Study of clinically important genetic polymorphisms of the human genome associated with cardiovascular disease

Cardiovascular diseases are the leading cause of death worldwide. The most frequent and severe cardiovascular disease is coronary artery disease resulting from coronary arteries’ atherosclerosis. Though many risk factors that increase the risk of coronary artery disease have been identified, such as high levels of LDL- and total cholesterol, hypertension, diabetes mellitus and smoking, researchers have focused on the genetic background of coronary artery disease by analyzing genetic factors that either independently or in interaction with known risk factors may contribute to the pathogenesis of the disease. Genetics of cardiovascular disease is a wide field consisting of association studies of gene polymorphisms with disease. Coronary artery disease is a multifactorial disease of complex genetic aetiology and thus there are reports on many gene candidates. The selection of studied gene polymorphisms is based on the physiology and pathophysiology of the involved system with coronary artery disease and the alterations that polymorphisms of these genes may cause. The association studies of gene polymorphisms with coronary artery disease evaluate and compare the frequency of gene polymorphisms between patients and healthy controls. The present study is an association study investigating the association of selected gene polymorphisms with coronary artery disease. Our aim is to determine the possible association of gene polymorphisms with coronary artery disease in Greek coronary artery disease patients and to analyze their interaction with patient characteristics, such as previous myocardial infarction, hypertension and diabetes or according to their smoking habits. We further assessed the synergistic effect on analyzed gene polymorphisms through gene score application. A total of 154 coronary artery bypass graft surgery patients and 155 non- coronary artery disease controls were included in the study. We have selected this patient atherosclerotic model, as coronary artery bypass graft surgery is a critical well documented endpoint of atherosclerotic coronary artery disease. The control group consisted of healthy individuals with no clinical evidence of coronary artery disease and also without medical record of coronary artery disease, myocardial infarction or other cardiovascular disease. In total of participants we have genotyped the following polymorphisms: M235T and T174M polymorphisms of angiotensinogen gene (AGT), I / D polymorphism of angiotensin I converting enzyme gene (ACE), A1166C polymorphism of angiotensin II type 1 receptor (AT1R), -786T> C and G894T polymorphisms of the endothelial synthase of nitric oxide gene (eNOS) and the alleles ε2, ε3 and ε4 of apolipoprotein E gene (apoE). AGT, ACE and AT1R gene polymorphisms lead to hyperactivity of renin-angiotensin-aldosterone system, eNOS gene polymorphisms are associated with reduced endothelial synthesis of NO, while ε2 and ε4 alleles of apoE affect cholesterol levels. All analyzed polymorphisms potentially affect the pathophysiology of coronary artery disease through altered physiology of the systems they are componets.Οι παθήσεις του καρδιαγγειακού συστήματος αποτελούν την κύρια αιτία θανάτου της σύγχρονης εποχής. Η κυριότερη σε βαρύτητα και συχνότητα καρδιαγγειακή νόσος είναι η στεφανιαία νόσος που απορρέει από την αθηροσκλήρωση των στεφανιαίων αγγείων. Πολλοί παράγοντες κινδύνου που αυξάνουν τον κίνδυνο εμφάνισης της στεφανιαίας νόσου έχουν προσδιοριστεί, όπως τα υψηλά επίπεδα LDL- και ολικής χοληστερόλης, η υπέρταση, ο σακχαρώδης διαβήτης και το κάπνισμα. Για τη διερεύνηση του γενετικού υπόβαθρου της στεφανιαίας νόσου, το ενδιαφέρον των ερευνητών έχει στραφεί στη μελέτη των γενετικών παραγόντων που -είτε ανεξάρτητα είτε σε αλληλεπίδραση με τους γνωστούς παράγοντες κινδύνου- μπορεί να συμβάλλουν στην εμφάνιση της νόσου. Το πεδίο της γενετικής των καρδιαγγειακών παθήσεων στηρίζεται στη μελέτη συσχέτισης γονιδιακών πολυμορφισμών με την εμφάνιση της νόσου. Η επιλογή των γονιδιακών πολυμορφισμών πραγματοποιείται σύμφωνα με την αλλαγή που προκαλούν στη φυσιολογία του συστήματος που συμμετέχουν και κατ’ επέκταση στην παθοφυσιολογία της νόσου. Οι μελέτες συσχέτισης γονιδιακών πολυμορφισμών με τη στεφανιαία νόσο μελετούν και συγκρίνουν τη συχνότητα εμφάνισης των γονιδιακών πολυμορφισμών ανάμεσα σε ασθενείς και υγιείς ως προς το φαινότυπο της νόσου. Η παρούσα μελέτη είναι μία μελέτη συσχέτισης επιλεγμένων γονιδιακών πολυμορφισμών με τη στεφανιαία νόσο. Σκοπός της εργασίας είναι ο προσδιορισμός της πιθανής συσχέτισης των γονιδιακών πολυμορφισμών με τη στεφανιαία νόσο στον Ελληνικό πληθυσμό και η ανάλυση της αλληλεπίδρασής τους με συνυπάρχουσες παθολογικές καταστάσεις των ασθενών, όπως η ύπαρξη προηγούμενου εμφράγματος του μυοκαρδίου, υπέρτασης, διαβήτη καθώς και σύμφωνα με τις καπνιστικές τους συνήθειες. Ένας επιπλέον στόχος της μελέτης είναι η εφαρμογή του μοντέλου του γενετικού αθροίσματος, ώστε να εκτιμήσουμε τη συνολική επίδραση του συνδυασμού των πολυμορφισμών με τη στεφανιαία νόσο. Στη μελέτη συμμετείχαν συνολικά 309 άτομα. Από αυτά, 154 συνιστούν την ομάδα των ασθενών και πρόκειται για ασθενείς με βαριά αθηροσκληρωτική στεφανιαία νόσο που έχουν υποβληθεί σε επέμβαση αορτοστεφανιαίας παράκαμψης. Επιλέχθηκαν ασθενείς που έχουν υποβληθεί σε επέμβαση αορτοστεφανιαίας παράκαμψης ως μοντέλο αθηροσκλήρωσης, καθώς αποτελούν μία καλά χαρακτηρισμένη ομάδα ασθενών ως προς το φαινότυπο της στεφανιαίας νόσου. Οι υπόλοιποι 155 συμμετέχοντες συνιστούν την ομάδα ελέγχου και πρόκειται για υγιή ως προς τη στεφανιαία νόσο άτομα, χωρίς κλινικά ευρήματα της νόσου ή ιατρικό ιστορικό άλλων καρδιαγγειακών παθήσεων σε αντιστοιχία φύλου με τους ασθενείς. Στο σύνολο των συμμετεχόντων αναλύθηκαν οι πολυμορφισμοί Μ235Τ και Τ174Μ του γονιδίου του αγγειοτενσινογόνου (AGT), I/D του γονιδίου του μετατρεπτικού ενζύμου της αγγειοτενσίνης Ι (ACE), A1166C του γονιδίου του υποδοχέα τύπου 1 της αγγειοτενσίνης ΙΙ (AT1R), -786T>C και G894T του γονιδίου της ενδοθηλιακής συνθετάσης του μονοξειδίου του αζώτου (eNOS) και τα αλληλόμορφα ε2, ε3 και ε4 του γονιδίου της απολιποπρωτεΐνης Ε (apoE). Οι πολυμορφισμοί των γονιδίων AGT, ACE και AT1R που μελετήθηκαν οδηγούν σε υπερενεργότητα του συστήματος Ρενίνης - Αγγειοτενσίνης - Αλδοστερόνης, οι πολυμορφισμοί του γονιδίου eNOS σχετίζονται με μειωμένη σύνθεση του ΝΟ στο ενδοθήλιο, ενώ τα αλληλόμορφα ε2 και ε4 της apoE επιδρούν στα επίπεδα χοληστερόλης. Στο σύνολό τους οι πολυμορφισμοί που μελετήθηκαν, μέσω των συστημάτων που εντοπίζονται, πιθανώς επηρεάζουν την παθοφυσιολογία της στεφανιαίας νόσου

Genotyping of CYP2C9 and VKORC1 in the Arabic Population of Al-Ahsa, Saudi Arabia

Polymorphisms in the genes encoding CYP2C9 enzyme and VKORC1 reductase significantly influence the dose variability of coumarinic oral anticoagulants (COAs). Substantial inter- and intraethnic variability exists in the frequencies of CYP2C9∗2 and ∗3 and VKORC1 –1639A alleles. However, the prevalence of CYP2C9 and VKORC1 genetic variants is less characterized in Arab populations. A total of 131 healthy adult subjects from the Al-Ahsa region of Saudi Arabia were genotyped for the CYP2C9∗2 and ∗3 and VKORC1 –1639G>A polymorphisms by PCR-RFLP method. The frequencies of the CYP2C9∗2 and ∗3 and VKORC1 –1639A alleles were 13.3%, 2.3%, and 42.4%, respectively, with no subjects carrying 2 defective alleles. The frequencies of the CYP2C9∗3 and VKORC1 –1639A alleles were significantly lower than those reported in different Arabian populations. None of the subjects with the VKORC1 –1639AA genotype were carriers of CYP2C9∗1/∗3 genotypes that lead to sensitivity to COAs therapy. The low frequency of the CYP2C9∗3 allele combined with the absence of subjects carrying 2 defective CYP2C9 alleles suggests that, in this specific population, pharmacogenetic COAs dosing may mostly rely upon VKORC1 genotyping

CYP2C9*2 allele increases risk for hypoglycemia in POR*1/*1 type 2 diabetic patients treated with sulfonylureas

It is previously shown that carriers of the defective allele CYP2C9*3 that leads to impaired sulfonylurea metabolism are at increased sulfonylurea-induced hypoglycemia risk due to diminished drug metabolism, whereas no effect of CYP2C9*2 allele was found. Recently, a polymorphism in P450 oxidoreductase (POR) gene, assigned as POR*28 allele, was associated with increased CYP2C9 activity. The aim of this study was to assess i) the effect of POR*28 allele on sulfonylurea-induced hypoglycemia risk and ii) the association of CYP2C9*2 allele with hypoglycemia risk in non-carriers of POR*28 allele. The study group consisted of 176 patients with diagnosed type 2 diabetes mellitus (T2DM) treated with sulfonylureas, of whom 92 patients had experienced at least one drug-associated hypoglycemic event (cases), while 84 had never experienced a hypoglycemic event (controls). POR*28 allele was detected by use of real-time TaqMan PCR. POR*28 allele was not associated with sulfonyl-induced hypoglycemia. In POR*1/*1 patients, CYP2C9*1/*2 genotype was more common in cases than in controls (32.7 vs. 14.3%, p=0.041). In a model adjusted for age, BMI, duration of T2DM and renal function, and POR*1/*1 entered as a selection variable, CYP2C9*2 allele increased the hypoglycemia risk in response to sulfonylurea (odds ratio: 3.218, p=0.031). In conclusion, our results suggest that POR*28 allele is masking the association of CYP2C9*2 allele with sulfonyl-induced hypoglycemia. Therefore, POR*28 allele is an important source of CYP2C9 activity variability and combined with CYP2C9 gene polyÂmorphisms may explain individual variability in the effect of sulfonylureas. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart New York

POR*28 SNP is associated with lipid response to atorvastatin in children and adolescents with familial hypercholesterolemia

BACKGROUND: In children and adolescents with familial hypercholesterolemia (FH) pharmacotherapy with statins is the cornerstone in the current regimen to reduce low-density lipoprotein cholesterol (LDLc) and premature coronary heart disease risk. There is, however, a great interindividual variation in response to therapy, partially attributed to genetic factors. The polymorphic enzyme POR transfers electrons from NADPH to CYP450 enzymes including CYP3A, which metabolize atorvastatin. POR*28 polymorphism is associated with increased CYP3A enzyme activity. We analyzed the association of POR*28 allele with response to atorvastatin. MATERIALS & METHODS: One hundred and five FH children and adolescents treated with atorvastatin at doses 10-40 mg were included in the study. Total cholesterol (TChol) and LDLc were measured at baseline and after 6 months of treatment. POR*28 allele was analyzed with TaqMan assay. CYP3A4*22, CYP3A5*3 and SLCO1B1 521T>C and 388A>G genotypes were also determined with TaqMan or PCR-RFLP methods. RESULTS: POR*28 carriers had significantly lower percent mean reduction of TChol (33.1% in *1/*1, 29.8% in *1/*28 and 25.9% in *28/*28 individuals, p = 0.045) and of LDLc (43.9% in *1/*1, 40.9% in *1/*28 and 30.8% in *28/*28 individuals, p = 0.013). In multivariable linear regression adjusted for confounding factors, POR*28 genotypes, additionally to baseline cholesterol level, accounted for an estimated 8.3% and 7.3% of overall variability in % TChol and LDLc reduction (β: 4.05; 95% CI: 1.73-6.37; p = 0.001 and β: 5.08; 95% CI: 1.62-8.54; p = 0.004, respectively). CYP3A4*22, CYP3A5*3 and SLCO1B1 521T>C and 388A>G polymorphisms were not associated with lipid reductions and did not modify the effect of POR*28 on atorvastatin response. CONCLUSION: In children with FH, carriage of POR*28 allele is associated with reduced effect of atorvastatin on TChol and LDLc and therefore identifies FH children that may require higher atorvastatin doses to achieve full therapeutic benefits. Additional studies in different populations are needed to replicate this association

Implementing pharmacogenetic testing in fluoropyrimidine-treated cancer patients: DPYD genotyping to guide chemotherapy dosing in Greece

Introduction: Dihydropyrimidine dehydrogenase (DPD), encoded by DPYD gene, is the rate-limiting enzyme responsible for fluoropyrimidine (FP) catabolism. DPYD gene variants seriously affect DPD activity and are well validated predictors of FP-associated toxicity. DPYD variants rs3918290, rs55886062, rs67376798, and rs75017182 are currently included in FP genetic-based dosing guidelines and are recommended for genotyping by the European Medicines Agency (EMA) before treatment initiation. In Greece, however, no data exist on DPYD genotyping. The aim of the present study was to analyze prevalence of DPYD rs3918290, rs55886062, rs67376798, rs75017182, and, additionally, rs1801160 variants, and assess their association with FP-induced toxicity in Greek cancer patients.Methods: Study group consisted of 313 FP-treated cancer patients. DPYD genotyping was conducted on QuantStudio ™ 12K Flex Real-Time PCR System (ThermoFisher Scientific) using the TaqMan® assays C__30633851_20 (rs3918290), C__11985548_10 (rs55886062), C__27530948_10 (rs67376798), C_104846637_10 (rs75017182) and C__11372171_10 (rs1801160).Results: Any grade toxicity (1-4) was recorded in 208 patients (66.5%). Out of them, 25 patients (12%) experienced grade 3-4 toxicity. DPYD EMA recommended variants were detected in 9 patients (2.9%), all experiencing toxicity (p = 0.031, 100% specificity). This frequency was found increased in grade 3-4 toxicity cases (12%, p = 0.004, 97.9% specificity). DPYD deficiency increased the odds of grade 3-4 toxicity (OR: 6.493, p = 0.014) and of grade 1-4 gastrointestinal (OR: 13.990, p = 0.014), neurological (OR: 4.134, p = 0.040) and nutrition/metabolism (OR: 4.821, p = 0.035) toxicities. FP dose intensity was significantly reduced in DPYD deficient patients (β = −0.060, p <0.001). DPYD rs1801160 variant was not associated with FP-induced toxicity or dose intensity. Triple interaction of DPYD*TYMS*MTHFR was associated with grade 3-4 toxicity (OR: 3.725, p = 0.007).Conclusion: Our findings confirm the clinical validity of DPYD reduced function alleles as risk factors for development of FP-associated toxicity in the Greek population. Pre-treatment DPYD genotyping should be implemented in clinical practice and guide FP dosing. DPYD*gene interactions merit further investigation as to their potential to increase the prognostic value of DPYD genotyping and improve safety of FP-based chemotherapy

Therapeutic Drug Monitoring (TDM) Implementation in Public Hospitals in Greece in 2003 and 2021: A Comparative Analysis of TDM Evolution over the Years

Therapeutic drug monitoring (TDM) is the clinical practice of measuring drug concentrations. TDM can be used to determine treatment efficacy and to prevent the occurrence or reduce the risk of drug-induced side effects, being, thus, a tool of personalized medicine. Drugs for which TDM is applied should have a narrow therapeutic range and exhibit both significant pharmacokinetic variability and a predefined target concentration range. The aim of our study was to assess the current status of TDM in Greek public hospitals and estimate its progress over the last 20 years. All Greek public hospitals were contacted to provide data and details on the clinical uptake of TDM in Greece for the years 2003 and 2021 through a structured questionnaire. Data from 113 out of 132 Greek hospitals were collected in 2003, whereas for 2021, we have collected data from 98 out of 122 hospitals. Among these, in 2003 and 2021, 64 and 51 hospitals, respectively, performed TDM. Antiepileptics and antibiotics were the most common drug categories monitored in both years. The total number of drug measurement assays decreased from 2003 to 2021 (153,313 ± 7794 vs. 90,065 ± 5698; p = 0.043). In direct comparisons between hospitals where TDM was performed both in 2003 and 2021 (n = 35), the mean number of measurements was found to decrease for most drugs, including carbamazepine (198.8 ± 46.6 vs. 46.6 ± 10.1, p p = 0.001), amikacin (147.3 ± 65.2 vs. 91.1 ± 71.4; p = 0.033), digoxin (783.2 ± 226.70 vs. 165.9 ± 28.9; p p = 0.004). Only for vancomycin, a significant increase in measurements was recorded (206.1 ± 96.1 vs. 789.1 ± 282.8; p = 0.012). In conclusion, our findings show that TDM clinical implementation is losing ground in Greek hospitals. Efforts and initiatives to reverse this trend are urgently needed