Cernunnos deficiency: Further delineation in 5 Egyptian patients.

Cernunnos deficiency is a rare genetic disorder characterized by immunodeficiency, microcephaly, growth retardation, bird-like facies, sensitivity to ionizing radiation, few autoimmune manifestations, premature aging of hematopoietic stem cells at an early age, and occasional myeloproliferative disease. Herein we present five Egyptian Cernunnos patients from 3 different families. We describe the patients' clinical phenotypes, their immunological profile as well as genetic results. Sequence analysis revealed three different mutations in the NHEJ1 gene: a nonsense variant c.532C > T; p.(Arg178Ter), an intronic variant c.178-1G > A and a frameshift insertion variant c.233dup; p.(Asn78LysfsTer14). In conclusion, Cernunnos deficiency can have a wide range of clinical features. The characteristic immune profile including a decrease in recent thymic emigrants and naive T cells, markedly elevated memory T cells together with normal to high IgM, and a decrease in IgG and IgA. This immune profile is highly suggestive of Cernunnos deficiency in T-B-NK + SCID patients especially surviving for older ages.

Chronic Granulomatous Disease: a Cohort of 173 Patients-10-Years Single Center Experience from Egypt.

PURPOSE: Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder of phagocytes, characterized by recurrent fungal and bacterial infections. Our aim is to describe the different clinical presentations, non-infectious auto-inflammatory features, types and sites of infections, and to estimate the mortality among our large cohort. METHODS: This is a retrospective study conducted at the Pediatric Department of Cairo University Children's Hospital in Egypt, including cases with a confirmed CGD diagnosis. RESULTS: One hundred seventy-three confirmed CGD patients were included. AR-CGD was diagnosed in 132 patients (76.3%) including 83 patients (48%) with p47phox defect, 44 patients (25.4%) with p22phox defect, and 5 patients (2.9%) with p67phox defect. XL-CGD was diagnosed in 25 patients (14.4%). The most common recorded clinical manifestations were deep-seated abscesses and pneumonia. Gram-negative bacteria and Aspergillus were the most frequently isolated species. Regarding the outcome, 36 patients (20.8%) were lost from follow-up. Among patients with known outcome, 94/137 patients (68.6%) are living, while 43/137 patients (31.4%) died. CONCLUSION: AR-CGD is predominant in Egypt; CGD must always be ruled out in any patient presenting with typical or atypical mycobacterial or BCG-disease.

Flow cytometry optimizing the diagnostic approach in inborn errors of immunity: experience from Egypt.

BACKGROUND: Human inborn errors of immunity (IEI) are a group of inherited genetic disorders of the immune system. IEI Patients suffer from severe repeated infections, autoimmunity, lymphadenopathy and/or increased susceptibility to malignancies. IEI are due to absence, disproportion, or loss of function of immune cells; mostly inherited in autosomal recessive manner, hence are more common in countries with high rate of consanguinity. Definite diagnosis of IEI is achieved by genetic analysis, however it is not always available. AIM: to report on different IEI categories and impact of expanding the use of flow cytometry (FCM) in diagnosis, categorization and follow up of IEI patients in a highly consanguineous population. METHODS: Retrospective chart review on different IEI categories diagnosed at the primary immunodeficiency center in Cairo University Specialized Pediatric hospital from 2011 to 2021 based on expanding the use of FCM. RESULTS: 1510 IEI patients were diagnosed; 480 were diagnosed genetically with FMF, 11 with cystic fibrosis and 1019 patients were diagnosed with other IEI disorders. Phagocytic defects were the commonest (30%) followed by severe combined immunodeficiency (22%) and combined immunodeficiency (18.3%). FCM testing properly diagnosed and categorized 73% of the cases. CONCLUSION: Using multi-color FCM to evaluate immune cells populations, subpopulations, functions, and intracellular proteins expression is proved a useful cost-effective method for screening, categorization and follow up of IEI patients. FCM can improve the diagnosis of IEI significantly when tests are properly targeted and well designed. This study presents a 10-year experience in diagnosis of IEI using FCM at a tertiary referral center in a setting of limited resources and yet high prevalence of IEI.

The Cause-Effect Dilemma of Hematologic Changes in COVID-19: One Year after the Start of the Pandemic.

COVID-19 is a systemic infection that leads to multisystem affection, including hematological changes. On the other hand, the patients who have certain hematological diseases are more susceptible to COVID-19 infection. The aim of this review is to examine the wide spectrum of hematological changes that are reported to occur due to COVID-19 infection. Most of the studies over the past year mainly show that most of these changes are mainly non-specific, but are of prognostic value. On the other hand, the susceptibility of hematological patients to COVID-19 infection and complications remains questionable. Patients with certain hematological diseases (including malignancy) and those who are treated by aggressive immunosuppressive therapy have shown higher rates of COVID-19 infection and complications. On the other hand, for most of the patients suffering from other chronic hematological conditions, no evidence has shown a greater risk of infection, compared to the general population.

Genetic Testing in Egyptian Patients with Inborn Errors of Immunity: a Single-Center Experience.

BACKGROUND: Inborn errors of immunity (IEI) are a group of heterogeneous disorders with geographic and ethnic diversities. Although IEI are common in Egypt, genetic diagnosis is limited due to financial restrictions. This study aims to characterize the genetic spectrum of IEI patients in Egypt and highlights the adaptation of the molecular diagnostic methods to a resource-limited setting. METHODS: Genetic material from 504 patients was studied, and proper diagnosis was achieved in 282 patients from 246 families. Mutational analysis was done by Sanger sequencing, next-generation sequencing (NGS) targeting customized genes panels, and whole-exome sequencing (WES) according to the patients' phenotypes and availability of genetic testing. RESULTS: A total of 194 variants involving 72 different genes were detected with RAG1/2 genes being the most encountered followed by DOCK8, CYBA, LRBA, NCF1, and JAK3. Autosomal recessive (AR) inheritance was detected in 233/282 patients (82.6%), X-linked (XL) recessive inheritance in 32/282 patients (11.3%), and autosomal dominant (AD) inheritance in 18/282 patients (6.4%), reflecting the impact of consanguineous marriages on the prevalence of different modes of inheritance and the distribution of the various IEI disorders. CONCLUSION: The study showed that a combination of Sanger sequencing in selected patients associated with targeted NGS or WES in other patients is an effective diagnostic strategy for IEI diagnosis in countries with limited diagnostic resources. Molecular testing can be used to validate other nonexpensive laboratory techniques that help to reach definitive diagnosis and help in genetic counseling and taking proper therapeutic decisions including stem cell transplantation or gene therapy.

Bone health in pediatric transfusion-dependent beta-thalassemia: Circulating osteoprotegerin and RANKL system.

INTRODUCTION: While the mechanism of bone disease in thalassemia is multifactorial and still under investigation, the receptor activator of nuclear factor kappa B (RANK), receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) have pivotal roles in regulating bone metabolism. This study aimed to measure RANKL and OPG serum levels, and to detect the incidence of RANKL rs9533156, OPG rs2073618, and OPG rs2073617 genotypes in pediatric ß-thalassemia patients and to assess their relation to bone mineral density. METHODS: Sixty patients with transfusion-dependent ß-thalassemia (TBT) patients ages 5 to 14 years were included, and 60 healthy, age- and sex-matched volunteers contributed as a control group. The patients were scanned for bone mineral density. RESULTS: The mean of spine dual-energy X-ray absorptiometry (DXA) Z-score in patients was -1.66 ± 1.02 standard deviation (SD). Twenty-four of them had low spine DXA Z-scores. The patients showed significantly lower OPG levels and OPG/RANKLs ratios than the control group (3.28 ± 9.11 ng/ml and 11.38 ± 14.93 ng/ml, and 0.01 ± 0.03 and 0.07 ± 0.09, respectively). The RANKL SNP rs9533156 TC heterozygous genotype was detected more with statistical significance in patients than controls. The incidence of OPG rs2073618 and OPG rs2073617 genotypes were 2.3 times and 1.9 times more frequent in patients than controls, respectively. CONCLUSION: The RANK/RANKL/OPG system may have an important role in regulating bone metabolism in TBT patients, although further studies are needed to clarify its role.

Fungal infections in primary immunodeficiency diseases.

Primary immunodeficiency diseases (PID), encompass a heterogeneous group of diseases, with increased susceptibility to recurrent, severe infections. Invasive fungal infections raise a serious concern related to their morbidity and mortality. Herein, we describe various fungal infections among different PID patients. Twenty-eight PID patients diagnosed with fungal infections were included; fourteen patients with chronic granulomatous disease, two with Hyper Immunoglobulin E syndrome, one with LRBA deficiency and one with MHC class II defect, one with unclassified immune dysregulation, one with CD4 lymphopenia and one patient with Immune dysregulation Polyendocrinopathy Enteropathy X-linked syndrome. Aspergillus species were the most common isolated causative organisms in 78% of patients, Candida species were the causative organisms in 32%, Pneumocystis jirovecii caused infections in 7% followed by Malassezia furfur, Fusarium spp., Mucormycosis, and Penicillium chrysogenium 3.5% for each. The mortality rate among our patients was 10/28 (35.7%). PID patients are at high risk of developing fungal infections.

Clinical Phenotypes and Immunological Characteristics of 18 Egyptian LRBA Deficiency Patients.

LPS-responsive beige-like anchor (LRBA) deficiency is an autosomal recessive primary immunodeficiency disorder, OMIM (#614700). LRBA deficiency patients suffer from variable manifestations including recurrent infections, immune dysregulation, autoimmunity, cytopenias, and enteropathy. This study describes different clinical phenotypes and immunological characteristics of 18 LRBA deficiency patients diagnosed from Egypt. T and B lymphocyte subpopulations, LRBA, and cytotoxic T lymphocyte-associated protein 4 (CTLA4) expression were evaluated in resting and stimulated T cells using flow cytometry. Next-generation sequencing was used to identify mutations in the LRBA gene. LRBA deficiency patients had significantly lower B cells and increased percentage of memory T cells. CTLA4 levels were lower in LRBA-deficient T regulatory cells in comparison to healthy donors at resting conditions and significantly increased upon stimulation of T cells. We identified 11 novel mutations in LRBA gene ranging from large deletions to point mutations. Finally, we were able to differentiate LRBA-deficient patients from healthy control and common variable immunodeficiency patients using a simple flow cytometry test performed on whole blood and without need to prior stimulation. LRBA deficiency has heterogeneous phenotypes with poor phenotype-genotype correlation since the same mutation may manifest differently even within the same family. Low LRBA expression, low numbers of B cells, increased numbers of memory T cells, and defective CTLA4 expression (which increase to normal level upon T cell stimulation) are useful laboratory tests to establish the diagnosis of LRBA deficiency. Screening of the siblings of affected patients is very important as patients may be asymptomatic at the beginning of the disease course.

MHC-II Deficiency Among Egyptians: Novel Mutations and Unique Phenotypes.

BACKGROUND: MHC class II deficiency leads to defective CD4+ T-cell function that results from impaired antigen presentation. A genetic disorder in 1 of 4 genes results in this syndrome that is associated with the clinical phenotype of combined immunodeficiency. OBJECTIVE: To describe the clinical, immunological, and molecular characteristics of 10 Egyptian patients from 9 different families having presented with MHC class II deficiency between 2012 and 2017. METHODS: An initial diagnosis based on the combination of clinical features and low HLA-DR expression by flow cytometry was confirmed by genetic analyses. RESULTS: Symptoms included failure to thrive (n = 9), persistent diarrhea (n = 5), and pneumonia (n = 8). Septicemia due to coagulase-negative staphylococci (n = 1) and Candida krusei (n = 1) was diagnosed. Nine patients orally received the live attenuated polio vaccine, of whom 3 developed acute flaccid paralysis thereafter. Nine patients received the BCG vaccine and none developed obvious signs of BCGitis. Four patients carried RFXANK gene mutations, 3 carried RFX5 gene mutations, 1 carried a CIITA gene mutation, and none carried RFXAP gene mutations. Six of the 7 detected mutations were previously unreported mutations: c.431T>C, c.247_250delTCAG, and c.600delG in the RFXANK gene; c.116+1G>A and c.715C>T in the RFX5 gene; and c.929delA in the CIITA gene. CONCLUSIONS: Given that Egypt is a North African country with a high rate of consanguinity, MHC class II deficiency is not rare. However, the molecular defects differ from those reported in nearby countries. Early diagnosis must be based on suspicious clinical signs and laboratory diagnosis because the defect can be missed by T-cell receptor excision circles based on neonatal screening.

Prevalence of α-Thalassemia in the Egyptian Population.

Hemoglobinopathies are the most common monogenic diseases in the world, causing many health problems worldwide. In Egypt, thalassemia is the most common cause of chronic hemolytic anemia and correlated with significant morbidity and mortality. One thousand Egyptian newborns were screened to detect α-thalassemia (α-thal) deletions using polymerase chain reaction (PCR)-based DNA analysis of cord blood samples. Ninety-one cases (9.1%) of the studied samples were proved to have at least one of the α genes deleted and 851 cases (85.1%) were normal by PCR analysis, while 58 samples (5.8%) failed to be amplified so further DNA analysis could not be done. In the studied group with α gene deletions, we found different types including silent carriers with only one α-globin gene deleted (3.1%), α-thal trait with two α-globin genes deleted (4.2%), Hb H disease with three α-globin genes deleted (1.8%) and no cases carrying Hb Bart's disease with loss of four α-globin genes. We determined the deletional spectrum of α-thal, which might be used in the future for molecular investigations of the disease in susceptible patients in our population.

Diagnosis of DOCK8 deficiency using Flow cytometry Biomarkers: an Egyptian Center experience.

In the past few years, several genes were shown to be implicated in various forms of the Hyper Immunoglobulin E syndrome. The present study is the first to describe a cohort of DOCK8 deficiency patients from Egypt. The study included 15 patients with features of combined immunodeficiency (CID) suggestive of DOCK8 deficiency. Flow cytometry was used for evaluation of DOCK8 expression and studying different immunological characteristics of those patients including evaluation of Th17, Tregs, T and B lymphocytes differentiation and the effect of the DOCK8 deficiency on the activation of the STAT3. Diagnosis was confirmed by mutational analysis. Profound defects in Th17 cells and Tregs were observed in all patients with impaired STAT3 phosphorylation, indicating that DOCK8 plays a pivotal role in the STAT3 signaling pathway. These findings together with decrease in memory B cells and defective DOCK8 expression by flow cytometry can confirm the diagnosis.

Genetic Counseling in Primary Immunodeficiency Disorders: An Emerging Experience in Egypt.

BACKGROUND: Primary immunodeficiency disorders (PIDs) are a heterogeneous group of diseases of the immune system leading to life-threatening infections, and, unless urgently treated with immune reconstitution, patients do not usually survive. With the continuing progress in molecular diagnosis, many mutations have been described in more than 300 genes. Genetic counseling has recently been considered an essential part of the management of PIDs. This study presents the experience of genetic counseling services in the largest PID center in Egypt, and reports on our management plan and the impact of prenatal diagnosis (PND) on families. METHODS: Based on the biochemical and molecular diagnosis of index cases, PND was offered for 10 families in 12 subsequent pregnancies. Five different genes were sequenced by Sanger sequencing in fetal samples. RESULTS: Seven fetuses were either normal or were carriers, while five fetuses were affected and human leukocyte antigen typing was performed, seeking a suitably related donor for stem cell transplantation. CONCLUSION: In spite of the genetic heterogeneity behind PIDs, genetic counseling should play a critical role in the management and future decisions of affected families.

Incidence and risk factors of acute kidney injury among the critically ill neonates.

Acute kidney injury (AKI) is a complex disorder with clinical manifestations ranging from mild dysfunction to complete kidney failure. The published literature on the incidence and outcome of AKI in the critically ill neonatal population is scarce. The aim of this study was to evaluate the types, the associated risk factors and short-term outcome of AKI in the critically ill neonates. A cohort study was conducted including 100 critically ill neonates successively admitted to the Neonatal Intensive Care Unit. The inclusion criteria were a gestational age ≥28 weeks and body weight ≥1 kg. Exclusion criteria included those with multiple congenital anomalies or on drugs altering glomerular filtration rate or AKI developing postoperatively. Neonates were evaluated for the development of AKI [creatinine >1.5 mg/dL and/or blood urea nitrogen (BUN) >20 mg/dL] and were assigned as group A (who developed AKI) and group B (who did not develop AKI). Forty-one patients developed AKI (group A) among whom nine (22%) showed oliguric AKI. The most common risk factors among group A patients were sepsis (75.6%) and nephrotoxic drug administration (75.6%), followed by shock (39%). There were no statistically significant differences between both groups except for male sex predominance and necrotizing enterocolitis (NEC), which were significantly higher among group A (P <0.05). Use of continuous positive airway pressure (CPAP) ventilation was significantly higher in neonates without AKI (13.6% vs 0.0%, P = 0.02). The mortality rate among group A reached 51.2%. Various risk factors including gender, gestational age, birth weight, shock, NEC, sepsis, nephrotoxic drugs, oliguria and mechanical ventilation were studied as regards outcome of group A, and all factors except gender and oliguria proved to be significantly higher in deceased neonates. Male sex and NEC were important risk factors for developing AKI that was predominantly non-oliguric. CPAP ventilation may have a protective effect against AKI. The mortality rate was more than three times higher in the AKI group.

Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt.

The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

Study of naïve and memory cells in a cohort of Egyptian chronic granulomatous disease patients.

CONTEXT: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by inherited defects in the NADPH oxidase complex which may be involved in important pathways that connect innate and adaptive immunity. OBJECTIVES: Characterize the naive and memory compartment of B and T lymphocytes in patients with CGD. METHODS: Twenty CGD patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow cytometric assessment of the naïve and memory compartments of peripheral blood lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. RESULTS: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males (47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical significant difference for the percentage and absolute count of CD19 + CD27+ memory B cell (p = 0.028 and p = 0.047 respectively), CD45RA cells (with p values of p = 0.000 and 0.033, respectively), the naïve compartment CD3 + CD45RA+ cells percentage and absolute counts (p = 0.005, 0.01respectively), CD3 + CD27 + cells percentage and absolute counts (p = 0.001, 0.012 respectively), CD3 + CD45RA + CD27+ cells percentage and absolute counts (p = 0.015, 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked group than control group. CONCLUSION: There was an altered naïve and memory B profile in CGD patients, this may increase susceptibility of the patients to opportunistic infections and autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the presence of infections.