, phenotypically, and immunologically normal with normal T, B, and natural killer cell counts. A second mutation was not identified in the patient or the mother. The patient has a single older male sibling who was not evaluated but was negative for relevant clinical history. FIG 1 A, Photograph showing skin rash. Diffuse skin rash was present on the extremities and, to a lesser extent, on the trunk of this patient with a heterozygous RAG1 mutation. B, Determination of recombinase activity level of wild-type and mutant RAG1. Stable ... TABLE I Lymphocyte subset quantitation by flow cytometry Further immunologic analysis revealed robust antibody responses to vaccine antigens such as tetanus and diphtheria toxoids, but significantly abnormal lymphocyte proliferation to mitogens (PHA, PWM) and antigens (Candida and tetanus toxoid) and stimulation with soluble anti-CD3. RAG1 function was determined by recombinase activity (Fig 1, B), and the 256–257delAA mutant had only 2.67% ± 0.58% activity as compared with wild-type RAG1. Although overexpression experiments have shown that this mutation can induce the production of an N-terminal–truncated protein by using a second internal ATG1 (which might explain the residual recombination activity), we failed to detect the expression of the mutant RAG1 on retrovirus-mediated transduction of Rag1−/− pro–B cells. Detailed B-cell subset analysis revealed low total memory B cells with low class-switched memory and marginal zone B cells with a significant increase in naive B cells (CD27−) and transitional B cells (CD19+CD24++CD38++). Functional B-cell analysis revealed that the patient’s B cells were capable of plasmablast differentiation on stimulation with CD40L and IL-21 or CpG, though it was decreased than that in a healthy control (data not shown). B-cell proliferation in response to CD40L + IL-21 was lower than in control B cells, but almost comparable to that in control with CpG. Also, the distribution of immunoglobulin light chains (kappa/lambda) was normal, arguing against significant defects of receptor editing of B cells. Therefore, it would appear that patient’s B cells are capable of responding to helper T-cell signals, and the reduced frequency of memory B cells is likely related to the low numbers of such T cells. There was evidence of autoantibody production with high titer of ANA, double-stranded DNA and SS-A antigens in addition to antibodies to 14 other autoantigens. Although there was no clinical evidence of specific autoimmune disease or granulomatous disease and renal function was preserved,2,3 the diffuse skin rash, which was responsive to steroids, suggests that a clear inflammatory process was present (prior to treatment). Molecular T-cell receptor repertoire analysis (T-cell receptor Vb spectratyping) revealed an overall polyclonal T-cell repertoire for most T-cell receptor Vb families with oligoclonality of a few (family 2 was absent and families 3–1, 6–4, 13, and 16 were oligoclonal with limited diversity). There was a significant increase in peripheral levels of IL-7, which is related to the presence of T-cell lymphopenia. Clinically, the patient responded well to wet dressings with ammonium lactate topical (AmLactin), a keratolytic, and treatment with triamcinolone. He was treated with prophylactic antibiotics with no evidence of infection. In a mouse model of a hypomorphic Rag1 variant, progressive T-cell lymphopenia with age was demonstrated,4 suggesting that heterozygosity for RAG defects may lead to premature immune senescence and T-cell dysfunction. This case is unique and exemplifies the magnitude of phenotypic variations that can be seen in molecular defects typically associated with SCID, or hypomorphic mutations in genes that are associated with OS,2 but in this case represents an idiopathic T-cell lymphopenia, adding to the spectrum of previously reported mutations.5 What is remarkable is the apparent adult-onset presentation of eosinophilia, skin rash, and low T-cell counts in this patient with only a single copy of a frameshift variation in the 5′ open reading frame of the RAG1 gene that usually causes an autosomal-recessive disorder. There is evidence that frameshift mutations in the 5′ end of RAG1 can maintain V(D)J recombination activity,1 and this may partially explain the late onset of the phenotype and normal B-cell numbers in the context of T-cell lymphopenia. As seen in the mouse model of hypomorphic Rag1 mutation,4 it is possible that the T-cell lymphopenia is due to an age-related decline in immunocompensatory mechanisms that permit the phenotype to be revealed later than typical. There are several differences between OS and idiopathic T-cell lymphopenia, with the former typically being clinically severe and characterized by elevated eosinophils and IgE along with erythrodermia, hepatosplenomegaly, lymphadenopathy, T-cell counts that are variable but generally oligoclonal in diversity, absent B cells, and normal natural killer cells.6 Patients with OS usually present early in life with recurrent and opportunistic infections, and the clinical course is fulminant and typically managed with hematopoietic cell transplantation. Adult-onset patients with idiopathic T-cell lymphopenia may have an overall milder clinical phenotype and are unlikely to require hematopoietic cell transplantation; nonetheless, these patients require careful monitoring to control significant infections as well as the development of autoimmunity and subsequent end-organ damage. Adult-onset ADA deficiency is the only other SCID-associated defect that has been reported with delayed manifestations in the context of heterozygous mutations in the ADA gene,7 resulting in partial ADA deficiency and a milder phenotype. In the partial ADA deficiency, immune function has been shown to decline over time, resulting in an immunologic and clinical phenotype. This phenomenon appears to be reproduced in this patient with the heterozygous RAG1 mutation. The presence of severe T-cell lymphopenia with dysregulated B-cell responses raises intriguing questions about the impact of hypomorphic mutations on immune function, and further the lack of an immunologic or clinical phenotype in the parent who is a carrier for this mutation suggests that there is an underlying additional genetic, epigenetic, or environmental modulation of RAG1 function in this patient.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.