Maria Cristina Pietrogrande

Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome

BACKGROUND The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells. OBJECTIVE To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. METHODS We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. RESULTS In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells. CONCLUSION We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.

Hereditary deficiency of gp91(phox) is associated with enhanced arterial dilatation: results of a multicenter study.

Background— NADPH oxidase is believed to modulate arterial tone, but its role in humans is still unclear. The objective of this study was to evaluate whether NADPH oxidase is involved in flow-mediated arterial dilation (FMD). Methods and Results— Twenty-five patients with hereditary deficiency of gp91phox, the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91phox, serum levels of nitrite and nitrate as markers of nitric oxide generation, oxidized low-density lipoprotein, and urinary excretion of isoprostanes as markers of oxidative stress were determined. Platelet gp91phox expression was downregulated in X-CGD patients (1.0±0.8 mean fluorescence; P<0.001) and upregulated in obese patients (4.1±2.2 mean fluorescence; P=0.01) compared with healthy subjects (2.9±1.7 mean fluorescence). Urinary excretion of isoprostanes was reduced in X-CGD patients (41.7±33.3 pg/mg creatinine; P=0.04) and increased in obese patients (154.4±91 pg/mg creatinine; P<0.001) compared with healthy subjects (69.5±52.4 pg/mg creatinine). Obese patients had higher serum oxidized low-density lipoprotein than healthy subjects (35.3±6.7 versus 24.8±9.8 U/L; P<0.001) and X-CGD patients (28.5±7.2 U/L; P<0.001). X-CGD patients had significantly higher FMD (14.7±5.9%) compared with healthy subjects (7.9±2.5%; P<0.001); obese patients had lower FMD (5.3±3.0%; P=0.028) compared with healthy subjects. Serum nitrite and nitrate levels were significantly higher in patients with X-CGD (36.0±10.8 μmol/L; P=0.016) and lower in obese patients (9.3±11.0 μmol/L; P=0.001) compared with healthy subjects (27.1±19.1 μmol/L). Serum nitrite and nitrate levels significantly correlated with FMD (Rs=0.403, P<0.001) and platelet gp91phox (Rs=−0.515, P<0.001). FMD inversely correlated with platelet gp91phox (Rs=−0.502, P<0.001) and isoprostanes (Rs=−0.513, P<0.001). Conclusion— This study provides the first evidence that, in humans, gp91phox is implicated in the modulation of arterial tone.

Reduced memory B cells in patients with hyper IgE syndrome

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

Intra-Erythrocyte Infusion of Dexamethasone Reduces Neurological Symptoms in Ataxia Teleangiectasia Patients: Results of a Phase 2 Trial

BackgroundAtaxia Teleangiectasia [AT] is a rare neurodegenerative disease characterized by early onset ataxia, oculocutaneous teleangiectasias, immunodeficiency, recurrent infections, radiosensitivity and proneness to cancer. No therapies are available for this devastating disease. Recent observational studies in few patients showed beneficial effects of short term treatment with betamethasone. To avoid the characteristic side effects of long-term administration of steroids we developed a method for encapsulation of dexamethasone sodium phosphate (DSP) into autologous erythrocytes (EryDex) allowing slow release of dexamethasone for up to one month after dosing. Aims of the study were: the assessment of the effect of EryDex in improving neurological symptoms and adaptive behaviour of AT patients; the safety and tolerability of the therapy.MethodsTwenty two patients (F:M = 1; mean age 11.2 ± 3.5) with a confirmed diagnosis of AT and a preserved or partially supported gait were enrolled for the study. The subjects underwent for six months a monthly infusion of EryDex. Ataxia was assessed by the International Cooperative Ataxia Rating Scale (ICARS) and the adaptive behavior by Vineland Adaptive Behavior Scales (VABS). Clinical evaluations were performed at baseline and 1, 3, and 6 months.ResultsAn improvement in ICARS (reduction of the score) was detected in the intention-to-treat (ITT) population (n = 22; p = 0.02) as well as in patients completing the study (per protocol PP) (n = 18; p = 0.01), with a mean reduction of 4 points (ITT) or 5.2 points (PP). When compared to baseline, a significant improvement were also found in VABS (increase of the score) (p < 0.0001, ITT, RMANOVA), with statistically significant increases at 3 and 6 months (p < 0.0001). A large inter-patient variability in the incorporation of DSP into erythrocytes was observed, with an evident positive effect of higher infusion dose on ICARS score decline. Moreover a more marked improvement was found in less neurologically impaired patients. Finally, a 19 month-extension study involving a subgroup of patients suggested that Erydex treatment can possibly delay the natural progression of the disease.EryDex was well tolerated; the most frequent side effects were common AT pathologies.ConclusionsEryDex treatment led to a significant improvement in neurological symptoms, without association with the typical steroid side effects.Trial registrationCurrent Controlled Trial2010-022315-19SpA

Clinical Features and Follow-Up in Patients with 22q11.2 Deletion Syndrome.

OBJECTIVE To investigate the clinical manifestations at diagnosis and during follow-up in patients with 22q11.2 deletion syndrome to better define the natural history of the disease. STUDY DESIGN A retrospective and prospective multicenter study was conducted with 228 patients in the context of the Italian Network for Primary Immunodeficiencies. Clinical diagnosis was confirmed by cytogenetic or molecular analysis. RESULTS The cohort consisted of 112 males and 116 females; median age at diagnosis was 4 months (range 0 to 36 years 10 months). The diagnosis was made before 2 years of age in 71% of patients, predominantly related to the presence of heart anomalies and neonatal hypocalcemia. In patients diagnosed after 2 years of age, clinical features such as speech and language impairment, developmental delay, minor cardiac defects, recurrent infections, and facial features were the main elements leading to diagnosis. During follow-up (available for 172 patients), the frequency of autoimmune manifestations (P = .015) and speech disorders (P = .002) increased. After a median follow-up of 43 months, the survival probability was 0.92 at 15 years from diagnosis. CONCLUSIONS Our data show a delay in the diagnosis of 22q11.2 deletion syndrome with noncardiac symptoms. This study provides guidelines for pediatricians and specialists for early identification of cases that can be confirmed by genetic testing, which would permit the provision of appropriate clinical management.

A prospective study on children with initial diagnosis of transient hypogammaglobulinemia of infancy: results from the Italian Primary Immunodeficiency Network.

Transient hypogammaglobulinemia of infancy (THI) is a heterogenous disorder characterized by reduced serum IgG levels in early infancy. A putative diagnosis is initially made after exclusion of other causes of hypogammaglobulinemia while a definitive diagnosis of THI can only be made a posteriori in patients with normalization of IgG levels. The aim of this study is to characterize clinical and immunological features of children with an initial diagnosis of THI in correlation to natural outcome, and to assess predictive laboratory parameters of clinical evolution for this disorder. We prospectively analysed clinical and immunological characteristics of 77 THI children at initial diagnosis and of 57 patients at follow-up. Memory B cell subsets and in vitro immunoglobulin production were evaluated. Seventy patients (91%) showed clinical symptoms. Patients suffered from infections (91%), allergies (47%) and autoimmune disease (4%). During follow-up 41/57 children (72%) normalized IgG values, mostly within 24 months of age (p<0.001), allowing the diagnosis of THI. The 16 children who did not normalize their IgG levels showed a higher frequency of severe infections and autoimmune disease (p<0.01). Moreover, they expressed a reduced frequency of IgM and switched memory B cells (p<0.01) and an inability to produce IgG in vitro (p<0.02). We conclude that most patients with an initial diagnosis of THI spontaneously recover within 24 months of age and have a benign clinical course, while a subgroup of children with undefined hypogammaglobulinemia share a clinical and immunological profile with other primary immunodeficiencies. Early recognition of children with hypogammaglobulinemia during infancy who are likely to suffer from permanent immunodeficiencies later in life would allow prompt and appropriate laboratory and clinical interventions.

A randomized trial of oral betamethasone to reduce ataxia symptoms in ataxia telangiectasia.

No controlled studies exist regarding the pharmaceutical reduction of ataxia symptoms in ataxia telangiectasia (A‐T). In a multicenter, double‐blind, randomized, placebo‐controlled crossover trial, oral betamethasone (BETA) and placebo were compared in terms of their reduction of ataxia symptoms as assessed with the International Cooperative Ataxia Rating Scale (ICARS). In this study of 13 A‐T children, betamethasone reduced the ICARS total score by a median of 13 points in the intent‐to‐treat population and 16 points in the per‐protocol population (ie, median percent decreases of ataxia symptoms of 28% and 31%, respectively). In conclusion, Oral betamethasone could be a promising therapy to relieve ataxia symptoms in A‐T patients; however, long‐term effectiveness and safety must be established. (Current Controlled Trials, number ISRCTN08774933.)

Pulmonary and sinus diseases in primary humoral immunodeficiencies with chronic productive cough.

Aims: To prospectively evaluate sinopulmonary disease in 24 patients with primary humoral immunodeficiency (11 with agammaglobulinaemia, nine with isolated IgA deficiency, and two with common variable immunodeficiency) and chronic productive cough, ascertain the usefulness of chest high resolution computed tomography (HRCT) in evaluating the progression of lung disease, and test a therapeutic approach to chronic sinusitis. Methods: Pulmonary abnormalities were evaluated using lung function tests and HRCT (Bhalla score); chronic sinusitis was diagnosed clinically and confirmed by flexible fibreoptic endoscopy. Sixteen patients entered the three year follow up. Results: Lung function testing revealed an obstruction in four patients; chest HRCT was abnormal in 16. There was a linear relation between the Bhalla score ⩾4 and the number of months with cough/year over the previous two years (clinical score), and between the difference in clinical scores during follow up and in the previous two years and the difference in Bhalla score. The pulmonary lesions did not significantly progress over a three year period. Thirteen patients had chronic sinusitis; 6/10 patients followed up were successfully treated with antibiotics plus topical therapy and two with nasal polypoid disease with endoscopic sinus surgery. Conclusions: In patients with primary humoral immunodeficiency and chronic productive cough, HRCT is very useful in delineating the extent of lung damage. The correlation between Bhalla score and clinical findings and the favourable outcome of the disease suggests that in most patients chest HRCT should not be repeated annually as previously suggested. Medical therapy seems to be effective in many cases of chronic sinusitis.

Molecular analysis of the pre-BCR complex in a large cohort of patients affected by autosomal-recessive agammaglobulinemia.

Autosomal-recessive agammaglobulinemia is a rare and heterogeneous disorder, characterized by early-onset infections, profound hypogammaglobulinemia of all immunoglobulin isotypes and absence of circulating B lymphocytes. To investigate the molecular basis of the disease, 23 patients with early-onset disease and no mutations in Bruton tyrosine kinase, the gene responsible for X-linked agammaglobulinemia, were selected and analyzed by direct sequencing of candidate genes. Two novel mutations in the μ heavy chain (μHC) gene (IGHM) were identified in three patients belonging to two unrelated families. A fourth patient carries a previously described G>A nucleotide substitution at the −1 position of an alternative splice site in IGHM; here, we demonstrate that this mutation is indeed responsible for aberrant splicing. Comparison of bone marrow cytofluorimetric profiles in two patients carrying different mutations in the IGHM gene suggests a genotype–phenotype correlation with the stage at which B-cell development is blocked. Several new single nucleotide polymorphisms (SNPs) both in the μHC and in the λ5-like/VpreB-coding genes were identified. Two unrelated patients carry compound heterozygous variations in the VpreB1 gene that may be involved in disease ethiology.

Does NADPH Oxidase Deficiency Cause Artery Dilatation in Humans

NADPH oxidase is known to modulate the arterial tone, but the role of its specific subunits is still unclear. The objective of this study was to compare the role of p47 and gp91phox (NOX2) on artery dilatation. We conducted a multicenter study enrolling 30 patients with chronic granulomatous disease (CGD) (25 with NOX2 deficiency and 5 with p47(phox) deficiency) and 30 healthy subjects (HS), matched for gender and age, in whom flow-mediated dilation (FMD), serum activity of NOX2 (soluble NOX2-derived peptide [sNOX2-dp]), urinary isoprostanes (8-iso-PGF2α), and platelet production of isoprostanes and NOX2 were determined. Compared to HS, patients with CGD had significantly higher FMD and lower sNOX2-dp and 8-iso-PGF2α levels. Compared to patients with NOX2 deficiency and HS, patients with p47(phox) hereditary deficiency had intermediate FMD and oxidative stress, that is, higher and lower FMD and lower and higher isoprostanes compared to HS and patients with NOX2 deficiency, respectively. In agreement with this finding, an ex vivo study showed higher inhibition of NOX2 activity and lower isoprostane formation in platelets from patients with NOX2 deficiency compared to platelets from ones with p47(phox) deficiency. Our observations lead to the hypothesis that oxidants are implicated in artery vasoconstriction.