Case Report: Analysis of Preserved Umbilical Cord Clarified X-Linked Anhidrotic Ectodermal Dysplasia With Immunodeficiency in Deceased, Undiagnosed Uncles.

Family history is one key in diagnosing inborn errors of immunity (IEI); however, disease status is difficult to determine in deceased relatives. X-linked anhidrotic ectodermal dysplasia with immunodeficiency is one of the hyper IgM syndromes that is caused by a hypomorphic variant in the nuclear factor kappa beta essential modulator. We identified a novel IKBKG variant in a 7-month-old boy with pneumococcal rib osteomyelitis and later found that his mother has incontinentia pigmenti. Genetic analysis of preserved umbilical cords revealed the same variant in two of his deceased maternal uncles. Analysis of preserved umbilical cord tissue from deceased relatives can provide important information for diagnosing IEI in their descendants.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

Ectodermal Dysplasia: Association with Anti-Basement Membrane Autoantibodies.

Ectodermal dysplasia (ED) is a group of several genetic conditions with absence or dysgenesis of at least two ectodermal derivatives: teeth, skin and its appendages including hair, nails, eccrine and sebaceous glands. The most important clinical findings in patients with ED are hypodontia, hypotrichosis, and hypohidrosis, which can lead to episodes of hyperthermia. Few reports have focused on the progressive keratopathy in ED. Cicatrizing conjunctivitis associated with anti-basement membrane autoantibodies has been described. We report a series of three ectodermal dysplasia patients with an ocular phenotype typically seen in ocular mucous membrane pemphigoid; conjunctival immunohistopathology revealed anti-basement membrane autoantibodies in all of them, and systemic immunosuppression proved to be effective in improving symptoms and helping to stabilize ocular surface disease.

Linear ubiquitin chain-binding domains.

Ubiquitin modification (ubiquitination) of target proteins can vary with respect to chain lengths, linkage type, and chain forms, such as homologous, mixed, and branched ubiquitin chains. Thus, ubiquitination can generate multiple unique surfaces on a target protein substrate. Ubiquitin-binding domains (UBDs) recognize ubiquitinated substrates, by specifically binding to these unique surfaces, modulate the formation of cellular signaling complexes and regulate downstream signaling cascades. Among the eight different homotypic chain types, Met1-linked (also termed linear) chains are the only chains in which linkage occurs on a non-Lys residue of ubiquitin. Linear ubiquitin chains have been implicated in immune responses, cell death and autophagy, and several UBDs - specific for linear ubiquitin chains - have been identified. In this review, we describe the main principles of ubiquitin recognition by UBDs, focusing on linear ubiquitin chains and their roles in biology.

Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency.

BACKGROUND: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis. OBJECTIVE: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID. METHODS: A disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts. RESULTS: Disease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin ß receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations. CONCLUSIONS: IκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID.

Interferon-gamma reduces the proliferation of M. tuberculosis within macrophages from a patient with a novel hypomorphic NEMO mutation.

X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by mutations in the nuclear factor-kappa B essential modulator (NEMO) gene. Here, we report the clinical and genetic features of a XL-EDA-ID patient who developed bacillus Calmette-Guérin infection. Patient lymphocytes failed to degrade IκB-α, and sequencing of NEMO identified the novel mutation c.1238A>C/p.H413P. Furthermore, patient monocyte-derived macrophages ingested Mycobacterium tuberculosis normally, but failed to control the intracellular proliferation of bacilli, a defect which was improved in the presence of interferon-gamma (IFN-γ). This work expands the genetic spectrum of XL-EDA-ID and demonstrates improvement in macrophage function in a NEMO-deficient patient by IFN-γ.

Rare Presentations of Epstein-Barr Virus--Associated Smooth Muscle Tumor in Children.

Epstein-Barr virus (EBV) has oncogenic potential and has been implicated in the etiology of a wide range of malignancies. Certain EBV-driven neoplasms, such as smooth muscle tumors (SMTs), manifest typically in immunocompromised patients. In children, these neoplasms have been encountered in the setting of primary immune disorders, specifically severe combined and common variable immunodeficiency syndromes. Human immunodeficiency virus infection and posttransplant immunosuppression, in particular liver and kidney transplantation, likewise increase the risk in the pediatric population. The location of these neoplasms appears related to the type of immunodeficiency: in acquired immunodeficiency syndrome they are frequently located intracranially or intraspinally, whereas after transplant they usually involve the liver or lung. We report 2 distinct cases of EBV-related SMT, unique through their coassociated immunosuppressive state or location: the 1st occurred in a patient with immunodeficiency secondary to NEMO gene mutation following hematopoietic stem cell transplantation; the 2nd developed in the orbit after heart transplant.

Unraveling the Link Between Ectodermal Disorders and Primary Immunodeficiencies.

Primary immunodeficiencies (PIDs) include a heterogeneous group of mostly monogenic diseases characterized by functional/developmental alterations of the immune system. Skin and skin annexa abnormalities may be a warning sign of immunodeficiency, since both epidermal and thymic epithelium have ectodermal origin. In this review, we will focus on the most common immune disorders associated with ectodermal alterations. Elevated IgE levels represent the immunological hallmark of hyper-IgE syndrome, characterized by severe eczema and susceptibility to infections. Ectodermal dysplasia (ED) is a group of rare disorders that affect tissues of ectodermal origin. Hypoidrotic ED (HED), the most common form, is inherited as autosomal dominant, autosomal recessive or X-linked trait (XLHED). HED and XLHED are caused by mutations in NEMO and EDA-1 genes, respectively, and show similarities in the cutaneous involvement but differences in the susceptibility to infections and immunological phenotype. Alterations in the transcription factor FOXN1 gene, expressed in the mature thymic and skin epithelia, are responsible for human and murine athymia and prevent the development of the T-cell compartment associated to ectodermal abnormalities such as alopecia and nail dystrophy. The association between developmental abnormalities of the skin and immunodeficiencies suggest a role of the skin as a primary lymphoid organ. Recently, it has been demonstrated that a co-culture of human skin-derived keratinocytes and fibroblasts, in the absence of thymic components, can support the survival of human haematopoietic stem cells and their differentiation into T-lineage committed cells.

Immune system disturbances in Clouston syndrome.

BACKGROUND: Clouston syndrome belongs to the family of ectodermal dysplasias. So far, a defective immune response has not been reported in Clouston syndrome. We report, for the first time, immunological particularities of a large multigenerational Polish family with Clouston syndrome. METHODS: Five members of the same family with Clouston syndrome, aged 6-76 years, and 20 healthy volunteers, aged 19-73 years, were enrolled in the study. In all participants, the ability of neutrophils to phagocytize opsonized Escherichia coli was assessed. Granulocyte oxidative burst was determined quantitatively, and an isolation of peripheral blood mononuclear cells and the detection of lymphocyte subsets were performed. All patients with Clouston syndrome underwent microscopic assessment of hair shafts, x-rays of the skull and hand bones, extra- and intraoral examination, and panoramic x-rays. RESULTS: Compared to the controls, all patients with Clouston syndrome presented with significantly reduced phagocytic activities of granulocytes and monocytes (P < 0.05). The percentages of granulocytes and monocytes being positive for oxidative burst were also significantly reduced in all patients with Clouston syndrome (P < 0.05). No disturbances in the percentages and absolute counts of T CD3+, T CD3+/CD4+, T CD3+/CD8+, natural killer, and B CD19+ cells were found. CONCLUSION: Although this study expands knowledge about Clouston syndrome, it also raises many questions. The results provide evidence of significantly reduced phagocytic activity and oxidative bursts of cells playing crucial roles in a nonspecific immune response. Further studies are required to understand the underlying mechanism of the hereby described abnormalities.

[Rifampicin-resistant Mycobacterium bovis BCG strain isolated from an infant with NEMO mutation]. / NEMO mutasyonlu bir bebekten izole edilen rifampisine dirençli Mycobacterium bovis BCG susu.

It is well known that disseminated Mycobacterium bovis BCG infection is developed after BCG vaccination in infants with congenital cellular immune deficiencies such as mutations in genes along the interleukin (IL)-12/interferon (IFN)-γ pathway and mutations in nuclear factor-kB essential modulator (NEMO). In this report, a rifampicin-resistant M.bovis BCG strain isolated from an infant with NEMO defect was presented. An 8-month-old male infant with NEMO defect admitted to the pediatric outpatient clinic of our hospital with fever, generalized lymphadenopathy and hepatosplenomegaly. Microscopic examination of the smears prepared from lymph node and liver biopsy specimens revealed abundant amount (3+) of acid-fast bacilli (AFB). Rifampicin-susceptible Mycobacterium tuberculosis complex (MTC) was detected by real-time PCR (GeneXpert MTB/RIF; Cepheid, USA) in the samples. The growth of mycobacteria was determined on the 20th day of culture performed in MGIT960 system (Becton Dickinson, USA). The isolate was identified as M.bovis BCG by GenoType MTBC kit (Hain Lifescience, Germany) and defined as M.bovis BCG [SIT 482 (BOV_1)] by spoligotyping. In the primary anti-tuberculosis drug susceptibility test performed by MGIT960 system, the isolate was found susceptible to rifampicin (RIF), isoniazid (INH), streptomycin (STM) and ethambutol (EMB). Then anti-tuberculosis treatment was started to the patient. However, the patient at the age of 2 years, re-admitted to the hospital with the complaint of hepatosplenomegaly. Smear of spontaneously draining abscess material obtained from subcutaneous nodules revealed intensive AFB positivity (3+) once again. In the present instance RIF-resistant MTC was detected with GeneXpert system in the specimen. The growth of mycobacteria was determined on the 13th day of culture and isolate was identified as M.bovis BCG. The present isolate was found susceptible to INH, STM and EMB but resistant to RIF. A mutation in the rpoB gene (codon 531, S531L) associated with RIF resistance was detected by using the partial sequencing of the rpoB gene. Patient died due to disseminated bovis BCG infection and multiple organ failure. To our knowledge, there are only six RIF-resistant M.bovis BCG strains isolated from patients in the literature. However, this is the first RIF-resistant M.bovis BCG strain isolated from a NEMO-deficient patient.

Novel hypomorphic mutation in IKBKG impairs NEMO-ubiquitylation causing ectodermal dysplasia, immunodeficiency, incontinentia pigmenti, and immune thrombocytopenic purpura.

NF-κB essential modulator (NEMO) is a component of the IKK complex, which participates in the activation of the NF-κB pathway. Hypomorphic mutations in the IKBKG gene result in different forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males without affecting carrier females. Here, we describe a hypomorphic and missense mutation, designated c.916G>A (p.D306N), which affects our patient, his mother, and his sister. This mutation did not affect NEMO expression; however, an immunoprecipitation assay revealed reduced ubiquitylation upon CD40-stimulation in the patient's cells. Functional studies have demonstrated reduced phosphorylation and degradation of IκBα, affecting NF-κB recruitment into the nucleus. The patient presented with clinical features of ectodermal dysplasia, immunodeficiency, and immune thrombocytopenic purpura, the latter of which has not been previously reported in a patient with NEMO deficiency. His mother and sister displayed incontinentia pigmenti indicating that, in addition to amorphic mutations, hypomorphic mutations in NEMO can affect females.

Rare mendelian primary immunodeficiency diseases associated with impaired NF-κB signaling.

Mendelian primary immunodeficiency diseases (MPIDs) are rare disorders affecting distinct constituents of the innate and adaptive immune system. Although they are genetically heterogeneous, a substantial group of MPIDs is due to mutations in genes affecting the nuclear factor-κB (NF-κB) transcription pathway, essential for cell proliferation and cell survival and involved in innate immunity and inflammation. Many of these genes encode for crucial regulatory components of the NF-κB pathway and their mutations are associated with immunological and developmental signs somehow overlapping in patients with MPIDs. At present, nine different MPIDs listed in the online mendelian inheritance in man (OMIM) are caused by mutations in at least nine different genes strictly involved in the NF-κB pathway that result in defects in immune responses. Here we report on the distinct function of each causative gene, on the impaired NF-κB step and more in general on the molecular mechanisms underlining the pathogenesis of the disease. Overall, the MPIDs affecting the NF-κB signalosome require a careful integrated diagnosis and appropriate genetic tests to be molecularly identified. Their discovery at an ever-increasing rate will help establish a common therapeutic strategy for a subclass of immunodeficient patients.

Quantitative proteomics of the human skin secretome reveal a reduction in immune defense mediators in ectodermal dysplasia patients.

In healthy human skin host defense molecules such as antimicrobial peptides (AMPs) contribute to skin immune homeostasis. In patients with the congenital disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result patients have recurrent skin infections. The disease is characterized by developmental abnormalities of ectodermal derivatives and absent or reduced sweating. We hypothesized that ED patients have a reduced skin immune defense because of the reduced ability to sweat. Therefore, we performed a label-free quantitative proteome analysis of wash solution of human skin from ED patients or healthy individuals. A clear-cut difference between both cohorts could be observed in cellular processes related to immunity and host defense. In line with the extensive underrepresentation of proteins of the immune system, dermcidin, a sweat-derived AMP, was reduced in its abundance in the skin secretome of ED patients. In contrast, proteins involved in metabolic/catabolic and biosynthetic processes were enriched in the skin secretome of ED patients. In summary, our proteome profiling provides insights into the actual situation of healthy versus diseased skin. The systematic reduction in immune system and defense-related proteins may contribute to the high susceptibility of ED patients to skin infections and altered skin colonization.

Correlating interleukin-12 stimulated interferon-γ production and the absence of ectodermal dysplasia and anhidrosis (EDA) in patients with mutations in NF-κB essential modulator (NEMO).

OBJECTIVE: Patients with hypomorphic mutations in Nuclear Factor-κB Essential Modulator (NEMO) are immunodeficient (ID) and most display ectodermal dysplasia and anhidrosis (EDA). We compared cytokine production by NEMO-ID patients with and without EDA. METHODS: PBMCs of NEMO-ID patients, four with EDA carrying E315A, C417R, D311N and Q403X, and three without EDA carrying E315A, E311_L333del and R254G, were cultured with PHA, PHA plus IL-12p70, LPS, LPS plus IFN-γ, TNF and IL-1ß. The production of various cytokines was measured in the supernatants. Fifty-nine healthy individuals served as controls. RESULTS: PBMCs of NEMO-ID patients without EDA produce subnormal amounts of IFN-γ after stimulation with PHA, but normal amounts of IFN-γ after PHA plus IL-12p70. In contrast, IFN-γ production by patients with EDA was low in both cases. Patients with EDA also generate lower PHA-stimulated IL-10 and IL-1ß than controls, whereas the production of these cytokines by patients without EDA was normal. CONCLUSION: Responses of PBMCs in NEMO-ID patients with EDA to PHA with and without IL-12p70 appear less robust than in NEMO-ID patients without EDA. This possibly indicates a better preserved NEMO function in our patients without EDA.

Autoimmune polyglandular syndrome type 3c with ectodermal dysplasia, immune deficiency and hemolytic-uremic syndrome.

Autoimmune polyglandular syndrome (APS) is a disorder which is associated with multiple endocrine gland insufficiency and also with non-endocrine manifestations. The pathophysiology of APS is poorly understood, but the hallmark evidence of APS is development of autoantibodies against multiple endocrine and non-endocrine organs. These autoantibodies are responsible for the dysfunction of the affected organs and sometimes may also cause non-endocrine organ dysfunction. The hemolytic-uremic syndrome (HUS) is a serious and life-threatening disease which develops due to many etiological factors including autoimmune disorders. Here, we present an unusual case of APS. Ectodermal dysplasia with immune deficiency and HUS occurred concomitantly in the same patient with APS type 3c. Once the autoantibody generation was initiated in the human body, development of multiple disorders due to organ dysfunction and also autoantibody-related diseases may have occurred.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

A novel gain-of-function IKBA mutation underlies ectodermal dysplasia with immunodeficiency and polyendocrinopathy.

PURPOSE: This study reports the identification of a novel heterozygous IKBA missense mutation (p.M37K) in a boy presenting with ectodermal dysplasia with immunodeficiency (EDA-ID) who had wild type IKBKG gene encoding NEMO. Our aim was to characterize the clinical course of this IκB-α gain-of-function mutant and to investigate if the p.M37K substitution affects NF-κB activation by interfering with IκB-α degradation, thus impairing NF-κB signaling and causing the EDA-ID phenotype. METHODS: NF-κB signaling was evaluated by measuring IκB-α degradation in patient fibroblasts. In addition, transiently transfected HeLa cells expressing either the M37K-mutant IκB-α allele, the previously characterized S36A-mutant IκB-α allele, or wild type IκB-α were evaluated for IκB-α degradation and NF-κB nuclear translocation following stimulation with TNF-α. RESULTS: Clinical findings revealed a classical ectodermal dysplasia phenotype complicated by recurrent mucocutaneous candidiasis, hypothyroidism, hypopituitarism, and profound combined immunodeficiency with decreased numbers of IL-17 T cells. IκB-α degradation after TNF-α and TLR agonist stimulation was abolished in patient fibroblasts as well as in HeLa cells expressing M37K-IκB-α similar to cells expressing S36A-IκB-α resulting in impaired nuclear translocation of NF-κB and reduced NF-κB dependent luciferase activity compared to cells expressing wild type IκB-α. Patient whole blood cells failed to secrete IL-6 in response to IL-1ß, Pam2CSK4, showed reduced responses to LPS and PMA/Ionomycin, and lacked IL-10 production in response to TNF-α. CONCLUSION: The novel heterozygous mutation p.M37K in IκB-α impairs NF-κB activation causing autosomal dominant EDA-ID with an expanded clinical phenotype.

[Reccurent mycobacterial diseases in patients with impaired axis IL-12/INF-gamma]. / Rola ukladu IL-12/INF-gamma w nawrotowych mykobakteriozach.

Mycobacteria is a large group of pathogens that are common in environment, in soil and tap water. Although mycobacteria [non tuberculosis mycobacteria] can inhabit body surface without causing any disease in the circumstances of primary or secondary immunodeficiency can cause clinically significant organ or systemic damage. Defect of IL-12/INFgamma axis is an example of primary immunodeficiency that predispose to mycobacterial infections while protection against other microorganisms is not damaged. We present review of known defects of IL-12/IFNgamma axis and brief presentation of our own experience.