MYSM1 attenuates DNA damage signals triggered by physiologic and genotoxic DNA breaks.

BACKGROUND: Patients with deleterious variants in MYSM1 have an immune deficiency characterized by B-cell lymphopenia, hypogammaglobulinemia, and increased radiosensitivity. MYSM1 is a histone deubiquitinase with established activity in regulating gene expression. MYSM1 also localizes to sites of DNA injury but its function in cellular responses to DNA breaks has not been elucidated. OBJECTIVES: This study sought to determine the activity of MYSM1 in regulating DNA damage responses (DDRs) to DNA double-stranded breaks (DSBs) generated during immunoglobulin receptor gene (Ig) recombination and by ionizing radiation. METHODS: MYSM1-deficient pre- and non-B cells were used to determine the role of MYSM1 in DSB generation, DSB repair, and termination of DDRs. RESULTS: Genetic testing in a newborn with abnormal screen for severe combined immune deficiency, T-cell lymphopenia, and near absence of B cells identified a novel splice variant in MYSM1 that results in nearly absent protein expression. Radiosensitivity testing in patient's peripheral blood lymphocytes showed constitutive γH2AX, a marker of DNA damage, in B cells in the absence of irradiation, suggesting a role for MYSM1 in response to DSBs generated during Ig recombination. Suppression of MYSM1 in pre-B cells did not alter generation or repair of Ig DSBs. Rather, loss of MYSM1 resulted in persistent DNA damage foci and prolonged DDR signaling. Loss of MYSM1 also led to protracted DDRs in U2OS cells with irradiation induced DSBs. CONCLUSIONS: MYSM1 regulates termination of DNA damage responses but does not function in DNA break generation and repair.

Advanced computational analysis of CD40LG variants in atypical X-linked hyper-IgM syndrome.

X-Linked Hyper-IgM Syndrome is caused by pathogenic variants in CD40LG. Three patients with atypical clinical and immunological features were identified with variants in CD40LG requiring further characterization. Flow cytometry was used to evaluate CD40L protein expression and binding capacity to a surrogate receptor, CD40-muIg. Though functional anomalies were observed, there was still a lack of clarity regarding the underlying mechanism. We developed structural models for wild-type and the three variants of CD40L protein observed in these patients (p. Lys143Asn, Leu225Ser and Met36Arg) to evaluate structural alterations by molecular mechanic calculations, and assess protein movement by molecular dynamic simulations. These studies demonstrate that functional analysis of variants of unknown significance in CD40LG can be supplemented by advanced computational analysis in atypical clinical contexts. These studies in combination identify the deleterious effects of these variants and potential mechanisms for protein dysfunction.

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.

Hematologically important mutations: X-linked chronic granulomatous disease (fourth update).

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe bacterial and fungal infections. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide and subsequently formed other reactive oxygen species (ROS) are instrumental in killing phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients in Europe and in about 20% in countries with a high ratio of parental consanguinity. This article lists all mutations identified in CYBB and should therefore help in genetic counseling of X-CGD patients' families. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of disease-causing mutations. In addition, we also include some mutations in G6PD, the gene on the X chromosome that encodes glucose-6-phosphate dehydrogenase, because inactivity of this enzyme may lead to shortage of NADPH and thus to insufficient activity of NADPH oxidase. Severe G6PD deficiency can induce CGD-like symptoms.

Lung function trajectories and bronchial hyperresponsiveness during childhood following severe RSV bronchiolitis in infancy.

BACKGROUND: Children with severe respiratory syncytial virus (RSV) bronchiolitis in infancy have increased risks of asthma and reduced lung function in later life. There are limited studies on the longitudinal changes of lung function and bronchial hyperreactivity from early to late childhood in infants hospitalized for RSV bronchiolitis. METHODS: In a prospective cohort of 206 children with their first episode of RSV-confirmed bronchiolitis in the first year of life, 122 had spirometry performed at least twice between 5-16 years of age. Methacholine bronchoprovocation was available in 127 and 79 children at 7 and 12 years of age, respectively. Longitudinal changes in FEV1 , FVC, and FEV1 /FVC z-scores and methacholine PC20 were analyzed. RESULTS: 55% of the study cohort (N = 122) were male, and 55% were Caucasian. During follow-up, longitudinal changes in z-scores for pre- and post-bronchodilator FEV1 (P < .0001) FVC (P < .0001) and FEV1 /FVC (P < .0001 for pre- and 0.007 for post-bronchodilator) from age 5 to 10-16 years were observed. Declined lung function in late childhood was significantly associated with gender, physician diagnosis of asthma, and allergic sensitization. PC20 geometric mean increased from 0.28 mg/mL at 7 years to 0.53 mg/mL at 12 years of age, and the frequency of abnormal bronchial hyperreactivity decreased from 96% to 78% (P = .0003). CONCLUSIONS: Following severe RSV bronchiolitis, there appear to be significant longitudinal changes in pre- and post-bronchodilator lung function during childhood. The study has several limitations including significant dropouts and the lack of a control group and post-bronchodilator measurements. Bronchial hyperreactivity is common in children following severe RSV bronchiolitis; however, it appears to decrease as they enter late childhood.

Snoring and sleep problems in children with and without allergic rhinitis: a case control study.

OBJECTIVE: To compare the sleep problems between children with and without allergic rhinitis (AR). MATERIAL AND METHOD: A case-control study was conducted in 65 children aged 6-15 years with allergic rhinitis and 104 control subjects matched individually by age, height and weight. Cases were recruited from the Pediatric Allergy Clinic at Thammasat University Hospital. The selection of cases was based on clinical history, physical examination and skin prick test. Matched healthy control children were recruited from the Thammasat primary school. Children and their caregivers who usually sleep with them completed the questionnaire. RESULTS: 86.2% of allergic rhinitis was classified as persistent rhinitis and 63.1% had moderate to severe disease. The most common presenting symptom was nasal blockage (66.2%). Allergic rhinitis patients had significant sleep problems with snoring, sleep apnea, restless, night sweating, mouth breathing, dry throat, morning headache, falling asleep in class, difficulty in waking up and not refreshed in the morning (p<0.05). Patients who categorized as blockers had significantly more restless sleep and dry mouth on waking up compared to that of non-blockers (p<0.05). CONCLUSION: There was a higher prevalence of sleep problems in children with AR then those without AR.

Scurvy revealed by difficulty walking: three cases in young children.

Scurvy is rare in developed countries but is known to cause lower-extremity pain and refusal to ambulate in children. Since the discovery of the link between scurvy and dietary deficiency of ascorbic acid, there has been a substantial decrease in its prevalence and recognition. Here we describe 3 cases of scurvy in young children presenting with difficulty walking. Only 1 of 3 patients had gingival lesions at the initial presentation. Two cases underwent an extensive evaluation for hematologic and rheumatologic diseases before the diagnosis of scurvy was made. Dietary histories eventually revealed that all 3 patients had sharply limited intake of fruits and vegetables secondary to oral aversion, and 1 patient had autism. Radiographic changes of long bones were observed in all patients. Interestingly, all patients had concomitant vitamin D deficiency. After replacement with vitamin C, all patients recovered and started to walk again with improved leg pain. These clinical manifestations and radiologic findings highlight the importance for rheumatologists to have a higher index of suspicion for scurvy in nonambulatory children.

Case Report: Insulin hypersensitivity in youth with type 1 diabetes.

Objective: Immediate type I, type III, and delayed type IV hypersensitivity reactions to insulin are rare, but potentially serious complications of exogenous insulin administration required for the treatment of type 1 diabetes (T1D). Methods: We present four cases of insulin hypersensitivity reactions occurring in youth with T1D and a literature review of this topic. Results: Insulin hypersensitivity reactions included types I, III, and IV with presentations ranging from localized urticaria, erythematous nodules, and eczematous plaques to anaphylaxis with respiratory distress. Reactions occurred in youth with newly diagnosed T1D and in those with long-standing T1D who were using both injection and insulin pump therapy. Multidisciplinary care involving pediatric endocrinology and allergy/immunology utilizing trials of many adjunct therapies yielded minimal improvement. Despite the use of various treatments, including antihistamines, topical therapies, immunosuppressant medications, desensitization trials, and intravenous immune globulin, cutaneous reactions, elevated hemoglobin A1c levels, and negative effects on quality of life remain persistent challenges. One patient became one of the youngest pancreas transplant recipients in the world at age 12 years due to uncontrollable symptoms and intolerable adverse effects of attempted therapies. Conclusion: Although rare, insulin hypersensitivity reactions negatively affect glycemic control and quality of life. These cases demonstrate the varying severity and presentation of insulin hypersensitivity reactions along with the limited success of various treatment approaches. Given the life-sustaining nature of insulin therapy, further studies are needed to better understand the underlying pathophysiology of insulin hypersensitivity and to develop targeted treatment approaches.

Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

The Heart of the Matter: Secondary Hypogammaglobulinemia and Constrictive Pericarditis.

Constrictive pericarditis is the final common result of a number of processes that affect the pericardium. Establishing the diagnosis and determining the underlying etiology of constrictive pericarditis are often a diagnostic rendezvous. Here, we describe a patient who presented to the general practitioner with edema, ascites, and weight gain and was found to have constrictive pericarditis secondary to an inflammatory myofibroblastic tumor of the mediastinum. Interestingly, she had a relative lack of cardiorespiratory complaints, and, aside from the edema and mildly elevated jugular venous pressure, she had an unremarkable cardiac and pulmonary examination. During the diagnostic evaluation for constrictive pericarditis, she was found to have hypogammaglobulinemia and profound lymphocytopenia. A stool α-1-antitrypsin level was sent and was elevated, which confirmed the diagnosis of protein-losing enteropathy, a rare but important complication of constrictive pericarditis. This case highlights important diagnostic considerations and management of these complications for the general practitioner.

Immunological Findings and Clinical Outcomes of Infants With Positive Newborn Screening for Severe Combined Immunodeficiency From a Tertiary Care Center in the U.S.

The implementation of severe combined immunodeficiency (SCID) newborn screening has played a pivotal role in identifying these patients early in life as well as detecting various milder forms of T cell lymphopenia (TCL). In this study we reviewed the diagnostic and clinical outcomes, and interesting immunology findings of term infants referred to a tertiary care center with abnormal newborn SCID screens over a 6-year period. Key findings included a 33% incidence of non-SCID TCL including infants with novel variants in FOXN1, TBX1, MYSM1, POLD1, and CD3E; 57% positivity rate of newborn SCID screening among infants with DiGeorge syndrome; and earlier diagnosis and improved transplant outcomes for SCID in infants diagnosed after compared to before implementation of routine screening. Our study is unique in terms of the extensive laboratory workup of abnormal SCID screens including lymphocyte subsets, measurement of thymic output (TREC and CD4TE), and lymphocyte proliferation to mitogens in nearly all infants. These data allowed us to observe a stronger positive correlation of the absolute CD3 count with CD4RTE than with TREC copies, and a weak positive correlation between CD4RTE and TREC copies. Finally, we did not observe a correlation between risk of TCL and history of prenatal or perinatal complications or low birth weight. Our study demonstrated SCID newborn screening improves disease outcomes, particularly in typical SCID, and allows early detection and discovery of novel variants of certain TCL-associated genetic conditions.

Impaired tumor necrosis factor-α secretion by CD4 T cells during respiratory syncytial virus bronchiolitis associated with recurrent wheeze.

BACKGROUND: Infants with severe respiratory syncytial virus (RSV) bronchiolitis have an increased risk of recurrent wheezing and asthma. We aimed to evaluate the relationships between regulatory T cell (Treg) percentage and cytokine production of in vitro-stimulated CD4+ T cells during acute bronchiolitis and the development of recurrent wheezing in the first 3 years of life. METHODS: We obtained peripheral blood from 166 infants hospitalized with their first episode of RSV-confirmed bronchiolitis. Granzyme B (GZB) expression, and interleukin-10, interferon-γ, tumor necrosis factor-α (TNF-α), IL-4, and IL-5 production by in vitro anti-CD3/CD28- and anti-CD3/CD46-activated CD4+ T cells, and percentage of peripheral Treg (CD4+CD25hi Foxp3hi ) cells were measured by flow cytometry. Wheezing was assessed every 6 months. Recurrent wheezing was defined as three or more episodes following the initial RSV bronchiolitis. RESULTS: Sixty-seven percent (n = 111) of children had wheezing after their initial RSV infection, with 30% having recurrent wheezing. The percentage of peripheral Treg (CD4+CD25hi Foxp3hi ) cells was not significantly different between the wheezing groups. Decreased TNF-α production from anti-CD3/CD28- and anti-CD3/CD46- activated CD4+ T cells was observed in the recurrent wheezers, compared with nonwheezers (p = .048 and .03, respectively). There were no significant differences in the GZB+ CD4+ T cells and production of other inflammatory cytokines between these groups. CONCLUSIONS: We demonstrated lower TNF-α production by in vitro stimulated CD4+ T cells during severe RSV bronchiolitis in children that subsequently developed recurrent wheezing, compared with children with no subsequent wheeze. These findings support the role of CD4+ T cell immunity in the development of subsequent wheezing in these children.