Purpose
Leukocyte adhesion deficiency (LAD) represents a rare group of inherited inborn errors of immunity (IEI) characterized by bacterial infections, delayed umbilical stump separation, and ...autoimmunity. This single-center study aimed at describing the clinical, immunological, and molecular characterizations of 34 LAD-I Egyptian pediatric patients.
Methods
Details of 34 patients’ personal medical history, clinical and laboratory findings were recorded; Genetic material from 28 patients was studied. Mutational analysis was done by Sanger sequencing.
Results
Omphalitis, skin and soft tissue infections with poorly healing ulcers, delayed falling of the umbilical stump, and recurrent or un-resolving pneumonia were the most common presentations, followed by chronic otitis media, enteropathy, periodontitis; and recurrent oral thrush. Persistent leukocytosis and neutrophilia were reported in all patients, as well as CD18 and CD11b deficiency. CD18 expression was < 2% in around 90% of patients. Sixteen different pathological gene variants were detected in 28 patients who underwent
ITGß2
gene sequencing, of those, ten were novel and six were previously reported. Three families received a prenatal diagnosis. Patients were on antimicrobials according to culture’s results whenever available, and on prophylactic Trimethoprim-Sulfamethoxazole 5 mg/kg once daily, with regular clinical follow up. Hematopoietic stem cell transplantation (HSCT) was offered for 4 patients. However due to severity of the disease and delay in diagnosis, 58% of the patients passed away in the first 2 years of life.
Conclusion
This study highlights the importance of early diagnosis and distribution of
ITGß2
gene mutation in Egyptian children. Further molecular studies, however, remain a challenging necessity for better disease characterization in the region.
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.
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.
Background
Caspase recruitment domain family, member 11 (CARD11) is an important protein which plays a fundamental role in the activation of NF-κβ pathway in lymphocytes. CARD11 deficiency can be ...inherited in either autosomal dominant or autosomal recessive forms and present with different phenotypes including combined immunodeficiency, atopic dermatitis, and other variable manifestations. The present report describes clinical phenotypes and immunological defects of two unrelated patients with missense homozygous variants in CARD11 presenting with combined immunodeficiency (CID) and atopic skin disease resembling that reported in dominant negative CARD11 deficiency. The patients underwent next generation sequencing, immunophenotyping of T and B subsets by flow cytometry, T cell stimulation, and evaluation of CARD11 expression.
Results
Both patients had features suggesting CID including repeated pneumoniae with ICU admissions, chronic diarrhea, and itchy atopic skin disease. Patient-1 has homozygous missense variant in the C terminal domain (c.2839G > A, p.Glu947Lys), and patient-2 has homozygous variant in the inhibitory domain (c.1073C > G, p.Pro568Arg). Both have profound defects in Tregs with normal recent thymic emigrants, memory, and naïve CD4+ T cells. However, in response to stimulation, T cells failed to upregulate the expression of CD25. CARD11 expression by flow cytometry was decreased rather than abolished as previously described in patients with autosomal recessive CARD11 deficiency. B cells showed marked deficiency of switched memory and increase in transitional B cells.
Conclusion
Missense variants causing CARD11 deficiency may affect the protein function rather than the expression and can result in a phenotype combining the atopic skin disease and the features of CID.
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.
Introduction
Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox),
NCF1
(p47-phox),
CYBA
(p22-phox),
NCF2
(p67-phox), or
NCF4
...(p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming.
Aim
The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients.
Materials and Methods
Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis.
Results
The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %).
Conclusion
In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.
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.
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.
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.
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.
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.
Primary immunodeficiency (PID) patients are prone to developing viral infections and should not be vaccinated with live vaccines. In such patients, prolonged excretion and viral divergence may occur ...and they may subsequently act as reservoirs in the community introducing mutated virus and jeopardizing polio eradication. One hundred and thirty PID cases were included for poliovirus detection in stool with assessment of divergence of detected polioviruses from oral polio vaccine (OPV) virus. Clinical presentations of PID patients with detectable poliovirus in stool specimens are described.
Six PID patients (4.5%) had detectable vaccine-derived poliovirus (VDPV) excretion in stool specimens; of these, five patients had severe combined immunodeficiency (two with acute flaccid paralysis, one with meningoencephalitis and two without neurological manifestations), and one patient had X-linked agammaglobulinemia (paralysis developed shortly after diagnosis of immunodeficiency). All six case-patients received trivalent OPV. Five case-patients had type 2 immunodeficiency-related vaccine-derived polioviruses (iVDPV2) excretion; one had concomitant excretion of Sabin like type 3 virus and one was identified as iVDPV1 excretor. Surveillance for poliovirus excretion among PID patients is critical as these patients represent a potential source to reseed polioviruses into populations.
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.
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.
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.
In spite of the genetic heterogeneity behind PIDs, genetic counseling should play a critical role in the management and future decisions of affected families.
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.
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