Gene regulation in inborn errors of immunity: Implications for gene therapy design and efficacy.

Inborn errors of immunity (IEI) present a unique paradigm in the realm of gene therapy, emphasizing the need for precision in therapeutic design. As gene therapy transitions from broad-spectrum gene addition to careful modification of specific genes, the enduring safety and effectiveness of these therapies in clinical settings have become crucial. This review discusses the significance of IEIs as foundational models for pioneering and refining precision medicine. We explore the capabilities of gene addition and gene correction platforms in modifying the DNA sequence of primary cells tailored for IEIs. The review uses four specific IEIs to highlight key issues in gene therapy strategies: X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (X-CGD), X-linked hyper IgM syndrome (XHIGM), and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). We detail the regulatory intricacies and therapeutic innovations for each disorder, incorporating insights from relevant clinical trials. For most IEIs, regulated expression is a vital aspect of the underlying biology, and we discuss the importance of endogenous regulation in developing gene therapy strategies.

A Registry Study of 240 Patients with X-Linked Agammaglobulinemia Living in the USA.

PURPOSE: To understand the natural history and clinical outcomes for patients with X-linked agammaglobulinemia (XLA) in the United States utilizing the United States Immunodeficiency Network (USIDNET) patient registry. METHODS: The USIDNET registry was queried for data from XLA patients collected from 1981 to 2019. Data fields included demographics, clinical features before and after diagnosis of XLA, family history, genetic mutation in Bruton's tyrosine kinase (BTK), laboratory findings, treatment modalities, and mortality. RESULTS: Data compiled through the USIDNET registry on 240 patients were analyzed. Patient year of birth ranged from 1945 to 2017. Living status was available for 178 patients; 158/178 (88.8%) were alive. Race was reported for 204 patients as follows: White, 148 (72.5%); Black/African American, 23 (11.2%); Hispanic, 20 (9.8%); Asian or Pacific Islander, 6 (2.9%), and other or more than one race, 7 (3.4%). The median age at last entry, age at disease onset, age at diagnosis, and length of time with XLA diagnosis was 15 [range (r) = 1-52 years], 0.8 [r = birth-22.3 years], 2 [r = birth-29 years], and 10 [r = 1-56 years] years respectively. One hundred and forty-one patients (58.7%) were < 18 years of age. Two hundred and twenty-one (92%) patients were receiving IgG replacement (IgGR), 58 (24%) were on prophylactic antibiotics, and 19 (7.9%) were on immunomodulatory drugs. Eighty-six (35.9%) patients had undergone surgical procedures, two had undergone hematopoietic cell transplantation, and two required liver transplantation. The respiratory tract was the most affected organ system (51.2% of patients) followed by gastrointestinal (40%), neurological (35.4%), and musculoskeletal (28.3%). Infections were common both before and after diagnosis, despite IgGR therapy. Bacteremia/sepsis and meningitis were reported more frequently before XLA diagnosis while encephalitis was more commonly reported after diagnosis. Twenty patients had died (11.2%). The median age of death was 21 years (range = 3-56.7 years). Neurologic condition was the most common underlying co-morbidity for those XLA patients who died. CONCLUSIONS: Current therapies for XLA patients reduce early mortality, but patients continue to experience complications that impact organ function. With improved life expectancy, more efforts will be required to improve post-diagnosis organ dysfunction and quality of life. Neurologic manifestations are an important co-morbidity associated with mortality and not yet clearly fully understood.

Immunogenicity of the mRNA-1273 COVID-19 vaccine in adult patients with inborn errors of immunity.

BACKGROUND: Patients with inborn errors of immunity (IEI) are at increased risk of severe coronavirus disease-2019 (COVID-19). Effective vaccination against COVID-19 is therefore of great importance in this group, but little is known about the immunogenicity of COVID-19 vaccines in these patients. OBJECTIVES: We sought to study humoral and cellular immune responses after mRNA-1273 COVID-19 vaccination in adult patients with IEI. METHODS: In a prospective, controlled, multicenter study, 505 patients with IEI (common variable immunodeficiency [CVID], isolated or undefined antibody deficiencies, X-linked agammaglobulinemia, combined B- and T-cell immunodeficiency, phagocyte defects) and 192 controls were included. All participants received 2 doses of the mRNA-1273 COVID-19 vaccine. Levels of severe acute respiratory syndrome coronavirus-2-specific binding antibodies, neutralizing antibodies, and T-cell responses were assessed at baseline, 28 days after first vaccination, and 28 days after second vaccination. RESULTS: Seroconversion rates in patients with clinically mild antibody deficiencies and phagocyte defects were similar to those in healthy controls, but seroconversion rates in patients with more severe IEI, such as CVID and combined B- and T-cell immunodeficiency, were lower. Binding antibody titers correlated well to the presence of neutralizing antibodies. T-cell responses were comparable to those in controls in all IEI cohorts, with the exception of patients with CVID. The presence of noninfectious complications and the use of immunosuppressive drugs in patients with CVID were negatively correlated with the antibody response. CONCLUSIONS: COVID-19 vaccination with mRNA-1273 was immunogenic in mild antibody deficiencies and phagocyte defects and in most patients with combined B- and T-cell immunodeficiency and CVID. Lowest response was detected in patients with X-linked agammaglobulinemia and in patients with CVID with noninfectious complications. The assessment of longevity of immune responses in these vulnerable patient groups will guide decision making for additional vaccinations.

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

Health-Related Quality of Life in 91 Patients with X-Linked Agammaglobulinemia.

PURPOSE: X-linked agammaglobulinemia (XLA) is a primary immunodeficiency (PID) caused by a defect in the gene encoding for Bruton tyrosine kinase (BTK). In the absence of a functional BTK, patients have low or absent circulating B cells and low or absent serum immunoglobulin. Despite gammaglobulin replacement and prompt use of antimicrobial agents, patients with XLA continue to experience infectious and non-infectious complications throughout their lifetime. The purpose of this study was to understand self-perceived health status of US-based patients with XLA, and examine the associations amongst clinical characteristics, treatment experience, and quality of life (QoL). METHODS: A 46 and 68 question survey, developed by the Immune Deficiency Foundation (IDF) and a Short Form-12item v2® (SF-12v2®) for adults and SF-10™ for children to assess QoL, were mailed by IDF to patients in 2017 and 2018. Those that self-identified as having XLA or males with agammaglobulinemia were selected for analysis. Mean physical and mental composite scores (PCS and MCS) from SF-12v2® and mean physical health component (PHS) and psychological health summary (PSS) from SF-10™ scores were compared to the US normative data. RESULTS: Ninety-one patients completed the surveys: 58 (63.7%) adults and 33 (36.3%) children. For the combined surveys, the overall median age at time of the survey was 28.5 years (yrs); Inter-Quartile-Range (IQR) 13-49.5 yrs; the median age at diagnosis was 2 yrs (IQR = 0-4 yrs) and the median number of years with XLA diagnosis was 23 (IQR 10.75-40yrs). Amongst adult patients, physical scores were noted to be below the general adult population but did not reach statistical significance. In contrast, 2 or more chronic conditions impacted both physical and mental QoL (p < .001) and hospitalization was associated with significantly decreased physical health QoL (p < .001); three or more infections in the past 12 months exhibited impact on physical health although was not found to be statistically significant. Adult patients with public insurance fared worse in mental health domains compared to those with combined public and private or those with private alone (p = 0.001). Employment status did not impact QoL. None of these variables met statistical significance nor demonstrated impact within the pediatric population in either physical or mental domains of health. CONCLUSION: Our study provides further insight into what factors impact both physical and mental domains of health amongst patients with XLA. Early detection to prevent the development of associated morbidity, as well as vigilant care to prevent hospitalizations and infections, can limit the impact this disease may have on the overall well-being of XLA patients.

SARS-CoV-2-Specific and Functional Cytotoxic CD8 Cells in Primary Antibody Deficiency: Natural Infection and Response to Vaccine.

PURPOSE: CD8 cytotoxic T cells (CTLs) play a critical role in the clearance of virally infected cells. SARS-CoV-2-specific CD8 T cells and functional CTLs in natural infections and following COVID-19 vaccine in primary antibody deficiency (PAD) have not been reported. In this study, we evaluated T cell response following COVID-19 or COVID-19 mRNA vaccination in patients with PADs by assessing SARS-CoV-2 tetramer-positive CD8 T cells and functional CTLs. METHODS: SARS-CoV-2-specific CD8 and functional CTLs were examined in a patient with X-linked agammaglobulinemia (XLA) and a patient with common variable immunodeficiency (CVID) following COVID-19 infection, and in 5 patients with CVID and 5 healthy controls 1 month following 2nd dose of COVID-19 mRNA vaccine (Pfizer-BioNTech). Cells were stained with SARS-CoV-2 spike protein-specific tetramers, and for functional CTLs (CD8+ CD107a+ granzyme B+ perforin+), with monoclonal antibodies and isotype controls and analyzed by flow cytometry. RESULTS: SARS-CoV-2-specific tetramer + CD8 T cells and functional CTLs in the patient with XLA following COVID-19 infection were higher, as compared to healthy control subject following COVID-19 infection. On the other hand, SARS-CoV2-tetramer + CD8 T cells and functional CTLs were lower in CVID patient following COVID19 infection as compared to healthy control following COVID-19 infection. SARS-CoV2-tetramer + CD8 T cells and functional CTLs were significantly lower in SARS-CoV2-naive CVID patients (n = 10) following vaccination when compared to SARS-CoV-2-naive healthy vaccinated controls (n = 10). CONCLUSIONS: CVID is associated with reduced SARS-CoV-2-specific CD8 T cells and functional CTLs in both natural SARS-CoV-2 infection and in response to SARS-CoV-2 mRNA vaccine, whereas natural infection in XLA is associated with a robust SARS-CoV-2-specific CD8 and functional CTL responses.

Specific T-cell responses for guiding treatment with convalescent plasma in severe COVID-19 and humoral immunodeficiency: a case report.

BACKGROUND: The immune response to SARS-CoV-2 virus, the cause of COVID-19, is complex. Antibody mediated responses are important for viral clearance but may also drive hyperinflammation in severe COVID-19. We present a case of an individual with a genetic inability to produce antibodies and severe COVID-19, receiving no other specific anti-viral treatment than convalescent COVID-19 plasma, illustrating that hyperinflammation can occur in the absence of a humoral anti-viral response. In addition, the case illustrates that the assessment of SARS-CoV-2 T cell responses can facilitate clinical decision making in patients with COVID-19 and weak or absent humoral immune responses. CASE PRESENTATION: A male with X-linked agammaglobulinemia on regular immunoglobulin replacement therapy, hospitalized for 35 days due to severe COVID-19. Systemic inflammatory parameters were highly elevated. After treatment with convalescent COVID-19 plasma he became afebrile and the fatigue diminished. He was discharged on day 42 and nasopharyngeal SARS-CoV-2 PCR eventually was negative on day 49. Evidence of SARS-CoV-2 specific T cells prior to administration of plasma therapy suggested that antibodies were crucial for viral clearance. Regular assessment showed robust and persistent SARS-CoV-2 specific T-cell responses after recovery suggested that prophylactic administration of convalescent COVID-19 plasma was unnecessary. CONCLUSION: Assessment of SARS-CoV-2T-cell responses can facilitate the clinical management of COVID-19 patients with humoral immunodeficiencies.

Immunogenicity of Pfizer-BioNTech COVID-19 vaccine in patients with inborn errors of immunity.

BACKGROUND: In mid-December 2020, Israel started a nationwide mass vaccination campaign against coronavirus disease 2019 (COVID-19). In the first few weeks, medical personnel, elderly citizens, and patients with chronic diseases were prioritized. As such, patients with primary and secondary immunodeficiencies were encouraged to receive the vaccine. Although the efficacy of RNA-based COVID-19 vaccines has been demonstrated in the general population, little is known about their efficacy and safety in patients with inborn errors of immunity (IEI). OBJECTIVE: Our aim was to evaluate the humoral and cellular immune response to COVID-19 vaccine in a cohort of patients with IEI. METHODS: A total of 26 adult patients were enrolled, and plasma and peripheral blood mononuclear cells were collected from them 2 weeks following the second dose of Pfizer-BioNTech COVID-19 vaccine. Humoral response was evaluated by testing anti-SARS-CoV-2 spike (S) receptor-binding domain and antinucleocapsid antibody titers and evaluating neutralizing ability by inhibition of receptor-binding domain-angiotensin-converting enzyme 2 binding. Cellular immune response was evaluated by using ELISpot, estimating IL-2 and IFN-γ secretion in response to pooled SARS-CoV-2 S- or M-peptides. RESULTS: Our cohort included 18 patients with a predominantly antibody deficiency, 2 with combined immunodeficiency, 3 with immune dysregulation, and 3 with other genetically defined diagnoses. Twenty-two of them were receiving immunoglobulin replacement therapy. Of the 26 patients, 18 developed specific antibody response, and 19 showed S-peptide-specific T-cell response. None of the patients reported significant adverse events. CONCLUSION: Vaccinating patients with IEI is safe, and most patients were able to develop vaccine-specific antibody response, S-protein-specific cellular response, or both.

Clinical characteristics and prenatal diagnosis for 22 families in Henan Province of China with X-linked agammaglobulinemia (XLA) related to Bruton's tyrosine kinase (BTK) gene mutations.

BACKGROUND: X-linked agammaglobulinaemia (XLA) is a rare immunodeficiency disease for which recurrent severe infection is the major clinical symptom. BTK is the main causative gene, with X chromosome recessive inheritance. However, the mutations reported to date do not fully explain the disorder. METHODS: We detected the percentage of CD19+ B cells and serum immunoglobulin (IgG, IgA, and IgM) levels by flow cytometry and rate scatter immunoturbidimetry, and investigated the BTK mutation profile in 22 XLA patients using Sanger sequencing and real-time PCR . RESULTS: We evaluated the clinical symptoms of 22 XLA patients and investigated genetic mutations present, identifying six novel mutations in the BTK gene: 2 missense mutations (c.23G > T and c.112 T > C), 2 frameshift mutations (c.522_523insC and c.1060delA), 1 large deletion (deletion of exon 2 to 5), and 1 splice-site mutation (c.1631 + 2 T > C). Prenatal diagnoses were performed in six families (F10, F11, F15, F18, F20 and F21), with the following results: the male fetus in Family 10 (F10) did not carry the c.922_923delGA mutation; the male fetus in Family 15 (F15) did not carry the c.1631 + 1G > T splicing mutation; the female fetus in Family 20 (F20) did not carry the c.1931 T > C mutation; the female fetus in Family 21 (F21) did not carry the large deletion mutation. Hence, these four fetuses are not likely to develop XLA. Male fetuses with c.1060delA and c.1684C > T mutations were identified in Family 11 and Family 18, respectively. The pregnant woman in F18 chose to terminate the pregnancy, whereas the pregnant woman in F11 chose to continue the pregnancy. CONCLUSION: We confirmed the diagnosis of 22 XLA patients from 22 unrelated families and detected six new pathogenic mutations. Prenatal diagnosis was performed in six families. Early genetic diagnosis and routine lifelong immunoglobulin replacement therapy can prevent and treat infections in XLA children, saving their lives.

Impaired polysaccharide responsiveness without agammaglobulinaemia in three patients with hypomorphic mutations in Bruton Tyrosine Kinase-No detection by newborn screening for primary immunodeficiencies.

Hypomorphic mutations in the gene encoding Bruton tyrosine kinase (BTK) may result in milder phenotypes and delayed diagnosis of B-cell related immunodeficiencies due to residual BTK function. Newborn screening for kappa-deleting-recombination-excision circles (KRECs) reliably identifies classical X-linked agammaglobulinaemia (XLA) patients with profound B-cell lymphopenia at birth but has not been evaluated in patients with residual BTK function. We aimed to evaluate clinical findings, BTK function and KREC copy numbers in three patients with BTK mutations presenting with impaired polysaccharide responsiveness without agammaglobulinaemia. One patient had an invasive pneumococcal infection at the age of 4 years. All three patients (two brothers) had visible tonsils, normal to slightly decreased immunoglobulin G levels, undetectable pneumococcal antibodies despite pneumococcal conjugate vaccinations, no antibody response after a diagnostic polysaccharide vaccination as well as profound B-cell lymphopenia with residual B-cell differentiation. BTK mutations were identified by Sanger sequencing. BTK staining and phosphorylation assays were performed on peripheral B cells. KREC copy numbers were determined from dried blood spots obtained within the first week of life as well as once at the age of 8, 6 and 3 years, respectively. BTK staining showed residual protein expression. Also, residual BTK activity could be demonstrated. KREC copy numbers from dried blood spots were above the threshold set for detection of patients with profound B-cell lymphopenia. Male patients with impaired polysaccharide responsiveness should be evaluated for B-cell lymphopenia followed by BTK analyses irrespective of immunoglobulin levels or tonsil size.

Disturbed Transcription of TLRs' Negative Regulators and Cytokines Secretion among TLR4- and 9-Activated PBMCs of Agammaglobulinemic Patients.

Toll-like receptors (TLRs) are inevitable elements for immunity development and antibody production. TLRs are in close interaction with Bruton's tyrosine kinase which has been found mutated and malfunctioned in the prototype antibody deficiency disease named X-linked agammaglobulinemia (XLA). TLRs' ability was evaluated to induce transcription of TLR-negative regulators, including suppressor of cytokine signaling 1 (SOCS1), interleukin-1 receptor-associated kinase 3 (IRAK-M), tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), and Ring finger protein 216 (RNF216), and Tumor necrosis factor-α (TNF-α) and Interferon-α (IFN-α) production via Lipopolysaccharides (LPS) and CpG-A oligodeoxynucleotides (CpG-A ODN). Measured by TaqMan real-time polymerase chain reaction (PCR), meaningfully increased transcripts of SOCS1 and RNF216 were found in XLA peripheral blood mononuclear cells (PBMCs). Also, TLR inductions of XLA have led to similar downregulations in the regulator's transcription which was different from that in healthy donors. Cytokine measurement by enzyme-linked immunosorbent assay (ELISA) revealed a significant lower TNF-α production both before and after LPS. By selected molecules in this study, TLRs' potential defectiveness range expands TLRs expression, downstream signaling, and cytokine production. The results show new potential elements that could play a part in TLRs defect and pathogenesis of agammaglobulinemia as well.

X-Linked Agammaglobulinaemia: Outcomes in the modern era.

Colonel Ogden Bruton reported X-Linked Agammaglobulinaemia in 1952 and treated the child with replacement immunoglobulin therapy. Over 60years later, the treatment for XLA has largely remained unchanged. Replacement immunoglobulin lacks the isotypes IgA and IgM, leading to concerns that patients continue to experience recurrent sinopulmonary tract infections and be at increased risk of bronchiectasis. There is potential hope of earlier diagnosis with newborn screening, and a potential cure for these patients, in the form of gene therapy. However, it is first necessary to evaluate current management and outcomes to aid decisions regarding further research and clinical trials. This article reviews current management and outcomes of XLA, whilst identifying gaps in our knowledge base that may need answering before we proceed with novel diagnostic methods and treatment for XLA.

Gastrointestinal Manifestations in X-linked Agammaglobulinemia.

PURPOSE: X-linked agammaglobulinemia is a primary humoral immunodeficiency characterized by hypogammaglobulinemia and increased susceptibility to infection. Although there is increased awareness of autoimmune and inflammatory complications in X-linked agammaglobulinemia (XLA), the spectrum of gastrointestinal manifestations has not previously been fully explored. METHODS: We present a case report of a family with two affected patients with XLA. Given the gastrointestinal involvement of the grandfather in this family, we performed a retrospective descriptive analysis of XLA patients with reported diagnoses of GI manifestations and inflammatory bowel disease (IBD) or enteritis registered at the United States Immunodeficiency Network, a national registry of primary immunodeficiencies. RESULTS: In this cohort of patients with XLA, we found that up to 35% had concurrent gastrointestinal manifestations, and 10% had reported diagnoses of IBD or enteritis. The most commonly reported mutations were missense, which have been associated with a less severe XLA phenotype in the literature. The severity of symptoms were wide ranging, and management strategies were diverse and mainly experimental. CONCLUSIONS: Patients with XLA may require close monitoring with particular attention for GI manifestations including IBD and infectious enteritis. Further studies are needed to improve diagnosis and management of GI conditions in XLA patients.

Respiratory Health and Related Quality of Life in Patients with Congenital Agammaglobulinemia in the Northern Region of the UK.

INTRODUCTION: Patients with congenital agammaglobulinemia, characterized by a defect in B lymphocyte differentiation causing B alymphocytosis, require life-long IgG replacement. There is scant literature regarding the effectiveness of IgG treatment at preventing mucosal (particularly sinopulmonary tract) infection and whether current management adequately restores "normal" health and quality of life (QoL). We aimed to document infective episodes pre- and post-commencing IgG replacement, determine any change in lung function and structure and assess respiratory status and QoL in a cohort of patients treated in Newcastle. METHODS: Clinical data were extracted from medical records of 15 patients identified from the immunology database, focusing on infective episodes, serial chest CT and spirometry results. Thirteen patients completed a selection of standardized and validated questionnaires assessing physical health, respiratory health and QoL. RESULTS: Pediatric patients on IgG therapy suffered fewer infections per patient year (0.74) than adults (2.13). 6/14 patients showed deteriorating respiratory status despite adequate therapy. Health questionnaires revealed a significant burden of respiratory disease on a patient's life. CONCLUSION: Clinical data showed patients with congenital agammaglobulinemia receiving immunoglobulin therapy retained a higher than average infection rate, most of which affected mucosal barriers. Most patients self-reported worse respiratory symptoms, a lower respiratory-related QoL and a lower general health QoL relative to a healthy population. Most participants had progressive structural lung damage and decreased lung function. These results suggest that current management is not entirely effective at preventing deterioration of respiratory health or restoring QoL.

X-Linked Agammagobulinemia in a Large Series of North African Patients: Frequency, Clinical Features and Novel BTK Mutations.

PURPOSE: X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. METHODS: Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2% peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. RESULTS: We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5% vs. 85%). No strong evidence for genotype-phenotype correlation was observed. CONCLUSIONS: This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

Characterization of all possible single-nucleotide change caused amino acid substitutions in the kinase domain of Bruton tyrosine kinase.

Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease-causing variations among the human protein kinases, still it is just 10% of all the possible single-nucleotide substitution-caused amino acid variations (SNAVs). In the BTK kinase domain (BTK-KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease-causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty-seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms.

Prevalence of BTK mutations in male Algerian patterns with agammaglobulinemia and severe B cell lymphopenia.

X linked agammaglobulinemia (XLA) is the first described primary immunodeficiency and the most common form of agammaglobulinemia. It is characterized by susceptibility to recurrent infections, profound decrease of all immunoglobulin isotypes and very low level of B lymphocytes in peripheral blood. The disorder is caused by mutations in the Bruton's Tyrosine Kinase (BTK). Nine male patients suspected to have XLA from nine unrelated families were enrolled in this study. We performed sequencing of the BTK gene in all nine patients, and in the patients' relatives when possible. The XLA diagnosis was confirmed for six patients with six different mutations; we identified a novel mutation (c.1522G>A) and five known mutations. One third of nine unrelated patients do not have mutations in BTK and thus likely suffer from autosomal recessive agammaglobulinemia in the setting of consanguinity. Our results support that the autosomal recessive agammaglobulinemia can be more common in Algeria.

High-throughput sequencing reveals an altered T cell repertoire in X-linked agammaglobulinemia.

To examine the T cell receptor structure in the absence of B cells, the TCR ß CDR3 was sequenced from DNA of 15 X-linked agammaglobulinemia (XLA) subjects and 18 male controls, using the Illumina HiSeq platform and the ImmunoSEQ analyzer. V gene usage and the V-J combinations, derived from both productive and non-productive sequences, were significantly different between XLA samples and controls. Although the CDR3 length was similar for XLA and control samples, the CDR3 region of the XLA T cell receptor contained significantly fewer deletions and insertions in V, D, and J gene segments, differences intrinsic to the V(D)J recombination process and not due to peripheral T cell selection. XLA CDR3s demonstrated fewer charged amino acid residues, more sharing of CDR3 sequences, and almost completely lacked a population of highly modified Vß gene segments found in control DNA, suggesting both a skewed and contracted T cell repertoire in XLA.