Pharmacogenetic considerations in therapy with novel antiplatelet and anticoagulant agents.

Antiplatelets and anticoagulants are extensively used in cardiovascular medicine for the prevention and treatment of thrombosis in the venous and arterial circulations. Wide inter-individual variability has been observed in response to antiplatelets and anticoagulants, which triggered researchers to investigate the genetic basis of this variability. Data from extensive pharmacogenetic studies pointed to strong evidence of association between polymorphisms in candidate genes and the pharmacokinetics and pharmacodynamic action and clinical response of the antiplatelets clopidogrel and the anticoagulant warfarin. In this review, we conducted an extensive search on Medline for the time period of 2009-2023. We also searched the PharmGKB website for levels of evidence of variant-drug combinations and for drug labels and clinical guidelines. We focus on the pharmacogenetics of novel antiplatelets and anticoagulants while excluding acetylsalicylic acid, warfarin and heparins, and discuss the current knowledge with emphasis on the level of evidence.

Aromatic Hydrocarbon Receptor Repressor (AHRR) is a biomarker of ambient air pollution exposure and Coronary Artery Disease (CAD).

Two hundred and twenty subjects were recruited while undergoing cardiac catheterization. AHRR cg05575921 methylation was shown to be significantly decreased in ever smokers compared to never smokers (Mean± SD = 64.2 ± 17.2 vs 80.1 ± 11.1 respectively; P < 0.0001). In addition, higher urinary levels of 2-OHNAP and 2-OHFLU were significantly associated with more AHRR cg05575921 hypomethylation, even after correcting for smoking (ß[95%CI]= -4.161[-7.553, -0.769]; P = 0.016 and -5.190[-9.761, -0.618]; P = 0.026, respectively) but not 1-OHPYR (ß[95%CI]= -3.545 [-10.935, 3.845]; P = 0.345). Additionally, hypomethylation of AHRR ROI was significantly associated with obstructive coronary artery disease (CAD) after adjusting for smoking, age, sex, diabetes and dyslipidemia (OR [95%CI] = 1.024[1.000 - 1.048]; P = 0.046). Results of this study necessitate further validation to potentially consider clinical incorporation of AHRR methylation status as an early predictive biomarker for the potential association between ambient air pollution and CAD.

Waterpipe and cigarette epigenome analysis reveals markers implicated in addiction and smoking type inference.

Waterpipe smoking is frequent in the Middle East and Africa with emerging prevalence worldwide. The epigenome acts as a molecular sensor to exposures and a crucial driver in several diseases. With the widespread use of waterpipe smoking, it is timely to investigate its epigenomic markers and their role in addiction, as a central player in disease prevention and therapeutic strategies. DNA methylome-wide profiling was performed on an exposure-rich population from the Middle East, constituting of 216 blood samples split equally between never, cigarette-only and waterpipe-only smokers. Waterpipe smokers showed predominantly distinct epigenetic markers from cigarette smokers, even though both smoking forms are tobacco-based. Moreover, each smoking form could be accurately (∼90 %) inferred from the DNA methylome using machine learning. Top markers showed dose-response relationship with extent of smoking and were validated using independent technologies and additional samples (total N = 284). Smoking markers were enriched in regulatory regions and several biological pathways, primarily addiction. The epigenetically altered genes were not associated with genetic etiology of tobacco use, and the methylation levels of addiction genes, in particular, were more likely to reverse after smoking cessation. In contrast, other epigenetic markers continued to feature smoking exposure after cessation, which may explain long-term health effects observed in former smokers. This study reports, for the first time, blood epigenome-wide markers of waterpipe smokers and reveals new markers of cigarette smoking, with implications in mechanisms of addiction and the capacity to discriminate between different smoking types. These markers may offer actionable targets to reverse the epigenetic memory of addiction and can guide future prevention strategies for tobacco smoking as the most preventable cause of illnesses worldwide.

Pharmacogenomics in practice: a review and implementation guide.

Considerable efforts have been exerted to implement Pharmacogenomics (PGx), the study of interindividual variations in DNA sequence related to drug response, into routine clinical practice. In this article, we first briefly describe PGx and its role in improving treatment outcomes. We then propose an approach to initiate clinical PGx in the hospital setting. One should first evaluate the available PGx evidence, review the most relevant drugs, and narrow down to the most actionable drug-gene pairs and related variant alleles. This is done based on data curated and evaluated by experts such as the pharmacogenomics knowledge implementation (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC), as well as drug regulatory authorities such as the US Food and Drug Administration (FDA) and European Medicinal Agency (EMA). The next step is to differentiate reactive point of care from preemptive testing and decide on the genotyping strategy being a candidate or panel testing, each of which has its pros and cons, then work out the best way to interpret and report PGx test results with the option of integration into electronic health records and clinical decision support systems. After test authorization or testing requirements by the government or drug regulators, putting the plan into action involves several stakeholders, with the hospital leadership supporting the process and communicating with payers, the pharmacy and therapeutics committee leading the process in collaboration with the hospital laboratory and information technology department, and healthcare providers (HCPs) ordering the test, understanding the results, making the appropriate therapeutic decisions, and explaining them to the patient. We conclude by recommending some strategies to further advance the implementation of PGx in practice, such as the need to educate HCPs and patients, and to push for more tests' reimbursement. We also guide the reader to available PGx resources and examples of PGx implementation programs and initiatives.

LINE-1 methylation mediates the inverse association between body mass index and breast cancer risk: A pilot study in the Lebanese population.

INTRODUCTION: Lebanon is among the top countries worldwide in combined incidence and mortality of breast cancer, which raises concern about risk factors peculiar to this country. The underlying molecular mechanisms of breast cancer require elucidation, particularly epigenetics, which is recognized as a molecular sensor to environmental exposures. PURPOSE: We aim to explore whether DNA methylation levels of AHRR (marker of cigarette smoking), SLC1A5 and TXLNA (markers of alcohol consumption), and LINE-1 (a genome-wide repetitive retrotransposon) can act as molecular mediators underlying putative associations between breast cancer risk and pertinent extrinsic (tobacco smoking and alcohol consumption) and intrinsic factors [age and body mass index (BMI)]. METHODS: This is a cross-sectional pilot study which includes breast cancer cases (N = 65) and controls (N = 54). DNA methylation levels were measured using bisulfite pyrosequencing on available peripheral blood samples (N = 119), and Multivariate Imputation by Chained Equations (MICE) was used to impute missing DNA methylation values in remaining samples. Multiple mediation analysis was performed to assess direct and indirect (via DNA methylation) effects of intrinsic and extrinsic factors on breast cancer risk. RESULTS: In relation to exposure, AHRR hypo-methylation was associated with cigarette but not waterpipe smoking, suggesting potentially different biomarkers of these two forms of tobacco use; SLC1A5 and TXLNA methylation were not associated with alcohol consumption; LINE-1 methylation was inversely associated with BMI (ß-value [95% confidence interval (CI)] = -0.04 [-0.07, -0.02]), which remained significant after adjustment for age, smoking and alcohol consumption. In relation to breast cancer, there was no detectable association between AHRR, SLC1A5 or TXLNA methylation and cancer risk, but LINE-1 methylation was significantly higher in breast cancer cases when compared to controls (mean ± SD: 72.00 ± 0.66 versus 70.89 ± 0.73, P = 4.67 × 10-14). This difference remained significant after adjustment for confounders (odds ratio (OR) [95% CI] = 9.75[3.74, 25.39]). Moreover, LINE-1 hypo-methylation mediated 83% of the inverse effect of BMI on breast cancer risk. CONCLUSION: This pilot study demonstrates that alterations in blood LINE-1 methylation mediate the inverse effect of BMI on breast cancer risk. This warrants large scale studies and stratification based on clinic-pathological types of breast cancer.

Effect of bisphenols on telomerase expression and activity in breast cancer cell lines.

Bisphenol A (BPA), a monomer of polycarbonates and resins, was shown to induce the expression of telomerase enzyme which has been associated with breast cancer development and progression. However, the effects of BPA analogues, bisphenol F (BPF) and bisphenol S (BPS) on telomere-linked pathway have not been evaluated. Herein, MCF-7 (estrogen receptor (ER)-positive) and MDA-MB-231 (ER-negative) cells were treated with BPA, BPF and BPS ± estrogen receptor inhibitor (ERI), for 24 and/or 48 h. RNA expression and enzymatic activity of telomerase were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and telomeric repeat amplification protocol (TRAP); respectively. Relative telomere length (RTL) was also measured using quantitative PCR. After 24 h, the three bisphenols resulted in a 2-3 folds increase in expression and activity of telomerase in MCF-7 but not in MDA-MB-231 cells, and this increase was prevented upon co-treatment with ERI. The observed increase in the expression and activity of telomerase after 24 h of treatment with bisphenols was associated with differential and modest ER-dependent lengthening in RTL at 48 h. Our results show that telomerase potentially mediates the effects of the three bisphenols in ER-positive breast carcinoma. Hence, further investigation is warranted to elucidate the telomerase-linked pathways that could underlie bisphenol-related effects.

The Interaction between Genetic Polymorphisms in FTO and TCF7L2 Genes and Dietary Intake with Regard to Body Mass and Composition: An Exploratory Study.

In contrast to the large number of genetic studies on obesity, there has been significantly less nutrigenetics investigation of the interaction between diet and single nucleotide polymorphisms (SNPs) in obesity, especially within Eastern Mediterranean populations. The aim of this study was to evaluate the potential interactions between three candidate SNPs, namely, rs1558902 and rs9939609 in the fat mass and obesity (FTO) gene and the rs7903146 variant of the Transcription factor 7 like 2 (TCF7L2) gene, and macronutrient intake with regard to obesity, body fat, and muscle composition. Three hundred and eight healthy Lebanese adults were included in this study. Data collection included a questionnaire for demographics and lifestyle in addition to a detailed dietary assessment using a culture-specific 80-item semi-quantitative food frequency questionnaire. This was coupled with anthropometric measurements and peripheral blood withdrawal for DNA and genotyping using Taqman allele discrimination assays. The two FTO candidate SNPs were not associated with risk of obesity in this population sample, yet there was a trend, though not a significant one, towards lower muscle mass among carriers of the risk allele of either FTO SNPs. To our knowledge, these results have not been previously reported. As for the TCF7L2 rs7903146 variant, results were congruent with the literature, given that individuals who were homozygous for the risk allele had significantly higher body mass index (BMI) and body fat despite lower intakes of saturated fat. Similar interactions, though not significant, were shown with muscle mass, whereby individuals who were homozygous for the risk allele had lower muscle mass with higher intakes of saturated fat, a result that, to our knowledge, has not been previously reported.

Distribution and Role of N-acetyltransferase 2 Genetic Polymorphisms in Bladder Cancer Risk in a Lebanese Population

Background: In Lebanon, bladder cancer (BC) has an unusually high prevalence. Individuals who are exposed to aromatic amines from smoking or certain occupations and carrying the slow N-acetyl transferase 2 (NAT2) acetylator' phenotype may be at a higher risk. Methods: Data and DNA from 115 Lebanese BC cases and 306 controls were examined. Ten NAT2 single nucleotide polymorphisms were genotyped, seven of which were then included in haplotype and phenotype analysis. Results: BC patients were more likely to be males (87.8% vs. 54.9%) and current smokers (60.9% vs. 26.5%) when compared to controls. In both groups, most participants had the slow NAT2 acetylator phenotype (66.1% of BC cases vs 62.7% of controls; P=0.302) with the NAT2*5B and *6A haplotypes being the most common. The odds ratio (95%CI) of having BC among slow NAT2 acetylators was 1.157 (0.738-1.815) and remained non-significant after adjustment [1.097 (0.666-1.806)]. Sensitivity analysis with a subgroup of 113 cases and 84 controls for which occupational history was available revealed a statistically significant association between slow NAT2 acetylators and BC in females only. The sample size was however very small and the CI quite wide. Conclusions: This is the first study to evaluate the distribution of NAT2 haplotypes and their potential role in BC in a Lebanese population. The absence of any significant association may be due to the relatively small sample size, the unavailability of matching by gender, and the lack of evaluation of genetic interactions with extent of active and passive smoking, exposure to environmental pollutants, diet, and other genes. The potential association limited to females needs further evaluation.

Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.

NUDT15 and TPMT genetic polymorphisms are related to 6-mercaptopurine intolerance in children treated for acute lymphoblastic leukemia at the Children's Cancer Center of Lebanon.

BACKGROUND: Interindividual variability in thiopurine-related toxicity could not be completely explained by thiopurine S-methyltransferase (TPMT) polymorphisms, as a number of patients who are homozygous wild type or normal for TPMT still develop toxicity that necessitates 6-mercaptopurine (MP) dose reduction or protocol interruption. Recently, few studies reported on an inherited nucleoside diphosphate-linked moiety X motif 15 (NUDT15) c.415C>T low-function variant that is associated with decreased thiopurine metabolism and leukopenia in childhood acute lymphoblastic leukemia (ALL) and other diseases. PROCEDURES: The aim of this study is to measure the frequency of TPMT and NUDT15 polymorphisms and assess whether they are predictors of MP intolerance in children treated for ALL. One hundred thirty-seven patients with ALL of whom 121 were Lebanese were evaluated. MP dose intensity was calculated as the ratio of the tolerated MP dose to planned dose during continuation phase to maintain an absolute neutrophil count (ANC) dose above 300 per µl. RESULTS: One patient was NUDT15 heterozygous TC and tolerated only 33.33% of the planned MP dose, which was statistically significantly different from the median-tolerated MP dose intensity of the rest of the cohort (76.00%). Three patients had the TPMT*3A haplotype and tolerated 40.00-66.66% of the planned MP dose, which was also statistically significantly different from the rest of the cohort. CONCLUSIONS: This is the first report on the association of TPMT and NUDT15 polymorphisms with MP dose intolerance in Arab patients with ALL. Genotyping for additional polymorphisms may be warranted for potential gene/allele-dose effect.

Comparative effect of 25(OH)D3 and 1,25(OH)2D3 on Th17 cell differentiation.

Vitamin D is a secosteroid hormone that plays an important regulatory role in calcium homeostasis and bone metabolism. Immune cells can both produce and respond to 1,25(OH)2D3. CD4+ T cells from vitamin D receptor (VDR) KO mice produce higher levels of IFN-γ and IL-17 than their wild type counterparts, and play a crucial role in the pathogenesis of autoimmune diseases (AID). We are particularly interested in studying the effect of vitamin D on pathogenic Th17 cells in humans. We investigated the in vitro effect of 1,25(OH)2D3 and 25(OH)D3 on the differentiation and cytokine production of primary CD4+ T cells from normal donors, and cultured in Th17 polarizing conditions. Both forms of vitamin D reduced the expression of pathogenic Th17 markers and their secretion of pro-inflammatory cytokines (IL-17A, IFN-γ). Furthermore, both vitamin D forms induced an expansion of CD25hi cells and upregulated their expression of CTLA-4 and Foxp3 regulatory markers.