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Introduction: Inborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. Methods: Research laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton’s tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussion: A phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgMinduced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-kB) activation pathway, normal IkBa degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed IkBa degradation and reduced calcium ion (Ca2+) influx occurs on anti-IgM stimulation in the patient’s B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain. [ABSTRACT FROM AUTHOR]

Background: The use of next-generation sequencing in inborn errors of immunity (IEI) has considerably increased the identification of novel gene variants, many of which are identified in patients without the described clinical phenotype or with variants of uncertain pathogenic significance in previously described genes. Properly designed functional and cellular assays, many necessarily accomplished by research-based laboratories, reveal the pathogenic consequences of the gene variants and contribute to diagnosis. Activated PI3Kδ syndrome (APDS) is a rare disease that can be divided into APDS1, caused by gain of function (GOF) mutations in PIK3CD gene, and APDS2, with loss of function (LOF) variants in the PIK3R1 gene. Both entities present hyperactivation of the PI3K pathway, which can be analyzed through Akt and S6 phosphorylation status., Methods: Our objective was to perform an accurate, robust, and reproducible functional assay to analyze the phosphorylation status of proteins in the PI3K-Akt-S6 pathway by flow cytometry, to contribute to diagnosis, to monitor treatments, and to establish intra-assay standardization., Results: We illustrate the robustness and reproducibility of our experimental procedure in patients with APDS who had high Akt and/or S6 phosphorylation levels at baseline, and after anti-IgM stimulation in B cells. We show the relevance of an appropriate cohort of samples from healthy donors, processed within the same conditions as the suspected samples, in particular the time frame for sample processing once blood is collected., Discussion: We highlight the importance of B cell stimulation through B cell receptor signaling, which is highly recommended, especially for samples that would be processed more than 24 hours after blood extraction. Also, having a defined experimental procedure is important, including the cytometer setup, which allows cytometer reproducibility for a period of time, enabling the comparison of a sample at different times., Competing Interests: LM and EG have received consultee fees and sponsored research grant by Pharming. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Pharming. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 del Pino Molina, Reche Yebra, Soto Serrano, Clemente Bernal, Avendaño-Monje, Ocejo-Vinyals, Rodríguez Pena and López Granados.)

Background: Real world data on the response to the SARS-CoV-2 vaccine in patients with immunomediated diseases (IMIDs) treated with immunesuppressants is of great interest because vaccine response may be impaired. The main aim was to study the humoral and cellular immune response after SARS-CoV-2 vaccination in patients with IMIDs treated with immunosuppressants. The secondary aim was to describe the frequency of SARS-CoV-2 infections after vaccination in these patients., Material and Methods: This is an observational study including 86 patients with IMIDs. All patients were treated with biologic or targeted synthetic disease-modifying antirheumatic drugs [b/tsDMARDs: TNF inhibitors (TNFi), rituximab, anti-interleukin 6 receptor (anti-IL6R) or JAK inhibitors (JAKi)]. Demographic and clinical information were collected. After 4-6 weeks of 2nd and 3rd vaccine doses, humoral response was assessed using the Thermo Scientific ELiA SARS-CoV-2-Sp1 IgG Test. Also, in patients with serum SARS-CoV-2 antibody levels under 100UI/ml, cellular response was analyzed using the QuantiFERON SARS-CoV-2 Starter Pack., Results: A total of 86 patients under b/tsDMARDs and 38 healthy controls were included. Most patients received TNFi (45 with TNFi, 31 with rituximab, 5 with anti-IL6R and 5 with JAKi). SARS-CoV-2 antibodies (Ab) were present in an 86% of patients with IMIDs and in 100% healthy controls (p = 0.017). However, 12 (14%) patients had undetectable SARS-CoV-2 Ab levels, all treated with rituximab. In addition, SARS-CoV-2 Ab (IU/ml) were statistically lower in patients (Mdn (IQR): 59.5 (17-163) in patients vs 625 (405-932) in controls, p < 0.001). Patients treated with rituximab had lower Ab levels than those treated with TNFi and controls (p < 0.001). The cellular response to SARS-CoV-2 vaccine was evaluated in 30 patients. Eleven patients had a positive cellular response, being more frequent in patients treated with rituximab (p = 0.03). SARS-CoV-2 infection was reported in 43% of patients and 34% of controls after vaccination. Only 6 (7%) patients required hospitalization, most of whom treated with rituximab (67%)., Conclusion: SARS-CoV-2 antibody levels were lower in patients than in controls, especially in patients treated with rituximab. A cellular response can be detected despite having a poor humoral response. Severe infections in vaccinated patients with IMIDs are rare, and are observed mainly in patients treated with rituximab., (© 2023. Canadian Society of Allergy & Clinical Immunology.)

Introduction: Inborn errors of immunity (IEI) are a heterogeneous group of diseases caused by intrinsic defects of the immune system. Estimating the immune competence of immunocompromised patients for an infection risk assessment or after SARS-CoV-2 vaccination constituted a challenge., Methods: The aim of this study was to determine the humoral responses of patients with IEI through a comprehensive analysis of specific receptor-binding domain-positive (RBD+) IgG+ memory B cells (MBCs) by flow cytometry, together with routine S-specific IgG antibodies and QuantiFERON SARS-CoV-2 (T-cell response), before the vaccine and 3 weeks after a second dose., Results and Discussion: We first analyzed the percentage of specific RBD+ IgG+ MBCs in healthy healthcare workers. Within the control group, there was an increase in the percentage of specific IgG+ RBD+ MBCs 21 days after the second dose, which was consistent with S-specific IgG antibodies.Thirty-one patients with IEI were included for the pre- and post-vaccination study; IgG+ RBD+ MBCs were not evaluated in 6 patients due to an absence of B cells in peripheral blood. We detected various patterns among the patients with IEI with circulating B cells (25, 81%): an adequate humoral response was observed in 12/25, consider by the detection of positive S-specific IgG antibodies and the presence of specific IgG+ RBD+ MBCs, presenting a positive T-cell response; in 4/25, very low S-specific IgG antibody counts correlated with undetectable events in the IgG+ RBD+ MBC compartment but with positive cellular response. Despite the presence of S-specific IgG antibodies, we were unable to detect a relevant percentage of IgG+ RBD+ MBCs in 5/25; however, all presented positive T-cell response. Lastly, we observed a profound failure of B and T-cell response in 3 (10%) patients with IEI, with no assessment of S-specific IgG antibodies, IgG+ RBD+ MBCs, and negative cellular response. The identification of specific IgG+ RBD+ MBCs by flow cytometry provides information on different humoral immune response outcomes in patients with IEI and aids the assessment of immune competence status after SARS-CoV-2 mRNA vaccine (BNT162b2), together with S-specific IgG antibodies and T-cell responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 del Pino Molina, Bravo Gallego, Nozal, Soto-Serrano, Martínez-Feito, Reche-Yebra, González-Torbay, Cuesta-Martín de la Cámara, Gianelli, Cámara, González-García, González-Muñoz, Rodríguez-Pena and López Granados.)

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients., (© 2022. The Author(s).)

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2., Competing Interests: Declaration of interests A.B. declares the filing of a patent application relating to the use of peptide pools in whole blood for detection of SARS-CoV-2 T cells (pending). The remaining authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Primary antibody deficiencies (PADs) are characterized by hypogammaglobulinemia and impaired B-cell differentiation. Patients with common variable immunodeficiency (CVID) present severe reductions in at least 2 serum immunoglobulins and impaired terminal differentiation of B cells. Most patients with CVID do not appear to present monogenic defects. Activated phosphoinositide 3-kinase delta syndrome (APDS), caused by gain-of-function mutations in the PIK3CD gene (p110δ), can present in patients with a CVID-like phenotype. Memory B-cell differentiation requires the orchestrated activation of numerous intracellular signaling pathways, which promote transcriptional programs required for long-term B-cell survival. The aim of this study was to develop a flow cytometry assay to trace the PI3K-Akt-mTOR pathway, a critical component of B-cell homeostasis, and analyze its status in PADs., Methods: We analyzed the intracellular expression of Akt and S6 by flow cytometry and their phosphorylation status in both baseline conditions and upon B-cell receptor activation with anti-IgM in various primary B-cell subsets of patients with CVID and APDS., Results: B cells from CVID patients showed reduced phosphorylation in Akt and S6 proteins after anti-IgM stimulation. Constitutive high baseline B-cell levels of Akt and S6 phosphorylation in a patient with APDS were reduced once m-TOR inhibition therapy was initiated., Conclusions: Intracellular flow cytometry can be routinely employed to explore alterations in the PI3K-Akt-mTOR pathway in B cells from patients with PADs. AKT and S6 phosphorylation levels are informative biomarkers that could be employed as mTOR inhibitors for monitoring therapies targeting this pathway., (© 2020 International Clinical Cytometry Society.)

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and different degrees of B cell compartment alteration. Memory B cell differentiation requires the orchestrated activation of several intracellular signaling pathways that lead to the activation of a number of factors, such as nuclear factor kappa B (NF-κB) which, in turn, promote transcriptional programs required for long-term survival. The aim of this study was to determine if disrupted B cell differentiation, survival and activation in B cells in CVID patients could be related to defects in intracellular signaling pathways. For this purpose, we selected intracellular readouts that reflected the strength of homeostatic signaling pathways in resting cells, as the protein expression levels of the Bcl-2 family which transcription is promoted by NF-κB. We found reduced Bcl-2 protein levels in memory B cells from CVID patients. We further explored the possible alteration of this crucial prosurvival signaling pathway in CVID patients by analysing the expression levels of mRNAs from anti-apoptotic proteins in naive B cells, mimicking T cell-dependent activation in vitro with CD40L and interleukin (IL)-21. BCL-XL mRNA levels were decreased, together with reduced levels of AICDA, after naive B-cell activation in CVID patients. The data suggested a molecular mechanism for this tendency towards apoptosis in B cells from CVID patients. Lower Bcl-2 protein levels in memory B cells could compromise their long-term survival, and a possible less activity of NF-κB in naive B cells, may condition an inabilityto increase BCL-XL mRNA levels, thus not promoting survival in the germinal centers., (© 2020 British Society for Immunology.)

Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients., Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube , standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD)., Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5 + CD38 +/++ CD21 het CD24 ++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5 + CD38 het CD21 + CD24 + (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21 - CD24 - (6.1 vs 0.74 cells/µl, p<0.0001) and CD21 - CD24 ++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD + (21 vs 32 cells/µl, p=0.03) and IgMD - (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%)., Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID., Competing Interests: JD, MB, TK, MP-A, EL-G, A-KK, EB, and AO each report being one of the inventors on the EuroFlow-owned patent PCT/NL 2015/050762 (Diagnosis of primary immunodeficiencies), which is licensed to Cytognos, a company that pays royalties to the EuroFlow Consortium., (Copyright © 2021 del Pino-Molina, López-Granados, Lecrevisse, Torres Canizales, Pérez-Andrés, Blanco, Wentink, Bonroy, Nechvatalova, Milota, Kienzler, Philippé, Sousa, van der Burg, Kalina, van Dongen and Orfao.)

X-linked Agammaglobulinemia is a primary immunodeficiency caused by mutations in BTK, a tyrosine kinase essential for B lymphocytes differentiation. Patients usually have very low or absent B lymphocytes and are not able to develop humoral specific responses. Here we present a boy, diagnosed with XLA due to a mutation on the promoter region of the gene, whose phenotype is characterised by low percentage of B cells, hypogammaglobulinemia, oscillating neutropenia, antibodies responses to some antigens after vaccination and IgE-mediated allergy. Additional technology as flow cytometry was needed to demonstrate the pathological status of the variant. We focus on the idea that XLA should be suspected in males with B lymphopenia and hypogammaglobulinemia, even if they make humoral specific responses. We also highlight the importance of sequencing BTK's promoter region, as mutations on it can be disease-causing., (© 2020 The Authors. Published by Elsevier Ltd.)

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints., (Copyright © 2019 Wentink, Kalina, Perez-Andres, del Pino Molina, IJspeert, Kavelaars, Lankester, Lecrevisse, van Dongen, Orfao and van der Burg.)

Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown., Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses., Methods: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs., Results: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA + PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA + PCs with mild versus severe smIgA + MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA + and smIgG + MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27 + MBCs with almost normal IgG 3 + MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG 2 + MBCs; and (6) with IgA 1 + MBCs., Conclusion: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naïve to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.

Good syndrome is an immunodeficiency presenting with thymoma, hypogammaglobulinemia and almost absent B cells. To investigate the origin of the B-cell lymphopenia in these patients, we studied B cell differentiation in the bone marrow of Good syndrome patients. We found very low numbers of precursor B cells in bone marrow of Good syndrome patients and a differentiation arrest after the pro-B-cell stage; this is different from other agammaglobulinemia patients with a defect in pre B-cell receptor signaling., (Copyright © 2018. Published by Elsevier Inc.)

Primary immunodeficiencies (PIDs) are immune disorders resulting from defects in genes involved in immune regulation, and manifesting as an increased susceptibility to infections, autoimmunity, and cancer. However, the molecular basis of some prevalent entities remains poorly understood. Epigenetic control is essential for immune functions, and epigenetic alterations have been identified in different PIDs, including syndromes such as immunodeficiency-centromeric-instability-facial-anomalies, Kabuki, or Wolf-Hirschhorn, among others. Although the epigenetic changes may differ among these PIDs, the reversibility of epigenetic modifications suggests that they might become potential therapeutic targets. Here, we review recent mechanistic advances in our understanding of epigenetic alterations associated with certain PIDs, propose that a fully epigenetically driven mechanism might underlie some PIDs, and discuss the possible prophylactic and therapeutic implications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and poor response to vaccines. Its diagnosis is made based on clinical and immunological criteria, after exclusion of other diseases that can cause similar phenotypes. Currently, less than 20% of cases of CVID have a known underlying genetic cause. We have analyzed whole-exome sequencing and copy number variants data of 36 children and adolescents diagnosed with CVID and healthy relatives to estimate the proportion of monogenic cases. We have replicated an association of CVID to p.C104R in TNFRSF13B and reported the second case of homozygous patient to date. Our results also identify five causative genetic variants in LRBA, CTLA4, NFKB1 , and PIK3R1 , as well as other very likely causative variants in PRKCD, MAPK8 , or DOCK8 among others. We experimentally validate the effect of the LRBA stop-gain mutation which abolishes protein production and downregulates the expression of CTLA4, and of the frameshift indel in CTLA4 producing expression downregulation of the protein. Our results indicate a monogenic origin of at least 15-24% of the CVID cases included in the study. The proportion of monogenic patients seems to be lower in CVID than in other PID that have also been analyzed by whole exome or targeted gene panels sequencing. Regardless of the exact proportion of CVID monogenic cases, other genetic models have to be considered for CVID. We propose that because of its prevalence and other features as intermediate penetrancies and phenotypic variation within families, CVID could fit with other more complex genetic scenarios. In particular, in this work, we explore the possibility of CVID being originated by an oligogenic model with the presence of heterozygous mutations in interacting proteins or by the accumulation of detrimental variants in particular immunological pathways, as well as perform association tests to detect association with rare genetic functional variation in the CVID cohort compared to healthy controls.

Primary antibody deficiencies (PADs), the most prevalent inherited primary immunodeficiencies (PIDs), are associated with a wide range of genetic alterations (both monogenic or polygenic) in B cell-specific genes. However, correlations between the genotype and clinical manifestations are not evident in all cases indicating that genetic interactions, environmental and epigenetic factors may have a role in PAD pathogenesis. The recent identification of key defects in DNA methylation in common variable immunodeficiency as well as the multiple evidences on the role of epigenetic control during B cell differentiation, activation and during antibody formation highlight the importance of investing research efforts in dissecting the participation of epigenetic defects in this group of diseases. This review focuses on the role of epigenetic control in B cell biology which can provide clues for the study of potential novel pathogenic defects involved in PADs.

Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology. Here we perform a high-throughput DNA methylation analysis of this disorder using a pair of CVID-discordant MZ twins and show predominant gain of DNA methylation in CVID B cells with respect to those from the healthy sibling in critical B lymphocyte genes, such as PIK3CD, BCL2L1, RPS6KB2, TCF3 and KCNN4. Individual analysis confirms hypermethylation of these genes. Analysis in naive, unswitched and switched memory B cells in a CVID patient cohort shows impaired ability to demethylate and upregulate these genes in transitioning from naive to memory cells in CVID. Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals.