Jolan E. Walter

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.

Abnormalities of T-cell receptor repertoire in CD4+ regulatory and conventional T cells in patients with RAG mutations: Implications for autoimmunity

Jared H. Rowe, MD, Brian D. Stadinski, PhD, Lauren A. Henderson, MD, Lisa Ott de Bruin, MD, Ottavia Delmonte, MD, Yu Nee Lee, PhD, M. Teresa de la Morena, MD, Rakesh K. Goyal, MD, Anthony Hayward, MD, PhD, Huang Chiung-Hui, PhD, Maria Kanariou, MD, Alejandra King, MD, Taco W. Kuijpers, MD, Jian Yi Soh, MD, Benedicte Neven, MD, PhD, Jolan E. Walter, MD, PhD, Eric S. Huseby, PhD, Luigi D. Notarangelo, MD

Natural killer cells from patients with recombinase-activating gene and non-homologous end joining gene defects comprise a higher frequency of CD56bright NKG2A+++ cells, and yet display increased degranulation and higher perforin content

Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag−/− natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16−/int CD57− cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype

Background: Mutations in recombination‐activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. Objective: Using a flow cytometry–based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. Methods: Abelson virus–transformed Rag2−/− pro‐B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild‐type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP‐expressing cells was evaluated by using flow cytometry, and high‐throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. Results: The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild‐type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. Conclusions: Our data support genotype‐phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease‐causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype. Graphical abstract Figure. No caption available.

Characterising RAG1 and RAG2 with predictive genomics

While widespread genome sequencing ushers in a new era of preventive medicine, the tools for predictive genomics are still lacking. Time and resource limitations mean that human diseases remain uncharacterised because of an inability to predict clinically relevant genetic variants. A strategy of targeting highly conserved protein regions is used commonly in functional studies. However, this benefit is lost for rare diseases where the attributable genes are mostly conserved. An immunological disorder exemplifying this challenge occurs through damaging mutations in RAG1 and RAG2 which presents at an early age with a distinct phenotype of life-threatening immunodeficiency or autoimmunity. Many tools exist for variant pathogenicity prediction but these cannot account for the probability of variant occurrence. Here, we present a method that predicts the likelihood of mutation for every amino acid residue in the RAG1 and RAG2 proteins. Population genetics data from approximately 146,000 individuals was used for rare variant analysis. Forty-four known pathogenic variants reported in patients and recombination activity measurements from 110 RAG1/2 mutants were used to validate calculated scores. Probabilities were compared with 98 currently known human cases of disease. A genome sequence dataset of 558 patients who have primary immunodeficiency but that are negative for RAG deficiency were also used as validation controls. We compared the difference between mutation likelihood and pathogenicity prediction. Our method builds a map of most probable mutations allowing pre-emptive functional analysis. This method may be applied to other diseases with hopes of improving preparedness for clinical diagnosis.While widespread genome sequencing ushers in a new era of preventive medicine, the tools for predictive genomics are still lacking. The greatest hurdle in diagnosis of rare disease is validation for variants of unknown significance. RAG deficiency presents at an early age with a distinct phenotype of combined immunodeficiency with granuloma and/or autoimmunity. Allele frequency of a SNV in the general population is an indicator of the functional or structural importance of a particular amino acid residue. However, rare diseases are often attributable to variants in genes which are highly conserved. Mutation of a conserved residue does not confirm pathogenicity and functional validation must be confirmed to correctly identify a monogenic disorders such as RAG deficiency. We present protein variants in RAG1 and RAG2 which are most likely to be seen clinically as disease-causing. Our method of mutation rate residue frequency builds a map of most probable mutations allowing pre-emptive functional analysis. We compare the accuracy of our predicted probabilities to previously established functional measurements.

Adult-Onset Myopathy in a Patient with Hypomorphic RAG2 Mutations and Combined Immune Deficiency

To the Editor: Severe loss of function mutations in recombination activating genes (RAG1 and RAG2), required for the initial phase of V(D)J recombination in T cell and B cell receptors, are associated with a T-B-NK+ form of severe combined immunodeficiency (SCID) [1]. However, hypomorphic mutations in the same RAG genes can lead to atypical (or Bleaky^) forms of SCID, which may present with a broad spectrum of phenotypes [1–5]. These include Omenn syndrome, which presents in infancy, and is characterized by erythroderma, eosinophilia, elevated IgE level, lymphadenopathy, and hepatosplenomegaly. Often milder and with later onset, a range of immune dysregulation syndromes can be seen. These can be associated with autoantibodies against self-antigens and cytokines [1, 6, 7], as well as defects in Tcell and B cell tolerance [1]. Some patients present with a combined immunodeficiency phenotype with granulomatous disease and autoimmunity, including autoimmune cytopenias, vitiligo, myasthenia gravis, and psoriasis [1–3, 6]. Patientswith hypomorphicmutations in SCID-associated genes may appear to have common variable immune deficiency (CVID). Of note, a sizeable proportion of patients with CVID present with autoimmunity (approximately 30%) and/or granulomatous disease (approximately 10%) [8]. While there can be significant overlap in laboratory and clinical manifestation of CVID and hypomorphic RAG mutations, features such as opportunistic infections, significant T and B cell defects, and the presence of autoantibodies, including those targeting cytokines and causing autoimmunity, should prompt investigation for an underlying monogenic defect, which could include hypomorphic RAG deficiency.We found two cases ofRAGmutation in the literature that were uncovered in patients previously diagnosed with CVIDwho developed opportunistic infections as well as autoimmunity (vitiligo and autoimmune cytopenias) [1, 9]) as well as two cases of RAGmutation in patients with selective antibody deficiency with impaired production of antibody against bacterial polysaccharide [10].

Identification of Autoantibodies in Carriers of X-Linked Chronic Granulomatous Disease by Autoantigen Microarray Analysis

describes the study design to facilitate the enrollment of patients treated in clinical settings. Study: All females who become pregnant during IgHy treatment (treated while pregnant or in the 30 days prior to conception) will be encouraged to participate. As soon as the patient becomes aware of the pregnancy, treatment with IgHy will be stopped. Subjects will receive an alternative gammaglobulin treatment and all other medical care will be performed as is standard. The overall duration of the study is approximately 6 years and there is no minimum sample size pre-specified. Data from medical records on the routine assessments during the pregnancy will be collected. In those subjects consenting to blood drawing, samples will be taken to assess antibodies against rHuPH20 every 3 months. Data will be assessed on the development of the fetus in utero, and on the growth and development of the infant for 2 years after delivery. Descriptive methods will be used to analyze and report data from this IgHy pregnancy registry. ESID-0098 Non-Interventional Post-Authorization Safety Study (PASS) on the Long-Term Safety of HYQVIA® (IGHY) K. Fielhauer, N. Nikolov, C. Hermann, A. Nagy, R.I. Schiff Bioscience, Baxter Healthcare, Vienna, Austria Bioscience, Baxter Healthcare SA, Zurich, Switzerland Bioscience, Baxter Healthcare, Westlake Village, USA Introduction: IgHy (recombinant human hyaluronidase [rHuPH20]-facilitated subcutaneous [SC] infusion of immunoglobulin [IG]) is a novel treatment that has been approved as a replacement therapy in adults (≥18 years) with primary immunodeficiency (PID) syndromes, myeloma, and chronic lymphocytic leukemia. This is a non-interventional, prospective, uncontrolled, multi-center, open label, post-authorization safety study to be conducted in the EEA to acquire additional data (including the assessment of anti-rHuPH20 antibodies) on the long-term safety of IgHy. This abstract describes the study design to facilitate the evaluation of patients treated in a

Regulatory T cell subsets, T cell receptor repertoire, and autoantibodies in RAG1-deficient Omenn syndrome

Continued B) In homozygous IGHG diplotypes only one of the two alternative allotypes is expressed, which result in restricted qualities of IgG molecules and B cells. IGHG gene frequencies and the allotypic IgG subclass levels, the activity of the genes, are available for children and adults of healthy Caucasians (Fig. 4). IGHG genes have impact on disease and immunotherapy. The function of IgG is related to the constant part of the heavy γ chains the Fcγ part of the molecule in parallel with the variable adaptive antibody binding site. Different effects of polysaccharide and protein vaccines, with different amounts of specific antibodies were recorded for different IGHG genes. They are also associated to different severity of bacterial and viral infections and can be used as predisposing, prognostic and sometimes carrier markers. The IgG molecules of IGHG genes are differently affected by viruses acting as FcγRs modifying the disease. Suscepetibility/resistence of various infections both bacterial and viral, allergens and tumour antigens are influenced by IGHG genes. The IGHG genes are associated with different phenotypes of allergic, autoimmune, infectious, immunodeficiency and malignt disorders. In treatments with IVIG, the levels of foreign allelic IgG subclasses are recognized and can be followed. This is the ultimate evidence of that the allelic IgG subclasses are unique. IGHG genes and allotypic IgG subclasses are predisposing or prognostic genetic markers in various infections, immunodeficiencies, different phenotypes of allergic disease, autoimmune disorders and malignant diseases. Fig.1Fig. 2Fig.3Fig. 4 29 IMMUNOPHENOTYPING OF B LYMPHOCYTES IN PATIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY (CVID) F. Mazzi, G. Patuzzo, A. Vella, R. Ortolani, A. Barbieri, A. Puccetti, E. Tinazzi, G. Marchi, O.M. Codella, R. Beri, C. Lunardi Department of Medicine, Unit of Autoimmune Diseases, University of Verona, Department of Pathology and Diagnosis, Section of Immunology, University of Verona, Verona, Immunopathology, Institute G. Gaslini and University of Genova, Genova, Italy Introduction: CVID is a primary immunodeficiency characterized by failure of B lymphocytes to differentiate in plasma cells, with deficient immunoglobulin secretion. The identified genetic defects account only for a minority of cases. Objective: The importance of B cells immunophenotyping in the classification of CVID is well known. This procedure can identify some alterations on cell surface molecule expression that could explain the immunological disorder at the basis of CVID. Moreover, some immunophenotipical aspects can correlate with clinical features, severity and prognosis of the disease. Aim: We studied a cohort of 16 patients affected by CVID, to identify alterations of B cells and to find correlations with clinical features. Methods: We studied circulating B cells in patients and controls by flow-cytometry, using a specific panel of antibodies for B cells. Conclusion: We compared the population of “switched memory” IgD-CD27+B lymphocytes, used in the Friburg classification, with the population of IgM-IgDCD23-CD27+B cells, we have analysed. IgM-IgDCD23-CD27+B cells were reduced in patients compared to healthy controls and in patients they were lower than IgD-CD27+B cells. The reduction of these lymphocytes was correlated more tightly than IgDCD27+B cells to lymphoadenopathy, splenomegaly, J Clin Immunol (2012) 32 (Suppl 1):S1–S379