Carolina Prando

Deep Dermatophytosis and Inherited CARD9 Deficiency

BACKGROUND Deep dermatophytosis is a severe and sometimes life-threatening fungal infection caused by dermatophytes. It is characterized by extensive dermal and subcutaneous tissue invasion and by frequent dissemination to the lymph nodes and, occasionally, the central nervous system. The condition is different from common superficial dermatophyte infection and has been reported in patients with no known immunodeficiency. Patients are mostly from North African, consanguineous, multiplex families, which strongly suggests a mendelian genetic cause. METHODS We studied the clinical features of deep dermatophytosis in 17 patients with no known immunodeficiency from eight unrelated Tunisian, Algerian, and Moroccan families. Because CARD9 (caspase recruitment domain-containing protein 9) deficiency has been reported in an Iranian family with invasive fungal infections, we also sequenced CARD9 in the patients. RESULTS Four patients died, at 28, 29, 37, and 39 years of age, with clinically active deep dermatophytosis. No other severe infections, fungal or otherwise, were reported in the surviving patients, who ranged in age from 37 to 75 years. The 15 Algerian and Tunisian patients, from seven unrelated families, had a homozygous Q289X CARD9 allele, due to a founder effect. The 2 Moroccan siblings were homozygous for the R101C CARD9 allele. Both alleles are rare deleterious variants. The familial segregation of these alleles was consistent with autosomal recessive inheritance and complete clinical penetrance. CONCLUSIONS All the patients with deep dermatophytosis had autosomal recessive CARD9 deficiency. Deep dermatophytosis appears to be an important clinical manifestation of CARD9 deficiency. (Funded by Agence Nationale pour la Recherche and others.).

Inherited IL-12p40 Deficiency: Genetic, Immunologic, and Clinical Features of 49 Patients From 30 Kindreds

AbstractAutosomal 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-&ggr;). 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 &bgr;1 (IL-12R&bgr;1) 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.

A novel form of human STAT1 deficiency impairing early but not late responses to interferons

Autosomal recessive STAT1 deficiency is associated with impaired cellular responses to interferons and susceptibility to intracellular bacterial and viral infections. We report here a new form of partial STAT1 deficiency in 2 siblings presenting mycobacterial and viral diseases. Both carried a homozygous missense mutation replacing a lysine with an asparagine residue at position 201 (K201N) of STAT1. This mutation causes the abnormal splicing out of exon 8 from most STAT1 mRNAs, thereby decreasing (by ~ 70%) STAT1 protein levels. The mutant STAT1 proteins are not intrinsically deleterious, in terms of tyrosine phosphorylation, dephosphorylation, homodimerization into γ-activating factor and heterotrimerization into ISGF-3, binding to specific DNA elements, and activation of the transcription. Interestingly, the activation of γ-activating factor and ISGF3 was impaired only at early time points in the various cells from patient (within 1 hour of stimulation), whereas sustained impairment occurs in other known forms of complete and partial recessive STAT1 deficiency. Consequently, delayed responses were normal; however, the early induction of interferon-stimulated genes was selectively and severely impaired. Thus, the early cellular responses to human interferons are critically dependent on the amount of STAT1 and are essential for the appropriate control of mycobacterial and viral infections.

A novel form of cell type-specific partial IFN-γR1 deficiency caused by a germ line mutation of the IFNGR1 initiation codon

IFN-gammaR1 deficiency is a genetic etiology of Mendelian susceptibility to mycobacterial diseases, and includes two forms of complete recessive deficiency, with or without cell surface expression, and two forms of partial deficiency, dominant or recessive. We report here a novel form of partial and recessive Interferon gamma receptor 1 (IFN-gammaR1) deficiency, which is almost as severe as complete deficiency. The patient is homozygous for a mutation of the initiation codon (M1K). No detectable expression and function of IFN-gammaR1 were found in the patients fibroblasts. However, IFN-gammaR1 expression was found to be impaired, but not abolished, on the EBV-transformed B cells, which could respond weakly to IFN-gamma. The mechanism underlying this weak expression involves leaky translation initiation at both non-AUG codons and the third AUG codon at position 19. It results in the residual expression of IFN-gammaR1 protein of normal molecular weight and function. The residual IFN-gamma signaling documented in this novel form of partial IFN-gammaR1 deficiency was not ubiquitous and was milder than that seen in other forms of partial IFN-gammaR1 deficiency, accounting for the more severe clinical phenotype of the patient, which was almost as severe as that of patients with complete deficiency.

Mycobacterial disease in patients with chronic granulomatous disease: A retrospective analysis of 71 cases

BACKGROUND Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by inborn errors of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex. From the first year of life onward, most affected patients display multiple, severe, and recurrent infections caused by bacteria and fungi. Mycobacterial infections have also been reported in some patients. OBJECTIVE Our objective was to assess the effect of mycobacterial disease in patients with CGD. METHODS We analyzed retrospectively the clinical features of mycobacterial disease in 71 patients with CGD. Tuberculosis and BCG disease were diagnosed on the basis of microbiological, pathological, and/or clinical criteria. RESULTS Thirty-one (44%) patients had tuberculosis, and 53 (75%) presented with adverse effects of BCG vaccination; 13 (18%) had both tuberculosis and BCG infections. None of these patients displayed clinical disease caused by environmental mycobacteria, Mycobacterium leprae, or Mycobacterium ulcerans. Most patients (76%) also had other pyogenic and fungal infections, but 24% presented solely with mycobacterial disease. Most patients presented a single localized episode of mycobacterial disease (37%), but recurrence (18%), disseminated disease (27%), and even death (18%) were also observed. One common feature in these patients was an early age at presentation for BCG disease. Mycobacterial disease was the first clinical manifestation of CGD in 60% of these patients. CONCLUSION Mycobacterial disease is relatively common in patients with CGD living in countries in which tuberculosis is endemic, BCG vaccine is mandatory, or both. Adverse reactions to BCG and severe forms of tuberculosis should lead to a suspicion of CGD. BCG vaccine is contraindicated in patients with CGD.

Human adaptive immunity rescues an inborn error of innate immunity

The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.

Paternal uniparental isodisomy of chromosome 6 causing a complex syndrome including complete IFN‐γ receptor 1 deficiency

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency associated with clinical disease caused by weakly virulent mycobacterial species. Interferon gamma receptor 1 (IFN‐γR1) deficiency is a genetic etiology of MSMD. We describe the clinical and genetic features of a 7‐year‐old Italian boy suffering from MSMD associated with a complex phenotype, including neonatal hyperglycemia, neuromuscular disease, and dysmorphic features. The child also developed necrotizing pneumonia caused by Rhodococcus equi. The child is homozygous for a nonsense mutation in exon 3 of IFNGR1 as a result of paternal uniparental disomy (UPD) of the entire chromosome 6. This is the first reported case of uniparental disomy resulting in a complex phenotype including MSMD.

Phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity in patients with inherited IFN-γR1 or IFN-γR2 deficiency

An understanding of protective immunity to mycobacterial infection is critical for the development of effective strategies to control tuberculosis (TB), a major public health problem worldwide. Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by clinical disease caused by weakly virulent mycobacteria, such as Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccines and nontuberculous, environmental mycobacteria (EM) (OMIM209950)1. Patients are also susceptible to M. tuberculosis. Nine genes have been found to be mutated in patients with MSMD (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, IRF8, ISG15, NEMO, and CYBB). All these genes are involved in interferon (IFN)-γ immunity, which is therefore essential for defense against mycobacterial infections in humans 2. X-linked recessive (XR)- MSMD caused by mutations in CYBB, results in impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in monocyte-derived macrophages (MDMs) and EBV-transformed B (EBV-B) cell lines, but not in monocytes or granulocytes3. By contrast, germline mutations in CYBB that impair NADPH oxidase activity in all cell types, result in X-linked chronic granulomatous disease (CGD)4. An increasing number of case reports from various countries have shown that BCG disease and TB are important features of CGD, particularly in countries in which BCG vaccine is routinely administered, TB is endemic, or both4. Macrophages are known to be the first line of defense against mycobacteria, generating the reactive oxygen species (ROS) and probably responsible for microbicidal activity 5. Phagocyte NADPH oxidase activity can be enhanced by treatment with IFN-γ and the corresponding genes can also be induced by IFN-γ 6. A contribution of NADPH oxidase deficiency to mycobacterial disease in patients with inborn errors of IFN-γ is however uncertain. The occurrence of BCG disease and TB in CGD patients and in patients with macrophage-tropic mutations of the NADPH oxidase complex suggests that impaired macrophage NADPH oxidase activity may contribute to both diseases in patients with IFN-γR deficiency 4.We therefore tested the hypothesis that the function of the NADPH oxidase complex might be partly dependent on IFN-γ, at least in human MDMs in vitro. We first evaluated NADPH oxidase activity in EBV-B cells from MSMD patients without cellular responses to IFN-γIFN-γR1/IFN-γR2 [c] complete deficiencies), patients without NADPH oxidase activity (CGD), and healthy controls. We selected EBV-B cells from patients with cIFN-γR1 (n=9) or cIFN-γR2 (n=6) deficiencies, XR-MSMD (n=6), or CGD (n= 18), and healthy controls. We assessed the ability of EBV-B cells to produce superoxide (O2-) in the cytochrome-c reduction assay and to release hydrogen peroxide (H2O2) by reducing 10-acetyl-3,7 dihydroxyphenoxazine (Resurfin or Amplex Red®). We also investigated the NADPH oxidase activity by luminal and isoluminol assays (supplemental figure 1 A-B). As previously reported3, the EBV-B cells of the healthy controls produced and released ROS following stimulation with a phorbol ester, such as phorbol myristate acetate (PMA), for two hours. All EBV-B cells from patients with cIFN-γR1/cIFN-γR2 had levels of O2- production similar to those of healthy controls (Figure 1A and supplemental figure 1A), unlike the EBV-B cells of CGD and XR-MSMD patients. However, H2O2 release from the EBV-B cells of all patients with cIFN-γR1/cIFN-γR2 deficiencies was severely impaired (Figure 1B and supplemental figure 1B). Figure 1 NADPH oxidase activity in human EBV-B cells, MDDCs and MDMs We further explored MDMs from patients with cIFN-γR1 (n=1) or cIFN-γR2 (n=2) deficiency. MDMs from these patients produced very small amounts of H2O2 after stimulation with IFN-γ or PPD, suggesting an impairment of NADPH oxidase activity (Figure 1 C, D). This is consistent with the findings for EBV-B cells (Figure 1B). We also assessed NADPH oxidase activity in monocyte-derived dendritic cells (MDDCs) from all patients. MDDCs from XR-MSMD patients have not been tested previously 3. We derived MDDCs in vitro by treatment with GM-CSF plus IL-13 7 and the respiratory burst was evaluated with Amplex Red®. MDDCs from the healthy controls released H2O2 and, as expected, MDDCs from CGD patients did not (Figure 1E). Notably, MDDCs from XR-MSMD patients (n=4), and from patients with cIFN-γR1 (n=1) or cIFN-γR2 (n=1) deficiency produced similar amounts of H2O2 than those obtained from healthy controls (Figure 1E). The impairment of NADPH oxidase activity was, therefore, cell-specific and restricted to MDMs in these patients. Conversely, the normal respiratory burst activity of granulocytes and monocytes from MSMD patients with cIFN-γR1 or cIFN-γR2 deficiency (data not shown), like that herein documented in MDDCs, may account for their protection against fungi and bacteria other than mycobacteria, whereas CGD patients are typically susceptible to these microbes. In conclusion, these results support a role for the IFN-γ pathway in the up-regulation of NADPH oxidase activity in MDMs and EBV-B cells, and suggest that impairment of the phagocyte respiratory burst contributes to BCG disease and TB in patients with inborn errors of IFN-γ immunity. IFN-γ can regulate NAPDH oxidase activity in different cell types 8. O2- is rapidly converted to H2O2 by spontaneous and enzymatic dismutation. Differences between the amounts of H2O2 and O2- produced in response to PMA were detected in the same cells. There are several possible reasons for this, including the role of endogenous superoxide dismutase in catalyzing the dismutation of O2- to H2O2, differences in kinetics and the techniques used to measure O2- and H2O2 levels, and variability due to differences in the pH of assay buffers. It was striking that some mycobacteria, e.g. M. tuberculosis, produced the enzyme such KatG, a catalase-peroxidase, that protect from killing by H2O2 but not O2- in mice 9. Probably NADPH oxidase activity contributes to cytokine production, granuloma genesis or autophagy more than killing of mycobacteria 5. The mechanisms affecting this activity in MDMs and EBV-B cells from MSMD patients remain unclear and require further investigation.

Letter to the editorPhagocyte nicotinamide adenine dinucleotide phosphate oxidase activity in patients with inherited IFN-γR1 or IFN-γR2 deficiency

An understanding of protective immunity to mycobacterial infection is critical for the development of effective strategies to control tuberculosis (TB), a major public health problem worldwide. Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by clinical disease caused by weakly virulent mycobacteria, such as Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccines and nontuberculous, environmental mycobacteria (EM) (OMIM209950)1. Patients are also susceptible to M. tuberculosis. Nine genes have been found to be mutated in patients with MSMD (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, IRF8, ISG15, NEMO, and CYBB). All these genes are involved in interferon (IFN)-γ immunity, which is therefore essential for defense against mycobacterial infections in humans 2. X-linked recessive (XR)- MSMD caused by mutations in CYBB, results in impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in monocyte-derived macrophages (MDMs) and EBV-transformed B (EBV-B) cell lines, but not in monocytes or granulocytes3. By contrast, germline mutations in CYBB that impair NADPH oxidase activity in all cell types, result in X-linked chronic granulomatous disease (CGD)4. An increasing number of case reports from various countries have shown that BCG disease and TB are important features of CGD, particularly in countries in which BCG vaccine is routinely administered, TB is endemic, or both4. Macrophages are known to be the first line of defense against mycobacteria, generating the reactive oxygen species (ROS) and probably responsible for microbicidal activity 5. Phagocyte NADPH oxidase activity can be enhanced by treatment with IFN-γ and the corresponding genes can also be induced by IFN-γ 6. A contribution of NADPH oxidase deficiency to mycobacterial disease in patients with inborn errors of IFN-γ is however uncertain. The occurrence of BCG disease and TB in CGD patients and in patients with macrophage-tropic mutations of the NADPH oxidase complex suggests that impaired macrophage NADPH oxidase activity may contribute to both diseases in patients with IFN-γR deficiency 4.We therefore tested the hypothesis that the function of the NADPH oxidase complex might be partly dependent on IFN-γ, at least in human MDMs in vitro. We first evaluated NADPH oxidase activity in EBV-B cells from MSMD patients without cellular responses to IFN-γIFN-γR1/IFN-γR2 [c] complete deficiencies), patients without NADPH oxidase activity (CGD), and healthy controls. We selected EBV-B cells from patients with cIFN-γR1 (n=9) or cIFN-γR2 (n=6) deficiencies, XR-MSMD (n=6), or CGD (n= 18), and healthy controls. We assessed the ability of EBV-B cells to produce superoxide (O2-) in the cytochrome-c reduction assay and to release hydrogen peroxide (H2O2) by reducing 10-acetyl-3,7 dihydroxyphenoxazine (Resurfin or Amplex Red®). We also investigated the NADPH oxidase activity by luminal and isoluminol assays (supplemental figure 1 A-B). As previously reported3, the EBV-B cells of the healthy controls produced and released ROS following stimulation with a phorbol ester, such as phorbol myristate acetate (PMA), for two hours. All EBV-B cells from patients with cIFN-γR1/cIFN-γR2 had levels of O2- production similar to those of healthy controls (Figure 1A and supplemental figure 1A), unlike the EBV-B cells of CGD and XR-MSMD patients. However, H2O2 release from the EBV-B cells of all patients with cIFN-γR1/cIFN-γR2 deficiencies was severely impaired (Figure 1B and supplemental figure 1B). Figure 1 NADPH oxidase activity in human EBV-B cells, MDDCs and MDMs We further explored MDMs from patients with cIFN-γR1 (n=1) or cIFN-γR2 (n=2) deficiency. MDMs from these patients produced very small amounts of H2O2 after stimulation with IFN-γ or PPD, suggesting an impairment of NADPH oxidase activity (Figure 1 C, D). This is consistent with the findings for EBV-B cells (Figure 1B). We also assessed NADPH oxidase activity in monocyte-derived dendritic cells (MDDCs) from all patients. MDDCs from XR-MSMD patients have not been tested previously 3. We derived MDDCs in vitro by treatment with GM-CSF plus IL-13 7 and the respiratory burst was evaluated with Amplex Red®. MDDCs from the healthy controls released H2O2 and, as expected, MDDCs from CGD patients did not (Figure 1E). Notably, MDDCs from XR-MSMD patients (n=4), and from patients with cIFN-γR1 (n=1) or cIFN-γR2 (n=1) deficiency produced similar amounts of H2O2 than those obtained from healthy controls (Figure 1E). The impairment of NADPH oxidase activity was, therefore, cell-specific and restricted to MDMs in these patients. Conversely, the normal respiratory burst activity of granulocytes and monocytes from MSMD patients with cIFN-γR1 or cIFN-γR2 deficiency (data not shown), like that herein documented in MDDCs, may account for their protection against fungi and bacteria other than mycobacteria, whereas CGD patients are typically susceptible to these microbes. In conclusion, these results support a role for the IFN-γ pathway in the up-regulation of NADPH oxidase activity in MDMs and EBV-B cells, and suggest that impairment of the phagocyte respiratory burst contributes to BCG disease and TB in patients with inborn errors of IFN-γ immunity. IFN-γ can regulate NAPDH oxidase activity in different cell types 8. O2- is rapidly converted to H2O2 by spontaneous and enzymatic dismutation. Differences between the amounts of H2O2 and O2- produced in response to PMA were detected in the same cells. There are several possible reasons for this, including the role of endogenous superoxide dismutase in catalyzing the dismutation of O2- to H2O2, differences in kinetics and the techniques used to measure O2- and H2O2 levels, and variability due to differences in the pH of assay buffers. It was striking that some mycobacteria, e.g. M. tuberculosis, produced the enzyme such KatG, a catalase-peroxidase, that protect from killing by H2O2 but not O2- in mice 9. Probably NADPH oxidase activity contributes to cytokine production, granuloma genesis or autophagy more than killing of mycobacteria 5. The mechanisms affecting this activity in MDMs and EBV-B cells from MSMD patients remain unclear and require further investigation.

II Brazilian Consensus on the use of human immunoglobulin in patients with primary immunodeficiencies

RESUMO Nos ultimos anos, novas imunodeficiencias primarias e defeitos geneticos tem sido descritos. Recentemente, produtos de imunoglobulina, com aprimoramento em sua composicao e para uso por via subcutânea, tornaram-se disponiveis em nosso meio. Com o objetivo de orientar o medico no uso da imunoglobulina humana para o tratamento das imunodeficiencias primarias, os membros do Grupo de Assessoria em Imunodeficiencias da Associacao Brasileira de Alergia e Imunologia produziram um documento que teve por base uma revisao narrativa da literatura e sua [...]