Differential Expression of Serum Proinflammatory Cytokine TNF-α and Genetic Determinants of TNF-α, CYP2C19*17, miR-423 Genes and Their Effect on Coronary Artery Disease Predisposition and Progression.

Coronary artery disease (CAD) is the leading cause of death and hospitalization worldwide and represents a problem for public health systems everywhere. In Saudi Arabia, the prevalence of CAD is estimated to be 5.5%. Risk factors for CAD include older age, male gender, obesity, high blood pressure, smoking, diabetes, hyperlipidemia, and genetic factors. Reducing the risk factors in susceptible individuals will decrease the prevalence of CAD. Genome wide association studies have helped to reveal the association of many loci with diseases like CAD. In this study, we examined the link between single nucleotide variations (SNVs) of TNF-α-rs1800629 G>A, CYP2C19*17 (rs12248560) C>T, and miR-423 rs6505162 C>A and the expression of TNF-α with CAD. We used the mutation specific PCR, ARMS-PCR, and ELISA. The results showed that the A allele of the TNF-α rs1800629 G>A SNP is linked to CAD with odd ratio (OR) (95% CI) = 2.10, p-value = 0.0013. The T allele of the CYP2C19*17 (rs12248560) C>T is linked to CAD with OR (95% CI) = 2.02, p-value = 0.003. In addition, the A allele of the miR-423 rs6505162 C>A SNV is linked to CAD with OR (95% CI) = 1.49, p-value = 0.036. The ELISA results indicated that the TNF-α serum levels are significantly increased in CAD patients compared to healthy controls. We conclude the TNF-α rs1800629 G>A, CYP2C19*17, and miR-423 rs6505162 C>A are potential genetic loci for CAD in the Saudi population. These findings require further verification in future studies. After being verified, our results might be utilized in genetic testing to identify individuals that are susceptible to CAD and, therefore, for whom reducing modifiable risk factors (e.g., poor diet, diabetes, obesity, and smoking) would result in prevention or delay of CAD.

Genetic Determinants of Cardiovascular Disease: The Endothelial Nitric Oxide Synthase 3 (eNOS3), Krüppel-Like Factor-14 (KLF-14), Methylenetetrahydrofolate Reductase (MTHFR), MiRNAs27a and Their Association with the Predisposition and Susceptibility to Coronary Artery Disease.

Coronary artery disease (CAD) is an important cause of death worldwide. CAD is caused by genetic and other factors including hypertension, hyperlipidemia, obesity, stress, unhealthy diet, physical inactively, smoking and Type 2 diabetes (T2D). The genome wide association studies (GWASs) have revealed the association of many loci with risk to diseases such as cancers, T2D and CAD. Nitric oxide (NO) is a potent vasodilator and is required for normal vascular health. It is produced in the endothelial cells in a reaction catalyzed by the endothelial NO synthase (eNOS). Methylenetetrahydrofolate reductase (MTHFR) is a very important enzyme involved in metabolism of folate and homocysteine, and its reduced function leads to cardiovascular disease. The Krüppel-like factor-14 (KLF-14) is an important transcriptional regulator that has been implicated in metabolic syndrome. MicroRNA (MiRNAs) are short non-coding RNAs that regulate the gene expression of proteins involved in important physiological processes including cell cycle and metabolism. In the present study, we have investigated the potential impact of germline pathogenic variants of endothelial eNOS, KLF-14, MTHFR, MiRNA-27a and their association with risk to CAD in the Saudi population. Methods: Amplification Refractory Mutation System (ARMS) PCR was used to detect MTHFR, KLF-14, miRNA-27a and eNOS3 genotyping in CAD patients and healthy controls. About 125 CAD cases and 125 controls were enrolled in this study and statistical associations were calculated including p-value, risk ratio (RR), and odds ratio (OD). Results: There were statistically significant differences (p < 0.05) in genotype distributions of MTHFR 677 C>T, KLF-14 rs972283 G>A, miRNAs27a rs895819 A>G and eNOS3 rs1799983 G>T between CAD patients and controls. In addition, our results indicated that the MTHFR-TT genotype was associated with increased CAD susceptibility with an OR 2.75 (95%) and p < 0.049, and the KLF14-AA genotype was also associated with increased CAD susceptibility with an OR of 2.24 (95%) and p < 0.024. Moreover, the miRNAs27a-GG genotype protects from CAD risk with an OR = 0.31 (0.016), p = 0.016. Our results also indicated that eNOS3 -GT genotype is associated with CAD susceptibility with an OR = 2.65, and p < 0.0003. Conclusion: The MTHFR 677C>T, KLF14 rs972283 G>A, miRNAs27a A>G, and eNOS3 rs1799983 G>T genotypes were associated with CAD susceptibility (p < 0.05). These findings require verification in future large-scale population based studies before these loci are used for the prediction and identification of individuals at risk to CAD. Weight control, physical activity, and smoking cessation are very influential recommendations given by clinicians to the at risk individuals to reduce or delay the development of CAD.

Clinical Utility of Amplification Refractory Mutation System-Based PCR and Mutation-Specific PCR for Precise and Rapid Genotyping of Angiotensin-Converting Enzyme 1 (ACE1-rs4646996 D>I) and Angiotensin-Converting Enzyme 2 (ACE2-rs4240157T>C) Gene Variations in Coronary Artery Disease and Their Strong Association with Its Disease Susceptibility and Progression

Background: Experimental clinical and research studies demonstrated that the renin−angiotensin system (RAS) affects the pathogenesis of atherosclerosis and the prognosis of coronary heart disease (CHD). The results show that ACE2 (angiotensin I-converting enzyme 2) might act as a protective protein for cardiovascular diseases; however, only a few studies in human populations have been carried out. The aim of this study was to develop, optimize, and validate a direct T-ARMS-based PCR assay for the precise and rapid genotyping of ACE1-rs4646996 D>I and ACE2-rs4240157T>C and study their association with coronary artery disease susceptibility and progression. Methodology: This study included 149 consecutive coronary artery disease patients and 150 healthy controls. We utilized T-ARMS for the precise and rapid genotyping of ACE2-rs4240157; rs4646994. Results: Our results indicated that the ACE1-rs4646996 D>I genotypes observed between CAD cases and controls were statistically significant (p < 0.008) and, similarly, the ACE2-rs4240157T>C genotypes observed were significant (p < 0.0001). Moreover, the frequency of the D allele (ACE1-D>I) and C allele (ACE2-rs4240157T>C) was found to be higher among CAD patients than the HC. Our results indicated that in the codominant model, the ACE2-ID genotype was strongly associated with increased CAD susceptibility in a codominant model with an OR of 2.37, (95%) CI = (1.023−5.504), and p < 0.04. Similarly, the ACE2-DD genotype was strongly associated with an increased CAD susceptibility with an OR of 3.48, (95%) CI = (1.49 to 8.117), and p < 0.003. Similarly, in allelic comparison, the D allele was strongly associated with CAD susceptibility with an OR of 1.59, (95%) CI = (1.12−2.24), and p < 0.003. Our results revealed that there was a significant correlation between ACE2-I/D genotypes and hypertension, T2D, and obesity (p < 0.05). The results of ACE2 rs4240157 genotyping indicated a strong association in the codominant model with an increased CAD susceptibility with an OR of 3.62, (95%) CI = (2.027 to 6.481), and p < 0.0001. Similarly, in a dominant inheritance model, a strong association is observed between the ACE2 rs4240157 (CT+CC) genotype with an OR of 6.34, (95%) CI = (3.741 to 10.749), and p < 0.0001. In allelic comparison, the T allele was strongly associated with CAD susceptibility with an OR of 5.56, (95% CI = (3.56 to 7.17), and p < 0.0001. Similarly, our results revealed that there was a significant association of the ACE2-rs4240157T>C genotypes with Triglycerides (mg/dL), HDL-C (mg/dL), total Cholesterol (mg/dL), and C-reactive protein (mg/L) in CAD. Conclusion: It was indicated that the ARMS technique and MS-PCR assay proved to be fast, accurate, and reliable for ACE2-rs4240157T>C and ACE1-rs4646996 D>I, respectively, and can be used as a potential molecular tool in the diagnosis of genetic diseases in undeveloped and developing countries­where there might be a shortage of medical resources and supplies. ACE1-I>D genotypes were strongly associated with T2D, hypertension, and obesity (p < 0.002). Besides the ACE2-rs4240157 CT heterozygosity genotype, the T allele was strongly associated with CAD susceptibility. Future longitudinal studies in different ethnic populations with larger sample sizes are recommended to validate these findings

Clinical Implications of Krüpple-like Transcription Factor KLF-14 and Certain Micro-RNA (miR-27a, miR-196a2, miR-423) Gene Variations as a Risk Factor in the Genetic Predisposition to PCOS.

Polycystic ovary syndrome (PCOS) is a disorder with a symptomatic manifestation of an array of metabolic and endocrine impairments. PCOS has a relatively high prevalence rate among young women of reproductive age and is a risk factor for some severe metabolic diseases such as T2DM, insulin insensitivity, and obesity, while the most dominant endocrine malfunction is an excess of testosterone showing hyperandrogenism and hirsutism. MicroRNAs have been implicated as mediators of metabolic diseases including obesity and insulin resistance, as these can regulate multiple cellular pathways such as insulin signaling and adipogenesis. Genome-wide association studies during the last few years have also linked the Krüpple-like family of transcription factors such as KLF14, which contribute in mechanisms of mammalian gene regulation, with certain altered metabolic traits and risk of atherosclerosis and type-2 DM. This study has characterized the biochemical and endocrine parameters in PCOS patients with a comprehensive serum profiling in comparison to healthy controls and further examined the influence of allelic variations for miRNAs 27a (rs895819 A > G), 196a2 (rs11614913 C > T), 423 (rs6505162C > A), and transcription factor KLF14 (rs972283 A > G) gene polymorphism on the risk and susceptibility to PCOS. The experimental protocol included amplification refractory mutation-specific (ARMS)-PCR to detect and determine the presence of these polymorphic variants in the study subjects. The results in this case−control study showed that most of the serum biomarkers, both biochemical and endocrine, that were analyzed in the study demonstrated statistically significant alterations in PCOS patients, including lipids (LDL, HDL, cholesterol), T2DM markers (fasting glucose, free insulin, HOMA-IR), and hormones (FSH, LH, testosterone, and progesterone). The distribution of Krüppel-like factor 14 rs972283 G > A, miR-27a rs895819 A > G, and miR-196a-2 rs11614913 C > T genotypes analyzed within PCOS patients and healthy controls in the considered population was significant (p < 0.05), except for miR-423 rs6505162 C > A genotypes (p > 0.05). The study found that in the codominant model, KLF14-AA was strongly associated with greater PCOS susceptibility (OR 2.35, 95% CI = 1.128 to 4.893, p < 0.022), miR-27a-GA was linked to an enhanced PCOS susceptibility (OR 2.06, 95% CI = 1.165 to 3.650, p < 0.012), and miR-196a-CT was associated with higher PCOS susceptibility (OR 2.06, 95% CI = 1.191 to 3.58, p < 0.009). Moreover, allele A of KLF-14 and allele T of miR-196a2 were strongly associated with PCOS susceptibility in the considered population.

Diet-derived small molecules (nutraceuticals) inhibit cellular proliferation by interfering with key oncogenic pathways: an overview of experimental evidence in cancer chemoprevention.

Discouraging statistics of cancer disease has projected an increase in the global cancer burden from 19.3 to 28.4 million incidences annually within the next two decades. Currently, there has been a revival of interest in nutraceuticals with evidence of pharmacological properties against human diseases including cancer. Diet is an integral part of lifestyle, and it has been proposed that an estimated one-third of human cancers can be prevented through appropriate lifestyle modification including dietary habits; hence, it is considered significant to explore the pharmacological benefits of these agents, which are easily accessible and have higher safety index. Accordingly, an impressive embodiment of evidence supports the concept that the dietary factors are critical modulators to prevent, retard, block, or reverse carcinogenesis. Such an action reflects the ability of these molecules to interfere with multitude of pathways to subdue and neutralize several oncogenic factors and thereby keep a restraint on neoplastic transformations. This review provides a series of experimental evidence based on the current literature to highlight the translational potential of nutraceuticals for the prevention of the disease through consumption of enriched diets and its efficacious management by means of novel interventions. Specifically, this review provides the current understanding of the chemopreventive pharmacology of nutraceuticals such as cucurbitacins, morin, fisetin, curcumin, luteolin and garcinol toward their potential as anticancer agents.

Molecular determination of progesterone receptor's PROGINS allele (Alu insertion) and its association with the predisposition and susceptibility to polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome, previously known as Stein-Leventhal syndrome, is associated with altered reproductive endocrinology, predisposing a young woman towards the risk of PCOS. It has a prevalence of 6-20% among the reproductive-age women. Progesterone is a key hormone in the pathophysiology of PCOS and patients show diminished response (progesterone resistance), implicating the role of progesterone receptor (PR) as a factor in the disease etiology and prognosis. In this case-control study, we have used mutation-specific PCR (confirmed by Sanger sequencing) to detect the presence of a pathologically significant PR polymorphic variant called as PROGINS. The variant has an Alu insertion in intron G and has two SNPs in exon 4 and exon 5, with all the three aberrations in complete disequilibrium. Our results demonstrated a statistically significant difference in the frequencies of PROGINS between the PCOS patients and healthy controls (p = 0.047). The frequencies of the genotypes CC (A1/A1), CT (A1/A2), and TT (A2/A2) in patients were 74.50%, 20.58%, and 4.90%, and in healthy controls they were 87.28%, 11%, and 1.69%, respectively. Our results put forward two determining factors with regard to PCOS: (i) the frequency of PROGINS allele was significantly higher among PCOS patients compared to the healthy matched controls (0.15 vs 0.07) in the studied population, (ii) the PROGIN allele was significantly associated with the lower levels of serum progesterone in PCOS patients (p < 0.003). The findings are conspicuous as these relate the PROGINS variant to the increased susceptibility of PCOS and might explain the progesterone resistance in patients.

Dietary molecules and experimental evidence of epigenetic influence in cancer chemoprevention: An insight.

The world-wide rate of incidence of cancer disease has been only modestly contested by the past and current preventive and interventional strategies. Hence, the global effort towards novel ideas to contain the disease still continues. Constituents of human diets have in recent years emerged as key regulators of carcinogenesis, with studies reporting their inhibitory potential against all the three stages vis-a-vis initiation, promotion and progression. Unlike drugs which usually act on single targets, these dietary factors have an advantage of multi-targeted effects and pleiotropic action mechanisms, which are effective against cancer that manifest as a micro-evolutionary and multi-factorial disease. Since most of the cellular targets have been identified and their consumption considered relatively safe, these diet-derived agents often appear as molecules of interest in repurposing strategies. Currently, many of these molecules are being investigated for their ability to influence the aberrant alterations in cell's epigenome for epigenetic therapy against cancer. Targeting the epigenetic regulators is a new paradigm in cancer chemoprevention which acts to reverse the warped-up epigenetic alterations in a cancer cell, thereby directing it towards a normal phenotype. In this review, we discuss the significance of dietary factors and natural products as chemopreventive agents. Further, we corroborate the experimental evidence from existing literature, reflecting the ability of a series of such molecules to act as epigenetic modifiers in cancer cells, by interfering with molecular events that map the epigenetic imprints such as DNA methylation, histone acetylation and non-coding RNA mediated gene regulation.

Potential Impact of MicroRNA Gene Polymorphisms in the Pathogenesis of Diabetes and Atherosclerotic Cardiovascular Disease.

MicroRNAs (miRNAs) are endogenous, small (18-23 nucleotides), non-coding RNA molecules. They regulate the posttranscriptional expression of their target genes. MiRNAs control vital physiological processes such as metabolism, development, differentiation, cell cycle and apoptosis. The control of the gene expression by miRNAs requires efficient binding between the miRNA and their target mRNAs. Genome-wide association studies (GWASs) have suggested the association of single-nucleotide polymorphisms (SNPs) with certain diseases in various populations. Gene polymorphisms of miRNA target sites have been implicated in diseases such as cancers, diabetes, cardiovascular and Parkinson's disease. Likewise, gene polymorphisms of miRNAs have been reported to be associated with diseases. In this review, we discuss the SNPs in miRNA genes that have been associated with diabetes and atherosclerotic cardiovascular disease in different populations. We also discuss briefly the potential underlining mechanisms through which these SNPs increase the risk of developing these diseases.