Gulbu Uzel

Combined Immunodeficiency Associated with DOCK8 Mutations

BACKGROUND Recurrent sinopulmonary and cutaneous viral infections with elevated serum levels of IgE are features of some variants of combined immunodeficiency. The genetic causes of these variants are unknown. METHODS We collected longitudinal clinical data on 11 patients from eight families who had recurrent sinopulmonary and cutaneous viral infections. We performed comparative genomic hybridization arrays and targeted gene sequencing. Variants with predicted loss-of-expression mutations were confirmed by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay and immunoblotting. We evaluated the number and function of lymphocytes with the use of in vitro assays and flow cytometry. RESULTS Patients had recurrent otitis media, sinusitis, and pneumonias; recurrent Staphylococcus aureus skin infections with otitis externa; recurrent, severe herpes simplex virus or herpes zoster infections; extensive and persistent infections with molluscum contagiosum; and human papillomavirus infections. Most patients had severe atopy with anaphylaxis; several had squamous-cell carcinomas, and one had T-cell lymphoma-leukemia. Elevated serum IgE levels, hypereosinophilia, low numbers of T cells and B cells, low serum IgM levels, and variable IgG antibody responses were common. Expansion in vitro of activated CD8 T cells was impaired. Novel homozygous or compound heterozygous deletions and point mutations in the gene encoding the dedicator of cytokinesis 8 protein (DOCK8) led to the absence of DOCK8 protein in lymphocytes. CONCLUSIONS Autosomal recessive DOCK8 deficiency is associated with a novel variant of combined immunodeficiency.

Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (MonoMAC) syndrome

The syndrome of monocytopenia, B-cell and NK-cell lymphopenia, and mycobacterial, fungal, and viral infections is associated with myelodysplasia, cytogenetic abnormalities, pulmonary alveolar proteinosis, and myeloid leukemias. Both autosomal dominant and sporadic cases occur. We identified 12 distinct mutations in GATA2 affecting 20 patients and relatives with this syndrome, including recurrent missense mutations affecting the zinc finger-2 domain (R398W and T354M), suggesting dominant interference of gene function. Four discrete insertion/deletion mutations leading to frame shifts and premature termination implicate haploinsufficiency as a possible mechanism of action as well. These mutations were found in hematopoietic and somatic tissues, and several were identified in families, indicating germline transmission. Thus, GATA2 joins RUNX1 and CEBPA not only as a familial leukemia gene but also as a cause of a complex congenital immunodeficiency that evolves over decades and combines predisposition to infection and myeloid malignancy.

Residual NADPH Oxidase and Survival in Chronic Granulomatous Disease

BACKGROUND Failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs) is the major defect in chronic granulomatous disease, causing recurrent infections and granulomatous complications. Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice, or deletion mutations in the genes for p22(phox), p40(phox), p47(phox), p67(phox) (autosomal chronic granulomatous disease), or gp91(phox) (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived ROIs. We hypothesized that residual ROI production might be linked to survival in patients with chronic granulomatous disease. METHODS We assessed the risks of illness and death among 287 patients with chronic granulomatous disease from 244 kindreds. Residual ROI production was measured with the use of superoxide-dependent ferricytochrome c reduction and flow cytometry with dihydrorhodamine oxidation assays. Expression of NADPH oxidase component protein was detected by means of immunoblotting, and the affected genes were sequenced to identify causal mutations. RESULTS Survival of patients with chronic granulomatous disease was strongly associated with residual ROI production as a continuous variable, independently of the specific gene affected. Patients with mutations in p47(phox) and most missense mutations in gp91(phox) (with the exception of missense mutations in the nucleotide-binding and heme-binding domains) had more residual ROI production than patients with nonsense, frameshift, splice, or deletion mutations in gp91(phox). After adolescence, mortality curves diverged according to the extent of residual ROI production. CONCLUSIONS Patients with chronic granulomatous disease and modest residual production of ROI have significantly less severe illness and a greater likelihood of long-term survival than patients with little residual ROI production. The production of residual ROI is predicted by the specific NADPH oxidase mutation, regardless of the specific gene affected, and it is a predictor of survival in patients with chronic granulomatous disease. (Funded by the National Institutes of Health.).

GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity.

Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.

Second messenger role for Mg2+ revealed by human T-cell immunodeficiency

The magnesium ion, Mg2+, is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca2+ does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg2+ influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg2+ influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca2+ influx in T cells but not B cells. These observations reveal a role for Mg2+ as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.Summary The magnesium ion, Mg2+, is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a role in intracellular signaling similar to Ca2+ is unknown. In this study, we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T lymphocyte activation. We demonstrate that a rapid transient Mg2+ influx is induced by antigen receptor stimulation in T cells or growth factor stimulation in non-lymphoid cells. MagT1 deficiency abrogates the Mg2+ influx leading to impaired responses to antigen receptor engagement including defective activation of phospholipase Cγ and a markedly impaired Ca2+ influx in T cells but not B cells. These observations reveal a role for Mg2+ as an intracellular second messenger and identify MagT1 as a possible target for novel therapeutics.

Autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses, and myelodysplasia

We identified 18 patients with the distinct clinical phenotype of susceptibility to disseminated nontuberculous mycobacterial infections, viral infections, especially with human papillomaviruses, and fungal infections, primarily histoplasmosis, and molds. This syndrome typically had its onset in adulthood (age range, 7-60 years; mean, 31.1 years; median, 32 years) and was characterized by profound circulating monocytopenia (mean, 13.3 cells/microL; median, 14.5 cells/microL), B lymphocytopenia (mean, 9.4 cells/microL; median, 4 cells/microL), and NK lymphocytopenia (mean, 16 cells/microL; median, 5.5 cells/microL). T lymphocytes were variably affected. Despite these peripheral cytopenias, all patients had macrophages and plasma cells at sites of inflammation and normal immunoglobulin levels. Ten of these patients developed 1 or more of the following malignancies: 9 myelodysplasia/leukemia, 1 vulvar carcinoma and metastatic melanoma, 1 cervical carcinoma, 1 Bowen disease of the vulva, and 1 multiple Epstein-Barr virus(+) leiomyosarcoma. Five patients developed pulmonary alveolar proteinosis without mutations in the granulocyte-macrophage colony-stimulating factor receptor or anti-granulocyte-macrophage colony-stimulating factor autoantibodies. Among these 18 patients, 5 families had 2 generations affected, suggesting autosomal dominant transmission as well as sporadic cases. This novel clinical syndrome links susceptibility to mycobacterial, viral, and fungal infections with malignancy and can be transmitted in an autosomal dominant pattern.

Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation–polyendocrinopathy–enteropathy–X-linked–like syndrome

BACKGROUND Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

Viral infections in interferon-γ receptor deficiency

Abstract Interferon-γ receptor deficiency is a recently described immunodeficiency that is associated with onset of severe mycobacterial infections in childhood. We describe the occurrence of symptomatic and often severe viral infections in 4 patients with interferon-γ receptor deficiency and mycobacterial disease. The viral pathogens included herpes viruses, parainfluenza virus type 3, and respiratory syncytial virus. We conclude that patients with interferon-γ receptor deficiency and mycobacterial disease have increased susceptibility to some viral pathogens. (J Pediatr 1999;135:640-3)

Immune deficiencies, infection, and systemic immune disordersDominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation–polyendocrinopathy–enteropathy–X-linked–like syndrome

BACKGROUND Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis.

BACKGROUND Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. OBJECTIVE We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. METHODS PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. RESULTS We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation. CONCLUSION Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.