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Background: According to the WHO's recommendation for developing countries, Bacillus Calmette-Guerin (BCG) vaccination has been implemented in some countries as part of national vaccination programs at birth. Although it is generally considered safe, some complications may occur; including BCGitis (local) or BCGosis (systemic), ranging from mild like local abscesses to fatal impediments like osteomyelitis and disseminated BCG infection. This study aimed to determine the spectrum of inborn errors of immunity (IEI) in BCG-vaccinated neonates experiencing local or systemic complications. Methods: In this cross-sectional study, we investigated Iranian children referred to the Immunology, Asthma, and Allergy Research Institute (IAARI) between 2007-2023 for suspected immunodeficiency. Medical history was recorded, and primary screening tests for immunodeficiency were conducted for all cases. For suspected cases, more advanced immunologic investigations were performed to reach a definitive diagnosis. Furthermore, the study incorporated the documented genetic findings of the patients under investigation. All patients with inborn error of immunity who had a history of BCG vaccine complications within the first year of vaccination were enrolled in the study. Results: We investigated 3,275 cases suspected of IEI, identifying197 patients with both IEI and BCG vaccine complications. Among these, 127 (64.5%) were male. Symptoms began at or before 3 months of age in 64.8% of the cases, and parental consanguinity was reported in 79.2%. Genetic diagnoses were confirmed in 108 patients. Of the 197 patients, 108 (54.8%) had BCGitis, while 89 (45.2%) experienced systemic complications (BCGosis). A family history of IEI, BCG-related complications, and unexplained deaths were observed in 20.3%, 12.2%, and 29.9% of cases, respectively. Furthermore, 46.2% had at least one of these three risk factors in their history. Conclusions: Early BCG vaccine complications may indicate an underlying immunodeficiency, particularly when there is a positive family history of BCG complications, immunodeficiency, or unexplained deaths. Nation-wide vaccination protocols should address this issue by delaying inoculation to allow for immunological screening of suspected immunodeficient patients, thereby preventing BCG vaccine-related morbidity and mortality. [ABSTRACT FROM AUTHOR]

Background: Primary immunodeficiency diseases (inborn errors of immunity) with partial albinism are a group of autosomal recessive syndromes including Chediak Higashi Syndrome (CHS), Griscelli Syndrome type 2 (GS2), Hermansky‐Pudlak Syndromes type 2 and 10 (HPS2, HPS10), Vici syndrome and P14/LAMTOR2 deficiency. Methods: Twenty‐five patients including 10 CHS, 10 GS2, and 5 HPS2 were evaluated in this study within the last 10 years. Five cases with oculocutaneous albinism (OCA) and 5 healthy subjects without albinism were used as two control groups. Genetic analyses were performed by whole exome or panel sequencing or targeted Sanger sequencing. Subsequently, leukocyte granules in peripheral blood smear and hair shaft were examined as screening tests. Results: Giant granules were only presented in the leukocytes cytoplasm of 10/10 CHS patients. The uneven cluster of pigments and giant melanin granules in hair samples were observed in 10/10 GS2 and 10/10 CHS patients, respectively. In both 5/5 OCA and 5/5 HPS2 patients, there were regular pigments in the middle of hair shafts. Genetic analyses were performed for all patients, revealing 7 novel variants in LYST gene for CHS patients and 4 novel variants in AP3B1 for HPS2 patients. Conclusion: Receiving hematopoietic stem cell transplantation (HSCT) in a timely manner is crucial in CHS and GS2 patients; therefore, screening tests may provide a vital clue for early diagnosis in these patients. However, the final confirmation of CHS, GS2, and HPS2 disorders is done by genetic assay. [ABSTRACT FROM AUTHOR]

Background: In order to support the comprehensive classification of Leukocyte Adhesion Deficiency‐I (LAD‐I) severity by simultaneous screening of CD11a/CD18, this study assessed clinical, laboratory, and genetic findings along with outcomes of 69 LAD‐I patients during the last 15 years. Methods: Sixty‐nine patients (40 females and 29 males) with a clinical phenotype suspected of LAD‐I were referred to Immunology, Asthma, and Allergy research institute, Tehran, Iran between 2007 and 2022 for further advanced immunological screening and genetic evaluations as well as treatment, were enrolled in this study. Results: The diagnosis median age of the patients was 6 months. Delayed umbilical cord separation was found in 25 patients (36.2%). The median diagnostic delay time was 4 months (min–max: 0–82 months). Forty‐six patients (66.7%) were categorized as severe (CD18 and/or CD11a: below 2%); while 23 children (33.3%) were in moderate category (CD18 and/or CD11a: 2%–30%). During the follow‐ups, 55.1% of children were alive with a mortality rate of 44.9%. Skin ulcers (75.4%), omphalitis (65.2%), and gingivitis (37.7%) were the most frequent complaints. Genetic analysis of the patients revealed 14 previously reported and three novel pathogenic mutations in the ITGB2 gene. The overall survival of patients with and without hematopoietic stem cell transplantation was 79.3% and 55.6%, respectively. Conclusion: Physicians' awareness of LAD‐I considering delayed separation of umbilical cord marked neutrophilic leukocytosis, and variability in CD11 and CD18 expression levels, and genetic analysis leads to early diagnosis and defining disease severity. Moreover, the prenatal diagnosis would benefit families with a history of LAD‐I. [ABSTRACT FROM AUTHOR]

Griscelli syndrome type 2 (GS2) is an autosomal recessive immunodeficiency characterized by hair hypopigmentation, recurrent fever, hepatosplenomegaly and pancytopenia. This study aims to find new genetic changes and clinical features in 18 children with GS2 caused by the RAB27A gene defect. In all, 18 Iranian children with GS2 who presented with silver grey hair and frequent pyogenic infection were included in this study. After recording demographic and clinical data, PCR sequencing of the RAB27A gene was performed for all exons and exon–intron boundaries. Two patients in this study were subjected to whole‐exome sequencing followed by Sanger sequencing. Light microscopy study of hair showed large irregular clumps of pigment with the absence of giant granules on the blood smear. Mutation analysis of the RAB27A gene identified two novel missense mutations as homozygous in a patient, one in exon 2, c.140G>C and another in exon 4, c.328G>T. In addition, for 17 other patients, 6 reported mutations were obtained including c.514_518delCAAGC, c.150_151delAGinsC, c.400_401delAA, c.340delA, c.428T>C and c.221A>G. The mutation c.514_518delCAAGC was the most frequent and found in 10 patients; this mutation may be considered a hotspot in Iran. Early diagnosis and treatment of RAB27A deficiency can contribute to better disease outcomes. In affected families, genetic results could be urgently needed to make a timely decision about haematopoietic stem cell transplantation and prenatal diagnosis. [ABSTRACT FROM AUTHOR]

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder due to a genetic defect in one of the components of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. This complex is composed of membrane-bound gp91-phox and p22-phox subunits, and cytosolic subunits consisting of p47-phox, p67-phox, and p40-phox. A mutation in CYBB gene encoding gp91-phox located on chromosome Xp21.1, leads to X-linked CGD. Herein, we report a 4-year-old Iranian boy presented with episodes of recurrent fever, cervical lymphadenopathy, and abdominal abscesses. Mutation analysis of the CYBB gene in the patient indicated a one-nucleotide deletion, c.316delT, resulting in p.W106GfsX. [ABSTRACT FROM AUTHOR]

Chronic granulomatous disease (CGD), a rare inherited primary immunodeficiency disorder, is caused by mutation in any one of the genes encoding components of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme. NCF2 gene (encoding P67-phox component) is one of them and its mutation is less common to cause CGD (around 5-6%). Here, we assessed mutation analysis of NCF2 in 4 CGD patients with p67-phox defect in Iran. These patients showed classical CGD symptoms. NCF2 sequence analyses revealed two different homozygous mutations including a nonsense mutation in exon 4, c.304C>T (Arg 102X) in one case and a CA deletion in exon 13 (Leu346fsX380) in one brother and sister; the latter is a new mutation which has not been reported in previous studies. In another patient in whom the attempts to amplify exon 2 individually from genomic DNA were unsuccessful, PCR amplification of exon 2 revealed no band of this exon on agarose gel. A PCR amplification mix of exon 2 and exon 7, with an internal control, confirmed the lack of exon 2 in this patient. Although a gross deletion in other exons of NCF2 has been previously reported, a large deletion encompassing exon 2 has been not reported yet. This abstract was also presented in ESID 2012, Florence, Italy. [ABSTRACT FROM AUTHOR]

Objective: Hemophagocytic lymphohistiocytosis (HLH) is a rare immunodeficiency disorder recognized by high fever without response to antibiotics, hepatosplenomegaly, cytopenia and hemophagocytosis. HLH can be classified into two major forms: primary and secondary. Familial hemophagocytic lymphohistiocytosis (FHL), a type of primary HLH in which mutations in PRF1 UNC13D, STX11, STXBP2 genes cause the disease. Since genetic studies in STX11 and PRF1 have been investigated in Iranian FHL patients and no mutations found in some of them, this study aims to include UNC13D gene to FHL diagnosis panel to help patients diagnose early and accurate and do prenatal and carrier status. Material and Methods: Patients with HLH diagnosis and without mutation in PRF1 and STX11 genes entered this study. Primers were designed for exon and exonintron regions of UNC13D gene. After genomic DNA was extracted from whole blood samples, PCR technique was done and followed by direct sequencing of the products. Finally, sequencing results were analyzed. Results: Mutation analysis of UNC13D gene revealed several mutations including one novel splicing mutation (c.2091+1G>A), one missense mutation (c.1933C>T), one frame shift deletion (c.627Tdel) and three single nucleotide polymorphisms (SNPs) (c.888G>C, c.2601A>G, c.3183A>G). Conclusion: Considering the mutations detected in UNC13D gene and its association with FHL, elucidation of genetic study of this gene is necessary for accurate diagnosis and appropriate treatment and can help us extend our knowledge of this heterogeneous disease. [ABSTRACT FROM AUTHOR]

Objective: Leukocyte adhesion deficiency (LAD) is one of the rare immunodeficiency diseases clinically characterized by recurrent severe bacterial infections, periodontitis, delayed umbilical cord separation, absence of pus formation, periodontitis, delayed wound healing, and often persistent leukocytosis. Three types of the disease were discussed. With autosomal recessive inheritance and a prevalence of 1 in 1,000,000 live births, Type I (LADI) is caused by mutations in the gene ITGB2, located on chromosome 21 (21q22.3), encoding for CD18 protein. More than 110 distinct mutations in ITGB2 gene have been identified and registered at Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk/ac/all.php).This study aims to find the disease causing mutations in ITGB2 gene for helping future life in patient families via genetic counseling and prenatal diagnosis. Material and Methods: All of the patients with LAD1 diagnosis in the base of Flow cytometry (defect in CD18 and CD11 a, b, c) who were referred to IARRI during 2 years entered the study. 2 mL EDTA blood was obtained from all patients and their parents and genomic DNA was extracted. Polymerase chain reaction (PCR) for encoding exons and exon-intron boundaries of ITGB2 gene was performed. PCR results were direct sequenced in both orientations. Results: Sequencing amplified regions by PCR revealed a total of five different new mutations in five exons. These included three missense mutations, (c.382G>A in exon 6, c.715G>A in exon 7 and c.850G>A in exon 8) and two deletions (c.1907delA in exon 15 and c.1590_1594delCGGGC in exon 13). Mutation c.1907delA was found in two unrelated families. The last mutation was not previously reported. In fact, four out of 5 mutations have been reported previously. However, we find the mutation c.1590_1594delCGGGC neither in HGMD nor in other human mutation databases. Deletion of 5 nucleotides in this mutation results in a frame shift in open reading frame of CD18 protein and causes a premature stop codon at that position, p.Tyr530*. In all cases, mutation found in the patients was observed in their parents. Conclusion: The found mutations in this study can be used for genetic counseling and prenatal diagnosis of LAD I. [ABSTRACT FROM AUTHOR]

Objective: Biobank is a collection of resources containing biological and genetic materials that help store valuable samples. Establishing a biobank makes easy to access samples for assessing their characteristics. Our biobank consists of four separate biobanks including DNA, HLA, serum, and cell banks. Our aim is to standardize banking the samples of patients and their families who referred to Immunology, Asthma and Allergy Research Institute (IAARI) and suffered from primary immunodeficiency (PID), asthma and allergy. Materials and Methods: IAARI's biobank was established in 2006 with DNA and serum banks, then cell and HLA banks were added in 2012. All of the cases with diagnosis of PID, asthma and allergy entered in this registry and after taking informed consent, their samples were stored in the biobank. Results: Up to now, we stored a total of 3288 DNA samples from PID patients and their relatives, 450 serums from allergic patients, 186 samples of asthmatic patients, more than 700 HLA samples of healthy donors' volunteers, and 43 cell samples from PID patients. Conclusion: This biobank can be used to early diagnosis of PID genetics, and finding matched hematopoietic stem cell transplantation (HSCT) candidates for them. In addition, it can be helpful for investigating prospective genetic and familial surveys. This also decreases need for cost and resampling from patients. [ABSTRACT FROM AUTHOR]

Objective: Primary immunodeficiency disorders (PID) are a heterogeneous group of genetic disorders resulting from quantitative or functional defects in immune cells. Recently, T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs) have been used for the detection of T and B cell lymphopenia in newborn screening programs based on region-specific cutoff levels. Here, we report the newborn screening program for obtaining the cutoff levels for TREC and KREC copy numbers that may be applied in our newborn screening program and as a diagnostic tool in infants with a family history of PID in Iran. Materials and methods: DNA was extracted from a single 3.2mm punch from dried blood spots collected from 2160 anonymized newborns, which were obtained from two major referral health centers for newborn screening between 2014 and 2016. Samples from thirty patients with a definite diagnosis of severe combined immunodeficiency, X-linked agammaglobulinaemia (XLA), ataxia telangiectasia (AT) and twenty-five healthy controls were used for determining the cutoffs. Samples from other patients including those with IgA deficiency (IgAD), Wiskott-Aldrich syndrome, Hyper-IgM syndromes (HIgM) and Dedicator of cytokinesis 2 (DOCK2) deficiency were considered as controls. TREC, KREC and beta-actin (ACTB) copy numbers were analyzed in all samples using triplex-quantitative real-time PCR. The diagnostic efficiency of this method was evaluated using ROC curve analysis. Results: Subsequent to statistical analysis, the cutoff values were calculated and obtained with a high sensitivity and specificity. Among the thirty cases with abnormal results (1.4%), twelve cases (0.6%) were retested and were subsequently shown to be in the normal range. Moreover, all of the positive, disease control patients were correctly identified. Conclusion: Determining cutoff levels with a high sensitivity and specificity for both TREC and KREC is essential for correctly identifying children with severe forms of PID in newborn screening programs, whilst minimising false positive results. Early diagnosis of patients with PID enables early implementation of appropriate preventative measures and definitive therapies such as hematopoietic stem cell transplantation. [ABSTRACT FROM AUTHOR]

Objective: Nowadays genetic information is need to diagnose, treat, and generally manage different diseases. So genetic testing of immunodeficiency (PID) disorders is also of importance, especially for decisive diagnosis as well as bone marrow transplantation. Registration of information based on genetics has potential benefits for families possessing individuals with that disease for both present and future. This registry aims to collect genetics information about PID patients and their families' members. Materials and Methods: All of the Iranian patients suspected to PID who referred to immunology, asthma and allergy research institute (IAARI) between 2007 and 2016have been investigated regarding to PID. After taking informed consent, screening tests for PID were performed completely. Genetics data of individuals after definite diagnosis were enrolled in the genetic registry. Results: Genetic testing of 15 different PID diseases has been performed for more than 450 cases and collected in this registry. Of these 258 cases and their families had decisive mutation and genetic diagnosis. The rate of diseases was: Chronic Granulomatous Disease 22.17%, Severe Combined Immunodeficiency 21.33%, Wiskott-Aldrich Syndrome 10.46%, Congenital Neutropenia 8.36%, Leukocyte Adhesion Deficiency 7.53%, Griscelli 5.85%, Hemophagocytic Lymphohistiocytosis 5.23%, Angioedema 5.23%, Cystic Fibrosis 3.76%, Hyper IgE 2.92%, Hyper IgM 2.92%, X-linked Agammaglobulinemia 2.72%, Hermansky-Pudlak Syndrome 0.83%, Ataxia-Telangiectasia 0.41% and WHIM Syndrome 0.20%. Conclusions: This registry can be valuable for making aware of patients including decisive diagnosis, genetic counseling, carrier detection and prenatal diagnosis. In addition, this is a bank of data for future research. [ABSTRACT FROM AUTHOR]

Background: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency (approximately 1 case in 200.000-250.000 newborns) caused by defect in one of the components of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme (membrane-bound glycoproteins gp91, and cytosolic subunits p47-phox, p67-phox, p40-phox and p22-phox). This leads to defective intracellular killing by phagocyte cells. One of the components of NADPH-oxidase is p22-phox which is encoded by CYBA gene. In this study were identified new genetic changes of CYBA in 22 Iranian patients with autosomal recessive-CGD (AR-CGD). Methods: Twenty two CGD patients based on defect in NADPH oxidase activity (by NBT slide test and DHR-123 assay) and their demographic data and clinical histories were entered this study. They were referred to Immunology, Asthma and Allergy Research Institute (IAARI) for diagnosis and treatment. All patients had p22-phox deficiency based on western blotting. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) and PCR followed by direct sequencing to find p22-phox mutations. Results: Mutation analysis of CYBA revealed 12 different mutations, including three novel mutations: one deletion in exon 1, and two transitions in exon 3 [c.136G>A (p.Gly46Ser)], and exon 6 [c.388C>T (p.Gln130X)]. Conclusions: Three new mutations of CYBA gene in 4 of 22 Iranian patients with AR-CGD were found. These 3 novel mutations can partly complete the data base of Human Gene Mutation Database (HGMD). It can also be helpful for further prenatal diagnosis of next children in the affected families. Also it will be useful for timely decision making in bone marrow transplantation as treatment for CGD patients. [ABSTRACT FROM AUTHOR]

Background: Leukocyte adhesion deficiency type I (LAD-I) is a rare, autosomal recessive inherited immunodeficiency disease. LAD-I is caused by mutations in the ITGB2 gene and characterized by recurrent severe bacterial infections, as well as impaired wound healing with lack of pus formation. Methods: In this study, we investigated /TG62 gene mutations in 12 patients and their parents. Genomic DNA was extracted from whole blood samples. All coding regions of the ITGB2 gene were amplified using PCR and followed by direct sequencing. Results: Genetic analysis revealed 12 different homozygous mutations, including six missense (c.382G>A, c.2146G>C, c.715G>A, C.691OC, C.1777C and new C.1686OA), two new nonsense (c.1336G>T and C.1821OA), three-frame shift (c.1143delc, c.1907delA and new c.474dupC) and a splice site (c.1877+2T>C). Flow cytometry analysis of CD11/CD18 expression on neutrophils revealed defect in CD18 in all twelve cases (1.4% to 42%), CD11a in ten cases (0.1% to 26.7%), CD11b in nine cases (1.2% to 58.8%), and CD11c in all cases (0% to 18.1 %). The patients' parents were both heterozygous carriers. Conclusion: Our findings showed four new mutations in the ITGB2 gene. These results can be used for decisive genetic diagnosis, genetic counseling, as well as prenatal diagnosis for all patients who are suspended to LADI. [ABSTRACT FROM AUTHOR]

Background: Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields., Objective: The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest., Materials and Methods: We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.mL -1 Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA., Results: A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030)., Conclusion: The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry., Competing Interests: The authors declare no conflict of interest, (Copyright: © 2021 The Author(s); Published by Iranian Journal of Biotechnology.)

Background: Leukocyte adhesion deficiency type I (LAD-I) is a rare, autosomal recessive inherited immunodeficiency disease. LAD-I is caused by mutations in the ITGB2 gene and characterized by recurrent severe bacterial infections, as well as impaired wound healing with lack of pus formation., Methods: In this study, we investigated ITGB2 gene mutations in 12 patients and their parents. Genomic DNA was extracted from whole blood samples. All coding regions of the ITGB2 gene were amplified using PCR and followed by direct sequencing., Results: Genetic analysis revealed 12 different homozygous mutations, including six missense (c.382G>A, c.2146G>C, c.715G>A, c.691G>C, c.1777C and new c.1686C>A), two new nonsense (c.1336G>T and c.1821C>A), three-frame shift (c.1143delc, c.1907delA and new c.474dupC) and a splice site (c.1877+2T>C). Flow cytometry analysis of CD11/CD18 expression on neutrophils revealed defect in CD18 in all twelve cases (1.4% to 42%), CD11a in ten cases (0.1% to 26.7%), CD11b in nine cases (1.2% to 58.8%), and CD11c in all cases (0 % to 18.1%). The patients' parents were both heterozygous carriers., Conclusion: Our findings showed four new mutations in the ITGB2 gene. These results can be used for decisive genetic diagnosis, genetic counseling, as well as  prenatal diagnosis for all patients who are suspended to LADI.