Fourth Update on the Iranian National Registry of Primary Immunodeficiencies: Integration of Molecular Diagnosis.

BACKGROUND: The number of inherited diseases and the spectrum of clinical manifestations of primary immunodeficiency disorders (PIDs) are ever-expanding. Molecular diagnosis using genomic approaches should be performed for all PID patients since it provides a resource to improve the management and to estimate the prognosis of patients with these rare immune disorders. METHOD: The current update of Iranian PID registry (IPIDR) contains the clinical phenotype of newly registered patients during last 5 years (2013-2018) and the result of molecular diagnosis in patients enrolled for targeted and next-generation sequencing. RESULTS: Considering the newly diagnosed patients (n = 1395), the total number of registered PID patients reached 3056 (1852 male and 1204 female) from 31 medical centers. The predominantly antibody deficiency was the most common subcategory of PID (29.5%). The putative causative genetic defect was identified in 1014 patients (33.1%) and an autosomal recessive pattern was found in 79.3% of these patients. Among the genetically different categories of PID patients, the diagnostic rate was highest in defects in immune dysregulation and lowest in predominantly antibody deficiencies and mutations in the MEFV gene were the most frequent genetic disorder in our cohort. CONCLUSIONS: During a 20-year registration of Iranian PID patients, significant changes have been observed by increasing the awareness of the medical community, national PID network establishment, improving therapeutic facilities, and recently by inclusion of the molecular diagnosis. The current collective study of PID phenotypes and genotypes provides a major source for ethnic surveillance, newborn screening, and genetic consultation for prenatal and preimplantation genetic diagnosis.

DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Level of Agreement between Children with Asthma and their Parents on Quality of Life.

BACKGROUND: Child-parent agreement is a controversial aspect of measuring health-related quality of life (HRQoL) in children and adolescents. The aim of this study was to assess the agreement between the child self-reports and parent proxy reports of the PedsQL 3.0 Asthma Module in Iranian children with asthma to evaluate HRQoL. Moreover, the psychometric properties of the child and parent reports of the PedsQL 3.0 Asthma Module were assessed in the present study. METHODS: Participants were 112 children with asthma and their parents, who completed the Farsi version of the PedsQL 3.0 Asthma Module. The multitrait-multimethod correlation matrix and factor analysis were used to test whether the child self-reports and the parent proxy reports measured the same construct. Additionally, convergent and discriminant validity and internal consistency were assessed using the Pearson correlation. RESULTS: The correlation between the child and parent HRQoL perceptions ranged between 0.13 and 0.36 across the same domains. Our factor analysis revealed that the child self-reports and the parent proxy reports measured 2 different constructs of HRQoL. Furthermore, our findings showed that both the child self-reports and the parent proxy reports of the PedsQL 3.0 Asthma Module had excellent internal consistency and acceptable convergent and discriminant validity. CONCLUSION: Although the child self-reports and the parent proxy reports of the Farsi version of PedsQL 3.0 Asthma Module showed good psychometric properties, they were not interchangeable. Our children with asthma and their parents evaluated child HRQoL from their own viewpoints.

Evaluation of T Cell Proliferation Using CFSE Dilution Assay: A Comparison between Stimulation with PHA and Anti-CD3/Anti-CD28 Coated Beads.

A decrease in T cell count or reduced T cell function can be indicative of T cell immunodeficiency. In the present study, T-cell function was assessed using Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution test after stimulation with commonly used Phytohaemagglutinin (PHA) or anti-CD3/anti-CD28 coated beads in pediatric patients with recurrent infections. Seven infants with recurrent infections and seven sex/age-matched healthy infants were included in this study. A blood cell count, immunophenotyping, and serum immunoglobulin level were performed. The proliferation of T cells was also assessed with CFSE dilution after stimulation with PHA or anti-CD3/anti-CD28 coated beads.  This study showed increased IgA, IgG, and IgM levels in patients compared to the controls. In contrast to the controls, the immunophenotyping results showed a significant decline in the number of CD4+ T cells in patients. Although there was no difference in CD3+ T cell proliferation between patients and controls, the CD4+ and CD8+ T cell proliferation rates were significantly decreased in patients when stimulated with PHA. As a mitogen with the potential for maximum proliferation of T cells, PHA is better able to distinguish between patients with recurrent infections and controls than anti-CD3/anti-CD28, which mimics only the TCR pathway for stimulation of T cells.

Pulmonary function test in transfusion-dependent ß-thalassemia major patients: a pilot study.

Lung involvement is one of known complications of thalassemia. The aim of this study was to determine predominant type of pulmonary dysfunction and its relationship to iron overload in ß-thalassemia children. Fifty thalassemia major children with treatment of regular blood transfusion and desferrioxamine participated in the study. Thirty-three boys and 17 girls (median age 12.5 years) with ß-thalassemia enrolled in the study. Other information including body mass index, hematocrit, and the number of years of blood transfusion were recorded. Serum ferritin level and hematocrit were 3346 ± 1667 mg/dL and 27.7 ± 2, respectively. Pulmonary function tests were performed in all subjects for detecting pulmonary dysfunction. Thirty-five patients (70%) with thalassemia had abnormal result of spirometry. Obstructive airway disease based on reduced forced expiratory volume in 1 second (FEV(1)) and FEV(1)/forced vital capacity (FVC) ratio <80% was detected in 4 patients (8%). Six patients (12%) showed restrictive pattern, as defined by a reduction FVC <80% and FEV(1)/FVC ratio ≥80%. In this study, small airway involvement based on presence of forced expiratory flow (FEF(25%-75%)) <60%, FEV(1)/FVC ratio >70%, and FVC >80% was detected in 25 subjects (50%). Decreased values of peak expiratory flow rate (PEF) were detected in 23 (46%) and low FEV(1) in 10 (20%) subjects. There was no significant correlation between abnormal pulmonary function test and serum ferritin level in children with thalassemia. This study showed small airway disease was predominant abnormality in thalassemia patients, although additional larger studies are needed to evaluate underlying mechanisms and validate these findings.

Characterization of 11 new cases of leukocyte adhesion deficiency type 1 with seven novel mutations in the ITGB2 gene.

BACKGROUND: Leukocyte adhesion deficiency type 1 (LAD I) is an autosomal recessive disorder caused by mutations in the ITGB2 gene, encoding the beta2 integrin family. Severe recurrent infections, impaired wound healing, and periodontal diseases are the main features of disease. METHODS: In order to investigate clinical and molecular manifestations of new LAD I cases, 11 patients diagnosed in one center during 7 years were studied. Patients were screened for the ITGB2 gene mutations, using polymerase chain reaction, followed by single-strand conformation polymorphism and sequencing. RESULTS: The most common first presenting feature of the patients was omphalitis. The mean age of cord separation was 19.9 +/- 1 days. The most common clinical manifestations of the patients during the follow-up period included omphalitis, skin ulcers with poor healing, sepsis, and otitis media. During the follow-up, eight patients died. Eight homozygous changes, including seven novel mutations, were detected: two splicing (IVS4-6C>A, IVS7+1G>A), three missense (Asp128Tyr, Ala239Thr, and Gly716Ala), and three frameshift deletions (Asn282fsX41, Tyr382fsX9, and Lys636fsX22). CONCLUSION: Our results indicate that different mutations underlie the development of LAD I. Definitive molecular diagnosis is valuable for genetic counseling and prenatal diagnosis. Regarding clinical presentations, it seems that omphalitis is the most consistent finding seen in LAD I infants.

Evaluation of myocardial function using the Tei index in patients with Kawasaki disease.

Myocarditis is a well-recognized component of Kawasaki disease, with left ventricular dysfunction occurring in more than half of the patients during the acute phase of the disease. The purpose of our study was to evaluate myocardial function in these patients using the myocardial performance index, also known as the Tei index. In a prospective study, 14 patients underwent echocardiographic evaluation, first at the time of diagnosis of the disease, in its acute phase before treatment with intravenous immunoglobulin and then 2 weeks later after treatment with immunoglobulin. We assessed the Tei-index, the ejection fraction, shortening fraction, and the presence of valvar regurgitation, pericardial effusion, or coronary arterial involvement. As a control, we also assessed 22 healthy children, matched for age and sex with the study population. Of the patients, half had an increased left ventricular Tei-index in the acute phase, as compared with the data obtained after treatment, the index changing from 0.43 +/- 0.08 to 0.35 +/- 0.06 (p equal to 0.003). An increased index for the right ventricle was found in 5 patients (36%), values being 0.30 +/- 0.05 as opposed to 0.26 +/- 0.04 (p equal to 0.009). Of the patients, 5 (35.7%) also had decreased ejection fractions and proportional shortening fractions during the acute phase, confirming left ventricular dysfunction. We concluded that the Tei-index, which measures combined systolic and diastolic function, is a simple, sensitive, and accurate tool for estimating global myocardial dysfunction in patients with Kawasaki disease.

Clinical and laboratory findings in hyper-IgM syndrome with novel CD40L and AICDA mutations.

BACKGROUND: Hyper-immunoglobulin M (HIGM) syndromes are a heterogeneous group of primary immunodeficiency disorders, characterized by recurrent infections associated with decreased serum levels of immunoglobulin G (IgG) and IgA and normal to increased serum levels of IgM. These patients have immunoglobulin class switch recombination defects, caused by mutations in several genes. METHODS: In order to investigate clinical and immunological manifestations of HIGM in Iran, 23 Iranian patients with an age range of 5 months to 35 years, who were followed up over a period of 17 years, were studied. Fourteen of the 23 patients were screened for CD40L, AICDA, UNG, and CD40 gene mutations, using polymerase chain reaction followed by direct sequencing. RESULTS: All patients, except one, initially presented with infectious diseases; the most common manifestations were respiratory tract infections. Six different CD40L mutations were identified, five were novel, one splicing (IVS1+2T>C), three missense (T254M, G167R, L161P), and two frame shift deletions (T29fsX36 and D62fsX79). In addition, one novel AICDA mutation (E122X) was detected. No mutation was found in six out of 14 analyzed patients. CONCLUSION: CD40L mutations comprise the most common type of immunoglobulin class switch recombination defects. There are several patients with HIGM phenotype, in which the underlying genetic defects remain to be identified. Other defects such as those in components of the mismatch repair mechanism could be potential candidates for the latter.

Comparison of Common Monogenic Defects in a Large Predominantly Antibody Deficiency Cohort.

BACKGROUND: Predominantly antibody deficiencies (PADs) are the most common primary immunodeficiencies, characterized by hypogammaglobulinemia and inability to generate effective antibody responses. OBJECTIVE: We intended to report most common monogenic PADs and to investigate how patients with PAD who were primarily diagnosed as suffering from agammaglobulinemia, hyper-IgM (HIgM) syndrome, and common variable immunodeficiency (CVID) have different clinical and immunological findings. METHODS: Stepwise next-generation sequencing and Sanger sequencing were performed for confirmation of the mutations in the patients clinically diagnosed as suffering from agammaglobulinemia, HIgM syndrome, and CVID. RESULTS: Among 550 registered patients, the predominant genetic defects associated with agammaglobulinemia (48 Bruton's tyrosine kinase [BTK] and 6 µ heavy chain deficiencies), HIgM syndrome (21 CD40 ligand and 7 activation-induced cytidine deaminase deficiencies), and CVID (17 lipopolysaccharides-responsive beige-like anchor deficiency and 12 atypical Immunodeficiency, Centromeric instability, and Facial dysmorphism syndromes) were identified. Clinical disease severity was significantly higher in patients with µ heavy chain and CD40 ligand mutations compared with patients with BTK (P = .003) and activation-induced cytidine deaminase (P = .009) mutations. Paralysis following live polio vaccination was considerably higher in patients with µ heavy chain deficiency compared with BTK deficiency (P < .001). We found a genotype-phenotype correlation among patients with BTK mutations regarding clinical manifestation of meningitis and chronic diarrhea. Surprisingly, we noticed that first presentations in most patients with Immunodeficiency, Centromeric instability, and Facial dysmorphism were respiratory complications (P = .008), whereas first presentations in patients with lipopolysaccharides-responsive beige-like anchor deficiency were nonrespiratory complications (P = .008). CONCLUSIONS: This study highlights similarities and differences in the clinical and genetic spectrum of the most common PAD-associated gene defects. This comprehensive comparison will facilitate clinical decision making, and improve prognosis and targeted treatment.

Severe Allergic Bronchopulmonary Mycosis and Long-Term Follow-Up.

Allergic bronchopulmonary aspergillosis (ABPA) is the most common immunologic reaction following fungal allergen exposure in asthmatic patients. A less frequent syndrome in response to other fungal species like candida is allergic bronchopulmonary mycosis (ABPM). This reaction is mostly associated with asthma exacerbation, changes in Immunoglobulin E levels, and nonspecific findings in high resolution computed tomography (HRCT). This study presents a 9-year-old girl, a known case of childhood asthma, resolved 4 years ago as a novel case of ABPM due to Candida albicans manifested by severe emphysema, bronchiectasis, and pneumothorax which consequently required long-term treatment to get relieved.

Sudden unilateral blindness in a girl with Kawasaki disease.

This article describes a 9-year-old girl with clinical and laboratory features of Kawasaki disease. On day 7 of her febrile illness, she developed sudden loss of vision in the right eye. Visual acuity was no light perception. Dilated funduscopy showed diffuse intense retinal whitening, narrowing retinal arterioles, and a pale swollen disk-but no cherry-red spot. These findings suggested ophthalmic artery obstruction. Kawasaki disease can be sight-threatening.

Bone mineral density in beta-thalassemia major and intermedia.

OBJECTIVE: This study was conducted to assess bone mineral density (BMD) and bone mineral content (BMC) in patients with thalassemia major and intermedia, and to correlate them with biochemical and hematological profile. DESIGN: 106 thalassemic patients (49 major and 57 intermedia) were scanned by dual energy xray absorptiometry technique for BMD and BMC at lumbar spine and femoral neck. The effects of sex, transfusion/chelation program as well as hemoglobin, calcium, phosphorus, alkaline phosphatase and serum ferritin level were also evaluated on BMD and BMC. RESULTS: Patients with thalassemia major and intermedia, younger than 20 yr, showed lower BMD and BMC in the lumbar region (p < 0.05). Both parameters correlated significantly with hemoglobin level; other biochemical and hematological parameters did not influence BMD and BMC values. CONCLUSION: Bone marrow density is a good index of bone status in patients with Thalassemia and should be done in these patients annually.

Bone mineral density in children wth systemic lupus erythematosus and juvenile rheumatoid arthritis.

BACKGROUND: Although there is increasing in bone metabolism in patients with rheumatic disorders, few data exist on bone mineral density (BMD) in children with rheumatic disorders or on the association of BMD with disease-related variables. We determined BMD in Iranian children with systemic lupus erythematosus (SLE) and juvenile rheumatoid arthritis (JRA) to evaluate the relationship between disease-related variables and BMD. PATIENTS AND METHODS: Twenty patients (13 girls and 7 boys) with SLE (n=15) and JRA (n=5) with a mean age of 13.10+/-3.29 years (range, 6-17 years), attending a pediatric rheumatology clinic and 20 healthy controls (matched for age and sex with each patient) were enrolled in a cross-sectional study between 2001 and 2003. BMD (g/cm(2)) of the femoral neck (BMD-F) and lumbar vertebrae (BMD-L) were measured by dual energy X-ray absorptiometry (DEXA). The correlation between BMD and cumulative dose of steroids, daily dose of steroid, disease duration, disease activity, height, weight, and age was investigated. RESULTS: BMD in the patients (BMD-F=0.72+/-0.15, BMD-L=0.70+/-0.19) was significantly lower than controls (BMD-F=0.95+/-0.17, BMD-L=0.98+/-0.20, P=<0.001). The severity of descreased BMD was more prominent in lumbar vertebrae than the femoral neck (P=0.04). None of the variables were consistently related to a decrease in BMD. CONCLUSION: BMD was significantly lower in patients compared with controls. It was more prominent in lumbar vertebrae (trabecular bone). Although cumulative dose of steroids and diseaese appeared to have some influence on BMD, none were independently correlated with BMD.

Atypical desquamation in a 2.5-year-old boy with Kawasaki disease: A case report.

Kawasaki disease (KD) is a vasculitis that mostly affects children under 5 years of age. This article presents a 2.5-year-old boy who presented with 6 days of fever, generalized maculopapular rash, bilateral non-exudative conjunctivitis, cracked lips, right cervical lymphadenopathy, erythematous extremities, and perianal desquamation. Laboratory studies showed leukocytosis and sterile pyuria. Because diagnosis of KD was proved, oral acetylsalicylic acid with the anti-inflammatory dose and intravenous immunoglobulin were started for him. On the seventh day of admission time, he developed desquamation and erythema on the site of his right cervical lymphadenopathy as well as periungual scaling. About three weeks after starting the treatment, scaling of the cervical lymphadenopathy and periungual area stopped. Echocardiography was performed for him three times: at the time of diagnosis, four weeks, and 6 months later and revealed normal coronary arteries. We report this sign, desquamation on the site of cervical lymphadenopathy, as a new finding.

Hodgkin lymphoma developing in a 4.5-year-old girl with hyper-IgE syndrome.

The authors report a case of Hodgkin lymphoma developing in a 4.5-year-old female child with hyper-IgE syndrome. This is one of the few cases of malignancy reported in this syndrome. The patient had severe atopic dermatitis, asthma, recurrent pneumonia, recurrent skin infections, and growth retardation. Immunologic evaluation revealed a high level of immunoglobulin E (7000 IU/mL) and peripheral eosinophilia. She was found to have normal values for serum IgG, IgM, IgA, WBC chemotaxis, serum complement function and normal sweat chloride test. The development of fatal Hodgkin lymphoma in this patient with hyper-IgE syndrome may suggest an increased risk for developing premature malignancies in hyper-IgE syndrome, although the precise immunologic defect is still unknown.

A fast and easy nitroblue tetrazolium method for carrier screening and prenatal detection of chronic granulomatous disease.

BACKGROUND: Analysis of the functional activity of neutrophils is of great importance in the differential diagnosis of patients with recurrent bacterial infections. It has long been established that stimulated polymorphonuclear leukocytes reduce nitroblue tetrazolium. Application of a simple and reliable nitroblue tetrazolium method that clearly differentiates the chronic granulomatous disease patients and heterozygote carriers in some groups suspected to have chronic granulomatous disease was investigated. METHODS: This study consisted of 197 samples taken from 100 children (2 - 24-month-old) and 81 neonates (aged < 2 months) referred to our center either due to a suspected bacterial infection or suspected immunodeficiency. The sample also included 16 cord blood samples. Fifty healthy adult individuals were enrolled in this study and were diagnosed as normal control. Neutrophil reduction of nitroblue tetrazolium can be stimulated in vitro by protein kinase agonists such as phorbol myristate acetate, resulting in release of superoxide anion. RESULTS: Phorbol myristate acetate is an exceptionally powerful stimulant and when used in conjunction with glass adherence, caused nearly all normal neutrophils to become transformed and reduced nitroblue tetrazolium to formazan deposits. Of 197 blood samples, 9 were diagnosed as having unrelated chronic granulomatous disease and 7 were carriers of X-linked or autosomal recessive chronic granulomatous disease. The carriers had a range of 15 - 75% stimulated neutrophils. CONCLUSION: We have established a phorbol myristate acetate-stimulated nitroblue tetrazolium test for detection of chronic granulomatous disease patients, which clearly differentiates the chronic granulomatous disease patients from heterozygote carriers. The results in cord fetal blood indicate that this test may be used for antenatal diagnosis of affected boys, carrier females, and autosomal recessive variants of chronic granulomatous disease. The technique is simple, fast, inexpensive, and requires only a few microliters of blood.

Initial intravenous gamma-globulin treatment failure in Iranian children with Kawasaki disease.

The purpose of this study was to determine the initial rates of intravenous gamma-globulin treatment (IVIG) failure in Kawasaki disease (KD) and their predisposing factors. This study was a retrospective analysis of the initial response to IVIG (2 g/kg), assessed from the medical reports of all patients admitted to Namazee Hospital pediatric ward, from March 1998 to March 2002, and who fulfilled the criteria for KD. Data were available for 64 patients, 58 of whom (90.6%) became afebrile 48 hours after completion of the initial dose of IVIG (Group I) and six (9.4%) who remained febrile (Group II). Two patients had a prompt response to a second dose of IVIG. In Group I, five patients (8.6%) developed coronary artery disease, seen on echocardiography. In Group II, two patients (33.3%) developed coronary artery disease. No significant difference was found in the prevalence of coronary artery disease between the two groups (p = 0.12), or in age or gender. The rate of initial treatment failure was 9.4% in this cohort of patients, which is comparable with previous reports. No predictive factors such as coronary artery disease, age or gender were found for initial treatment failure in KD.

Down-regulation of TLR2, 3, 9 and Signaling Mediators, MyD88 and TRIF, Gene Transcript Levels in Patients with Kawasaki Disease Treated with IVIG.

Kawasaki disease (KD) is an acute febrile systemic vasculitis of childhood characterized by elevated levels of inflammatory mediators at the acute stage. High-dose intravenous immunoglobulin (IVIG) is well accepted as a conventional therapy for KD. The aim of the present study was to determine the expression level of Toll like receptors (TLRs) and their corresponding signaling mediators in PBMCs of IVIG-treated KD patients. TLR2, 3, 9 and signaling mediators, MyD88 and TRIF transcript levels were determined in PBMCs from 31 KD patients, before (acute phase), 2 weeks later (sub-acute phase) and 6 weeks later (convalescent phase) of IVIG therapy using real time PCR. The mean age of the patients was 3.6 years and 65% of subjects were male and 35% were female. 20 age-matched irrelevant febrile patients and 20 healthy subjects were included as control groups. Elevated levels of TLR2, MyD88, and TRIF gene transcripts were observed in the PBMCs at acute phase of untreated KD patients in compression with normal subjects. IVIG therapy resulted in significant decrease in TLR2, 3 and 9 (60-90%) as well as MyD88 and TRIF (60-70%) transcripts following 2 and 6 weeks. With Regard to significant up-regulation of MyD88 and TRIF at the acute phase of KD, our findings suggest TLR signaling pathway potential in KD pathogenesis and may also support the assumption of an infectious background in KD. Down-regulation of TLR members and corresponding mediators in IVIG treated patient suggest general TLR pathway suppression as a novel anti-inflammatory mechanism of IVIG.