Manal S. Fawzy

Rapid Detection of β-Thalassemia Alleles in Egypt Using Naturally or Amplified Created Restriction Sites and Direct Sequencing: A Step in Disease Control

β-Thalassemia (thal), the most common genetic disorder in Egypt, is a major health problem with an estimated carrier rate of 9–10%. This study, aimed at describing the β-globin gene mutations in the Suez Canal area, an important Egyptian region, to provide a foundation for a disease control program. We studied 44 β-thalassemic patients (and their relatives) from 35 families living in this region. The commonest mutations were genetically diagnosed using naturally or amplified created restriction sites. Less frequent mutations were characterized by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. Twelve different mutations were identified in 51 unrelated chromosomes. The three most frequent mutations were IVS-I-110 (G→A), IVS-I-1 (G→A) and IVS-I-6 (T→C). The spectrum of rarer mutations was heterogeneous and differed from that reported in other areas of Egypt. We also identified the first homozygous case of a rare mutation, codon 24 (−G; +CAC), displaying a thalassemia major phenotype. Parental consanguinity was high (60.6%) with 35.7% of the compound heterozygous patients having consanguineous parents. These data provide insights for the distribution of β-thal alleles in this region, and could be used as a basis for genetic counseling and prenatal diagnosis.

MicroRNA-196a2 Biomarker and Targetome Network Analysis in Solid Tumors.

IntroductionMicroRNAs (miRNAs) have been linked to cancer development and progression. The molecular mechanisms underlying the genetic associations of the miRNA single nucleotide polymorphism with cancer vary by cancer site. As there are no previous studies on the miR-196a2 variant or expression in any type of cancer among our population, we aimed to determine the expression profile of mature miR-196a2 in various types of solid tumors and to analyze the impact of its polymorphism (rs11614913; C/T) on the expression levels.Materials and MethodsThe study included 230 cancer patients (including 17 types of cancer), 26 patients with pre-cancer lesions, and 100 unrelated controls. Archived formalin-fixed, paraffin-embedded specimens (n = 197) were available for both miRNA expression analysis and single nucleotide polymorphism identification. Venous blood was collected from 59 histologically confirmed sporadic cancer patients and the study controls for single nucleotide polymorphism identification. Real-time polymerase chain reaction analysis was performed for allelic discrimination and relative quantification of miR-196a2 in the study samples. In silico target gene prediction and network analysis was performed.ResultsWe found that individuals with the T variant were associated with cancer risk under all genetic association models, especially in colorectal, esophageal, skin, lung, thyroid, and renal cancer. Overall and stratified analysis showed miR-196a2 over-expression in most of the current malignant tumor samples relative to their corresponding cancer-free tissues. Carriers of the C allele had significantly higher expression levels of miR-196a2. Correlation with the clinicopathological features of cancer showed organ-specific effects. Gene enrichment analysis of predicted and validated targets speculated the putative role of miR-196a2 in cancer-associated biology.ConclusionsWe highlighted cancer-type specific expression profiles of miR-196a2, which was correlated with the clinicopathological features in various types of cancer. Taken together, our results suggest that the miRNA signature could have promising diagnostic and prognostic significance.

Plasma thrombin-activatable fi brinolysis inhibitor levels and Thr325Ile polymorphism as a risk marker of myocardial infarction in Egyptian patients

Objective The objective of this study was to investigate whether thrombin activatable fibrinolytic inhibitor (TAFI) Thr325Ile polymorphism and TAFI antigen (Ag) levels could constitute a risk marker of myocardial infarction (MI) in Egyptian patients. Study population and results The study included forty-six patients with acute MI (mean age 55.7 ± 8.1 years, 33 men, 13 women) compared with age and sex-matched healthy volunteers (n = 54) as a control group. Clinical examination, laboratory investigations, electrocardiography (ECG) and/or echocardiography were done. TAFI Thr325Ile (reference sequence: rs1926447) polymorphism was genotyped in both studied groups using TaqMan SNP (single nucleotide polymorphism) genotyping assay. The genotypes of the high-risk allele [Thr/Ile (CT) and Ile/Ile (TT)] were significantly more frequent in patients compared with the control group (54.4% and 32.6% vs. 51.8% and 5.6%, respectively) and were also associated with an increased risk of MI [OR = 4.95, (95% CI: 1.80 - 13.63); P= 0.0001]. Ile325 allele carriers were more frequent in cases than in control subjects (60.0% vs. 31.5%) [OR = 3.26, (95% CI = 1.82 - 5.83), P= 0.001]. The Thr325Ile SNP significantly correlated with TAFI antigen levels with the C/C genotype corresponding with the highest and the T/T genotype with the lowest TAFI antigen levels (P< 0.001). No statistically significant relation was found between TAFI Thr325Ile polymorphism and either the type or the site of MI. Conclusions TAFI Thr325Ile and its respective plasma protein level could have a contribution to MI risk in the Egyptian population. This could be helpful in refining a risk profile for coronary heart disease (CHD) patients.

Association of MicroRNA-196a2 Variant with Response to Short-Acting β2-Agonist in COPD: An Egyptian Pilot Study

Chronic obstructive pulmonary disease (COPD) is a multifactorial chronic respiratory disease, characterized by an obstructive pattern. Understanding the genetic predisposition of COPD is essential to develop personalized treatment regimens. MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that modulate the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules. Emerging evidences demonstrated the potential use of miRNAs as a disease biomarker. This pilot study aimed to investigate the association of the MIR-196a2 rs11614913 (C/T) polymorphism with COPD susceptibility, the clinical outcome and bronchodilator response to short-acting β2-agonist. Genotyping of rs11614913 polymorphism was determined in 108 COPD male patients and 116 unrelated controls using real-time polymerase chain reaction technology. In silico target prediction and network core analysis were performed. COPD patients did not show significant differences in the genotype distribution (p = 0.415) and allele frequencies (p = 0.306) of the studied miRNA when compared with controls. There were also no associations with GOLD stage, dyspnea grade, disease exacerbations, COPD assessment test for estimating impact on health status score, or the frequency of intensive care unit admission. However, COPD patients with CC genotype corresponded to the smallest bronchodilator response after Salbutamol inhalation, the heterozygotes (CT) had an intermediate response, while those with the TT genotype showed the highest response (p < 0.001). In conclusion MIR-196a2 rs11614913 polymorphism is associated with the bronchodilator response of COPD in our sample of the Egyptian population, generating hypothesis of the potential use of MIR-196a2 variant as a pharmacogenetic marker for COPD.

Atherosclerotic and thrombotic genetic and environmental determinants in Egyptian coronary artery disease patients: a pilot study

BackgroundCoronary artery disease (CAD) is the leading cause of morbidity and mortality worldwide. Multiple genetic variants in combination with various environmental risk factors have been implicated. This study aimed to investigate the association of twelve thrombotic and atherosclerotic gene variants in combination with other environmental risk factors with CAD risk in a preliminary sample of Egyptian CAD patients.MethodsTwenty three consecutive CAD patients undergoing diagnostic coronary angiography and 34 unrelated controls, have been enrolled in the study. Genotyping was based on polymerase chain reaction and reverse multiplex hybridization. Five genetic association models were tested. Data distribution and variance homogeneity have been checked by Shapiro-Wilk test and Levene test, respectively; then the appropriate comparison test was applied. Spearman’s rank correlation coefficient was used for correlation analysis and logistic regression has been performed to adjust for significant risk factors. Clustering the study participants according to gene-gene and gene-environment interaction has been done by Detrended Correspondence Analysis (DCA).ResultsThe univariate analysis indicated that the five variants; rs1800595 (FVR2; factor 5), rs1801133 (MTHFR; 5,10-methylenetetrahydrofolate reductase), rs5918 (HPA-1; human platelet antigen 1), rs1799752 (ACE; angiotensin-converting enzyme), and rs7412 and rs429358 (ApoE; apolipoprotein E) were significantly associated with CAD susceptibility under different genetic models. Multivariate analysis revealed clustering of the study population into three patient groups (P) and one control group. FVR2 was the most variant associated with CAD patients, combined with the factor V Leiden (FVL) variant in P1 cluster and with both ACE and MTHFR 667C > T in P2. Whereas, P3 was mostly affected by both MTHFR 667C > T and FXIII (factor 13) V89L mutations. When combined with traditional risk factors, P1 was mostly affected by dyslipidemia, smoking and hypertension, while P2 was mostly affected by their fasting blood sugar levels and ApoE variant.ConclusionsTaken together, these preliminary results could have predictive value to be applied in refining a risk profile for our CAD patients, in order to implement early preventive interventions including specific antithrombotic therapy. Further large scale and follow-up studies are highly recommended to confirm the study findings.

Association of MIR-499a expression and seed region variant (rs3746444) with cardiovascular disease in Egyptian patients

Abstract Background: Circulating microRNAs could be powerful markers of acute myocardial infarction (MI) and its functional genetic variants could increase susceptibility to cardiovascular disease (CVD). The current study aimed to quantify the microRNA (miR)-499a levels in serum of MI patients compared to hypertensive and healthy subjects and to investigate the association of its A/G variant rs3746444 with CVD in a sample of an Egyptian population. Methods: Serum miR-499a relative expressions were measured in 110 acute MI patients, 76 hypertensive patients, and 121 healthy controls by Real-time quantitative polymerase chain reaction. MIR-499a genotyping was performed for an additional 107 coronary artery disease patients by Real-time allele discrimination assay. Results: Acute MI patients showed high relative expression of miR-499a (> 105-fold, p < .001), and it was nearly undetectable in healthy controls and hypertensive patients. It showed an area under the curve of 0.953, with a sensitivity of 97.2% and a specificity of 75.0%. ST-elevation MI (STEMI) patients had higher miR-499a serum levels than patients with Non-STEMI. There was a significant association of MIR-499a variant with acute MI but not with hypertension under all genetic models tested. As a new finding, in overall and stratified analysis, the miR-499a variant was not correlated with its expression profile. Conclusions: Circulating miR-499a levels could be a useful biomarker, discriminating acute MI within 12 hours from healthy subjects. Its variant rs3746444 A/G is associated with increased susceptibility to acute MI and CAD in Egyptian population.

Evaluation of miRNA-196a2 and apoptosis-related target genes: ANXA1, DFFA and PDCD4 expression in gastrointestinal cancer patients: A pilot study

Previous reports have suggested the significant association of miRNAs aberrant expression with tumor initiation, progression and metastasis in cancer, including gastrointestinal (GI) cancers. The current preliminary study aimed to evaluate the relative expression levels of miR-196a2 and three of its selected apoptosis-related targets; ANXA1, DFFA and PDCD4 in a sample of GI cancer patients. Quantitative real-time PCR for miR-196a2 and its selected mRNA targets, as well as immunohistochemical assay for annexin A1 protein expression were detected in 58 tissues with different GI cancer samples. In addition, correlation with the clinicopathological features and in silico network analysis of the selected molecular markers were analyzed. Stratified analyses by cancer site revealed elevated levels of miR-196a2 and low expression of the selected target genes. Annexin protein expression was positively correlated with its gene expression profile. In colorectal cancer, miR-196a over-expression was negatively correlated with annexin A1 protein expression (r = -0.738, p < 0.001), and both were indicators of unfavorable prognosis in terms of poor differentiation, larger tumor size, and advanced clinical stage. Taken together, aberrant expression of miR-196a2 and the selected apoptosis-related biomarkers might be involved in GI cancer development and progression and could have potential diagnostic and prognostic roles in these types of cancer; particularly colorectal cancer, provided the results experimentally validated and confirmed in larger multi-center studies.

Mobile phones electromagnetic radiation and NAD+-dependent isocitrate dehydrogenase as a mitochondrial marker in asthenozoospermia

NAD+-dependent Isocitrate Dehydrogenase (NAD+-IDH) could be one of the cell phone radiation targets. Enzyme activity alteration may lead to decline in sperm motility during radio-frequency electromagnetic waves (RF-EMW) exposure. The current case control study aimed to investigate the possible relationship between mitochondrial NAD+-IDH activity in human seminal plasma and sperm motility among asthenozoospermic cellular phone users. A total number of ninety idiopathic infertile males referred from the Department of Dermatology and Andrology, were enrolled in this study. NAD+-IDH activity was measured in human seminal plasma by spectrophotometer. Computer-aided sperm analysis (CASA) following WHO criteria has been used for semen analyses. The results showed that IDH activity was increased in patients with prolonged cell phone daily use ≥4 h/day. Its level, correlated negatively with either the motility ratio percentages (r = −0.46, p < 0.001) or the progressive motility percentages (r = −0.50, p < 0.001) in the study groups. The current study suggests that NAD+-IDH in human seminal plasma could be one of seminal plasma biomarkers reflecting the mitochondrial function of spermatozoa. Alteration of its level could reflect the defective motility of sperms among some cases of cellular phone users.

A passenger strand variant in miR-196a2 contributes to asthma severity in children and adolescents: A preliminary study

There is emerging evidence to support the role of microRNAs in allergic airway diseases and inflammation. Genetic variants in microRNA genes might affect microRNA-mediated cell regulation. This preliminary study was designed to investigate the association of the microRNA-196a2 rs11614913 (C/T) polymorphism with susceptibility to asthma and clinical outcomes in children and adolescents. Genotyping of rs11614913 polymorphism was determined in 96 patients with bronchial asthma (6-18 years of age) and 96 unrelated controls, using real-time polymerase chain reaction technology. In-silico target prediction and network core analyses were performed. The asthmatics did not show significant differences in genotype distribution (p = 0.609) and allele frequencies (p = 0.428) compared with the controls. There were also no associations with disease duration, age at onset, asthma phenotype, asthma control, therapeutic level, airway hyper-responsiveness, or biochemical parameters in the blood. However, the CC genotype was associated with a more severe degree of asthma (p = 0. 023) and higher frequency of nocturnal asthma (p = 0.002). Carriers for CC were 17 times more likely to develop nocturnal asthma, and had a more than 2.5-fold increased risk for poor disease outcome compared with CT and TT individuals. In conclusion, microRNA-196a2 rs11614913 polymorphism might be associated with asthma severity in our sample of the Egyptian population. Further investigations in studies with a larger sample size and functional tests are needed to validate our findings and to explore the detailed biological mechanisms.

Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study

This study aimed to investigate thrombin-activatable fibrinolysis inhibitor (TAFI) Thr325Ile polymorphism and TAFI antigen (Ag) levels in breast cancer (BC) in the Egyptian population to clarify their role in relation to BC. A group of 300 females was recruited in this study; of these 150 unrelated patients with different stages of BC and 150 age-matched healthy controls. Plasma TAFI Ag was measured by ELISA and TAFI Thr325Ile (rs1926447) polymorphism was genotyped using TaqMan single nucleotide polymorphism (SNP) genotyping assay. The results showed the genotypes of the minor allele; Thr/Ile (CT) and Ile/Ile (TT) were significantly more frequent in patients compared to control group (50.0% and 22.0% vs. 42.0% and 13.3%, respectively) and were also associated with BC susceptibility [OR = 1.9 and 2.6; 95% CI: (1.1–3.3) and (1.3–5.5), respectively P = 0.01]. Ile325 allele carriers were more frequent in cases than in controls (47.0% vs. 34.0%) [OR = 1.7, (95% CI = 1.2–2.4), P = 0.001]. However, TAFI Thr325Ile polymorphism was not associated with BC stage or other clincopathological characteristics. TAFI Ag levels were correlated with advanced stages of BC, poor prognosis and risk of recurrence (P = 0.02, P = 0.04 and P < 0.001, respectively) and Thr325Ile SNP was significantly correlated with TAFI antigen levels with the C/C genotype corresponding to the highest and the T/T genotype to the lowest TAFI antigen levels (P < 0.001) in the study groups. In conclusion, this study showed for the first time that TAFI Thr325Ile polymorphism could have a contribution to BC susceptibility in our population. Furthermore, high TAFI plasma levels may serve as a predictor of poor prognosis in patients with BC.