Lung disease in STAT3 hyper-IgE syndrome requires intense therapy.

BACKGROUND: Pulmonary complications are responsible for high morbidity and mortality rates in patients with the rare immunodeficiency disorder STAT3 hyper-IgE syndrome (STAT3-HIES). The aim of this study was to expand knowledge about lung disease in STAT3-HIES. METHODS: The course of pulmonary disease, radiological and histopathological interrelations, therapeutic management, and the outcome of 14 STAT3-HIES patients were assessed. RESULTS: The patients' quality of life was compromised most by pulmonary disease. All 14 patients showed first signs of lung disease at a median onset of 1.5 years of age. Lung function revealed a mixed obstructive-restrictive impairment with reduced FEV1 and FVC in 75% of the patients. The severity of lung function impairment was associated with Aspergillus fumigatus infection and prior lung surgery. Severe lung tissue damage, with reduced numbers of ATP-binding cassette sub-family A member 3 (ABCA3) positive type II pneumocytes, was observed in the histological assessment of two deceased patients. Imaging studies of all patients above 6 years of age showed severe airway and parenchyma destruction. Lung surgeries frequently led to complications, including fistula formation. Long-term antifungal and antibacterial treatment proved to be beneficial, as were inhalation therapy, chest physiotherapy, and exercise. Regular immunoglobulin replacement therapy tended to stabilize lung function. CONCLUSIONS: Due to its severity, pulmonary disease in STAT3-HIES patients requires strict monitoring and intensive therapy.

Hematopoietic Stem Cell Transplantation as Treatment for Patients with DOCK8 Deficiency.

BACKGROUND: Biallelic variations in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency with eczema, recurrent bacterial and viral infections, and malignancy. Natural disease outcome is dismal, but allogeneic hematopoietic stem cell transplantation (HSCT) can cure the disease. OBJECTIVE: To determine outcome of HSCT for DOCK8 deficiency and define possible outcome variables. METHODS: We performed a retrospective study of the results of HSCT in a large international cohort of DOCK8-deficient patients. RESULTS: We identified 81 patients from 22 centers transplanted at a median age of 9.7 years (range, 0.7-27.2 years) between 1995 and 2015. After median follow-up of 26 months (range, 3-135 months), 68 (84%) patients are alive. Severe acute (III-IV) or chronic graft versus host disease occurred in 11% and 10%, respectively. Causes of death were infections (n = 5), graft versus host disease (5), multiorgan failure (2), and preexistent lymphoma (1). Survival after matched related (n = 40) or unrelated (35) HSCT was 89% and 81%, respectively. Reduced-toxicity conditioning based on either treosulfan or reduced-dose busulfan resulted in superior survival compared with fully myeloablative busulfan-based regimens (97% vs 78%; P = .049). Ninety-six percent of patients younger than 8 years at HSCT survived, compared with 78% of those 8 years and older (P = .06). Of the 73 patients with chimerism data available, 65 (89%) had more than 90% donor T-cell chimerism at last follow-up. Not all disease manifestations responded equally well to HSCT: eczema, infections, and mollusca resolved quicker than food allergies or failure to thrive. CONCLUSIONS: HSCT is curative in most DOCK8-deficient patients, confirming this approach as the treatment of choice. HSCT using a reduced-toxicity regimen may offer the best chance for survival.

Efficacy and safety of trimodulin, a novel polyclonal antibody preparation, in patients with severe community-acquired pneumonia: a randomized, placebo-controlled, double-blind, multicenter, phase II trial (CIGMA study).

PURPOSE: The CIGMA study investigated a novel human polyclonal antibody preparation (trimodulin) containing ~ 23% immunoglobulin (Ig) M, ~ 21% IgA, and ~ 56% IgG as add-on therapy for patients with severe community-acquired pneumonia (sCAP). METHODS: In this double-blind, phase II study (NCT01420744), 160 patients with sCAP requiring invasive mechanical ventilation were randomized (1:1) to trimodulin (42 mg IgM/kg/day) or placebo for five consecutive days. Primary endpoint was ventilator-free days (VFDs). Secondary endpoints included 28-day all-cause and pneumonia-related mortality. Safety and tolerability were monitored. Exploratory post hoc analyses were performed in subsets stratified by baseline C-reactive protein (CRP; ≥ 70 mg/L) and/or IgM (≤ 0.8 g/L). RESULTS: Overall, there was no statistically significant difference in VFDs between trimodulin (mean 11.0, median 11 [n = 81]) and placebo (mean 9.6; median 8 [n = 79]; p = 0.173). Twenty-eight-day all-cause mortality was 22.2% vs. 27.8%, respectively (p = 0.465). Time to discharge from intensive care unit and mean duration of hospitalization were comparable between groups. Adverse-event incidences were comparable. Post hoc subset analyses, which included the majority of patients (58-78%), showed significant reductions in all-cause mortality (trimodulin vs. placebo) in patients with high CRP, low IgM, and high CRP/low IgM at baseline. CONCLUSIONS: No significant differences were found in VFDs and mortality between trimodulin and placebo groups. Post hoc analyses supported improved outcome regarding mortality with trimodulin in subsets of patients with elevated CRP, reduced IgM, or both. These findings warrant further investigation. TRIAL REGISTRATION: NCT01420744.

TNFRSF1A and MEFV mutations in childhood onset multiple sclerosis.

To investigate frequency and phenotype of TNFRSF1A and MEFV mutations in childhood-onset multiple sclerosis (MS). Twenty-nine clinically well characterized patients were investigated for mutations in exons 2, 3, 4, and 6 of the TNFRSF1A gene and in exons 2, 3, 9, 10 of the MEFV gene. Standardized morbidity ratio (SMR) was used to assess whether the number of observed mutations was higher than expected. Eleven out of 29 patients tested positive for mutations. Heterozygosity for the TNFRSF1A R92Q (rs4149584) variant was found in 6/11 mutation-positive patients. The SMR for R92Q in our pediatric MS population was 4.6 (95% CI 1.7-10.0), 7.0 (95% CI 2.6-15.2), and 13.6 (95% CI 5.0-29.7), depending on reference population. Six patients carried at least one heterozygous MEFV mutation with SMRs of 21.4 (95% CI 7.9-46.6) and 14.6 (95% CI 5.4-31.9). Clinical characteristics of childhood MS patients with or without mutations did not differ significantly. Conclusion One third of our childhood MS patients had a heterozygous mutation in the TNFRSF1A and/or MEFV gene. This proportion by far exceeds the number of mutations expected and was higher than in adult MS patients, suggesting that these mutations might contribute to the pathogenesis of childhood MS.

Outcome of hematopoietic cell transplantation for DNA double-strand break repair disorders.

BACKGROUND: Rare DNA breakage repair disorders predispose to infection and lymphoreticular malignancies. Hematopoietic cell transplantation (HCT) is curative, but coadministered chemotherapy or radiotherapy is damaging because of systemic radiosensitivity. We collected HCT outcome data for Nijmegen breakage syndrome, DNA ligase IV deficiency, Cernunnos-XRCC4-like factor (Cernunnos-XLF) deficiency, and ataxia-telangiectasia (AT). METHODS: Data from 38 centers worldwide, including indication, donor, conditioning regimen, graft-versus-host disease, and outcome, were analyzed. Conditioning was classified as myeloablative conditioning (MAC) if it contained radiotherapy or alkylators and reduced-intensity conditioning (RIC) if no alkylators and/or 150 mg/m2 fludarabine or less and 40 mg/kg cyclophosphamide or less were used. RESULTS: Fifty-five new, 14 updated, and 18 previously published patients were analyzed. Median age at HCT was 48 months (range, 1.5-552 months). Twenty-nine patients underwent transplantation for infection, 21 had malignancy, 13 had bone marrow failure, 13 received pre-emptive transplantation, 5 had multiple indications, and 6 had no information. Twenty-two received MAC, 59 received RIC, and 4 were infused; information was unavailable for 2 patients. Seventy-three of 77 patients with DNA ligase IV deficiency, Cernunnos-XLF deficiency, or Nijmegen breakage syndrome received conditioning. Survival was 53 (69%) of 77 and was worse for those receiving MAC than for those receiving RIC (P = .006). Most deaths occurred early after transplantation, suggesting poor tolerance of conditioning. Survival in patients with AT was 25%. Forty-one (49%) of 83 patients experienced acute GvHD, which was less frequent in those receiving RIC compared with those receiving MAC (26/56 [46%] vs 12/21 [57%], P = .45). Median follow-up was 35 months (range, 2-168 months). No secondary malignancies were reported during 15 years of follow-up. Growth and developmental delay remained after HCT; immune-mediated complications resolved. CONCLUSION: RIC HCT resolves DNA repair disorder-associated immunodeficiency. Long-term follow-up is required for secondary malignancy surveillance. Routine HCT for AT is not recommended.

Mutation in an exonic splicing enhancer site causing chronic granulomatous disease.

In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.

Treatment of Infantile Inflammatory Bowel Disease and Autoimmunity by Allogeneic Stem Cell Transplantation in LPS-Responsive Beige-Like Anchor Deficiency.

Inflammatory bowel disease (IBD) in young children can be a clinical manifestation of various primary immunodeficiency syndromes. Poor clinical outcome is associated with poor quality of life and high morbidity from the complications of prolonged immunosuppressive treatment and malabsorption. In 2012, mutations in the lipopolysaccharide-responsive beige-like anchor (LRBA) gene were identified as the cause of an autoimmunity and immunodeficiency syndrome. Since then, several LRBA-deficient patients have been reported with a broad spectrum of clinical manifestations without reliable predictive prognostic markers. Allogeneic hematopoietic stem cell transplantation (alloHSCT) has been performed in a few severely affected patients with complete or partial response. Herein, we present a detailed course of the disease and the transplantation procedure used in a LRBA-deficient patient suffering primarily from infantile IBD with immune enteropathy since the age of 6 weeks, and progressive autoimmunity with major complications following long-term immunosuppressive treatment. At 12 years of age, alloHSCT using bone marrow of a fully matched sibling donor-a healthy heterozygous LRBA mutant carrier-was performed after conditioning with a reduced-intensity regimen. During the 6-year follow-up, we observed a complete remission of enteropathy, autoimmunity, and skin vitiligo, with complete donor chimerism. The genetic diagnosis of LRBA deficiency was made post-alloHSCT by detection of two compound heterozygous mutations, using targeted sequencing of DNA samples extracted from peripheral blood before the transplantation.

Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells in human subjects, causing hemolytic anemia linked to impaired nicotinamide adenine dinucleotide phosphate (NADPH) production and imbalanced redox homeostasis in erythrocytes. Because G6PD is expressed by a variety of hematologic and nonhematologic cells, a broader clinical phenotype could be postulated in G6PD-deficient patients. We describe 3 brothers with severe G6PD deficiency and susceptibility to bacterial infection. OBJECTIVE: We sought to study the molecular pathophysiology leading to susceptibility to infection in 3 siblings with severe G6PD deficiency. METHODS: Blood samples of 3 patients with severe G6PD deficiency were analyzed for G6PD enzyme activity, cellular oxidized nicotinamide adenine dinucleotide phosphate/NADPH levels, phagocytic reactive oxygen species production, neutrophil extracellular trap (NET) formation, and neutrophil elastase translocation. RESULTS: In these 3 brothers strongly reduced NADPH oxidase function was found in granulocytes, leading to impaired NET formation. Defective NET formation has thus far been only observed in patients with the NADPH oxidase deficiency chronic granulomatous disease, who require antibiotic and antimycotic prophylaxis to prevent life-threatening bacterial and fungal infections. CONCLUSION: Because severe G6PD deficiency can be a phenocopy of chronic granulomatous disease with regard to the cellular and clinical phenotype, careful evaluation of neutrophil function seems mandatory in these patients to decide on appropriate anti-infective preventive measures. Determining the level of G6PD enzyme activity should be followed by analysis of reactive oxygen species production and NET formation to decide on required antibiotic and antimycotic prophylaxis.

Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.

Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guérin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism (22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms (6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A-producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis.

The extended phenotype of LPS-responsive beige-like anchor protein (LRBA) deficiency.

BACKGROUND: LPS-responsive beige-like anchor protein (LRBA) deficiency is a primary immunodeficiency caused by biallelic mutations in LRBA that abolish LRBA protein expression. OBJECTIVE: We sought to report the extended phenotype of LRBA deficiency in a cohort of 22 LRBA-deficient patients. METHODS: Clinical criteria, protein detection, and genetic sequencing were applied to diagnose LRBA deficiency. RESULTS: Ninety-three patients met the inclusion criteria and were considered to have possible LRBA deficiency. Twenty-four patients did not express LRBA protein and were labeled as having probable LRBA deficiency, whereas 22 were genetically confirmed as having definitive LRBA deficiency, with biallelic mutations in LRBA. Seventeen of these were novel and included homozygous or compound heterozygous mutations. Immune dysregulation (95%), organomegaly (86%), recurrent infections (71%), and hypogammaglobulinemia (57%) were the main clinical complications observed in LRBA-deficient patients. Although 81% of LRBA-deficient patients had normal T-cell counts, 73% had reduced regulatory T (Treg) cell numbers. Most LRBA-deficient patients had low B-cell subset counts, mainly in switched memory B cells (80%) and plasmablasts (92%), with a defective specific antibody response in 67%. Of the 22 patients, 3 are deceased, 2 were treated successfully with hematopoietic stem cell transplantation, 7 are receiving immunoglobulin replacement, and 15 are receiving immunosuppressive treatment with systemic corticosteroids alone or in combination with steroid-sparing agents. CONCLUSION: This report describes the largest cohort of patients with LRBA deficiency and offers guidelines for physicians to identify LRBA deficiency, supporting appropriate clinical management.

Key findings to expedite the diagnosis of hyper-IgE syndromes in infants and young children.

BACKGROUND: Hyper-IgE syndromes (HIES) are primary immunodeficiency disorders characterized by elevated serum IgE, eczema, and recurrent infections. Despite the availability of confirmatory molecular diagnosis of several distinct HIES entities, the differentiation of HIES particularly from severe forms of atopic dermatitis remains a challenge. The two most common forms of HIES are caused by mutations in the genes STAT3 and DOCK8. METHODS: Here, we assess the clinical and immunologic phenotype of DOCK8- and STAT3-HIES patients including the cell activation, proliferation, and cytokine release after stimulation. RESULTS: Existing HIES scoring systems are helpful to identify HIES patients. However, those scores may fail in infants and young children due to the age-related lack of clinical symptoms. Furthermore, our long-term observations showed a striking variation of laboratory results over time in the individual patient. Reduced memory B-cell counts in concert with low specific antibody production are the most consistent findings likely contributing to the high susceptibility to bacterial and fungal infection. In DOCK8-HIES, T-cell lymphopenia and low IFN-gamma secretion after stimulation were common, likely promoting viral infections. In contrast to STAT3-HIES, DOCK8-HIES patients showed more severe inflammation with regard to allergic manifestations, elevated activation markers (HLA-DR, CD69, CD86, and CD154), and significantly increased inflammatory cytokines (IL1-beta, IL4, IL6, and IFN-gamma). CONCLUSION: Differentiating HIES from other diseases such as atopic dermatitis early in life is essential for patients because treatment modalities differ. To expedite the diagnosis process, we propose here a diagnostic workflow.

Diagnostic and Treatment Options for Severe IBD in Female X-CGD Carriers with Non-random X-inactivation.

BACKGROUND AND AIMS: X-linked chronic granulomatous disease [X-CGD] due to hemizygous mutations in CYBB is characterised by invasive bacterial and fungal infections and granulomatous inflammation. Inflammatory bowel disease [IBD] is an additional or isolated manifestation. Allogeneic haematopoietic stem cell transplantation [alloHSCT] is the standard curative treatment. X-CGD carriers are usually healthy but those with non-random X-chromosome inactivation [XCI] may develop infectious or autoinflammatory manifestations. METHODS AND RESULTS: We report on two female patients with severe treatment-refractory Crohn-like IBD manifesting at age 23 and 8 years, respectively. NADPH-oxidase activity testing and molecular genetics proved X-CGD carrier status with non-random XCI. As in CGD, histopathology from colonic biopsies disclosed pigment-laden macrophages and reduced CD68(+) macrophages. Following submyelo-ablative conditioning, the younger patient was treated with alloHSCT at age 20 years. She came into remission within 3 months after transplantation and shows complete mucosal healing after 16 months off all medications. CONCLUSIONS: We suggest that children and young adults with refractory IBD should mandatorily be tested for CGD. AlloHSCT should be considered as curative therapy in severely diseased female carriers of X-CGD with non-random XCI.

Nijmegen Breakage Syndrome: Clinical and Immunological Features, Long-Term Outcome and Treatment Options - a Retrospective Analysis.

PURPOSE: Nijmegen Breakage Syndrome (NBS) is a rare inherited condition, characterized by microcephaly, chromosomal instability, immunodeficiency, and predisposition to malignancy. This retrospective study, characterizing the clinical and immunological status of patients with NBS at time of diagnosis, was designed to assess whether any parameters were useful in disease prognosis, and could help determine patients qualified for hematopoietic stem cell transplantation. METHODS: The clinical and immunological characteristics of 149 NBS patients registered in the online database of the European Society for Immune Deficiencies were analyzed. RESULTS: Of the 149 NBS patients, 91 (61%), of median age 14.3 years, remained alive at the time of analysis. These patients were clinically heterogeneous, with variable immune defects, ranging from negligible to severe dysfunction. Humoral deficiencies predisposed NBS patients to recurrent/chronic respiratory tract infections and worsened long-term clinical prognosis. Eighty malignancies, most of lymphoid origin (especially non-Hodgkin's lymphomas), were diagnosed in 42% of patients, with malignancy being the leading cause of death in this cohort. Survival probabilities at 5, 10, 20 and 30 years of age were 95, 85, 50 and 35%, respectively, and were significantly lower in patients with than without malignancies. CONCLUSIONS: The extremely high incidence of malignancies, mostly non-Hodgkin's lymphomas, was the main risk factor affecting survival probability in NBS patients. Because treatment of NBS is very difficult and frequently unsuccessful, the search for an alternative medical intervention such as hematopoietic stem cell transplantation is of great clinical importance.

Chronic Candida albicans Meningitis in a 4-Year-Old Girl with a Homozygous Mutation in the CARD9 Gene (Q295X).

A 4-year-old Turkish girl of consanguineous parents was hospitalized for the evaluation of headaches and recurrent febrile episodes of unknown origin. Her medical history was unremarkable except for a few episodes of uncomplicated oral thrush. Meningitis was diagnosed, and Candida albicans was the only pathogen identified by polymerase chain reaction and culture. Despite systemic antifungal multidrug therapy, a prolonged course of 16 months of therapy was necessary to clear C. albicans from the cerebrospinal fluid. Molecular genetic analysis revealed a homozygous caspase recruitment domain 9 (CARD9) mutation (Q295X), which was reported to predispose to chronic mucocutaneous candidiasis. Immunologic workup excluded predisposing B-cell and T-cell defects. In addition, T cells producing interleukin-17 were repeatedly measured within the normal range. Analyses of neutrophils demonstrated normal nicotinamide adenine dinucleotide phosphate oxidase activity in response to various stimuli including Staphylococcus aureus and C. albicans. Additional neutrophilic functional testing, however, showed a decreased cytotoxicity to nonopsonized C. albicans, indicating an impaired killing mechanism against Candida spp. independent from the production of reactive oxygen species by the nicotinamide adenine dinucleotide phosphate oxidase system. Because this defect was only demonstrated in the absence of opsonins, it might especially predispose to chronic C. albicans infections in the central nervous system where opsonin concentrations are usually low. We, therefore, suggest that due to an additional neutrophil dependent defect CARD9 deficiency predisposes not only to chronic mucocutaneous candidiasis, but also to invasive chronic Candida infections, especially of the central nervous system.

Clinical picture and treatment of 2212 patients with common variable immunodeficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is an antibody deficiency with an equal sex distribution and a high variability in clinical presentation. The main features include respiratory tract infections and their associated complications, enteropathy, autoimmunity, and lymphoproliferative disorders. OBJECTIVE: This study analyzes the clinical presentation, association between clinical features, and differences and effects of immunoglobulin treatment in Europe. METHODS: Data on 2212 patients with CVID from 28 medical centers contributing to the European Society for Immunodeficiencies Database were analyzed retrospectively. RESULTS: Early disease onset (<10 years) was very frequent in our cohort (33.7%), especially in male subjects (39.8%). Male subjects with early-onset CVID were more prone to pneumonia and less prone to other complications suggesting a distinct disease entity. The diagnostic delay of CVID ranges between 4 and 5 years in many countries and is particularly high in subjects with early-onset CVID. Enteropathy, autoimmunity, granulomas, and splenomegaly formed a set of interrelated features, whereas bronchiectasis was not associated with any other clinical feature. Patient survival in this cohort was associated with age at onset and age at diagnosis only. There were different treatment strategies in Europe, with considerable differences in immunoglobulin dosing, ranging from 130 up to 750 mg/kg/mo. Patients with very low trough levels of less than 4 g/L had poor clinical outcomes, whereas higher trough levels were associated with a reduced frequency of serious bacterial infections. CONCLUSION: Patients with CVID are being managed differently throughout Europe, affecting various outcome measures. Clinically, CVID is a truly variable antibody deficiency syndrome.

Very early onset inflammatory bowel disease associated with aberrant trafficking of IL-10R1 and cure by T cell replete haploidentical bone marrow transplantation.

PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.

Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study.

BACKGROUND: In chronic granulomatous disease allogeneic haemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients. METHODS: This prospective study was done at 16 centres in ten countries worldwide. Patients aged 0-40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centres. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m(2) [infants <9 kg 1·2 mg/kg]; one dose per day on days -8 to -3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days -4 to -1; or thymoglobuline 2·5 mg/kg, one dose per day on days -5 to -3]; or low-dose alemtuzumab [<1 mg/kg on days -8 to -6]), and low-dose (50-72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45-65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days -5 to -3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centres (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related-donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. FINDINGS: 56 patients (median age 12·7 years; IQR 6·8-17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14-39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16-22) for neutrophils and 21 days (IQR 16-25) for platelets. At median follow-up of 21 months (IQR 13-35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86·46-99·09) and of EFS was 91% (79·78-96·17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III-IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. INTERPRETATION: This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease. FUNDING: None.

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.

Lung parenchyma surgery in autosomal dominant hyper-IgE syndrome.

PURPOSE: Autosomal dominant hyper-IgE syndrome (AD-HIES) due to heterozygous STAT3 mutation is a primary immunodeficiency characterized by eczema, elevated serum IgE, recurrent infections, and connective tissue and skeletal findings. Healing of pneumonias is often abnormal with formation of pneumatoceles and bronchiectasis. We aimed to explore whether healing after lung surgery is also aberrant. METHODS: We retrospectively analyzed the medical records of 32 patients with AD-HIES who received lung surgery for the management of pulmonary infections from 1960 to 2011. We collected information including patient demographics, STAT3 mutation status, clinical history, surgical and medical procedures performed, complications, related medical treatments, and outcomes. RESULTS: More than 50% of lung surgeries had associated complications, with the majority being prolonged bronchopleural fistulae. These fistulae often led to empyemas that necessitated additional interventions including prolonged antibiotics, prolonged thoracostomy tube drainage and re-operations. CONCLUSION: Lung surgery in AD-HIES patients is associated with high complication rates. STAT3 mutations likely lead to abnormalities in tissue remodelling that are further exacerbated by infection.

Defective actin accumulation impairs human natural killer cell function in patients with dedicator of cytokinesis 8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) mutations are responsible for a rare primary combined immunodeficiency syndrome associated with severe cutaneous viral infections, increased IgE levels, autoimmunity, and malignancy. Natural killer (NK) cells are essential for tumor surveillance and defense against virally infected cells. NK cell function relies on Wiskott-Aldrich syndrome protein for filamentous actin (F-actin) accumulation at the lytic NK cell immunologic synapse. DOCK8 activates cell division cycle 42, which, together with Wiskott-Aldrich syndrome protein, coordinates F-actin reorganization. Although abnormalities in T- and B-cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is unclear. OBJECTIVES: We sought to understand the role of DOCK8 in NK cell function to determine whether NK cell abnormalities explain the pathogenesis of the clinical syndrome of DOCK8 deficiency. METHODS: A cohort of DOCK8-deficient patients was assembled, and patients' NK cells, as well as NK cell lines with stably reduced DOCK8 expression, were studied. NK cell cytotoxicity, F-actin content, and lytic immunologic synapse formation were measured. RESULTS: DOCK8-deficient patients' NK cells and DOCK8 knockdown cell lines all had decreased NK cell cytotoxicity, which could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency impaired F-actin accumulation at the lytic immunologic synapse without affecting overall NK cell F-actin content. CONCLUSIONS: DOCK8 deficiency results in severely impaired NK cell function because of an inability to form a mature lytic immunologic synapse through targeted synaptic F-actin accumulation. This defect might underlie and explain important attributes of the DOCK8 deficiency clinical syndrome, including the unusual susceptibility to viral infection and malignancy.