Noninfectious complications in patients with pediatric-onset common variable immunodeficiency correlated with defects in somatic hypermutation but not in class-switch recombination.

BACKGROUND: Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired immunoglobulin production and usually presents with a normal quantity of peripheral B cells. Most attempts aiming to classify these patients have mainly been focused on T- or B-cell phenotypes and their ability to produce protective antibodies, but it is still a major challenge to find a suitable classification that includes the clinical and immunologic heterogeneity of these patients. OBJECTIVE: In this study we evaluated the late stages of B-cell differentiation in a heterogeneous population of patients with pediatric-onset CVID to clinically correlate and assess their ability to perform somatic hypermutation (SHM), class-switch recombination (CSR), or both. METHODS: We performed a previously reported assay, the restriction enzyme hotspot mutation assay (IgκREHMA), to evaluate in vivo SHM status. We amplified switch regions from genomic DNA to investigate the quality of the double-strand break repairs in the class-switch recombination process in vivo. We also tested the ability to generate immunoglobulin germline and circle transcripts and to upregulate the activation-induced cytidine deaminase gene through in vitro T-dependent and T-independent stimuli. RESULTS: Our results showed that patients could be classified into 2 groups according to their degree of SHM alteration. This stratification showed a significant association between patients of group A, severe alteration, and the presence of noninfectious complications. Additionally, 60% of patients presented with increased microhomology use at switched regions. In vitro activation revealed that patients with CVID behaved heterogeneously in terms of responsiveness to T-dependent stimuli. CONCLUSIONS: The correlation between noninfectious complications and SHM could be an important tool for physicians to further characterize patients with CVID. This categorization would help to improve elucidation of the complex mechanisms involved in B-cell differentiation pathways.

Naturally occurring mutation affecting the MyD88-binding site of TNFRSF13B impairs triggering of class switch recombination.

Mutations in the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) were previously found to be associated with hypogammaglobulinemia in humans. It has been shown that proliferation inducing ligand (APRIL) elicits class switch recombination (CSR) by inducing recruitment of MyD88 to a TACI highly conserved cytoplasmic domain (THC). We have identified a patient with hypogammaglobulinemia carrying a missense mutation (S231R) predicted to affect the THC. Aiming to evaluate the relevance of this novel mutation of TACI in CSR induction, we tested the ability of TACI, TLR9, or/and CD40 ligands to trigger CSR in naive B cells and B-cell lines carrying S231R. IgG secretion was impaired when triggered by TACI or/and TLR9 ligands on S231R-naive B cells. Likewise, these stimuli induced less expression of activation-induced cytidine deaminase, I(γ)1-C(µ), and I(γ)1-C(µ), while induction by optimal CD40 stimulation was indistinguishable from controls. These cells also showed an impaired cooperation between TACI and TLR9 pathways, as well as a lack of APRIL-mediated enhancement of CD40 activation in suboptimal conditions. Finally, after APRIL ligation, S231R-mutated TACI failed to colocalize with MyD88. Collectively, these results highlight the requirement of an intact MyD88-binding site in TACI to trigger CSR.

Immunological characteristics and two novel mutations in TACI in a cohort of 28 pediatric patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired immunoglobulin production. Mutations in the gene encoding TACI (TNFRSF13B) were previously found to be associated with CVID. Previous studies have identified a variety of sequence variants in TACI where A181E and C104R were the most common, with variable frequencies in different ethnic populations. So far, no mutations were identified in the recently reported "TACI highly conserved" (THC) cytoplasmic domain, important for the induction of class switch recombination. Our study evaluated immunological and clinical data on a cohort of 28 Argentinean pediatric CVID patients and allowed the identification of two novel mutations in TNFRSF13B, including one, S231R, affecting the highly conserved THC domain. In contrast, none of the patients presented with A181E and C104R mutations.

Novel syntaxin 11 gene (STX11) mutation in three Argentinean patients with hemophagocytic lymphohistiocytosis.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease with major diagnostic and therapeutic difficulties, basically comprising two different conditions: primary and secondary forms. Recent advances regarding molecular diagnosis may be useful to distinguish from one another, especially in sporadic cases starting in early infancy. MATERIALS AND METHODS: In this report, we evaluated three Argentinean patients with clinical suspicion of HLH, but without family history. We excluded mutations in the perforin gene but identified in the three patients a novel homozygous deletion (c. 581_584delTGCC; p.Leu194ProfsX2) in the gene-encoding syntaxin 11 (STX11), causing a premature termination codon. RESULTS AND CONCLUSION: Each parent from the three unrelated families resulted heterozygous for this deletion confirming the diagnosis of familial hemophagocytic lymphohistiocytosis type 4. Patients shared the same single-nucleotide polymorphism profile in STX11 gene, and genotyping at ten microsatellites surrounding this gene support the presence of a single-haplotype block carrying the novel mutation.

Genetic, molecular and functional analyses of complement factor I deficiency.

Complete deficiency of complement inhibitor factor I (FI) results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation leading to susceptibility to infections. Current genetic examination of two patients with near complete FI deficiency and three patients with no detectable serum FI and also close family members revealed homozygous or compound heterozygous mutations in several domains of FI. These mutations were introduced into recombinant FI and the resulting proteins were purified for functional studies, while transient transfection was used to analyze expression and secretion. The G170V mutation resulted in a protein that was not expressed, whereas the mutations Q232K, C237Y, S250L, I339M and H400L affected secretion. Furthermore, the C237Y and the S250L mutants did not degrade C4b and C3b as efficiently as the WT. The truncated Q336x mutant could be expressed, in vitro, but was not functional because it lacks the serine protease domain. Furthermore, this truncated FI was not detected in serum of the patient. Structural investigations using molecular modeling were performed to predict the potential impact the mutations have on FI structure. This is the first study that investigates, at the functional level, the consequences of molecular defects identified in patients with full FI deficiency.

Clinical and molecular analysis of 49 patients with X-linked agammaglobulinemia from a single center in Argentina.

INTRODUCTION: Argentina has a large number of patients with definite diagnosis of X-linked agammaglobulinemia reported in the Latin-American registry. Forty-nine of them were seen in our referral pediatric hospital, between 1987 and 2005. RESULTS AND DISCUSSION: A retrospective study of clinical, laboratory, and molecular data showed that respiratory tract infections were the most frequent initial clinical presentation and the most common among all manifestations prior to diagnosis (69%). Up to diagnosis, we found a high frequency of severe infections (sepsis, 14% and meningitis, 16%) and a high proportion of patients with chronic lung disease. During follow-up, the development of chronic lung disease was significantly related with age at diagnosis and inappropriate treatment. CONCLUSION: Although molecular diagnosis has been available in our center for the past 10 years, there is no doubt that awareness for early recognition of immunodeficiency should be improved through broader and more comprehensive education programs emphasizing characteristics of patients with immunodeficiencies.

Wiskott-Aldrich syndrome in Argentina: 17 unique, including nine novel, mutations.

Wiskott-Aldrich syndrome (WAS), is an X-linked immunodeficiency disease caused by mutations of the WAS protein (WASP) gene, characterized by thrombocytopenia, eczema and recurrent infections. X-linked thrombocytopenia (XLT) is a milder form with only platelet abnormalities. Cumulative mutation data have revealed that WASP genotypes are highly variable among WAS patients. By SSCP analysis, we determined the location of the mutation in 23 WAS patients from 17 unrelated families with variable clinical phenotypes. Direct sequence analysis of genomic DNA showed 9 novel mutations (Q52H, G70W, 393del7, Ex 7 Ex11del, IVS 8+1G-->C, 925delG, 959ins38, 1380del8, and IVS 2+2T-->C) and 8 known mutations distributed throughout the WAS gene. This is the first report of WAS gene mutations from a Latin American country.

Bruton tyrosine kinase gene mutations in Argentina.

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.

Quantification of Epstein-Barr virus load in Argentinean transplant recipients using real-time PCR.

BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplant recipients that is caused by suppression of T-cell function. OBJECTIVE: Evaluation of the diagnostic value of EBV DNA load in non-fractionated whole blood samples (n = 297) from 110 pediatric transplant patients by real-time PCR. RESULTS AND CONCLUSIONS: Patients with PTLD had a median viral load of 1.08 x 10(5) copies/ml blood (n = 24), which was significantly higher compared with patients without PTLD (median: 50 copies/ml blood, n = 273, P < 0.0001). From receiver operating characteristic (ROC) curve analysis we obtained a cut-off value of 6215 copies/ml blood with a sensitivity of 95.8%, specificity of 71.4%, negative predictive value (NPV) of 99.5% and positive predictive value (PPV) of 22.8%. Thus, real-time PCR proved to be more useful in ruling out than in indicating the presence of PTLD. Further analysis showed that patients without PTLD but developing a post-transplant EBV-primary infection had associated high viral loads that were indistinguishable from those of the PTLD group (statistically not significant). Similarly, the presence of clinical symptoms of disease in patients without PTLD was associated with higher viral loads than in patients that were asymptomatic (P < 0.0001), but the difference was much less significant when compared with the PTLD group of patients (P = 0.0391). These patients who had a high viral load may benefit from a close follow-up of the viral burden.

X-Linked Chronic Granulomatous Disease: First Report of Mutations in Patients of Argentina.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91, the largest component of the oxidase. METHODS: The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS: The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS: No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91°-CGD phenotype. The lack of gp91 protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

Beta 2-adrenergic polymorphisms and total serum IgE levels in children with asthma from Argentina.

BACKGROUND: Beta 2-Adrenergic receptor polymorphisms occurring at amino acid positions 16 (arginine/glycine) and 27 (glutamine/glutamic acid) are known to be functionally relevant. Associations with several asthma-related phenotypes, such as total serum IgE, have been investigated with different results. OBJECTIVE: To determine the contribution of polymorphisms and haplotypes of beta 2-adrenergic receptor with serum IgE levels in children from Argentina with mild, moderate, and severe asthma. METHODS: Beta 2-Adrenergic receptor polymorphisms were analyzed in 124 white asthmatic children using polymerase chain reaction during a 3-year period (January 1, 2005, through December 31, 2007). Total serum IgE level was measured by standard methods in all study participants, and age-adjusted values were determined for each individual. RESULTS: Serum levels of IgE were 4.3-fold higher than age-adjusted normal values in the study population. No association was found in regard to asthma severity. A significant difference of IgE serum levels was observed among polymorphisms at position 16, with the highest IgE level in the arginine/arginine group (P = .04). At position 27, even though median levels of IgE in homozygous glutamine were 2.2 times higher than homozygous glutamic acid, this increase did not reach statistical significance. When the population was stratified according to the most common homozygous haplotypes (arginine-arginine 16/glutamine-glutamine 27, glycine-glycine 16/glutamine-glutamine 27, and glycine-glycine 16/glutamic acid-glutamic acid 27), no association was found in relation to the serum levels of IgE. CONCLUSIONS: Beta 2-Adrenergic receptor polymorphisms, especially homozygous arginine 16, were associated with higher serum IgE levels in children with asthma. These genetic variants appear to contribute to the IgE level in asthmatic children from Argentina.

Clinical follow-up of 11 Argentinian CD40L-deficient patients with 7 unique mutations including the so-called "milder" mutants.

CD40 ligand (CD40L) deficiency is an X-linked combined immunodeficiency characterized by impaired class switch recombination. We analyzed clinical and molecular findings in 11 Argentinian patients from seven unrelated families. The mean age at onset of symptoms was 1.1 years (0.5-3.0 years) and the 10 alive patients have a median age of 17 years. We identified two nonsense mutations, including R11X reported as a "hypomorphic" defect, four missense mutations, and one point deletion. Although R11X was associated herein with parvovirus B19-anemia and higher Igs levels as previously described, histoplasmosis and Pneumocystis jiroveci pneumonia were also present. Other so-called "milder" mutation, T254M, was present in three related patients clinically and immunologically undistinguishable from the rest of the cohort. Furthermore, 10 of the 11 patients, having heterogeneous mutations, never had persistent neutropenia, none presented Cryptosporidium sp. infection nor developed liver-biliary tract disease, highlighting the debatable concept of "milder" mutations.

Characterization of immunodeficiency in a patient with growth hormone insensitivity secondary to a novel STAT5b gene mutation.

STAT5 proteins are components of the common growth hormone and interleukin 2 family of cytokines' signaling pathway. Mutations in the STAT5b gene, described in 2 patients, lead to growth hormone insensitivity that resembles Laron syndrome. Clinical immunodeficiency was also present, although immunologic defects have not been well characterized thus far. Here we describe a 16-year-old girl who suffered generalized eczema and recurrent infections of the skin and respiratory tract since birth. She also suffered severe chronic lung disease and multiple episodes of herpetic keratitis. Clinical features of congenital growth hormone deficiency were observed, such as persistently low growth rate, severely delayed bone age, and postnatal growth failure resulting from growth hormone resistance. This combined phenotype of growth hormone insensitivity and immunodeficiency was attributable to a homozygous C-->T transition that resulted in a nonsense mutation at codon 152 in exon 5 of the STAT5b gene. This novel mutation determined a complete absence of protein expression. The main immunologic findings were moderate T-cell lymphopenia (1274/mm3), normal CD4/CD8 ratio, and very low numbers of natural killer (18/mm3) and gammadelta T (5/mm3) cells. T cells presented a chronically hyperactivated phenotype. In vitro T-cell proliferation and interleukin 2 signaling were impaired. CD4+ and CD25+ regulatory T cells were significantly diminished, and they probably contributed to the signs of homeostatic mechanism deregulation found in this patient. This new case, in accordance with 2 previously reported cases, definitely demonstrates the significant role of the STAT5b protein in mediating growth hormone actions. Furthermore, the main immunologic findings bring about an explanation for the clinical immunodeficiency features and reveal for the first time the relevant role of STAT5b as a key protein for T-cell functions in humans.

X-linked chronic granulomatous disease: first report of mutations in patients of Argentina.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91(phox), the largest component of the oxidase. METHODS: The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS: The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS: No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91(o)-CGD phenotype. The lack of gp91(phox) protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

Prognostic value of soluble interleukin 2 receptor levels in Langerhans cell histiocytosis.

We investigated the prognostic significance of soluble interleukin 2 receptor (sIL-2r) levels in the pre- and post-treatment serum of paediatric patients with Langerhans cell histiocytosis (LCH). Serum levels of sIL-2r from 32 LCH patients and 14 healthy controls were determined using enzyme-linked immunosorbent assay. The LCH patients were classified, evaluated and treated according to the Histiocyte Society's protocols. The following clinical stages were considered: single-system disease (A) divided into single-site (A1; n=4), multiple-site (A2; n=9), and multisystem disease (B) without organ dysfunction (B1; n=5) and with organ dysfunction (B2; n=14). Pretreatment concentrations of sIL-2r were markedly increased at diagnosis in LCH patients compared with controls [in pg/ml, median (range) 9200 (1124-40000) versus 610 (343-800)], P < 0.0001. Levels differed significantly between stages A [3250 (1124-11000)] and B [22750 (3400-40000)], P < 0.05, and between substages A2 and B2, P < 0.05. There was a significant correlation between clinical stages and sIL-2r serum levels, r=0.7996 (P < 0.0001). Patients with > or = 17500 pg/ml of sIL-2r had a 30-month survival of 0.417 (SEM: 0.142) compared with those with levels < 17500 pg/ml, who presented a 30-month survival of 0.848 (SEM: 0.100) (log-rank, P < 0.0001). In multivariate analysis, sIL-2r levels > or = 17500 pg/ml were found to have greater predictive strength than other well-known prognostic factors.

Serum levels of interleukin-1 receptor antagonist and tumor necrosis factor-alpha are elevated in children with Langerhans cell histiocytosis.

PURPOSE: Langerhans cell histiocytosis (LCH) is a rare disease with variable prognosis in which lesions and clinical features suggest that pro- and anti-inflammatory cytokines may be involved in its pathogenesis. The authors wished to evaluate whether serum levels of interleukin-1 receptor agonist (IL-1Ra) and tumor necrosis factor-alpha (TNF-alpha) are elevated in children with LCH and decrease after chemotherapy. PATIENTS AND METHODS: Circulating levels of IL-1Ra and TNF-alpha were measured in 23 and 8 children with LCH, respectively, and 7 pediatric controls using commercially available ELISA kits. All patients fulfilled the Histiocyte Society LCH Protocols criteria for diagnosis, stratification, and treatment. RESULTS: Pretreatment concentrations of IL-1Ra and TNF-alpha were found to be significantly elevated in patients with LCH compared with controls. Among LCH substages, a significant difference in IL-1Ra values was observed between individuals with single-system single-site disease vs. multisystem disease with risk-organ dysfunction. In all eight patients evaluated, IL-1Ra levels decreased after 6 weeks of chemotherapy. Lower values of TNF were observed in three patients after treatment. A positive and significant correlation between IL-1Ra and TNF serum concentrations was found. CONCLUSIONS: Patients with LCH have elevated levels of IL-1Ra and TNF, which decrease after chemotherapy.