Regulatory T-cell dysfunction and cutaneous exposure to Staphylococcus aureus underlie eczema in DOCK8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8)-deficient patients have severe eczema, elevated IgE, and eosinophilia, features of atopic dermatitis (AD). OBJECTIVE: We sought to understand the mechanisms of eczema in DOCK8 deficiency. METHODS: Skin biopsy samples were characterized by histology, immunofluorescence microscopy, and gene expression. Skin barrier function was measured by transepidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous sensitization with ovalbumin (OVA) or cutaneous application of Staphylococcus aureus. RESULTS: Skin lesions of DOCK8-deficient patients exhibited type 2 inflammation, and the patients' skin was colonized by Saureus, as in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory (Treg) cells and a normal skin barrier. Dock8-/- and mice with an inducible Dock8 deletion in Treg cells exhibited increased allergic skin inflammation after epicutaneous sensitization with OVA. DOCK8 was shown to be important for Treg cell stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Treg cells. Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific, inducible Treg cells suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels after topical exposure to Saureus. Both were attenuated after adoptive transfer of WT but not DOCK8-deficient Treg cells. CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to Saureus to drive eczema in DOCK8 deficiency.

Profound differences in IgE and IgG recognition of micro-arrayed allergens in hyper-IgE syndromes.

BACKGROUND: The specificities of IgE and IgG for allergen molecules in patients with inborn errors of immunity (IEI) have not been investigated in detail. OBJECTIVE: To study IgE and IgG antibody specificities in patients with defined hyper-IgE syndromes (HIES) using a comprehensive panel of allergen molecules. METHODS: We used chips containing micro-arrayed allergen molecules to analyze allergen-specific IgE and IgG levels in sera from two groups of HIES patients: Autosomal recessive mutations in phosphoglucomutase-3 (PGM3); Autosomal dominant negative mutations of STAT3 (STAT3); and age-matched subjects with allergic sensitizations. Assays with rat basophil leukemia cells transfected with human FcεRI were performed to study the biological relevance of IgE sensitizations. RESULTS: Median total IgE levels were significantly lower in the sensitized control group (212.9 kU/L) as compared to PGM3 (5042 kU/L) and STAT3 patients (2561 kU/L). However, PGM3 patients had significantly higher allergen-specific IgE levels and were sensitized to a larger number of allergen molecules as compared to STAT3 patients. Biological relevance of IgE sensitization was confirmed for PGM3 patients by basophil activation testing. PGM3 patients showed significantly lower cumulative allergen-specific IgG responses in particular to milk and egg allergens as compared to STAT3 patients and sensitized controls whereas total IgG levels were comparable to STAT3 patients and significantly higher than in controls. CONCLUSION: The analysis with multiple micro-arrayed allergen molecules reveals profound differences of allergen-specific IgE and IgG recognition in PGM3 and STAT3 patients which may be useful for classification of IEI and clinical characterization of patients.

Diagnostic challenge in a series of eleven patients with hyper IgE syndromes.

Hyper IgE syndromes (HIES) is a heterogeneous group of Inborn Errors of Immunity characterized by eczema, recurrent skin and lung infections associated with eosinophilia and elevated IgE levels. Autosomal dominant HIES caused by loss of function mutations in Signal transducer and activator of transcription 3 (STAT3) gene is the prototype of these disorders. Over the past two decades, advent in genetic testing allowed the identification of ten other etiologies of HIES. Although Dedicator of Cytokinesis 8 (DOCK8) deficiency is no more classified among HIES etiologies but as a combined immunodeficiency, this disease, characterized by severe viral infections, food allergies, autoimmunity, and increased risk of malignancies, shares some clinical features with STAT3 deficiency. The present study highlights the diagnostic challenge in eleven patients with the clinical phenotype of HIES in a resource-limited region. Candidate gene strategy supported by clinical features, laboratory findings and functional investigations allowed the identification of two heterozygous STAT3 mutations in five patients, and a bi-allelic DOCK8 mutation in one patient. Whole Exome Sequencing allowed to unmask atypical presentations of DOCK8 deficiency in two patients presenting with clinical features reminiscent of STAT3 deficiency. Our study underlies the importance of the differential diagnosis between STAT3 and DOCK8 deficiencies in order to improve diagnostic criteria and to propose appropriate therapeutic approaches. In addition, our findings emphasize the role of NGS in detecting mutations that induce overlapping phenotypes.

Genetic Approaches for Definitive Diagnosis of Agammaglobulinemia in Consanguineous Families.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

Lessons from Genetic Studies of Primary Immunodeficiencies in a Highly Consanguineous Population.

During the last decades, the study of primary immunodeficiencies (PIDs) has contributed tremendously to unravel novel pathways involved in a variety of immune responses. Many of these PIDs have an autosomal recessive (AR) mode of inheritance. Thus, the investigation of the molecular basis of PIDs is particularly relevant in consanguineous populations from Middle East and North Africa (MENA). Although significant efforts have been made in recent years to develop genetic testing across the MENA region, few comprehensive studies reporting molecular basis of PIDs in these settings are available. Herein, we review genetic characteristics of PIDs identified in 168 patients from an inbred Tunisian population. A spectrum of 25 genes involved was analyzed. We show that AR forms compared to X-linked or autosomal dominant forms are clearly the most frequent. Furthermore, the study of informative consanguineous families did allow the identification of a novel hyper-IgE syndrome linked to phosphoglucomutase 3 mutations. We did also report a novel form of autoimmune lymphoproliferative syndrome caused by homozygous FAS mutations with normal or residual protein expression as well as a novel AR transcription factor 3 deficiency. Finally, we identified several founder effects for specific AR mutations. This did facilitate the implementation of preventive approaches through genetic counseling in affected consanguineous families. All together, these findings highlight the specific nature of highly consanguineous populations and confirm the importance of unraveling the molecular basis of genetic diseases in this context. Besides providing a better fundamental knowledge of novel pathways, their study is improving diagnosis strategies and appropriate care.

A founder mutation underlies a severe form of phosphoglutamase 3 (PGM3) deficiency in Tunisian patients.

Phosphoglucomutase 3 (PGM3) protein catalyzes the conversion of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6-P) to N-acetyl-d-glucosamine-1-phosphate (GlcNAc-1-P), which is required for the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) an important precursor for protein glycosylation. Mutations in PGM3 gene have been recently shown to underlie a new congenital disorder of glycosylation often associated to elevated IgE. Herein, we report twelve PGM3 deficient patients. They belong to three highly consanguineous families, originating from a rural district in the west central Tunisia. The patient's clinical phenotype is characterized by severe respiratory and cutaneous infections as well as developmental delay and severe mental retardation. Fourteen patients died in early infancy before diagnosis supporting the severity of the clinical phenotype. Laboratory findings revealed elevated IgE, CD4 lymphopenia and impaired T cell proliferation in most patients. Genetic analysis showed the presence, of a unique homozygous mutation (p.Glu340del) in PGM3 gene leading to reduced PGM3 abundance. Segregating analysis using fifteen polymorphic markers overlapping PGM3 gene showed that all patients inherited a common homozygous haplotype encompassing 10-Mb on chromosome 6. The founder mutational event was estimated to have occurred approximately 100 years ago. To date, (p.Glu340del) mutation represents the first founder mutation identified in PGM3 gene. These findings will facilitate the development of preventive approaches through genetic counselling and prenatal diagnosis in the affected families.

Activation induced cytidine deaminase mutant (AID-His130Pro) from Hyper IgM 2 patient retained mutagenic activity on SHM artificial substrate.

Activation induced cytidine deaminase (AID) is an essential enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) during secondary immune response. Mutations in the AICDA gene are responsible for Hyper IgM 2 syndrome where both CSR and SHM or only CSR are affected. Indeed, triggering either of the two mechanisms requires the DNA deamination activity of AID. Besides, different domains of AID may be differentially involved in CSR and SHM through their interaction with specific cofactors. Herein, we studied the AID-induced SHM activity of the AID-His130Pro mutant identified in a patient with Hyper IgM 2 syndrome. AID mutagenic activity was monitored by the reversion of nonsense mutations of the EGFP gene assessed by flow cytometry. We found that the His130Pro mutation, which affects CSR, preserves AID mutagenic activity. Indeed, the His130 residue is located in a putative specific CSR region in the APOBEC-like domain, known to involve CSR specific cofactors that probably play a major role in AID physiological activities.

A Founder Effect of c.257 + 2T > C Mutation in NCF2 Gene Underlies Severe Chronic Granulomatous Disease in Eleven Patients.

Chronic granulomatous disease (CGD) is the prototypic functional neutrophil disorder caused by genetic defects in one of the five genes encoding the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase subunits of phagocytes. Mutations causing the most prevalent form of CGD in western populations are located in the X-linked-CYBB gene. The four remaining autosomal recessive (AR) forms collectively account for one-third of CGD cases. We investigated the clinical and molecular features of eleven patients with CGD from 6 consanguineous families, originating from contiguous regions in the west of Tunisia. The patients' clinical phenotype is characterized by a high incidence of mycobacterial infections. Five out of the eleven patients died despite treatment arguing in favor of a severe clinical form of CGD. These findings correlated with the absence of functional p67phox protein as well as the absence of residual reactive oxygen intermediates (ROI) production. Genetic analysis showed the presence, in all patients, of a unique mutation (c.257 + 2T > C) in NCF2 gene predicted to affect RNA splicing. Segregating analysis using nine polymorphic markers overlapping the NCF2 gene revealed a common haplotype spanning 4.1 Mb. The founder event responsible for this mutation was estimated to have arisen approximately 175 years ago. These findings will facilitate the implementation of preventive approaches through genetic counseling in affected consanguineous families.

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

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

Novel and recurrent AID mutations underlie prevalent autosomal recessive form of HIGM in consanguineous patients.

Immunoglobulin class switch recombination deficiencies (Ig-CSR-D) are characterized by normal or elevated serum IgM level and absence of IgG, IgA, and IgE. Most reported cases are due to X-linked CD40L deficiency. Activation-induced cytidine deaminase deficiency is the most frequent autosomal recessive form, whereas CD40 deficiency is more rare. Herein, we present the first North African study on hyper IgM (HIGM) syndrome including 16 Tunisian patients. Phenotypic and genetic studies allowed us to determine their molecular basis. Three CD40LG mutations have been identified including two novels (c.348_351dup and c.782_*2del) and one already reported mutation (g.6182G>A). No mutation has been found in another patient despite the lack of CD40L expression. Interestingly, three AICDA mutations have been identified in 11 patients. Two mutations were novel (c.91T>C and c.389A>C found in one and five patients respectively), and one previously reported splicing mutation (c.156+1T>G) was found in five patients. Only one CD40-deficient patient, bearing a novel mutation (c.109T>G), has been identified. Thus, unlike previous reports, AID deficiency is the most frequent underlying molecular basis (68%) of Ig-CSR-D in Tunisian patients. This finding and the presence of specific recurrent mutations are probably due to the critical role played by inbreeding in North African populations.

Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels.

BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.

A 1,100-year-old founder effect mutation in IL12B gene is responsible for Mendelian susceptibility to mycobacterial disease in Tunisian patients.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder predisposing apparently healthy individuals to infections caused by weakly virulent mycobacteria such as bacille Calmette-Guerin (BCG), environmental mycobacteria, and poorly virulent Salmonella strains. IL-12p40 deficiency is the first reported human disease due to a cytokine gene defect and is one of the deficiencies that cause MSMD. Nine mutant alleles only have been identified in the IL12B gene, and three of them are recurrent mutations due to a founder effect in specific populations. IL-12p40 deficiency has been identified especially in countries where consanguinity is high and where BCG vaccination at birth is universal. We investigated, in such settings, the clinical, cellular, and molecular features of six IL-12p40-deficient Tunisian patients having the same mutation in IL12B gene (c.298_305del). We found that this mutation is inherited as a common founder mutation arousing ~1,100 years ago. This finding facilitates the development of a preventive approach by genetic counseling and prenatal diagnosis especially in affected families.

Inherited IL-12p40 deficiency: genetic, immunologic, and clinical features of 49 patients from 30 kindreds.

Autosomal recessive interleukin (IL)-12 p40 (IL-12p40) deficiency is a rare genetic etiology of mendelian susceptibility to mycobacterial disease (MSMD). We report the genetic, immunologic, and clinical features of 49 patients from 30 kindreds originating from 5 countries (India, Iran, Pakistan, Saudi Arabia, and Tunisia). There are only 9 different mutant alleles of the IL12B gene: 2 small insertions, 3 small deletions, 2 splice site mutations, and 1 large deletion, each causing a frameshift and leading to a premature stop codon, and 1 nonsense mutation. Four of these 9 variants are recurrent, affecting 25 of the 30 reported kindreds, due to founder effects in specific countries. All patients are homozygous and display complete IL-12p40 deficiency. As a result, the patients lack detectable IL-12p70 and IL-12p40 and have low levels of interferon gamma (IFN-γ). The clinical features are characterized by childhood onset of bacille Calmette-Guérin (attenuated Mycobacterium bovis strain) (BCG) and Salmonella infections, with recurrences of salmonellosis (36.4%) more common than recurrences of mycobacterial disease (25%). BCG vaccination led to BCG disease in 40 of the 41 patients vaccinated (97.5%). Multiple mycobacterial infections were rare, observed in only 3 patients, whereas the association of salmonellosis and mycobacteriosis was observed in 9 patients. A few other infections were diagnosed, including chronic mucocutaneous candidiasis (n = 3), nocardiosis (n = 2), and klebsiellosis (n = 1). IL-12p40 deficiency has a high but incomplete clinical penetrance, with 33.3% of genetically affected relatives of index cases showing no symptoms. However, the prognosis is poor, with mortality rates of up to 28.6%. Overall, the clinical phenotype of IL-12p40 deficiency closely resembles that of interleukin 12 receptor ß1 (IL-12Rß1) deficiency. In conclusion, IL-12p40 deficiency is more common than initially thought and should be considered worldwide in patients with MSMD and other intramacrophagic infectious diseases, salmonellosis in particular.

Clinical, immunological and genetic findings of a large tunisian series of major histocompatibility complex class II deficiency patients.

INTRODUCTION: Major histocompatibility complex class II (MHC-II) expression deficiency is a combined primary immunodeficiency leading to the impairment of the cellular and humoral immune responses. A majority of affected patients belong to consanguineous families particularly from the Maghreb, where a founder effect for a highly frequent mutation (named c.338-25_338del26) in the RFXANK gene was reported. Herein, we report the largest single Maghrebian country series of MHC-II deficient patients. PATIENTS AND METHODS: In Tunisia, among 551 PIDs diagnosed from 1993 to 2011, 54 had an MHC-II deficiency. The clinical features and immunological investigations were retrospectively analyzed in 34 children of them belonging to 28 kindred. The genetic study included the c.338-25_338del26 screening by the amplification of the affected region using polymerase chain reaction (PCR) followed by direct sequencing. RESULTS: Consanguinity was present in 22 out of 28 families. Mean age at the first infection was 6.1 months. Chronic diarrhea with failure to thrive and pulmonary infections were the most common manifestations occurring in 26 and 28 patients respectively. The most specific laboratory findings were the defect of MHC-II (HLA-DR) expression in all patients. The c.338-25_338del26 mutation was identified in 25 of them. CONCLUSION: In Maghrebian settings, pediatricians should definitely consider this diagnosis in the presence of an early onset of severe and recurrent infections of the respiratory and intestinal tracts, particularly protracted diarrhea with a failure to thrive. The founder effect for the c.338-25_338del26 mutation in the RFXANK gene is also confirmed, facilitating prenatal diagnosis as a preventive approach in the Tunisian affected families with severe forms, particularly in the context of limited access to bone marrow transplantation.