SARS-CoV-2 genome variations and evolution patterns in Egypt: a multi-center study.

A serious global public health emergency emerged late November 2019 in Wuhan City, China, by a new highly pathogenic virus, SARS-CoV-2. The virus evolution spread has been tracked by three developing databases: GISAID, Nextstrain and PANGO to understand its circulating variants. In this study, 110 diagnosed positive COVID-19 patient's samples, were collected from Kasr Al-Aini Hospital and the Children Cancer Hospital Egypt 57357 between May 2020 and January 2021, with clinical severity ranging from mild to severe. The viral genomes were sequenced by next generation sequencing, and phylogenetic analysis was performed to understand viral transmission dynamics. According to Nextstrain clades, most of our sequenced samples belonged to clades 20A and 20D, which in addition to clade 20B were present from the beginning of sample collection in May 2020. Clades 19A and 19B, on the other hand, appeared in the mid and late 2020 respectively, followed by the disappearance of clade 20B at the end of 2020. We identified a relatively high prevalence of the D614G spike protein variant and novel patterns of mutations associated together and with different clades. We also identified four mutations, spike H49Y, ORF3a H78Y, ORF8 E64stop and nucleocapsid E378V, associated with higher disease severity. Altogether, our study contributes genetic, phylogenetic, and clinical correlation data about the spread of the SARS-CoV-2 pandemic in Egypt.

Expression pattern and prognostic implication of SALL4 gene in myeloid leukemias: a case-control study.

SALL4 is a transcription factor that retains stem cells in an undifferentiated state and promotes its self-renewal. In addition, it is implicated in leukemogenesis via its effect on leukemic stem cells. This study aimed to characterize the expression pattern of SALL4 gene in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) at different progression phases of the leukemic process and to assess its prognostic significance. Real-time PCR was used in 106 patients: 54 AML patients; 43 de novo and 11 in complete remission (CR), 52 CML patients; 31 in chronic phase (CP), 11 in deep molecular response (MR4) and 10 in accelerated/blastic phase (AP/BP); and in 21 nonmalignant bone marrow samples. SALL4 gene expression was elevated in AML, AML-CR and CML-CP (median = 5.180, 4.604 and 14.125 fold changes, respectively). Elevated SALL4 gene expression among AML de novo patient was associated with poor disease-free survival (DFS) rates (p = .022). Among CML patients, the highest percentage of patients with a high SALL4 (p = .033) was among CML-CP. SALL4 has a role in leukemogenesis; high SALL4 expression was associated with poor DFS among AML patients.

Interleukin-23R gene polymorphism in pediatric Egyptian patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia is an acquired autoimmune disorder caused by the production of antiplatelet antibodies. These autoantibodies opsonize platelets for splenic clearance, resulting in low levels of circulating platelets. The current case-control study aimed at detecting the frequency of interleukin-23 receptor rs1884444 single nucleotide polymorphism in Egyptian children with primary immune thrombocytopenia and its possible role as a genetic marker for disease risk. Interleukin-23 receptor rs1884444 single nucleotide polymorphism was studied in 50 patients with primary immune thrombocytopenia and 100 healthy age and sex-matched controls by polymerase chain reaction amplification of the target gene followed by allele-specific restriction enzyme digestion. Regarding the distribution of the genotypes of the interleukin-23 receptor rs1884444 polymorphism, no statistically significant difference was found between cases and control groups. The variant genotypes (GT/TT) frequency was 10% in primary immune thrombocytopenia cases versus 7% in the control groups [P value = 0.755, odds ratio (OR): 0.326, 95% confidence interval (CI): 0.099-1.076]. Similarly, no difference was found between acute and chronic cases. The variant genotypes GT/TT frequency was 10.7% in acute versus 9.1% in chronic primary immune thrombocytopenia (P value = 0.849). The variant genotypes GT/TT were not found to be a risk factor for acute primary (P value = 0.807, OR: 0.641, 95% CI: 0.16-2.563) or chronic primary immune thrombocytopenia (P value = 0.914, OR: 0.762, 95% CI: 0.153-3.797). Our study suggests the possibility that interleukin-23 receptor gene polymorphism may not contribute to the susceptibility of development of primary immune thrombocytopenia in Egyptian children.

Association between Duffy antigen receptor expression and disease severity in sickle cell disease patients.

OBJECTIVES: Sickle cell disease (SCD) is associated with a pro-inflammatory state, characterized by an elevated baseline leukocyte count and inflammatory cytokines. Inflammation, white blood cell (WBC) adhesion to vascular endothelium with subsequent endothelial injury, and repeated ischemia-reperfusion injury contribute to disease pathogenesis. Identification of genetic polymorphisms that may modulate disease severity in SCD is becoming a field of interest. The Duffy blood group antigen has been identified as a receptor for various chemokines involved in neutrophil activation and trafficking. This study aimed at investigating the effect of RBCs' Duffy antigen expression and its genetic polymorphisms on modulating disease severity and its complications among Egyptian sickle cell patients. Methods We analyzed the association of Duffy genotypes and phenotypes with clinical expression of SCD in 100 Egyptian patients. The Duffy phenotype expression was detected by indirect anti-globulin test while Duffy genotyping was conducted with polymerase chain reaction-restriction fragment length polymorphism-based assay. Results Total WBC count was strongly associated with Duffy genotype. WBCs were significantly higher in Duffy-positive patients (P = 0.002). No statistical significance was evident between individual measures of disease severity (pulmonary dysfunction, avascular necrosis, central nervous system dysfunction, kidney dysfunction, and leg ulcers) and Duffy genotype. Conclusion Our study suggests that RBC Duffy expression increases levels of WBCs in SCD patients and that Duffy genotype may not be a potential biomarker for end-organ damage in SCD.

The clinical relevance and prognostic significance of adenosine triphosphate ATP-binding cassette (ABCB5) and multidrug resistance (MDR1) genes expression in acute leukemia: an Egyptian study.

AIM: Multidrug resistance (MDR1) represents a major obstacle in the chemotherapeutic treatment of acute leukemia (AL). Adenosine triphosphate ATP-binding cassette (ABCB5) and MDR1 genes are integral membrane proteins belonging to ATP-binding cassette transporters superfamily. PURPOSE: The present work aimed to investigate the impact of ABCB5 and MDR1 genes expression on the response to chemotherapy in a cohort of Egyptian AL patients. The study included 90 patients: 53 AML cases and 37 ALL cases in addition to 20 healthy volunteers as controls. METHODS: Quantitative assessment of MDR1 and ABCB5 genes expression was performed by quantitative real-time polymerase chain reaction. Additional prognostic molecular markers were determined as internal tandem duplications of the FLT3 gene (FLT3-ITD) and nucleophosmin gene mutation (NPM1) for AML cases, and mbcr-abl fusion transcript for B-ALL cases. RESULTS: In AML patients, ABCB5 and MDR1 expression levels did not differ significantly between de novo and relapsed cases and did not correlate with the overall survival or disease-free survival. AML patients were stratified according to the studied genetic markers, and complete remission rate was found to be more prominent in patients having low expression of MDR1 and ABCB5 genes together with mutated NPM1 gene. In ALL patients, ABCB5 gene expression level was significantly higher in relapsed cases and MDR1 gene expression was significantly higher in patients with resistant disease. CONCLUSION: In conclusion, the results obtained by the current study provide additional evidence of the role played by these genes as predictive factors for resistance of leukemic cells to chemotherapy and hence treatment outcome.

FcγRIIa and FcγRIIIa genetic polymorphisms in a group of pediatric immune thrombocytopenic purpura in Egypt.

Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder caused by the production of antiplatelet antibodies. The current case-control study aimed at detecting the frequency of FcγRIIa-131H/R and FcγRIIIa-158F/V genes polymorphism in Egyptian children with ITP as genetic markers for ITP risk, and to clear out their possible role in choosing the treatment protocols of ITP. To achieve this aim, FcγRIIa genotyping was tested by PCR-restriction fragment length polymorphism (RFLP) technique, whereas FcγRIIIa genotyping was tested by nested PCR followed RFLP analysis. The current case-control study was conducted on 92 children with ITP; 12 acute and 80 chronic cases and 90 controls. The V allele and FcγRIIIa FV heterotype were significantly higher in ITP patients and conferred increased ITP risk [odds ratio (OR) = 1.96 and 2.55, respectively]. The frequency of FcγRIIa H allele was significantly higher among chronic ITP patients. In conclusion, FcγRIIIa gene polymorphism may contribute to susceptibility to ITP. Moreover, analysis of the FcγR polymorphisms in ITP patients could influence the effectiveness of medications and selection of the line of treatment.

A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic leukemia.

AML1 is among the most frequent targets of chromosomal rearrangements in human leukemias. We report here the molecular analysis of a t(4;21)(q28;q22) that has disrupted AML1 in a patient with de novo T-cell acute lymphoblastic leukemia. By using 3'-RACE analysis, we show that this rearrangement results in the fusion of a novel gene immediately downstream of exon 5 or exon 6 of AML1, indicating that the AML1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. The sequence of the novel gene, located at 4q28, does not have any significant homology with any of the known genes in the human GenBank DNA database. However, the first 118 bases are identical to a part of a human ovarian EST. Also, its high homology with mouse and rat sequences suggests that this sequence most probably represents a part of a novel gene, which we named FGA7 (Fused Gene 7 to AML1). Following the AML1 open reading frame, the FGA7 sequence encodes an unknown protein of 27 amino acids. We isolated three bacterial artificial chromosome (BAC) clones that contain the FGA7 sequence and confirmed the breakpoint of the gene on the patient's metaphase spreads by fluorescence in situ hybridization using these BACs as probes. RT-PCR and Northern blot analyses revealed that FGA7 is expressed in ovarian and skeletal muscle tissues. The predicted AML1-FGA7 chimeric proteins contained a limited number of residues fused to AML1 in a situation similar to that reported for the AML1-EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full-length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor activates.

A new translocation that rearranges the AML1 gene in a patient with T-cell acute lymphoblastic leukemia.

The AML1 gene (also known as RUNX1 or CBFA2), located in chromosome band 21q22, encodes a transcription factor which heterodimerizes with the CBFbeta protein forming a complex called human core binding factor (CBF). The CBF complex appears to regulate a number of genes important for hematopoiesis. AML1 is one of the most common targets of chromosomal rearrangements in human leukemias and has been involved in 14 chromosomal translocations to date. Here we report a new chromosomal translocation, t(4;21)(q31;q22) that disrupts the AML1 gene in a 12-year-old boy with newly diagnosed T-cell acute lymphoblastic leukemia (ALL). This is the first reported chromosomal translocation where AML1 is rearranged in childhood T-cell ALL. By metaphase fluorescence in situ hybridization analysis, the AML1 breakpoint was mapped using recombinant phage clones, and shown to be either immediately upstream or downstream of exon 5.