Analysis of Clinical, Immunological and Molecular Features of Leukocyte Adhesion Deficiency Type I in Egyptian Children.

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.

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.

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.

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.

Association of DNA Methyltransferase 3B Promotor Polymorphism With Childhood Chronic Immune Thrombocytopenia.

BACKGROUND: DNA methylation is an epigenetic process that refers to chromatin-based mechanisms in the regulation of gene expression without DNA alternation. It is mediated by DNA methyltransferases (DNMTs). The DNA methyltransferase 3B (DNMT3B) gene contains a C-to-T single nucleotide polymorphism (SNP; rs2424913) in the Promotor region, 149 base pairs from the transcription start site, which is reported to significantly increase the Promotor activity. OBJECTIVE: To investigate the prevalance of rs2424913 single nucleotide polymorphism located in the DNMT3B gene Promotor. METHODS: In the present study, we investigated the prevalence of rs2424913 single nucleotide polymorphism located in DNMT3B gene Promotor by restriction fragment length polymorphism (PCR-RFLP) in Egyptian pediatric chronic immune thrombocytopenia (ITP) patients and controls. RESULTS: The homozygous genotype (TT) was significantly higher in our patient and conferred almost 3-fold increased risk of chronic ITP when compared to controls. CONCLUSION: The present study shows that DNMT3B rs2424913 promotor polymorphism represents a genetic risk factor that may play an important role in understanding the pathogenesis of chronic ITP.

Susceptibility and progression of end stage renal disease are not associated with angiotensin II type 1 receptor gene polymorphism.

CONTEXT: The role of the angiotensin II type 1 receptor (AT1R) gene polymorphism, A1166C, has been shown to be associated with end stage renal disease (ESRD) and its progression. There is also some evidence that HLA class II alleles are associated with ESRD independent of other factors. OBJECTIVE: To examine the association between AT1R gene polymorphism in the susceptibility and progression to ESRD in patients with chronic renal failure and to investigate if the AT1R genotypes and HLA-DR alleles predict the time to ESRD. MATERIALS AND METHODS: Genotyping was performed in 50 ESRD patients and 44 control subjects for the AT1R A1166C gene polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). ESRD patients were examined for HLA-DRB1 alleles according to a reverse hybridization line probe assay. RESULTS: Allele and genotype frequencies of the AT1R polymorphism did not differ significantly between ESRD patients and controls. Furthermore, there was no association between the AT1R gene polymorphism or HLA-DRB1 alleles with the time to the occurrence of end stage failure. DISCUSSION AND CONCLUSION: We concluded that the AT1R genotype does not contribute to the genetic susceptibility of ESRD and is not associated with progression of chronic kidney failure to ESRD.

Increased DNA damage in hepatitis C virus-related hepatocellular carcinoma.

One consequence of hepatitis C virus (HCV) infection is an elevated cancer risk. During chronic viral infection, deoxyribonucleic acid (DNA) damage is being induced by reactive oxygen and nitrogen species, which may play a pathogenic role in HCV-induced carcinogenesis. The study investigated DNA damage in peripheral blood lymphocytes from patients with hepatocellular carcinoma (HCC) and those with HCV infection with and without associated cirrhosis and normal controls. As a measure for genomic damage, the comet assay (single cell gel electrophoresis) was applied, which detects single- and double-strand breaks and alkali-labile sites through electrophoretic mobility of the resulting fragments. The levels of DNA damage were significantly higher in HCC and HCV-associated cirrhosis compared to HCV without cirrhosis and the control group. Patients presenting with DNA damage more than mean+two standard deviation of the controls had a 3.6-fold risk of having HCC more than those with undamaged DNA. HCV disease progression was the only discriminator predicting the extent of DNA damage. The accumulation of DNA damage is important in HCC evolution. DNA damage indicating intracellular oxidative and nitrative stress may lead to mutagenesis and consequently malignant transformation, which emphasizes the need to optimize the therapy for reducing the degree of genomic damage.

CD4(+) CD25(+) cells in type 1 diabetic patients with other autoimmune manifestations.

The existence of multiple autoimmune disorders in diabetics may indicate underlying primary defects of immune regulation. The study aims at estimation of defects of CD4(+) CD25(+high) cells among diabetic children with multiple autoimmune manifestations, and identification of disease characteristics in those children. Twenty-two cases with type 1 diabetes associated with other autoimmune diseases were recruited from the Diabetic Endocrine and Metabolic Pediatric Unit (DEMPU), Cairo University along with twenty-one normal subjects matched for age and sex as a control group. Their anthropometric measurements, diabetic profiles and glycemic control were recorded. Laboratory investigations included complete blood picture, glycosylated hemoglobin, antithyroid antibodies, celiac antibody panel and inflammatory bowel disease markers when indicated. Flow cytometric analysis of T-cell subpopulation was performed using anti-CD3, anti-CD4, anti-CD8, anti-CD25 monoclonal antibodies. Three cases revealed a proportion of CD4(+) CD25(+high) below 0.1% and one case had zero counts. However, this observation did not mount to a significant statistical difference between the case and control groups neither in percentage nor absolute numbers. Significant statistical differences were observed between the case and the control groups regarding their height, weight centiles, as well as hemoglobin percentage, white cell counts and the absolute lymphocytic counts. We concluded that, derangements of CD4(+) CD25(+high) cells may exist among diabetic children with multiple autoimmune manifestations indicating defects of immune controllers.