Generation of Dual-Color FISH probes targeting 9p21, Xp21, and 17p13.1 loci as diagnostic markers for some genetic disorders and cancer in Egypt Amal M. Mohamed, Maha Eid, Ola Eid, Shymaa H Hussein, Wael Mahmoud, Rana Mahrous, Khaled Refaat, Marwa Farid Journal of Genetic Engineering and Biotechnology, 2025 INTRODUCTION: The fluorescence in situ hybridization (FISH) is a very important technique, as it can diagnose many genetic disorders and cancers. Molecular cytogenetic analysis (FISH) can diagnose numerical chromosome aberrations, sex chromosomes anomalies, and many genetic disorders. AIM: With the limited number of commercially available probes that do not cover all research needs and the high prices of the commercial probes, our goal is to apply recent technologies to produce FISH probes that can accurately and sensitively diagnose genetic diseases and cancer in Egypt and establishing the inhouse production of different FISH probes. We intend to adhere to the published guidelines and validation procedures to ensure the production of accurate FISH probes for clinical diagnosis. METHODS: We used specific DNA segments extracted from BAC clones, and we performed nick translation to label the segment with fluorescence labeled dye. The second method involved the use of specific primers for the centromere of certain chromosomes and using PCR technique for amplification and labeling. The probes were tested on metaphase and interphase cells derived from cultured human peripheral blood samples. We followed standard guidelines to test the adequacy of probe slide hybridization, proper probe localization, probe sensitivity and specificity, probe reproducibility, cut-off values, and overall probe validation. RESULTS: In this research, we presented the generation of three dual-color probes, each probe has a control locus. We offered three dual-color probes targeted 9p21, Xp21 and 17p13.1 loci. chromosome 9p21probe for diagnosis of structural abnormalities in chromosome 9, the Xp21 to test for structural abnormalities of chromosome X, and the 17p13.1 for TP53 gene to detect the loss of p53. We also produced probes for Down syndrome specific region, Rb gene and centromeres for chromosomes X, 17, and 18. CONCLUSION: The produced probes are specific and sensitive and can be produced at the commercial level in the laboratory. The production of FISH probes in Egypt can be used as a powerful diagnostic marker for genetic disorders and cancers and our work can be consider as a base to start national project to produce our needs of FISH probes.
In vitro evaluation of antioxidant, biochemical and antimicrobial properties of biosynthesized silver nanoparticles against multidrug-resistant bacterial pathogens Wael Mahmoud, Ahmed M. Elazzazy, Enas N. Danial Biotechnology and Biotechnological Equipment, 2017 Interest in nanoparticles has increased rapidly over the last few years, becoming one of the most compelling scopes of research fields. The microbial methods utilized for biomediated nanoparticle synthesis are the most favourable and well-established substitute for the classic chemical and physical methods. In this study, silver nanoparticles (AgNPs) were biosynthesized by reducing Ag+ ions using a cell-free supernatant derived from Bacillus aerius culture and silver nitrate (AgNO3) solution as a precursor. The reaction mixture exhibited a colour change from yellow to brown, and ultraviolet-visible spectroscopy showed a surface plasmon resonance peak at 420 nm. The nanoparticles were monodispersed and spherical with an average particle size of 20.12–29.48 nm as determined by transmission electron microscopy. The Fourier transform infrared spectrum revealed the capping of AgNPs with biomolecular compounds that were responsible for the reduction of AgNO3 to AgNPs. The biosynthesized AgNPs had powerful and potent antibacterial activity against many multidrug-resistant bacterial pathogens. Moreover, the in vitro dose-dependent antioxidant activity of the aqueous extract of AgNP components showed good antioxidant activity as compared to the standard antioxidant ascorbic acid. These outcomes support the advantages of green techniques for synthesizing AgNPs that can be utilized effectively in the production of potential antioxidant and antibacterial AgNPs for commercial application.
The impact of silver nanoparticles produced by Bacillus pumilus as antimicrobial and nematicide Wael M. Mahmoud, Tamer S. Abdelmoneim, Ahmed M. Elazzazy Frontiers in Microbiology, 2016 This study evaluates the potential application of silver nanoparticles (AgNPs) as antimicrobial or nematicidal agents produced by the extremophile Bacillus pumilus, which was isolated from the alkaline Wadi El-Natrun Lake in Egypt. The AgNPs were characterized by ultraviolet–visible absorption spectroscopy, transmission electron microscopy, and energy dispersive x-ray spectroscopy. The size of AgNPs formed ranged from 20.12 to 29.48 nm. Panagrellus redivivus was exposed to different concentrations (0, 50, 100, 150, and 200 µg/mL) of AgNPs in a 5 mL nematode suspension (1×103 mL-1). The best result occurred at AgNP concentrations of 150 and 200 µg/mL, with death rates of 80% and 91%, respectively, following 48 h of exposure. AgNPs also exhibited potent antimicrobial properties when using Gram-negative and Gram-positive human pathogens, with MIC and MBC values of 5 μg/mL and 10 μg/mL, respectively. These laboratory assays prove that biologically synthesized AgNPs are an ecofriendly material that can be used in lieu of solvents or toxic chemicals.
Sex chromosome mosaicism in the gonads of DSD patients: A karyotype/ phenotype correlation Alaa K. Kamel, Hoda M. Abd El-Ghany, Mona K. Mekkawy, Manal M. Makhlouf, Inas M. Mazen, Nabil El Dessouky, Wael Mahmoud, Shereen A. Abd El Kader Sexual Development, 2015 Sex chromosome mosaicism results in a large clinical spectrum of disorders of sexual development (DSD). The percentage of 45,X cells in the developing gonad plays a major role in sex determination. However, few reports on the gonadal mosaic status have been published, and the phenotype is usually correlated with peripheral lymphocyte karyotypes, which makes the phenotype prediction imprecise. This study was conducted on 7 Egyptian DSD patients to demonstrate the effect of sex chromosome constitution of both blood lymphocytes and gonadal tissues on the phenotypic manifestations. Conventional cytogenetic and FISH analyses of blood lymphocytes were conducted, and laparoscopy with gonadal biopsy was performed for histopathologic examination and FISH analysis. Gonosomal mosaicism was detected in 3 patients who had a non-mosaic chromosome pattern in blood lymphocytes. Two patients showed the same type of sex chromosome mosaicism in both the blood and gonadal tissues but with different distributions. Two other patients revealed a non-mosaic pattern in both tissues. The present study elucidates the importance of examining sex chromosome mosaicism in gonadal tissues of DSD patients and highlights the critical role of 45,X mosaicism which can lead to serious effects during early gonadal organogenesis.
Methylenetetrahydrofolate reductase gene polymorphisms in Egyptian Turner Syndrome patients Manal F. Ismail, Waheba A. Zarouk, Mona O. Ruby, Wael M. Mahmoud, Randa S. Gad Acta Biochimica Polonica, 2015 BACKGROUND: Folate metabolism dysfunctions can result in DNA hypomethylation and abnormal chromosome segregation. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) encoding gene (C677T and A1298C) reduce MTHFR activity, but when associated with aneuploidy, the results are conflicting. Turner Syndrome (TS) is an interesting model for investigating the association between MTHFR gene polymorphisms and nondisjunction because of the high frequency of chromosomal mosaicism in this syndrome. OBJECTIVE: To investigate the association of MTHFR gene C677T and A1298C polymorphisms in TS patients and their mothers and to correlate these polymorphisms with maternal risk of TS offspring. SUBJECTS AND METHODS: MTHFR C677T and A1298C polymorphisms were genotyped in 33 TS patients, their mothers and 15 healthy females with their mothers as controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing technique. RESULTS: Genotype and allele frequencies of both C677T and A1298C were not significantly different between TS cases and controls. There were no significant differences in C677T genotype distribution between the TS mothers and controls (p=1). The MTHFR 1298AA and 1298AC genotypes were significantly increased in TS mothers Vs. control mothers (p=0.002). The C allele frequency of the A1298C polymorphism was significantly different between the TS mothers and controls (p=0.02). The association of A1298C gene polymorphism in TS patients was found to increase with increasing age of both mothers (p=0.026) and fathers (p=0.044) of TS cases. CONCLUSION: Our findings suggest a strong association between maternal MTHFR A1298C and risk of TS in Egypt.
Effect of biosynthesized silver nanoparticles on physiological parameters of vicia faba infected by bean yellow mosaic virus Journal of Pure and Applied Microbiology, 2014
Novel mutations in the displacement loop of mitochondrial DNA are associated with acute lymphoblastic leukemia: A genetic sequencing study Haitham Ahmed Yacoub, Wael Mahmoud Mahmoud, Hatim Alaa El-Din El-Baz, Ola Mohamed Eid, Refaat Ibrahim ELfayoumi, Salem Mohamed Elhamidy, Maged M. Mahmoud Asian Pacific Journal of Cancer Prevention, 2014 BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and represents approximately 25% of cancer diagnoses among those younger than 15 years of age. MATERIALS AND METHODS: This study investigated alterations in the displacement loop (d-loop) region of mitochondrial DNA (mtDNA) as a risk factor and diagnostic biomarker for early detection and diagnosis of acute lymphoblastic leukemia. Using mtDNA from 23 subjects diagnosed with acute lymphoblastic leukemia, the first 450 bp of the d-loop region were amplified and successfully sequenced. RESULTS: This revealed 132 mutations at 25 positions in this region, with a mean of 6 alterations per subject. The d-loop alterations in mtDNA in subjects were all identified as single nucleotide polymorphisms in a homoplasmic distribution pattern. Mutant alleles were observed in all subjects with individual frequency rates of up to 95%. Thirteen mutant alleles in the d-loop region of mtDNA occurred with a high frequency. Novel alleles and locations were also identified in the d-loop of mtDNA as follows: 89 G insertions (40%), 95 G insertions (13%), 182 C/T substitutions (5%), 308 C insertions (19%), and 311 C insertions (80%). The findings of this study need to be replicated to be confirmed. CONCLUSIONS: Further investigation of the relationship between mutations in mitochondrial d-loop genes and incidence of acute lymphoblastic leukemia is recommended.
New haplotypes of the ATP synthase subunit 6 gene of mitochondrial DNA are associated with acute lymphoblastic leukemia in Saudi Arabia Haitham Ahmed Yacoub, Wael Mahmoud Mahmoud, Hatim Alaa-Eldeen El-Din El-Baz, Ola Mohamed Eid, Refaat Ibrahim El-Fayoumi, Maged Mostafa Mahmoud, Steve Harakeh, Osama H.A. Abuzinadah Asian Pacific Journal of Cancer Prevention, 2014 BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and represents approximately 25% of cancer diagnoses among those younger than 15 years of age. AIM AND OBJECTIVES: This study investigated substitutions in the ATP synthase subunit 6 gene of mitochondrial DNA (mtDNA) as a potential diagnostic biomarker for early detection and diagnosis of acute lymphoblastic leukemia. Based on mtDNA from 23 subjects diagnosed with acute lymphoblastic leukemia, approximately 465 bp of the ATP synthase subunit 6 gene were amplified and sequenced. RESULTS: The sequencing revealed thirty-one mutations at 14 locations in ATP synthase subunit 6 of mtDNA in the ALL subjects. All were identified as single nucleotide polymorphisms (SNPs) with a homoplasmic pattern. The mutations were distributed between males and females. Novel haplotypes were identified in this investigation: haplotype (G) was recorded in 34% in diagnosed subjects; the second haplotype was (C) with frequency of 13% in ALL subjects. Neither of these were observed in control samples. CONCLUSIONS: These haplotypes were identified for the first time in acute lymphoblastic leukemia patients. Five mutations able to change amino acid synthesis for the ATP synthase subunit 6 were associated with acute lymphoblastic leukemia. This investigation could be used to provide an overview of incidence frequency of acute lyphoblastic leukemia (ALL) in Saudi patients based on molecular events.
