Detection of AZFc gene deletion in a cohort of Egyptian patients with idiopathic male infertility.

BACKGROUND: The deletions of azoospermic factor regions (AZF) are considered risk factor of spermatogenic failure. AZF duplications or complex copy number variants (CNVs) were rarely studied because STS-PCR could not always detect these changes. The application of multiplex ligation-dependent probe amplification (MLPA) as a valuable test for detection of the deletion and or duplication was introduced to investigate the AZF sub-region CNVs. The MLPA technique is still not applied on a large scale, and the publications in this area of research are limited. The aim of this work was to evaluate the efficacy of MLPA assay to detect AZF-linked CNVs in idiopathic spermatogenic failure patients and to evaluate its importance as a prognostic marker in the reproduction outcome. RESULTS: Forty infertile men (37 with azoospermia and 3 with severe oligozoospermia) and 20 normal fertile men were subjected to thorough clinical, pathological, and laboratory assessment, chromosomal study, MLPA, STS-PCR assays, histopathology study, and testicular sperm retrieval (TESE). Out of the 40 patients, 7 patients have shown CNV in the AZFc region, 6 patients have partial deletion, and one patient has partial duplication. Only one of the normal control has AZFc duplication. STS-PCR was able to detect the deletion in only 4 out of the 7 positive patients and none of the control. CONCLUSION: We concluded that MLPA should be applied on a larger scale for the detection of Y chromosome microdeletion as a rapid, efficient, and cheap test.

Association of Exosomal miR-34a with Markers of Dyslipidemia and Endothelial Dysfunction in Children and Adolescents with T1DM

Objective: Dyslipidemia and endothelial dysfunction are common disorders and major causative factors for atherosclerosis in patients with type 1 diabetes mellitus (T1DM). However, their pathophysiology in young patients with T1DM is still under evaluated. We aimed, for the first time, to assess the expression of exosomal micro-RNA 34a (miR-34a) in serum of children and adolescents with T1DM and correlate this expression with markers of dyslipidemia and endothelial dysfunction. Methods: The study included 120 T1DM patients and 100 control subjects. Assessment of miR-34a was performed using quantitative real-time polymerase chain reaction. Lipid profile was assessed on an automated analyzer and serum endoglin and intracellular adhesion molecule (ICAM) concentrations were measured using immunometric methods. Results: Relative expression of miR-34a and serum endoglin and ICAM concentrations were higher in patients than controls (p=0.001) and in patients with dyslipidemia (42 patients) compared to patients without dyslipidemia (78 patients) (p=0.01). Linear regression analysis revealed a strong independent association between exosomal miR-34a expression and total cholesterol, low-density lipoprotein, serum endoglin and serum ICAM after adjustment for other cofactors. The utility of miR-34a as an indicator for associated dyslipidemia was tested using receiver operator characteristic curve analysis which revealed area under the curve: 0.73 with confidence interval: 0.63-0.83 and p=0.001. Conclusion: This was the first study to show the altered expression of exosomal miR-34a among children and adolescents with T1DM. Moreover, association of miR-34a with markers of dyslipidemia and endothelial dysfunction was identified, suggesting that it could play a role in regulation of lipid metabolism and endothelial function in T1DM.

Expression of miRNAs-122, -192 and -499 in end stage renal disease associated with acute myocardial infarction.

INTRODUCTION: New diagnostic tools are needed to accurately detect acute myocardial infarction (AMI) in patients with end stage renal disease (ESRD) presenting with ischemic chest pain. We aimed in this study to investigate circulating miR-122, -192 and -499 expression levels in patients with AMI on top of ESRD and evaluate the potential of these miRNAs as blood-based biomarkers for AMI in patients with ESRD. MATERIAL AND METHODS: The study included 80 ESRD patients without AMI, 80 patients with ESRD associated with AMI and 60 healthy subjects. Assessment of microRNAs was done using SYBR Green based real-time PCR. RESULTS: Levels of miR-122 were 28-fold and 20-fold higher in controls than in ESRD patients with or without AMI respectively (p < 0.001), while no differences were detected between the two patient groups (p = 0.9). Levels of miR-192 showed a marked increase in ESRD patients with and without AMI compared to the control group (> 500-fold, > 8000-fold respectively, p ≤ 0.001). Patients who developed AMI had lower expression than ESRD patients without AMI (p < 0.001). Non-significant miR-499 elevation was found in ESRD patients without cardiac disease compared to the control group, while highly significant elevation of miR- 499 was demonstrated in ESRD patients who developed AMI compared to other ESRD patients and the control group (> 100-fold, > 350-fold respectively, p = 0.001). CONCLUSIONS: Altered expression of miR-122 and -192 may contribute in pathogenesis of ESRD. MiR-192 and -499 may serve as potential biomarkers for AMI in ESRD. Further studies are needed to correlate these miRNAs with disease progression and outcome.

Micro-RNA 196a2 expression and miR-196a2 (rs11614913) polymorphism in T1DM: a pilot study.

Background Recent emerging evidence supports the role of miR-196a2 in various human diseases. However, its role in type 1 diabetes mellitus (T1DM) is still underestimated. We aimed, for the first time, to investigate the expression of miR-196a2 in T1DM and the association of miR-196a2 (rs11614913) polymorphism with susceptibility of T1DM in a sample of patients from Cairo, Egypt. Methods The study included 150 patients and 150 healthy subjects. Evaluation of rs11614913 genotypes and miR-196a2 expression was done using the allelic discrimination and quantitative reverse transcriptase polymerase chain reaction (PCR) method, respectively. Results The Hardy-Weinberg equilibrium of single nucleotide polymorphism(SNP) was detected among controls (p = 0.2). Our results revealed that the TT genotype was more frequent in patients (22.6%) than controls (10%) while the CC genotype was more frequent in controls (47.3%) than patients (39.3%) (p = 0.01). The frequency of the T allele was significantly higher in patients than in controls (41.7 vs. 31.3%), while the C allele was more frequent in controls (p = 0.008). After adjustment for traditional risk factors, the association of the TT genotype with T1DM remained significant (TT vs. CC, odds ration [OR] = 3.2, 95% confidence interval [CI]: 1.4-7.4, p = 0.005). Power analysis of the data yielded a statistical power of 80% for the miR-196a2 rs11614913 with T1DM. Relative expression of miR-196a2 showed significant decrease in patients compared to controls (median = 0.09, 0.5, interquartile range [IQR] = 0.03-1.6, 0.1-2.1). However, miR-196a2 expression showed no significant difference between different rs11614913 genotypes (p = 0.5). Conclusions Our findings demonstrated that miR-196a rs11614913 is associated with T1DM and decreased expression of miR-196a2 may play a role in pathogenesis of T1DM.

Altered expression of WFS1 and NOTCH2 genes associated with diabetic nephropathy in T2DM patients.

AIM: The increased incidence of type 2 diabetes mellitus (T2DM) and the importance of early identification and management of its complications, especially diabetic nephropathy (DN), have spotted the light on genetic factors that increase risk of T2DM and its related nephropathy. The present study aimed at investigating expression of (KCNJ11, ABCC8, JAZF1, WFS1, PPARG, NOTCH2 and EXOSC4) genes in peripheral blood of T2DM patients. METHOD: The study included 30 non-complicated T2DM patients, 30 patients with DN and 40 healthy controls. Quantitative Real Time PCR Array was used to study gene expression. RESULTS: NOTCH2 showed higher expression while KCNJ11, JAZF1, WFS1 and PPARG genes showed lower expression in DN patients compared to non-complicated patients. KCNJ11, JAZF1, WFS1, PPARG, and EXOSC4 expression showed significant negative correlation with microalbumin, while NOTCH2 expression was significantly positively correlated with microalbumin. AS regard HbA1c and studied genes expression, there was significant negative correlation between WFS1 expression and HbA1c, while NOTCH2, KCNJ11, JAZF1, PPARG, EXOSC4 expression didn't show significant correlation with HbA1c. Risk ratio of studied genes expression showed that WFS1 and NOTCH2 had highest risk ratio (30) and highest sensitivity and specificity, in relation to DN and they were the best predictors in the group of studied genes at cut off value of ≤0.861 for WFS1 and ≥0.678 for NOTCH2. CONCLUSION: Altered expression of WFS1 and NOTCH2 genes may play a role in pathogenesis and development of DN in patients with T2DM. These results may contribute in early identification and management of DN.

Novel adipokines vaspin and irisin as risk biomarkers for cardiovascular diseases in type 2 diabetes mellitus.

AIMS: Vaspin and irisin are novel cytokines proposed as potential new biomarkers of insulin resistance and endothelial dysfunction. This work is to investigate circulating levels of vaspin and irisin in patients with type 2 diabetes mellitus (T2DM) with and without cardiovascular disease (CVD) to study potential association with disease risk. MATERIALS AND METHODS: Circulating levels of vaspin and irisin were assayed in serum from 135 T2DM patients (with and without CVD) and 70 control subjects by ELISA. RESULTS: Vaspin levels were significantly higher in T2DM patients than in control subjects (6798 ±â€¯3540 pg/ml vs. 3215 ±â€¯3209 pg/ml, p = 0.001) and in CVD patients than in non-CVD patients (7417.3 ±â€¯3507.6 pg/ml vs. 6017.3 ±â€¯3606.4 pg/ml, p = 0.001), with significant positive correlations with BMI, FPG, serum insulin and HOMA-IR. Irisin levels were significantly lower in T2DM patients than in controls (71.15 ±â€¯67.57 ng/ml vs.127 ±â€¯71.57 ng/ml, p = 0.004), and in CVD patients than in non-CVD patients (55.77 ±â€¯54.82 ng/ml vs. 115.5 ±â€¯67 ng/ml, p = 0.003), with significant correlations with HbA1c, HOMA-IR and BMI in diabetic patients, and with HbA1c and TG in CVD patients. Elevated levels of vaspin was associated with 1.7 times increased CVD risk (p = 0.001, OR = 1.7, 95%CI = 1.21-2.39), while lower levels of irisin associated with 1.6 times increased CVD risk (p = 0.007, OR = 1.6, 95%CI = 1.45-2.28). ROC analysis indicated serum vaspin and irisin as independent CVD risk biomarkers with sensitivity, 94% and 73.7%, and specificity, 74% and 74.1%; respectively. CONCLUSION: Our results indicate that circulating vaspin and irisin are potential new independent CVD risk biomarkers in T2DM.

Serum adipokines and vitamin D levels in patients with type 1 diabetes mellitus.

INTRODUCTION: Adiponectin, leptin and resistin are adipokines that play important roles in the regulation of lipid and carbohydrate metabolism in type 2 diabetes (T2DM). However, their influence in type 1 diabetes mellitus is still unknown. The aim of this study was to measure serum adiponectin, leptin and resistin levels and to investigate their relationships with vitamin D and other clinical and laboratory parameters in patients with type 1 diabetes. MATERIAL AND METHODS: Fifty subjects with type 1 diabetes and 50 healthy age- and sex-matched subjects were selected from the Endocrinology Outpatient Clinic of Cairo University Pediatrics Hospital. Enzyme-linked immunosorbent assay was used to measure the levels of leptin, adiponectin and resistin. Vitamin D levels were measured using electro-chemiluminescence immunoassay. RESULTS: There were no significant differences in adiponectin and leptin levels between diabetic and control subjects (p = 0.6 and p = 0.5 respectively). Resistin levels were significantly higher in the diabetic group compared to controls (p < 0.001) and in postpubertal patients compared to prepubertal patients (p < 0.04). Serum resistin in type 1 diabetes showed a negative correlation with vitamin D (p < 0.001) and a positive correlation with glycated hemoglobin (HbA1c) (p = 0.006), while other adipokines were not interrelated. CONCLUSIONS: These results strongly support a role of resistin and vitamin D deficiency in the pathophysiology of type 1 diabetes. Vitamin D may be involved in resistin regulation through an unknown mechanism. Further studies are recommended to understand resistin regulation in type 1 diabetes.