الفهرس 
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Abstract
Hypertension (HTN) is one of the most prevalent diseases involving cardiovascular system. Uncontrolled HTN is associated with many risks, including chronic renal failure, myocardial infarction, stroke, congestive heart failure, and death. Patients show wide variability in the response to antihypertensive medications, where only about 50% of the treated patients have controlled blood pressure. Genetics can play a role in explaining this variability.
Personalized medicine involves the use of an individual’s genetic data to guide decisions made in regard to the prevention, diagnosis, and management of the disease. Thus, helping offer the right patient the right dose of the right drug.
β-blockers are commonly prescribed antihypertensive medications that act as competitive antagonists of the Beta-1 adrenergic receptor that is present mainly in the kidney and heart. β-blockers, like other antihypertensives, show wide variability in efficacy that may be explained by genetic variability; hence novel genetic determinants are to be investigated.
Epigenetics focuses on studying the heritable changes in the function of the gene that occur with no change in the DNA sequence. DNA methylation and histone modifications are major epigenetic mechanisms. DNA methylation is considered one of the novel genetic determinants of drug response, especially after it has become possible to quantify DNA methylation at the individual site level, using pyrosequencing.
Micro RNAs (miRNAs) are small, non-protein coding single stranded RNAs that function as gene regulators at the post-transcriptional level, via inhibiting mRNA translation or inducing its degradation. miRNAs circulating in the body fluids, like plasma and urine, are considered potential genetic determinants for drug response because they are easily accessible and stable. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard for profiling miRNA expression. However, to get reliable miRNA expression results, the qRT-PCR data have to be normalized. Unfortunately, there is no universal normalizer and the suitable normalizer(s) should be identified for each experimental conditions. There is a need to identify the suitable normalizer for plasma miRNA expression data in HTN as there is no report about normalizers for such experimental conditions.
The aim of this work was to study whether the novel genetic determinants DNA methylation and miRNA expression are associated with antihypertensive response to β-blockers and to identify the most suitable normalizers for qRT- PCR data of plasma miRNA expression in HTN.
Samples from responder and non-responder hypertensive patients to β-blockers were obtained from the clinical trials PEAR and PEA-2. Healthy control samples were obtained from a University of Florida Institutional Review Board-approved study in which the participants signed a consent declaring their approval to the use of their samples for future genetic research.
from PEAR2 study, blood samples from 45 responders and 45 non-responders to metoprolol were tested for DNA methylation in 4 sites in the beta-1 adrenergic gene (ADRB1), using pyrosequencing.
To identify suitable normalizer(s) for plasma miRNA qRT-PCR data in HTN, 5 candidate normalizers expression levels were measured by qRT-PCR in 18 hypertensive patients’ plasma samples from PEAR and 10 plasma samples from healthy controls. NormFinder, DataAssist, and GeNorm software algorithms were used to determine the best normalizer(s). The 5 candidates expression levels were also analyzed in 9 hypertensive patients’ urine samples and compared to the plasma- generated data to check for consistency.
Expression levels of 22 miRNAs targeting β-blockers pharmacodynamics-related genes were assessed in baseline plasma samples from 30 good responders and 30 poor responders to metoprolol from the PEAR-2 clinical trial, using qRT-PCR (Discovery phase). MiRNAs that exhibited significantly different expression were profiled in baseline plasma samples from 25 good responders and 25 poor responders to atenolol from the PEAR clinical trial (Replication phase).
These current studies showed that:
• A specific DNA methylation site (site number 4) on ADRB1 gene was significantly associated with antihypertensive response to β-blocker, with P-value = 0.011. When antihypertensive response was assessed as change in heart rate, the same methylation site (site number 4) was significantly associated with the antihypertensive response to β-blocker (P-value = 0.023).
• Among the tested candidate normalizers for plasma miRNA qRT-PCR data in HTN, miR-92a was the best (first rank), followed by miR-21 and miR-16 (with a mean rank value of 2.67 each). The last was miR-223 (last rank)
• In the discovery phase of the study investigating the association of miRNA expression with antihypertensive response to β-blockers, the following miRNAs had significant difference in expression between responders and non-responders to metoprolol: hsa-miR-27a (P=0.018), hsa-miR-19a (P=0.024), hsa-miR-22 (P=0.027), hsa-miR-101 (P=0.032), and hsa-let-7e (P=0.038). In the replication phase, hsa-miR-19a exhibited significant differential expression between responders and non-responders to atenolol (P=0.017). Logistic regression showed miR-19a to be associated with the antihypertensive response to atenolol significantly, with odds ratio (OR) of 1.99 and 95% confidence interval (95% CI) of 1.17 – 3.37. MiR-19a was still significant after covariate adjustment, with OR (95% CI) of 2.15 (1.06 – 4.36).
In conclusion, DNA methylation and miRNA expression may help optimize HTN management and achieve the goals of personalized medicine.