8^th Santorini Conference Systems Medicine and Personalised Health and Therapy

Session I – FROM SYSTEMS BIOLOGY TO SYSTEMS MEDICINE

Session II – SYSTEMS PHARMACOGENOMICS AND MECHANISMS OF DRUG ACTION

Session III – CELLULAR TARGETS IN ONCO-HEMATOLOGY

Session IV – GENOMICS AND PROTEOMICS OF ALZHEIMER’S DISEASE

Session V – OMICS STUDIES OF HUMAN PHENOTYPES AND ENVIRONMENT

Session VI – NUTRITION AND METABOLIC HEALTH

Selected Oral Presentation

Session VII – PHARMACOGENOMICS AND PERSONALISED/STRATIFIED THERAPY

Session VIII – Genomic biomarkers and management of metabolic and inflammatory diseases

Session IX – New biomarkers and companion diagnostics

A Phenome wide association study shows novel pleiotropic effects of the rs644234 on ABO gene

AA ALDASORO ARGUIÑANO¹, NC NDIAYE¹, S SIEST¹

¹UMR INSERM U1122; IGE-PCV ‘Interactions Gène-Environnement en Physiopathologie Cardiovasculaire’, Faculté de Pharmacie – Université de Lorraine, Nancy, F-54000, France

Background: The Genome wide association studies (GWAS) have revolutionary improved discovery of novel genetic biomarkers determining genetic susceptibility to human diseases but the development of other methodological approaches is necessary in order to detect additional important information linked to the identified variants. Such an approach is the Phenome-wide association studies (PheWAS), which by analysing multiple phenotypes eventually linked to a single genetic variant lead to the identification of novel genotype-phenotype relationships and genetic pleiotropic discovery.

Objective: In this work we focused on the ABO gene, which produces the Histo-blood group ABO system transferase enzyme. This enzyme will modify the oligosaccharides of the glycoproteins, located in the cell surface, determining the type of blood group of one individual. Recent GWAS analyses have identified polymorphisms on this gene to be related with the thrombosis process, risk of suffering myocardial infarction and with multiple cardiovascular risk biomarkers. Especially the SNP rs644234, located in the intron 1 of the ABO gene, has been related with the levels of soluble E-selectin (sE-selectin).

Design: We performed a PheWAS analysis with the rs644234 and different inflammatory and lipid metabolism phenotypes available in the STANISLAS cohort, in order to investigate possible novel genotype-phenotype associations of the ABO polymorphism.

Results: The results showed pleiotropic effects of the rs644234. Indeed, we replicated previous known associations with sE-selectin (p-value: 6.6x10^-12), angiotensin I converting enzyme (ACE) (p-value: 0.008) and soluble intercellular adhesion molecule (sICAM) (p-value: 0.01). Moreover, new pleiotropic associations related with lipid phenotypes were found. The minor allele of rs644234 was associated with decreased ApoB (p-value: 0.019) and ApoE (p-value: 0.037) levels, and increased HDL levels (p-value: 0.012).

Conclusions: Previous GWAS studies have associated polymorphisms on ABO gene with the risk of suffering myocardial infarction, by increasing thrombotic risk as well as soluble adhesion molecules such as sE-selectin, ACE and sICAM. However, our results showed that the ABO gene has wider effects on lipid phenotypes, showing another pathway by which the ABO enzyme can impact on myocardial infarction. The pleiotropy showed for the ABO gene suggests that it has a complex role in the development of cardio-vascular diseases, and underline the importance of further studies in order to understand the exact mechanisms.

Using metabolomics to gain insight into major depressive disorder: a targeted approach on tryptophan pathway metabolites

P Masson¹, E Werner¹, C Boursier-Neyret¹, B Walther¹, L Ripoll², B Fève³, P Chanson⁵, E Corruble⁶, L Becquemont⁴

¹Technologie Servier, Orléans, France. ²Institut de Recherches Internationales Servier, Suresnes, France. ³UPMC Univ Paris 06, INSERM UMR S938, Centre de Recherche Saint-Antoine, AP-HP, Hôpital Saint-Antoine, Paris, France. ⁴Département de Pharmacologie, Faculté de médecine Paris-Sud, Université Paris-Sud, AP-HP, Hôpital Bicêtre, Le Kremlin Bicêtre, France. ⁵Service d’Endocrinologie et des Maladies de la Reproduction, AP-HP, Hôpital Bicêtre, Le Kremlin Bicêtre, France. ⁶Univ Paris Sud, INSERM UMR 1178, Service de Psychiatrie, équipe "Dépression et Antidépresseurs", AP-HP, Hôpital Bicêtre, Le Kremlin Bicêtre, France

Background: Major Depressive Disorder (MDD) is a heterogeneous disorder with variable courses and responses to treatment, and for which the understanding of underlying biological mechanisms is still incomplete. Metabolomics could provide new insights on MDD mechanisms, as the metabolome reflects biochemical alterations in the pathophysiological state of biological systems. Tryptophan-derived metabolites raise particular interest, as an imbalance between neuroprotective and neurodegenerative metabolites in this pathway might be involved in major depression¹.

Objective: To investigate the modulation of tryptophan pathway metabolites in the plasma of MDD patients compared to healthy controls

Design: We analyzed plasma samples from patients included in the METADAP study (a 6-month prospective, multicentric, real-world treatment study of 624 patients with a MDD diagnosis and a current major depressive episode) and from healthy volunteers (HV) who participated in the cross-sectional multicentric VARIETE study. A targeted UPLC-MS method was used to monitor 23 metabolites in the tryptophan pathway.

Results: Among the 14 metabolites that could be detected in the plasma samples, several were lower in MDD patients at inclusion (HDRS ≥ 18) compared to matched HVs, including serotonin, kynurenic acid, picolinic acid and indoxyl sulfate. Over METADAP study period (6 months of antidepressant treatment, during which a global decrease in the HDRS score was observed), plasma serotonin levels decreased, while the neuroprotective kynurenic and picolinic acids, as well as indoxyl sulfate, increased, approaching HV levels. Interestingly, the increase in kynurenic acid was more pronounced for the remitters than for the non-remitters.

Conclusions: these results support the hypothesis that an impairment in the neuroprotective components of the tryptophan pathway may be related to major depression.

Acknowledgements:

1.Myint AM, Kim YK, Verkerk R, Scharpé S, Steinbusch H, Leonard B. J Affect Disord 2007 Feb;98(1-2):143-51

Effects of Mediterranean diet in obese patients with metabolic syndrome and depression

V Dimitrijevic-Sreckovic1,4, H Janeski1 B Sreckovic2, I Soldatovic3,4, N. Nesovic1, M.Vujovic1

¹Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia, ²Clinical Center Bezanijska Kosa, ³Institute for Medical Statistics and Informatics, ⁴Faculty of Medicine, Belgrade University, Serbia

Introduction: Central obesity is the source of inflammatory factors, causing endothelial dysfunction. In central nervous system, inflammatory factors mediate neurotransmitter metabolism promoting excitotoxicity, increasing oxidative stress, and major clinical symptom is depression. Mediterranean diet (MD) resulted as a good prevention method for depression.

Aim: To examine the relationship of depression with abdominal obesity and metabolic syndrome (MS) criteria, blood pressure, lipids, glycaemia and inflammation factors. Analyzing effects of MD on body weight, abdominal obesity and score points in Hamilton scale for depression.

Material and methods: Study included 36 adolescents and youth (16-30 years) and 22 adults over 30 years, overweight and obese patients with MS, diagnosed with depression using Hamilton scale. MS was diagnosed using ATP III classification criteria. The following parameters were observed: BMI, waist circumference (WC), blood pressure, lipids, CRP and basal glycaemia and insulin. Seven day scheduled MD was used by patients.

Results: Correlation is found between Hamilton scale score and body weight, BMI, WC (p<0.01) and CRP (p<0.05) in adolescents and youth. Implementation of MD in adolescents and youth resulted in reduction of body weight and WC (p<0.05), Hamilton scale score (p<0.001), insulinemia, HOMA-IR and CRP (p>0.5). Hamilton scale and Hamilton scale score correlated with insulin (p<0.05) and glycaemia (p<0.05), basal insulin correlated with CRP (p<0.05) in adults. After MD, values of Hamilton score, WC, insulin and CRP were lower (p>0.5) in adults.

Conclusions: Mediterranean diet resulted in reduced values of Hamilton scale score, waist circumference, insulinemia and CRP in patients with MS and depression. Positive correlation of Hamilton scale score with glycaemia and insulinemia suggest importance of insulin resistance and glucoregulation disorder on occurrence and stage of depression in obese patients with metabolic syndrome. CRP is useful marker of low grade inflammation in obese patients with MS and depression.

Keywords: metabolic syndrome, obesity, depression, Mediterranean diet, hyperinsulinism

Reference: Raison CL, Capuron L, Miller AH. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol 2006;27:24–31

The development of assays to evaluate Nrf2 regulatory mechanisms in cancer

FP Charles Cano¹, G Wells²

¹UCL Genetics Institute and ²UCL School of Pharmacy, University College London, London, United Kingdom

Background: Nrf2 is a cap’n’collar basic leucine zipper transcription factor that regulates the expression of antioxidant response element genes. These genes share a cis-acting enhancer where Nrf2 asserts its effect and regulates redox homeostasis, energy metabolism and DNA repair.

The activity of Nrf2 is maintained at a low expression levels under normal homeostatic conditions, but increases rapidly in response to redox, electrophile stressors and growth factors. Nrf2 is regulated independently by two ubiquitination pathways via interactions with either Keap1 or βTrCP. Interaction with the WD40 domain of the latter protein is mediated by DSGIS and DSAPGS motifs in Nrf2.

It has been widely proposed that Nrf2 plays a key disease-modifying role in conditions such as cancer, inflammatory conditions and neurodegenerative diseases. Therefore, there is interest in developing molecules that utilise Nrf2 as a therapeutic target.

Objective: The aim of this study was to identify mutations within the binding domain of βTrCP that can affect its interaction with Nrf2 and develop an in vitro assay to evaluate the interaction of βTrCP with small molecule and peptide ligands.

Design: Germline and somatic mutations were identified in the COSMIC, 1000 genome project, Ensembl, DisGeNET and MutDB databases. Fluorescence polarization and thermal shift assays were developed to rank ligands based on their IC₅₀s.

Results: 148 genetic variants were identified from the selected databases and scientific literature, of these 37% were synonymous, 60% missense and 3% stop-gained variants. Predictions of the consequences of amino acid substitution where performed using online tools. Molecular dynamics analysis was also performed to assess the impact of mutations on βTrCP-substrate interactions.

Conclusions: We have identified several mutations that may have a direct impact on the β-TrCP interaction with Nrf2 and therefore regulate its activity. The work has potential utility in understanding the role of germline and somatic mutations of βTrCP in various disease states and upon the feasibility of targeting βTrCP activity to modulate Nrf2 transcription in such conditions. Subsequently, by developing in vitro assays to study βTrCP interactions we can screen compound libraries that could enter pharmaceutical development.

Reference: 1. Chowdhry S, et al. Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. Oncogene; 2013;32(32):3765–81.

Variant within the apolipoprotein B and its role in pseudo-FH development

Hubacek J.A.^1,Fellnerova A. ¹, Dlouha D. ¹, Tichy L.², Fajkusova L.², Freiberger T.³

¹Center for Experimental Medicine, IKEM, Prague; ²Centre of Molecular Biology and Gene Therapy, University Hospital Brno; ³Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno

Background: Familiar hypercholesterolemia (FH) is a serious disease, leading (if not treated) to premature myocardial infarction. FH is caused predominantly by mutations within the LDL receptor and APOB genes.

Objective: Recently, gene score from 12 SNPs within different genes have been defined, suggesting the existence of “pseudo-FH”, clinically indistinguishable from classical FH, but without one causal mutation. We have analyzed one from these SNPs in Czech patients with FH.

Design:APOB rs1367117 (G˃A) variant was genotyped using TaqMan technology on an AB 7300 RT PCR cycler in 298 FH patients without the causal mutation and in 296 patients with the LDL receptor mutation.

Results: Frequencies of the individual genotypes significantly differ between two analyzed groups (P<0.006). Minor A allele homozygotes occurs more often among the FH patients without the causal mutation than in patients with LDL receptor mutation (17.8% vs. 9.5%); with OR (95%CI) 2.10 (1.29 - 3.43; P<0.003).

Conclusion: The results from our pilot study underline the importance of common APOB rs1367117 variant in the “pseudo-FH” development. Analysis of further SNPs is necessary to complete the real genetic causality of “FH” in patients without the detected mutation.

Supported by Ministry of Health of the Czech Republic, grant nr. 15-28277A. All rights reserved.

The effect of acute Mastiha supplementation on the metabolites profile and oxidative stress in healthy humans

Efstathia Papada1, Aristea Gioxari1, Vincent Brieudes2, Nikos Lemonakis2, Hermann Stuppner3, Maria Halabalaki2,3, Emmanuel Mikros2, Leandros A Skaltsounis2, Andriana C. Kaliora1*

¹Laboratory of Food Chemistry-Biochemistry, Department of Dietetics and Nutritional Science, Harokopio University, 70 El Venizelou ave., 17671, Athens, Greece

²Laboratory of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, University of Athens, Panepistimioupoli Zografou, 15771 Athens, Greece

³University of Innsbruck, Institute of Pharmacy/Pharmacognosy CCB-Center for Chemistry and Biomedicine Innrain 80-82, 6020 Innsbruck, Austria

Background: Mastiha is the natural resin harvested from the trunk of Mastiha tree (Pistacia lentiscus, var. Chia) growing exclusively in Chios Island, Greece. It consists mainly of terpenoids and polyphenols, with established antioxidant and anti-inflammatory properties (Paraschos et al., 2012), however the effect of its supplementation in a postprandial phase remains unexplored.

Objective: a) to assess the bioavailability of Mastiha’s phytochemicals in the plasma and urine of healthy subjects, b) to explore metabolites profile and metabolism mechanisms after Mastiha consumption using NMR and LC-HRMS based metabolomics c) to evaluate oxidative stress markers in a postprandial phase.

Design: Seventeen healthy non-obese and male volunteers, aged 20-40 years old, followed a low in phytochemicals diet for 5 days (e.g. no fruits, vegetables, legumes, coffee, cocoa, teas, wine). On the experiment day and after 12h fasting, volunteers consumed 10g of Mastiha powder dispersed in water. Blood samples were collected on time points 0h (before Mastiha ingestion) and 0.5h, 1h, 2h, 4h, 6h, 24h (post-ingestion). Urine samples were collected on time points 0h, 4h, 8h and 24h. Volunteers consumed nothing per os until time point 8h. Two different platforms, NMR & LC-HRMS were employed for the high throughput analysis of urine and plasma, respectively. Serum resistance to oxidation was measured with the CuSO₄ technique. oxLDL and GSTp were measured in serum samples applying sandwich ELISA assays.

Results: Mastiha’s compounds were detected and identified in LC-HRMS profiles of plasma indicating that constituents such as moronic and isomasticadienonic acids are bioavailable. Also, multivariate spectroscopic data from NMR metabolomic profiles of urine samples were analyzed revealing certain clustering patterns of all the different time points. Moreover, several metabolites of triterpenic acids of mastiha were identified. Serum resistance to oxidation was expressed as the difference (ΔT) of tLAG of each time point from tLAG 0h. Increase was statistically significant on time point 4h (402.3 ± 65.0sec), reached a peak on time point 6h (524.6 ± 62.9sec) and remained statistically significant until 24h post-ingestion (424.2 ± 48.0sec). oxLDL levels (expressed as % change from 0h), were reduced significantly from time point 1h until time point 6h post-ingestion. No significant changes in GSTp were detected.

Conclusions: It is evident that Mastiha exhibits metabolic alterations in healthy individuals and regulates markers of oxidative stress in the dynamic and unsteady postprandial phase. The present results provide new insights in understanding the metabolism pathways of Mastiha’s constituents.

Reference: Paraschos S, Mitakou S, Skaltsounis AL. Curr Med Chem. 2012;19(14):2292-302.

Homocysteine- significant marker of metabolic syndrome and atherosclerosis risk- importance of si MS risk score

B Sreckovic¹, V Dimitrijevic-Sreckovic^2,4, I Soldatovic^3,4, H Janeski², N Janeski⁵, J Gacic¹

1. Clinical Center Bezanijska Kosa, 2. Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia, 3. Institute for Medical Statistics and Informatics, 4. Faculty of Medicine, Belgrade University, 5. Clinical Center Zemunska bolnica, Serbia

Background: Obesity is followed by metabolic changes in form of insulin resistance (IR) and low grade inflammation. In patients with metabolic syndrome (MS) and clinicaly evident vascular complications homocysteine values are higher. Hyperhomocysteinemia correlates with hyperinsulinemia and IR, resulting in oxidative stress, which causes endothelial lesions and dysfunction, promoting hypertension and atherosclerosis.

Objectives: To examine homocysteine levels in patients with and without MS, and find correlation between factors of MS and homocysteine values.

Methods: The study included 76 obese individuals (age over 30, body mass index (BMI) >25 kg/m2) classified into two groups: I- with MS (35 patients); II- without MS (41 patients). OGTT was used to evaluate the extent of glucoregulation disorder. IDF classification was applied for diagnosing MS. Si MS risk score by Soldatovic I et all. was used. IR was determined by homeostatic model assessment (HOMA IR). Serum CRP was measured by immunometric assay. Microalbuminuria was determined immuno-nephelometrically. Homocistein was determined by immunoassay FPIA-Abbot.

Results: Patients with MS had increased WC: (I-110.6±15.4, II±15.4 cm), BMI: (I-35.3±6.6, II-30.4±7.4 kg/m2), blood pressure (I-136.4±13.9 /90.5±9.5, II-118.4±12.2/78.1±9.7mmHg), glycaemia (I-5.4±1.6, II-4.8±0.8mmol/l), HOMA IR (I-8.8±9.4, II-5.3±3.8 mmol/µU/ml), triglycerides (I-2.17±0.95, II-1.45±0.7mmol/l), CRP(I-7.0±0.0, II- 3.7±3.8mg/l), microalbuminuria (I-87.4±81, II-56.4±56.9mg/24h), homocysteine (I-13.0±3.2, II-11.8±3.7 µmol/l) and decreased HDL (I-1.07±0.2, II-1.35±0.35mmol/l). Statistical significance between groups was found for WC, BMI, systolic and diastolic pressure, triglycerides, HDL-cholesterol (p<0.01) and CRP, Apo B, HOMA IR (p<0.05). Correlations: Homocysteine with systolic and diastolic pressure, Apo B and hyperlipoproteinemia (p<0.05) . Si MS risk score with homocysteine (p<0.01), r=0.263.

Conclusion: Abdominal obesity, hypertension, hypertriglyceridemia, inflammation factors, IR, homocysteine and microalbuminuria as markers of endothelial dysfunction were increased in patients with MS. Correlation of homocysteine values with si MS risk score indicates that it is significant co-founding factor of MS. Correlation of homocysteine with hypertension and hyperlipoproteinemia indicates importance of homocysteine as significant marker for atherosclerosis.

Reference: Soldatovic I, Vukovic R, Culafic Dj, Gajic M, Dimitrijevic Sreckovic V. si MS score: Simple method for Quantifying Metabolic Sindrome. PLOS ONE (2016), vol 11. No1

Risk factors for NODAT under immunossuppressants in renal transplanted children

Pauline Lancia¹, Wei Zhao^{1, 2}, Anne Maisin³, Dominique Simon⁴, Véronique Baudouin³, Valéry Elie¹, Evelyne Jacqz-Aigrain^{1, 2, 5}

1. Departments of Pediatric Pharmacology and Pharmacogenetics, Hôpital Robert Debré, APHP, Paris, France 2. Clinical Investigation Center CIC1426, INSERM, Paris, France 3. Pediatric Nephrology, Hôpital Robert Debré, APHP, Paris, France 4. Pediatric Endocrinology, Hôpital Robert Debré, APHP, Paris, France 5. Université Paris Diderot, Press Sorbonne Paris Cité, Paris, France

Background: New Onset Diabetes After Transplantation (NODAT) is a major complication of immunossuppressive therapy and a risk factor for transplant rejection. Its incidence ranges from 4 to 25% in adults. In 2013, L. Elens and coworkers reported the association of two genetic variants PPARα rs4253728 and POR*28 rs1057868 with the occurrence of NODAT in renal transplanted adults treated with tacrolimus, MMF and corticosteroïds.

Objective : Our objective was to assess the impact of these two genetics variants along with additional risk factors (age, sex, HLA missmatches…) on the occurrence of NODAT among renal transplanted children treated with tacrolimus, MMF, and corticoids.

Design: Two hundred thirty eight renal transplanted children were identified between 1990 and 2013 in Robert Debré hospital. After DNA extraction, genotypes were determined using allelic discrimination technique TaqMan. The statistical analysis have been performed using SPSS v15.

Results: One hundred and nine renal transplanted children were treated with tacrolimus and were included in the study (63 boys/46 girls). The mean age at time of transplantation was 10.8 years (± 0.5) and mean duration of folow-up was 4 years (± 0.3). A significant association of NODAT was evidenced with POR*28 (p=0.010), number of HLA mismatches (p=0.004) and donor’s CMV serology (p=0.026) but not with PPARα (p=0.434).

Conclusions : This pilot study validated the association between the SNP POR*28 and the occurrence of NODAT in renal transplanted children treated with tacrolimus. Early identification of patients carrying POR*28 genetic variant might allow to prevent the risk of NODAT and improve patient’s care.

Acknowledgements : This research project is associated to Global Research in Peadiatrics (GRIP).

Reference: Elens L, Sombogaard F, Hesselink DA, van Schaik RH, van Gelder T. Single-nucleotide polymorphisms in P450 oxidoreductase and peroxisome proliferator-activated receptor-α are associated with the development of new-onset diabetes after transplantation in kidney transplant recipients treated with tacrolimus. Pharmacogenet Genomics. 2013 Dec;23(12):649-57

Impression cytology of the conjunctival epithelial cells in patients with Graves orbitopathy

M Mrugacz¹, I Kasacka²

¹Department of Pediatric Ophthalmology Medical University of Bialystok, Washington Street 17, Bialystok, Poland

²Department of Histology and Embryology Medical University of Bialystok, Kilinski Street 1, Bialystok, Poland

Background: Graves’ orbitopathy (GO) is rare in pediatric patients, however is the most common extrathyroid manifestation of Graves’ disease (GD), being present in 30-67% of patients. GO is an autoimmune inflammatory disorder involving orbital connective and fatty tissues as well as the extraocular muscles. In children, GO is less common and less severe than in adults. The most common symptoms are upper eyelid retraction, conjunctival injection, and proptosis and periorbital edema. Severe complications include dysthyroid optic neuropathy, corneal ulceration and eyeball subluxation.

Objective: The aim of this study was evaluating goblet cell population and conjunctival epithelial morphology in patients with GO.

Design: Twenty GO patients (9 males and 11 females, age range= 7 - 23 years) and twenty controls (10 males and 10 females, age range= 8 -22 years) underwent conjunctival impression cytology. Conjunctival epithelial cells were analyzed microscopically, with regard to their shape, size, the nucleus-to-cytoplasm ration and nuclear chromatin condensation using an Olympus Bx50 light microscope.

Results: Impression cytology showed conjunctival squamous metaplasia and goblet cells loss in patients with GO.

Conclusions: Reduced goblet cells numbers and squamous metaplasia may be indicative of a higher degree of epithelial damage of conjunctival epithelial cells in GO patients, and the presence of lymphocytes and neutrophiles is a strong sign for an inflammatory background of this disease. In view of the simple, noninvasive nature of impression cytology, this technique may prove to be an important tool for the diagnosis and monitoring of dry eye changes in GO patients.

Acknowledgements: No

Reference: 1. Vilar-González S, Lamas-Oliveira C, Fagúndez-Vargas MA, Núñez-Quintanilla AT, Pérez-Rozos A, Merayo-Lloves J, Escobar-Barranco JJ, Rico-Pérez JM. Endocrinol Nutr 2015;62(4):188-99.

Increased Sleep Bruxism Risk is Associated with the HTR2A rs2770304 SNP

G H Oporto V.^{1, 2, 3, 4}; C Rodríguez-Niklitschek⁵; T Bornhardt², V Iturriaga², & LA. Salazar^{1,<supspace>3}

¹Centro de Biología Molecular y Farmacogenética, Departamento de Ciencias Básicas, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.

²Departamento de Odontología Adultos, Facultad de Odontología, Universidad de La Frontera, Temuco, Chile.

³Núcleo Científico y Tecnológico en Biorecursos (BIOREN), Universidad de La Frontera, Temuco, Chile.

⁴ Centro de Investigación en ciencias Odontológicas (CICO), Facultad de Odontología, Universidad de La Frontera, Temuco, Chile.

⁵ Escuela de Odontología, Universidad Mayor Campus Temuco, Temuco, Chile.

Background: Bruxism (BRX) is a condition of interest for researchers and clinicians in both dental and medical areas. BRX has two circadian manifestations, it can occur during sleep (sleep bruxism; SB) or during wakefulness (awake bruxism; WB). However, it can be suffered together as well (S&WB). Researchers suggest that central nervous system neurotransmitters and its related genes could be involved in the genesis of BRX. Serotonin is responsible for the circadian rhythm, maintaining arousal, regulating the stress response, muscle tone and breathing. Thus, serotonin could be associated with the pathogenesis of BRX.

Objective: We aimed to evaluate the frequency of genetic polymorphisms within HTR1A (rs6295), HTR2A (rs1923884, rs4941573, rs6313, rs2770304), HTR2C (rs17260565) and MAOA (rs12843268) genes in patients undergoing Bruxism treatment.

Design: Were included 179 patients submitted to BRX treatment, who were classified according to their diagnosis in SB (33 patients), WB (82 patients) and S&WB (63 patients). The control group included 135 patients. Genomic DNA was extracted from blood leukocytes using the method described by Salazar et al. (2001). Genotypes of HTR1A, HTR2A, HTR2C and MAOA were evaluated using TaqMan® SNP genotyping assay (Applied Biosystem, USA), in a StepOne™ Real-Time PCR system (Applied Biosystem, USA).

Results: We found differences in allelic frequencies for the HTR2A rs2770304 SNP. The C allele was associated with increased SB risk (odds ratio=1.96, 95% confidence interval: 1.34 – 3.39, p=0.02, Fisher´s exact test).

Conclusions: Our results indicate that the HTR2A rs2770304 SNP is involved in SB. Therefore; it becomes necessary to continue exploring the genetic basis of circadian manifestations of BRX to increase the current understanding of BRX physiopathology.

Reference: 1. Oporto G, Lagos J, Bornhardt T, Fuentes R, Salazar LA. Are there Genetic Factors Involved in Bruxism? Int J Odontostomat; 2012;6:249-54.

Global DNA methylation is associated with circadian manifestations of Bruxism

GH Oporto V^{1, 2, 3, 4}, T Bornhardt², V Iturriaga² & LA Salazar^{1, 3}

¹Centro de Biología Molecular y Farmacogenética, Departamento de Ciencias Básicas, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile, ²Departamento de Odontología Adultos, Facultad de Odontología, Universidad de La Frontera, Temuco, Chile, ³Núcleo Científico y Tecnológico en Biorecursos (BIOREN), Universidad de La Frontera, Temuco, Chile, ⁴ Centro de Investigación en Ciencias Odontológicas (CICO), Facultad de Odontología, Universidad de La Frontera, Temuco, Chile.

Background: Bruxism (BRX) is defined as a repetitive jaw-muscle activity characterized by clenching or grinding of the teeth and/or by bracing or thrusting of the mandible, having two main circadian manifestations; it can occur during sleep (SB) or during wakefulness (WB). However, it may be suffered together (W&SB). Relatives of people suffering BRX are also affected by this parafunction, therefore, possible genetic and/or epigenetic factors may be involved. BRX etiology includes alcohol consumption, smoking and neurotransmitters alterations. In addition, DNA methylation is a physiological epigenetic modification, occurring primarily by the addition of a methyl group to a CpG dinucleotide in a DNA sequence that regulates gene transcription. Altered global DNA methylation content is a feature of several diseases, and aberrant methylation is considered a mechanism by which environmental risk factors (e.g., tobacco, alcohol, and diet) may influence disease risk. Thus, circadian manifestations of BRX could be associated to aberrant DNA methylation.

Objective: We aimed to quantify global DNA methylation in patients under BRX treatment and controls.

Design: Subjects undergoing BRX treatment were classified in WB (42 patients), SB (32 patients) and W&SB (42 patients). The control group included 42 healthy patients. To quantify global DNA methylation, we used the ELISA kit MethylFlash Methylated DNA Quantification Kit (Colorimetric assay, Epigenetic Group Inc., NY, USA).

Results: We found significant differences in the content of methylated DNA in all circadian manifestations of BRX compared with the control group (SB 0.95 ± 2.02 ng/μL; WB 0.87 ± 2.1 ng/μL; W&SB 0.17 ± 0.25 ng/μL; Control 1.69 ±1.6 ng/μL; Kuskal-Wallis test [p=0.0001] followed by Dunn`s test [p<0.05]).

Conclusions: DNA in BRX patients was hypomethylated compared with the control group. Our results suggest that DNA methylation might be a novel etiologic factor involved in BRX etiology. Further research must be performed exploring the role of epigenetics modifications in circadian manifestations of BRX.

Acknowledgments: This work was funded by grants from Dirección de Investigación & Desarrollo, Universidad de La Frontera, Temuco, Chile (DI14-0059).

Reference: 1. Oporto G, Lagos J, Bornhardt T, Fuentes R, Salazar LA. Are there Genetic Factors Involved in Bruxism? Int J Odontostomat; 2012; 6:249-54.

CLOCK GENES AND SLEEP DISTURBANCES IN AUTISM SPECTRUM DISORDER

Ballester P.¹,², Martínez E.¹, Muriel J.¹, López-Gil V.¹, López-Gil S.¹,Planelles B.², Inda M.M.², Peiró A.M³

(1) Occupational Observatory, University Miguel Hernandez (UMH), Spain;

(2) Foundation for the Promotion of Health and Biomedical Research of the Valencian Community (FISABIO), Spain;

(3) General Hospital of Alicante (HGUA), Spain;

Introduction: Subjects with Autism Spectrum Disorder (ASD) have the circadian cycle affected. Sleep-related problems (SRP) affect up to the 84%. The molecular mechanisms of the circadian cycle involve clock genes and melatonin route genes. SRP encompass a wide range of conditions that affect to sleep parameters and have been categorized in the International Classification of Sleep Disorders.

Aim: Evaluate the relationship between SNPs in ASMT, Per1, Npas 2, and MTNR1A genes and SRP tracked with actigraphy.

Methods: A case control study was performed in subjects with ASD (n=25, 80%♂, mean age of 32±2 years) matched with controls (n=26, 62% ♂, mean age of 33±9 years). Sleep parameters (Sleep Onset Latency (SOL), Sleep Efficiency (SE) and Total Time Sleep (TTS)) were measured by actigraphy and sleep diaries during a week. DNA was extracted from peripheral blood and genotyping of the ASMT (rs444690 and rs5989681), Per1 (rs6416892 and rs885747), Npas2 (rs1811399), MTNR1A (rs28383652) genes was performed by Multiplex PCR.

Results: Significant differences (p< 0.05) were found in sleep parameters from ASD compared to controls (SOL 67±14 vs 17±2 minutes; TTS 465±100 vs 404±58 minutes and SE 79% vs 93%). Recessive genetic model from rs5989681 and overdominant genetic model from rs6416892 and rs885747 show a relation between genotype and SOL (p 0.04; p 0.02 and p 0.02).

Conclusions: Actigraphy is an objective and useful tool to analyze SRP in ASD population. Apparently, there is a relation between SOL Per1 and ASMT, but further genetic analysis is required.

Multi-omics, multi-tissue association study identifies novel epigenetic candidate loci and genomic structural variants implicated in dilated cardiomyopathy and heart failure

AE Posch1, J Haas2, F Sedaghat-Hamedani2, S Mester2, C Dietrich 1, Alan Lai2, E Kayvanpour2, D Pils3, K Frese2, E Wirsz1, M Wuerstle1, M Matzas1, A Keller4, HA Katus2, B Meder2

¹Siemens Healthcare, Strategy and Innovation, Erlangen, Germany, ²University Hospital Heidelberg, Department of Cardiology, Heidelberg, Germany, ³Siemens AG, Corporate Technology, Vienna, Austria, ⁴Saarland University, Chair for Clinical Bioinformatics, Saarbruecken, Germany

Background: Dilated Cardiomyopathies (DCM) can be caused by single nucleotide variations (SNV) in one of more than 40 different genes. Besides SNVs and small coding insertions and deletions, it is postulated that epigenetic modification as well as larger structural variants (SV) could be involved in the pathogenesis of DCM¹.

Objective: The objective was to identify novel susceptibility regions both on the structural genomic and epigenetic level that are linked to myocardial dysfunction and heart failure as well as potential biomarker candidates.

Design: To elucidate the contribution of structural variants to DCM, we performed whole-genome sequencing in a cohort of n=50 consecutive patients with primary DCM. Additionally, we performed mRNA transcriptome sequencing and DNA methylation profiling using the Illumina 450k chip of the same myocardial specimens of the left ventricle and from whole blood in each patient and control (n=31).

Results: Using a stringent multi-omics, multi-tissue approach and by validation in independent samples, a set of high-confidence candidate loci that are epigenetically regulated in DCM could be identified. Besides known disease-causing point mutations in 12 patients, we also detected 3,898 genomic structural variations. Additionally, we could show the functional effect of SVs on important cardiac genes and novel DCM candidates.

Conclusions: The present study provides to our knowledge the most comprehensive mapping of DNA methylation in human heart tissue and identifies novel loci associated with heart failure and DCM using a comprehensive approach covering genetic variation, DNA methylation and whole-transcriptome analyses. Additionally, we demonstrate the effect of SVs on cardiac gene transcription and its association with human Dilated Cardiomyopathy. It will be interesting to dissect large-scale cohorts to define exact frequencies of SVs that we could link to human Dilated Cardiomyopathy for the first time as well as to validate the identified epigenetic biomarker candidates.

Reference: 1. Haas et al. Atlas of the clinical genetics of human dilated cardiomyopathy. Eur Heart J. 2015 May 7;36(18)

Metabolomics as a new key player in systems biology for better understanding nutrition-health relationship

E Pujos-Guillot1,2, M Pétéra2, M Brandolini2, Y Liu1, F Giacomoni2, B Comte1

¹INRA, UMR1019, Mapping, F-63000 Clermont-Ferrand, France; ²INRA, UMR1019, PFEM, F-63000 Clermont-Ferrand, France

Background: Human body responses to nutrition and chronic metabolic disorders represent complex features that require integrated systems biology approach to better understand their relationship. Metabolomics, as a powerful phenotyping tool offers now the possibility of characterizing global alterations associated with nutritional exposure and disease conditions, and of identifying early and/or predictive biomarkers of disease development [1].

Objective: The objective of this work was to perform a proof of concept study of the use of untargeted metabolomics as part of an integrated strategy for identifying predictive biomarkers of the evolution toward Metabolic Syndrome (MetS), and bring new knowledge about this pathological state.

Design: A nested case-control approach was used within the French GAZEL cohort (n~20,000): at risk male subjects (n=112, 54–64 years old) with high body mass index (BMI, 25 ≤ BMI<30), free of MetS at baseline, were selected. Cases who developed MetS at the follow-up (5 years later) were compared with Controls (matched for BMI and age) for several parameters (clinical, biochemical parameters, and food habits). Baseline serum samples were analyzed using mass spectrometry-based untargeted metabolomics. A full bioinformatics workflow was optimized to process the data. In particular, data mining methods were used to select the best candidate for prediction. Metabolomic data were then integrated with the different parameters from the database to determine whether multidimensional models improve prediction and impact subject stratification.

Results: Metabolomic data allowed improving prediction capacity compared to the use of clinical data only. The multidimensional model integrating metabolomics with anthropometric, biochemical and nutritional data showed the best prediction performances. The analysis of the misclassified subjects revealed sub-phenotypes and generated hypotheses about the disease associated feature space, that need further characterization in order to propose adequate patient sub-stratification and prevention.

Conclusion: Metabolomics has the potential to become a key player in systems diagnosis strategies. Integration of metabolic profiles into multidimensional models can be an essential tool for a patient-centered personalized prevention.

[1]Ramautar R, Berger R, van der Greef J, Hankemeir T. Curr Opin Chem Biol. 2013;17(5):841-46.

Role of Enterococcus faecalis in endodontic treatment prognosis of devitalized teeth

CA Rodríguez-Niklitschek¹, GH Oporto V ^2,3, L Salazar².

¹Escuela de Odontología, Facultad de Ciencias, Universidad Mayor, Temuco, Chile; ²Centro de biología molecular y farmacogenética, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile, ³Centro de Investigación en Ciencias Odontológicas (CICO), Facultad de Odontología, Universidad de La Frontera, Temuco, Chile.

Background:Enterococcus faecalis (EF) has been associated with endodontic treatment failure (ETF) in devitalized teeth. Antimicrobial resistance is a worldwide issue. Has been reported that EF would be resistant to medications used into root canals (RC) during endodontic treatments. Thus, EF has the ability to remain in quiescent state into RC. Researchers suggest that these characteristics are due to the expression of certain genes and the operation of a proton bomb. The ability of EF to survive in unfavorable environments and in presence of intra-canal medications, support its role in ETF. Thereby, is important to know the prevalence of EF in teeth that needs an endodontic treatment.

Objective: To determine the presence of EF in devitalized teeth with endodontic treatment indication.

Design: Were obtained 85 samples of RC of devitalized teeth from dental teaching clinics of Universidad Mayor, Temuco, Chile and from a private dental clinic in Temuco, Chile. The samples were classified according to diagnosis in dental pulp necrosis (DPN), periapical granuloma (PG), acute periapical abscess (APA), chronic periapical abscess (CPA) and endodontic failure. Bacterial culture was performed using Enterococcocel agar medium and Potassium Tellurite medium.

Results: The pulpal diagnosis of teeth treated in University were different compared with treatment performed in the private clinic (p=0.01, chi-square test), DPN and PG were more frequent in the University (p=0.007, Fisher´s exact test) and CPA was more common in the private clinic (p=0.003, Fisher´s exact test). The highest frequency of EF was obtained in PG, and the lowest in APA (p=0.035, chi-square test).

Conclusions: EF is associated to PG diagnosis. An effective elimination of EF is necessary prior to RC filling, to reduce the risk of ETF. Further researches must be performed to develop more effective intra-canal medications. Besides, is mandatory to determine the mechanisms involved in resistance of EF to medications used in endodontics.

Acknowledgements: Fondo de Investigación y Desarrollo, Universidad Mayor (FIDUM 100901).

Reference: 1. Rodriguez-Niklitschek C., Oporto VGH. Clinical implications of Enterococcus faecalis microbial contamination in root canals of devitalized teeth: Literature Review. Rev Odont Mex 2015;19:177-182.

Different distribution of dopamine receptor D2 (DRD2) gene polymorphisms between smokers and drug dependent subjects among Malay male population in Kelantan, Malaysia

AB Ruzilawati¹, MS Deeza-Syafikah¹, MSK Sophia¹ and A Imran²

¹Department of Pharmacology and ²Department of Family Medicine,

School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia.

Background: Drug addiction and cigarette smoking remain worldwide problems, and their negative impacts on society are increasing. A common pathway for the reinforcing properties of most addictive drugs is the mesolimbic brain dopamine system. The system has also been implicated in the reinforcement of the effects of nicotine in smoking. The systems also have been shown to be involved in the reward pathway of drugs of abuse. One of the genes, dopamine receptor D2 (DRD2) gene has been reported to be one of the plausible candidate genes for nicotine dependence as well as genes involved in the rewarding effects of drugs of abuse.

Objective: The objective of the present study was to compare the allelic frequencies and genotypes of DRD2 gene polymorphisms between smokers and drug dependent subjects among Malay male population in Kelantan, Malaysia.

Design: 130 Malay male smokers and 100 Malay male male drug dependent subjects were recruited. All subjects were given informed consent form to participate in the study. The study was approved by Ethics Committee of School of Medical Sciences, Universiti Sains Malaysia. Malaysia. DNA was extracted from leucocytes and the allele was determined by a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). The PCR product was digested with restriction enzymes TaqI. The statistical analyses was performed using the SPSS package version 20.0.

Results: Interesting difference in gene distribution between the two groups was discovered. 49% of the smokers were found to have homozygous DRD2 A1/A1 genotype while 55% of drug dependent subjects were found to have heterozygous DRD2 A1/A2 genotype. There existed significant differences both in the frequencies of alleles and genotypes between subjects.

Conclusions: To conclude, it appears that there was a difference in gene distribution of dopamine receptor D2 (DRD2) between smokers and drug dependent subjects among Malay male population in Kelantan, Malaysia

Acknowledgements: This study was financially supported by Fundamental Research Grant Scheme, Ministry of Higher Education, Malaysia (grant no. 203/PPSP/6171125) and Research University Grant, Universiti Sains Malaysia, Malaysia (1001/PPSP/812171).

Reference: 1. Missale C, Nash RS, Robinson SW, Mohamed J, Marc GC. Dopamine receptors: from structure to function. Physiol Rev 1998; 78: 189-225

MiRNAs as potential salivary biomarkers in periodontitis

LS Santos¹, L Alves¹, D Martins², N Rosa¹, M Barros¹, MJ Correia¹.

¹Universidade Católica Portuguesa, Centro de Investigação Interdisciplinar em Saúde, Instituto de Ciências da Saúde, 3504-505 Viseu, Portugal. ²Departamento de genética e biotecnologia, Universidade de Trás os Montes e Alto Douro, 5001-801 Vila Real, Portugal.

Background: MiRNAs are small noncoding-RNAs that regulate gene expression. Their expression is frequently altered in disease, translating into distinct miRNA expression profiles. MiRNAs are present in extracellular fluids such as saliva, where they also correlate with various physiopathological conditions. Specific salivary miRNA signatures could thus be defined and used as risk, diagnostic, prognostic or treatment response biomarkers.

Objective:Through systematic literature review and a computational approach, we aimed to define a comprehensive set of miRNAs with potential use as salivary biomarkers for periodontitis. Design:PUBMED and Web-of-Science databases were searched using the expression (MicroRNA* OR mirna* OR miR) AND (Periodont* OR Gingiv*) AND Inflam*. Additional articles were selected from the reference lists. After applying predefined inclusion/exclusion criteria, the resulting 19 studies were searched for miRNAs with differential expression (p<0.05 on qRT-PCR) in periodontitis patients or cell cultures (upon bacterial stimulation). Presence in saliva was checked from an external database[1]. MiRWalk was used to identify periodontitis-associated validated targets and other miRNAs targeting periodontitis-associated genes. Results:42 miRNAs were differentially expressed, 36 of which had validated periodontitis-associated targets. All but one were previously detected in saliva. hsa-miR-146a-5p was the most promising candidate: it fulfils all three criteria and is differentially expressed in all types of studies, with agreeing replication. Consistent evidence also exists for hsa-miR-146b-5p and hsa-miR-223-3p. 406 other miRNAs are present in saliva and target other periodontitis-associated genes: 37 of these have 4 or more validated periodontitis-associated targets.

Conclusions:We provide a comprehensive set of miRNAs that 1)are differentially expressed either in periodontitis or in cell cultures upon bacterial stimulation, 2)are present in saliva and 3)target periodontitis-associated genes. Validation of these miRNA as salivary biomarkers for periodontitis will require further studies to confirm differential disease-associated salivary expression and to assess the corresponding sensitivity and specificity in clinical context. Translation of saliva-based biomarkers to clinical practise will allow the development of fast, noninvasive and inexpensive omics-based screening tests for early diagnosis and personalised periodontal management.

Reference:[1]Bahn JH, Zhang Q, Li F, Chan TM, Lin X, Kim Y et al. The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva. Clin Chem 2015;61(1):221-30.

Identification of miRNAs with potential utility as salivary biomarkers for type II diabetes

A Martins¹, LS Santos¹, N Rosa¹, M Barros¹, MJ Correia¹.

¹Universidade Católica Portuguesa, Centro de Investigação Interdisciplinar em Saúde, Instituto de Ciências da Saúde, 3504-505 Viseu, Portugal.

Background:MiRNAs are small noncoding RNAs that mediate post-transcriptional gene expression regulation. They play a central role in diverse biological processes and may be present in extracellular fluids such as saliva. Since the spectrum of salivary miRNAs correlates with physiopathological state, well-defined panels of salivary miRNAs could be used as risk, diagnostic, prognostic or treatment response biomarkers. Salivary miRNAs are therefore are highly attractive for clinical application.

Objective:Through systematic literature review and a computational approach, we aimed to define a set of miRNAs to be evaluated as potential biomarkers for type II diabetes mellitus (T2DM).

Design:PUBMED and Web-of-Science databases were searched using a combination of the MeSH terms “Diabetes Mellitus, Type2” and its synonyms AND MicroRNAs OR Insulin resistance OR Hyperinsulinemia OR Glucose intolerance OR Hyperglycaemia. 776 articles were retrieved, 31 of which complied with predefined inclusion/exclusion criteria. MiRNAs differentially expressed between T2DM patients and normal glucose tolerance (NGT) controls (p<0.05 on qRT-PCR validation) were identified, excluding conflicting results. An external database[1] was used to check for miRNAs presence in saliva and miRWalk was used to identify T2DM-associated validated targets of such miRNAs. miRWalk was also used to identify other salivary miRNAs targeting T2DM-associated genes. Results:49 miRNAs were differentially expressed in T2DM. Among these, according to miRWalk, 19 have a T2DM-relevant disease ontology annotation, regulating 11 different genes. All but one of these miRNAs were previously detected in saliva, making them suitable as salivary biomarkers. 162 other salivary miRNAs also target T2DM-associated genes: 21 of these have 2 or more validated T2DM-associated targets.

Conclusions:We provide a comprehensive set of miRNAs that are differentially expressed in T2DM patients, target T2DM-associated genes and were already detected in saliva. Validation of these miRNAs as salivary biomarkers for T2DM will require further studies to confirm differential salivary expression in T2DM and to assess sensitivity and specificity in clinical context. Translation of saliva-based biomarker assessment to clinical practise will allow the development of fast, noninvasive and inexpensive omics-based screening tests for early diagnosis and personalised T2DM management.

Reference:[1]Bahn JH, Zhang Q, Li F, Chan TM, Lin X, Kim Y et al. The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva. Clin Chem 2015;61(1):221-30.

The Study of Protective Effects of Grape Seed Extract Against Damage of the Organism Caused by Heavy Metals in Rattus Norvegicus

J. Sochor¹, L. Tomaskova¹, M. Baron¹

¹Department of Viticulture and Enology, Faculty of Horticulture, Mendel University in Brno, Valticka 337, Lednice CZ-691 44, Czech Republic, European Union

Background: Vitis vinifera seeds contains very high content of antioxidants [1]. They have a higher antioxidant potential than common kind of fruits and vegetables. Cadmium (Cd) is one of the most prevalent toxic metal.

Objective: The study explored the protective effects of grape seed extract (GSE) against Cd toxicity in the organism of Rattus norvegicus.

Design: Male adult Wistar rats (n=16) were devided into four equal groups. The first group was control, the second was treated with GSE (0.5 g of lyophilized powder/rat/per day), third was treated with CdCl₂ (10 mg CdCl₂ /rat/per day), and fourth CdCl₂ (10 mg CdCl₂ /rat/per day) plus GSE (0.5 g of lyophilized powder/rat/per day). Content of Total Bilirubin, Amino Aspartate Transferase (AST), Alkaline Phosphatase, Alanine Aminotransferase (ALT), Albumine, Glutathione Peroxidase (GPx), Superoxide Dismutase (SOD), Catalase and Antioxidant Activity were determined in blood of rats.

Results: After 28 days, results significantly showed damage of organism of Rattus threated by CdCl₂. Content of Total Bilirubin, Liver Enzymes, Antioxidant enzymes and Albumin were increased in the Cd groups. The results showed that Cd could increase oxidative stress. Among the most significant indicator appears ALT, AST, GPx and SOD where group with CdCl₂ had more than 3 times higher content in blood. GSE had a protective effect itself, results indicate decreased levels of antioxidant enzymes in the blood of Rattus. Add Cd together with GSE caused, that GPx and SOD was only 50% higher than control group.

Conclusions: This study concluded that GSE has beneficial protective effects against damage of the organism caused by heavy metals in Rattus norvegicus.

Acknowledgements: This work was funded through the International grant project of Mendel University in Brno

Reference: 1. Garcia-Jares C, Vazquez, A. Antioxidant White Grape Seed Phenolics: Pressurized Liquid Extracts from Different Varieties. Antiox DEC 2015; 4040737: 737-49.

A multimarker approach for the prediction of 30-day major adverse cardiac events in patients with ST-segment elevation myocardial infarction: cost-effectiveness analysis

S. Stankovic¹, D. Trifunovic², M. Asanin²

¹Center for Medical Biochemistry, Clinical Center of Serbia, 11 000 Belgrade, Serbia

²Clinic of Cardiology, School of Medicine University of Belgrade, Clinical Center of Serbia, 11 000 Belgrade, Belgrade, Serbia

Background: Despite advances in treatment, acute coronary syndromes, which consist mainly of ST-segment elevation myocardial infarction (STEMI) and unstable angina/non-STEMI, present an enormous medical, social, and economic burden worldwide. Primary percutaneous coronary intervention (pPCI) is a therapy of choice for the management of patients with acute STEMI. Despite the improvement in morbidity and mortality in these patients, groups at high risk of complications and adverse clinical events remain. Identification of patients at risk for major adverse cardiovascular events (MACE) might help selecting candidates for aggressive treatment which may improve outcome or early discharge after pPCI. With expansion of the number and types of circulating biomarkers available, the opportunity to improve risk stratification continues to grow.

Objective:To explore discriminative abilities of several biomarkers of inflammation and hemodynamic stress as predictors for MACE in patients with STEMI treated by pPCI. Also, to assess their cost-effectiveness compared with the RISK-PCI score for the prediction of MACE during a 30-day follow-up after pPCI.

Design: Using a decision model, the costs, accuracy, and cost-effectiveness of each model was evaluated. The RISK-PCI score was used as the baseline model. Other models were formed with the consecutive addition of selected markers: myeloperoxidase (MPO), high sensitive C-reactive protein, interleukin-6, adiponectin, oxidized LDL, B-type natriuretic peptide (BNP), N-terminal-proBNP to the baseline model. A best-case model was formed from a combination of biomarkers to yield the best patient stratification algorithm. All models were assessed by their predictive probabilities using receiver operating characteristic curves. One hundred fifty STEMI patients treated by pPCI was included in the study. Composite 30-day MACE was defined as cardiac death, non-fatal reinfarction, and target vessel revascularization. The analysis was performed from a third-party payer perspective.

Results: Only two strategies had outstanding discriminative abilities: the best-case model (RISK-PCI score+BNP+MPO) and RISK-PCI score plus BNP with area under the curve (AUC) values of 0.869 and 0.851, respectively. The cost-effectiveness ratio varied between 5199 € per AUC for the baseline model to RSD 9011€ per AUC for RISK-PCI score+NT-proBNP. After elimination of dominant strategies, the incremental cost-effectiveness ratio (ICER) for the remaining three strategies (baseline, RISK-PCI score plus BNP, and the best-case model) were calculated. For the RISK-PCI score plus BNP, the ICER (compared with the baseline model) was 18106 € per additional accuracy calculated for 100 analyses. The ICER for the best-case model (compared with the baseline model) was 84961€ per additional accuracy calculated for 100 analyses. Strategy involving hemodynamic stress biomarker BNP was more cost-effective than strategies involving inflammatory markers. Sensitivity analysis indicated that results were robust.

Conclusion: The obtained results support the feasibility of a multimarker approach for MACE prediction in STEMI patients treated by pPCI. The introduction of BNP in the clinical laboratory would be convenient and cost-effective.

Reference: 1. Mrdovic I, Savic L, Krljanac G, et al. Predicting 30-day major adverse cardiovascular events after primary percutaneous coronary intervention. The RISK-PCI score. Int J Cardiol 2013;162(3):220-7.

The VEGF Consortium

MG Stathopoulou¹, B Alizadeh², J Casas³, G Dagher⁴, P Deloukas⁵, F Innocenti⁶, J Lamont⁷, J Marc⁸, J-L Merlin⁹, J Schneider¹⁰, M Simmaco¹¹, RHN Van Schaik¹², M Ciullo¹³, S Seshadri¹⁴, S Siest¹

1. UMR INSERM U1122; IGE-PCV, Nancy, France

2. University Medical Center of Groningen, Groningen, The Netherlands

3. University College London, London, UK

4. Biobanking and Biomolecular Resources Research Infrastructure, Paris, France

5. Queen Mary University of London, London, UK

6. University of North Carolina at Chapel Hill, North Carolina, USA

7. Randox Laboratories Limited, Crumlin, UK

8. Univerza V Ljubljani, Ljubljana, Slovenia

9. Institut de Cancérologie de Lorraine, Nancy, France

10. University of Luxembourg, Luxembourg, Luxembourg

11. Universita degli studi di Roma, Rome, Italy

12. European Society of Pharmacogenomics and Personalised Therapy (ESPT), Nancy, France

13. Genetics of Complex Traits Laboratory at IGB- National Research Council of Italy, Naples, Italy.

14. Boston University, Boston, USA

Background: The Vascular Endothelial Growth Factor (VEGF) is a pivotal mediator of angiogenesis, known to impact both on physiological processes and on the development of complex chronic diseases.

The Vascular Endothelial Growth Factor European Genomic Federation - VEGF Consortium (http://www.vegfconsortium.org aims to develop a transnational collaborative network dedicated to large integrative and multidisciplinary genomic studies on VEGF in order to generate actionable knowledge for medical practice of chronic diseases.

Objectives: To identify VEGF ‘-omics’ profiling in health and disease to be used for patient stratification.

Design: Fourteen partners from 8 countries are involved in the consortium, representing ten internationally recognized cohorts totaling >15000 individuals. These partners are among the worldwide leaders in the fields of genomics, transcriptomics, epigenomics, “-omics” methodologies and analyses, chronic diseases prediction and prevention, personalised medicine, personalised therapy, pharmacogenomics, clinical implementation, and communication.

Results: Through two Genome-Wide Association Studies, we have identified 10 genetic variants, collectively explaining up to 50% of VEGF phenotypic variation^{1, 2}. We have also demonstrated associations between these polymorphisms and cardiovascular (CVD) risk factors such as blood circulating lipid levels, as well as epistatic (gene×gene) interactions. Eleven working groups have been formed.

Conclusions: The VEGF consortium brings together a large number of high-level researchers and academics, along with their expertise and resources. It is expected to provide robust results on the role of VEGF in chronic diseases prediction, prevention and personalized therapy. It is an original concept open to further collaborations in order to advance research on this challenging field.

References:

1.Debette S*, Visvikis-Siest S*, Chen MH, Ndiaye NC, Song C, Destefano A, Safa R, Azimi Nezhad M, Sawyer D, Marteau JB, Xanthakis V, Siest G, Sullivan L, Pfister M, Smith H, Choi SH, Lamont J, Lind L, Yang Q, Fitzgerald P, Ingelsson E, Vasan RS, Seshadri S. Identification of cis- and trans-acting genetic variants explaining up to half the variation in circulating vascular endothelial growth factor levels.

Circ Res. 2011 Aug 19;109(5):554-63.

*equal first authors

2.Choi SH*, Ruggiero D*, Sorice R*, Song C, Nutile T, Vernon Smith A, Concas MP, Traglia M, Barbieri C, Ndiaye NC, Stathopoulou MG, Lagou V, Maestrale GB, Sala C, Debette S, Kovacs P, Lind L, Lamont J, Fitzgerald P, Tönjes A, Gudnason V, Toniolo D, Pirastu M, Bellenguez C, Vasan RS, Ingelsson E, Leutenegger AL, Johnson AD, DeStefano AL, Visvikis-Siest S, Seshadri S**, Ciullo M**. Six Novel Loci Associated with Circulating VEGF Levels Identified by a Meta-analysis of Genome-Wide Association Studies. PLoS Genet. 2016 Feb 24;12(2):e1005874.

*equal first authors

**equal last authors

SERT, DAT1 and COMT Polymorphisms in Psychiatric Patients

M Štefanović¹, J Culej¹, D Karlović²

¹Clinical Institute of Chemistry and ²Department of Psychiatry, Sestre milosrdnice University Hospital Center, Zagreb, Croatia

Background:

Serotonin transporter is connected to serotoninergic neurotransmitter pathway. Best studied is the promoter region polymorphism, 5-HTTLPR, a repetitive element of varying length (short or S allele, or long or L allele) in the promoter of SLC6A4. S allele was shown to be associated with a variety of emotional disorders and psychopathological traits such as depression, suicidality, bipolar affective disorder and some others, but the findings were inconsistent.

On the other side, dopaminergic pathway indicators connected with psychiatric disorders include Dopamine Transporter (DAT) and Catechol-O-methyl Transferase (COMT). DAT transports dopamine into presynaptic neurons, while COMT is involved into dopamine degradation. There are two most investigated polymorphisms which were reported to affect dopaminergic pathway and psychiatric disorders: DAT1 VNTR (rs28363170) and COMT Val158Met (rs4680). Research of DAT1 VNTR showed relationship of 9 or 10 repeats allele with higher gene expression levels that leads to higher DAT protein availability. COMT Val158Met polymorphism showed to be related to higher levels of dopamine (Met allele).

Objective: As research results of genetic polymorphisms for all three genes were found to be inconsistent across literature, when trying to relate them to psychiatric disorders, we tried to investigate these relationships in context of our healthy subjects and patient’s data diagnosed with depression and schizophrenia.

Design: We compared genetic polymorphism distribution in healthy subjects vs patients diagnosed with depression and schizophrenia for three genes: 5-HTTLPR, S/L polymorphism (Healthy subjects=303, Depression=298), DAT1 VNTR, alleles 6, 9, 10, 11 (Healthy subjects=295, Schizophrenia=303) and COMT, Val158Met (Healthy subjects=306, Schizophrenia=299)

Results: For 5-HTTLPR, S/L polymorphism when compared depression vs schizophrenia vs control group we found 60(38%) vs 118(40%) vs 105(35%) LL genotypes; 81(42%) vs 120(40%) vs 145(48%) LS genotypes and 16(10%) vs 59(20%) vs 53(18%) SS genotypes; with no statistical significance among groups of subjects.

For DAT1 VNTR, alleles 6, 9, 10, 11 when compared schizophrenia vs control we found 153(51%) vs 146(50%) 10/10 genotypes; 129(43%) vs 119(40%) 9/10 genotypes; 14(5%) vs 25(9%) 9/9 genotypes and 7(2%) vs 5(2%) other (9/6, 9/11, 10/6, 10/11) genotypes, with no statistical significance among groups of subjects.

For COMT, Val158Met polymorphism we found 87(28%) vs 78(26%) Met/Met genotypes; 140(46%) vs 158(53%) Val/Met genotypes; 79(26%) vs 63(21%) Val/Val genotypes, also with no statistical significance among groups of subjects.

Conclusions: Unlike some other studies, we found no statistical significance of investigated genetic polymorphism relationship with any psychiatric disorders for all of three studied genes.

Serum fibroblast growth factor-21 is associated with renal sinus fat increasment independently of total intraabdominal obesity

Krievina G.^1,2, Tretjakovs P.^1,2, Skuja I.¹, Silina V.¹, Bahs G.¹

¹ Riga Stradins University

² University of Latvia; Institute of Cardiology and Regenerative Medicine

Pathways through which obesity might cause renal disease are not well understood. Recent studies have associated ectopic lipid accumulation in the kidney with obesity-related renal disease. Human studies indicate that circulating levels of fibroblast growth factor-21 (FGF21) increased in obese individuals. FGF21 was found to closely associated with renal dysfunction in end-stage renal disease subjects. We hypothesised that renal sinus (RS) fat volume may be independently associated with increased level of FGF21.

The study included 110 subjects (60/50 F/M; age of 39.8±5.8). CT images were captured and RS fat accumulation was measured using the 3D-Doctor software. Both kidneys and RS fat were measured, and ratio left kidney sinus fat/left kidney (LS/LK) and right kidney sinus fat/right kidney (RS/RK) were calculated. Intraabdominal (IA) fat volume was measured at the level of renal hilus. FGF21 serum level was detected by ELISA assay.

Partial rank correlation was used to adjust the association between LS/LK, RS/RK and FGF21 after accounting for the IA fat volume. According on sex-specific 75^th percentiles of FGF21 levels all measurements were divided into two groups and analysed by Mann-Whitney U test.

FGF21 correlated with both LS/LK and RS/RK ratios (r=0.50, p<0.001; and r=0.45, p<0.05). There were significant (p<0.05) increasment of both LS/LK (0.0075; (0.0019; 0.0145) and 0.0142 (0.0077; 0.0332))* and RS/RK (0.0027; (0.0003; 0.0051) and 0.0084 (0.0017; 0.0311))* ratios when data were divided according on sex-specific 75^th percentiles (234.32 pg/ml) of FGF21.

Taken together, these results suggest that serum FGF21 level may be increased in individuals with reduced renal function because of the increased fat accumulation in the renal sinuses.

* Data are presented as median (25^th, 75^th percentiles)

Funding by the grant No. 2014.10-4/VPP-5/1 (2014-2017) BIOMEDICINE of the framework of the Latvian National Program

Nutrigenomics and epigenetics in obesity treatment strategy

Iwona Wybranska

Jagiellonian University, Medical College, Kraków, Poland

Recently it has been suggested the existence of mammalian polyphenism in energy metabolism which might have implications for strategies to limit the obesity epidemic. Epigenetic marks, including CpG methylation, have been usually considered stable in somatic cells, although it has been observed that some environmental factors can cause variation or reversibility in the DNA methylation patterns in postnatal life. Epigenetic mechanisms might contribute to the worldwide increase in the prevalence of obesity. Thus, the pattern changes with ageing in a tissue-specific fashion, and an age-related increase in methylation is negatively associated with the expression of genes, suggesting that DNA methylation could be involved in increasing age dependent susceptibility to metabolic complications.

Epigenetic marks are tissue specific and include DNA methylation and histone modifications which mediate biological processes such as imprinting. As many imprinted genes are growth factors, or regulators of gene expression controlling growth, imprinting disorders often feature obesity as one of their clinical characteristics. The mechanisms by which nutritional challenges affect the risk of disease in later life are poorly understood. However, evidence indicates that the establishment of the epigenome can be affected by environmental factors during critical developmental periods. Possible disturbances of methylation may arise during fetal development due to lack of availability of dietary methyl donors. Potential interactions between the environment and epigenetic mechanisms mediating the expression of genes associated with increased BMI and adiposity, may also be possible as suggested for; the FTO locus is a DNA-demethylase enzyme, the MC4R gene which has reduced methylation following long-term exposure to a high fat diet, the PPARγ protein which interacts with histone acetyltransferases during adipogenesis and on the effect of diet on methylation of POMC and leptin. In the adult state, some examples of diet-induced epige-netic changes have also been reported. In this sense, folic acid (38,39) has been linked to DNA methylation in a dose-dependent manner, as reviewed by Kim et al. (40). Other dietary factors involved in DNA methylation within

the gastrointestinal tract and colorectal cancer develop-ment are alcohol (39), vitamin B 6 (41,42), vitamin A and some minerals (43). Selenium deficiency also seems to be an important modifier of methyl metabolism in some tissues (44), while arsenite deprivation hypomethylates Caco-2 cells (45).Finally, not only micronutrients can induce epigenetics changes. A threefold increase in the expression of the human manganese superoxide dismutase (MnSOD) gene has been associated with a decreased CpG methylation comparing a vegetarian with an omnivore group (46). Other dietary factors such as fatty acids are likely to par-ticipate in epigenetic regulation by DNA methylation (47). For instance, butyrate induced demethylation of RARbeta2 in cancer cells (48). Tocopherols have been related to epi-genetic modifications of histones (49). Other compounds present in different plant foods such as the isofl-avone genistein (50), tea polyphenols (51) and garlic’s diallyl disulfide (52) are also able to regulate DNA Other factors that are able to alter DNA methylation patterns such as inflammation, oxidative stress and hypoxia are exacerbated in adipose tissue of obese subjects. In this regard, the relationship between obesity and the epigenetic regulation of gene expression has been recently reported by our group, showing that the successful response to a hypocaloric diet could be related to a lower methylation of the FTO promoter in PBMC cells. CpG hypomethylation of FTO promoter has recently been also associated with resistance to T2DM.

Our next major goal is to see whether different diet or therapy may modify this process, whether we can turn the disease switch “on” or “off” by supplementing diet, minimizing stress, or giving epigenetically relevant pro-drugs.

Epistatic interactions between VEGF, LSR and APOE on blood lipid traits

T Xie¹, MG Stathopoulou¹, S Akbar², T Oster², FT Yen², Sophie Siest¹

1. UMR INSERM U1122; Interactions Gène-Environnement en Physiopathologie Cardio-Vasculaire (IGE-PCV), Université de Lorraine, Nancy, France.

2. EA3998 INRA USC 0340 UR AFPA, Université de Lorraine, Nancy, France

Background: The Vascular Endothelial Growth Factor (VEGF) is a pivotal factor for angiogenesis and is involved in different diseases such as tumor growth and vascular disorders. We have previously identified 10 single nucleotide polymorphisms (SNPs) that explain up to 50% of VEGF variability^1,2. We have also found associations between these SNPs and the levels of blood lipids. The levels of blood lipids, especially plasma cholesterol and triglycerides (TG) are risk factors for cardiovascular diseases (CVD). Apolipoproteins play an important role in the regulation of the processing and clearance of lipoproteins. Apolipoprotein E (ApoE) has been considered as an important risk factor for CVD. The lipolysis stimulated lipoprotein receptor (LSR) plays an important role in the removal of apoE-containing TG-rich lipoproteins during the postprandial phase.

Objectives: To investigate the role of potential epistasis of the GWAS published VEGF-related SNPs with LSR polymorphisms and with the established polymorphism of APOE on blood lipids levels in childhood and adulthood in healthy populations.

Design: Two populations from the STANISLAS Family Study (SFS) were used: unrelated healthy adults (n=432) and children (n=428, <18 years old).

Results: Epistatic interactions were identified in the models adjusted for age, gender, BMI and smoking between VEGF-related SNPs and LSR rs916147 for triglycerides in both children and adults, for apolipoprotein A1, CIII and E only in children, and for triglycerides, and apolipoprotein CIII only in adults. The interactions of VEGF SNPs with APOE were significant only in adults (triglycerides, cholesterol, apolipoprotein E).

Conclusions: These results indicating relationships between VEGF, APOE, and LSR reveal possible existence of complex molecular links between these three molecules for the regulation of lipid levels that is under investigation.

References:

1.Debette S*, Visvikis-Siest S*, Chen MH, Ndiaye NC, Song C, Destefano A, Safa R, Azimi Nezhad M, Sawyer D, Marteau JB, Xanthakis V, Siest G, Sullivan L, Pfister M, Smith H, Choi SH, Lamont J, Lind L, Yang Q, Fitzgerald P, Ingelsson E, Vasan RS, Seshadri S. Identification of cis- and trans-acting genetic variants explaining up to half the variation in circulating vascular endothelial growth factor levels.

Circ Res. 2011 Aug 19;109(5):554-63.

*equal first authors

2.Choi SH*, Ruggiero D*, Sorice R*, Song C, Nutile T, Vernon Smith A, Concas MP, Traglia M, Barbieri C, Ndiaye NC, Stathopoulou MG, Lagou V, Maestrale GB, Sala C, Debette S, Kovacs P, Lind L, Lamont J, Fitzgerald P, Tönjes A, Gudnason V, Toniolo D, Pirastu M, Bellenguez C, Vasan RS, Ingelsson E, Leutenegger AL, Johnson AD, DeStefano AL, Visvikis-Siest S, Seshadri S**, Ciullo M**. Six Novel Loci Associated with Circulating VEGF Levels Identified by a Meta-analysis of Genome-Wide Association Studies. PLoS Genet. 2016 Feb 24;12(2):e1005874.

*equal first authors

**equal last authors

Decrease of insulin sensitivity, from obesity through pre-diabetes - importance of Matsuda index

V Dimitrijevic-Sreckovic^1,4, H Janeski¹, B Sreckovic², I Soldatovic^3,4, N. Pajevic¹, P Djordjevic⁴

1. Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia, 2. Clinical Center Bezanijska Kosa, 3. Institute for Medical Statistics and Informatics, 4. Faculty of Medicine, Belgrade University, Serbia

Background: Increased insulin resistance (IR) and decreased insulin sensitivity (IS) characterise obesity and early glycoregulation disorders: impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). In this study we used Matsuda index of whole-body insulin sensitivity derived from the oral glucose tolerance test (OGTT), which represents a composite of both hepatic and peripheral tissue sensitivity to insulin.

Objectives: To examine IS and IR in obese and pre-diabetic patients, and analyze thrombotic, inflammatory factors, microalbuminuria and HbA1C as the risk factors for developing atherosclerotic complications.

Methods: The study included 434 obese individuals (age over 45, body mass index (BMI) >25 kg/m2) classified into three groups: I-obese (245); II-IFG (103); III- IGT (86). OGTT was used to evaluate the extent of disorder. IDF classification was applied for diagnosing MS, IR was determined by homeostatic model assessment (HOMA IR). IS was determined by Matsuda index of whole-body insulin sensitivity.

Results: Blood pressure, HbA1C and HOMA IR values followed the progression of disorder. WC: (I-103.9±15.5,II-105.7±17.9,III-100.5±16.4 cm), HbA1C (I-5.6±0.8,II-5.7±0.5,III 5.8±0.5), CRP (I-6.2±6.0,II-5.9±6.0,III-8.1±11.6mg/l), PAI-1(I-5.3±.2.3,II-4.5±3.4,III-4.6±2.0U/ml) and microalbuminuria (I-49.7±72.5,II-61.9±68.0,III-41.0±39.6mg/24h). Correlations: Statistical significance has been proved between groups: HOMA IR (p<0.0l), Matsuda index (p<0.0l), HbA1C (p<0.0l), microalbuminuria (p<0.105). MS was found (I-61%, II- 84.4%, III- 80.5%). In advanced stage of the disease patients with MS had increased IR and decreased IS: HOMA IR (I-6.0±5.3, II-6.1±3.7, III-8.8±9.5), Matsuda index (I-5.1±3.5, II-4.6±2.7, III-2.6±1.7).

Conclusion: Decreased IS presented as decreased Matsuda index, exists in obesity and decreases more in pre-diabetes. Correlations of insulinemia with WC and HOMA IR confirm the importance of abdominal obesity in disorder etiopathogenesis. Highly important correlation of 0’, 30’ and 120’ glycemia with HbA1C, HOMA IR confirms the connection of increased IR and decreased IS with the progression of glycoregulation disorder. Our early results indicate beneficial effects of Mediterranean diet on obesity, IS, glycoregulation, lipid status, blood pressure and antioxidant protection in primary prevention of diabetes mellitus type 2 and atherosclerosis.

Reference: Gutch M, Kumar S, Razi SM, Gupta KK, Gupta A. Assessment of insulin sensitivity/resistance. Indian J Endocrinol Metab. 2015 Jan-Feb;19(1):160-4.

The Role of ABCB1 (MDR1, P-glycoprotein) in the Disposition of Ciprofloxacin

Olga Efimkina^1*, Tiphaine Adam de Beaumais^2*, Catharina Scholl^3,4, Tatjana Lauck^3,4, Michael Steffens^3,4, Evelyne Jacqz-Aigrain², Julia Stingl^3,4on behalf of the TINN consortium

* : participated equally to the work

1: Institute for Pharmacology of Natural Products and Clinical Pharmacology, University Ulm, Germany

2: Department of Paediatric Pharmacology and Pharmacogenetics, Robert Debré hospital, Paris, France

3: Faculty Center for Translational Medicine, University Bonn Medical Faculty, Germany

4: Research Division, Federal Institute for Drugs and Medical Devices, Bonn, Germany

Background: The international survey “Treat Infections in Neonates” (TINN) aimed to optimize the ciprofloxacin treatment of neonates and particularly the vulnerable group of premature neonates. In these patients, ciprofloxacin is an antibiotic prescribed off-label for the treatment of severe infections due to multi-resistant Gram negative bacteria. Its pharmacokinetics is highly variable and crucial for drug efficacy and its safety profile is unknown in neonates.

Objective: P-glycoprotein (P-gp), also known as multidrug resistance protein 1 (MDR1), is an ubiquitously efflux pump and its role in ciprofloxacin pharmacokinetics is still to be determined. In the present study, we investigated in human lymphocytes the impact of P-gp expression and polymorphism on ciprofloxacin intracellular concentrations and cell sensitivity. The influence of P-gp polymorphism was also studied on pharmacokinetic parameters calculated in neonates treated by ciprofloxacin.

Design:In vitro sensitivity of lymphoblastoid cell lines (LCLs) was evaluated by growth inhibition assay: 100 LCLs were incubated for 72 h with different concentrations of ciprofloxacin (10, 100, 300 and 600 µM). Intracellular concentrations of ciprofloxacin were analyzed by HPLC-UV. P-gp expression was evaluated by quantitative PCR and genetic variants (rs1045642, rs2032582, rs1128503, rs3842, rs1922242, rs2032583) were tested using a real-time TaqMan allelic discrimination method. Clearance (CL) and volume of distribution at steady state (Vss) calculated by population modeling were determined in 50 neonates included in the pharmacokinetic TINN study.

Results: The sensitivity of LCLs to ciprofloxacin (IC₅₀) was dependent on the basal expression of P-gp: cells with low P-gp expression showed significant higher sensitivity to ciprofloxacin, compared to cells with higher gene expression (p= 0.038). This sensitivity of LCLs was associated with one variant of P-gp (rs1045642): cells carrying the genotype CC showed higher sensitivity to ciprofloxacin than those with wild-type genotype. The same tendency was observed with the variant rs2032583 (p=0.052). Furthermore IC50 correlated with intracellular maximum concentration (Cmax) of ciprofloxacin (p=0.013) and both P-gp variants described above impacted C_maxof ciprofloxacin.

In neonates, a different P-gp rs3842 impacted significantly ciprofloxacin clearance (p=0.04) and volume of distribution at steady-state (p=0.02).

Conclusion: P-gp polymorphism has a significant impact on intracellular concentrations of ciprofloxacin in vitro and neonatal clearance in vivo. Pharmacogenetics should be explored as a tool to optimize drug efficacy in neonatal treatment.

Acknowledgements: This work was funded through the TINN project.

Reference: Zhao, W., et al., Population pharmacokinetics of ciprofloxacin in neonates and young infants less than three months of age. Antimicrob Agents Chemother, 2014. 58(11): p. 6572-80.

Insights into the biology and therapeutic implications of mediators and effectors of pro-metastatic focal adhesion signaling

Moulay Alaoui-Jamali, Chia-Hao Chang, Krikor Bijian

Departments of Medicine, Oncology, and Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal, Canada

Early stage progression of primary cancer to metastatic forms is initiated by excessive cancer cell locomotion in response to chemotactic signals received from cancer tissue microenvironment. In general, invasive motile cancer cells exhibit excessive finger-like plasma membrane protrusions that are stabilized by focal adhesions (FAs), a network of dynamic protein complexes formed near leading edges of cell protrusions to couple cell matrix receptors to the actin cytoskeleton. Synchronous assembly/disassembly of FAs at cell protrusions is essential for the generation of traction forces necessary for cancer cell locomotion and spread. In preclinical models, metastatic breast cancer cells express rapid disassembly rates of FA proteins at leading edges of cell protrusions. We identified members of the Rab-GTPase family, which are amplified in a subset of breast cancer subtypes, to contribute to accelerated FA disassembly via their recruitment to lysosomal and autophagosomal compartments for degradation, leading to a rapid FA turnover and excessive cell locomotion and invasion. Using genetic and pharmacological (small molecule inhibitors) approaches we established that inhibition of Rabs and autophagy prevented FA disassembly and efficiently inhibited cancer cell locomotion, as well as cancer progression to metastasis in animal models. The translational implications of these findings will be presented. Supported by the Quebec Breast Cancer Foundation and the Canadian Institutes for Health Research.

Novel genetic variations in the promoter regions of solute carrier transporter genes within the Cape Admixed Population of South Africa

B Pearce, CW Jacobs, N Hoosain and M Benjeddou

Pharmacogenetics Laboratory, Department of Biotechnology, University of the Western Cape

Background:Human solute carrier transporters (SLCs) are important determinants of organic cation and anion uptake from the circulation into the renal proximal tubules. These organic an/cations include clinical drugs such as metformin, lamivudine, cimetidine, cisplatin and the neurotoxin 1-methyl-4-phenyl-pyridinium. Considerable interindividual variation exists in drug responses and toxicity. Genetic polymorphisms in drug transporters are increasingly being recognized as a possible mechanism explaining this variation in drug disposition and response. Genetic variants in the basal promoter region, in particular, may have significant effects on the expression levels of drug transporter genes. This may result in changes in the activity levels of drugs that are substrates for these transporters and possible reduction in the associated clinical responses.

Objective:The aim of this study was to sequence the basal promoter region of SLC22A1, SLC22A3, SLC47A1, SLC47A2, SLCO1B1 and SLCO1B3 genes and to assess the extent of genetic variation in these genes within the Cape Admixed (CA) population.

Materials and methods:Ninety six unrelated healthy subjects From the CA population were recruited for the study. The 5'-UTR regions of selected SLC genes were amplified using specifically designed PCR primers. The PCR products were sequenced using Sanger sequencing.

Results:Sequence analysis revealed 26 variations, of which 7 were novel mutations. Nineteen of the SNPs identified in this study were already reported and listed in the dbSNP database. The allele frequencies for these variants were compared to those observed in other admixed, African, Asian and Caucasian populations.

Conclusion:This study represents the first report of novel polymorphisms in slected SLC genes within the CA population. The data observed suggest that drugs which are substrates for these SLC transporters with novel genetic variants may exhibit different response profiles within this population.

Length variations of 3′-UTRs of ABC-drug transporters and consequences in microRNA regulation and pharmacotherapy resistances

O Bruhn¹, M Lindsay¹, K Drerup¹, C Röder², I Cascorbi¹

¹Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel, Germany, ²Division of Molecular Oncology, Institute for Experimental Cancer Research, University Hospital Schleswig-Holstein, Campus Kiel, Germany

Background: The 3′-UTR is an important regulatory motif of gene expression and the main binding site for microRNAs. In silico analysis of the 3′-UTRs of major ABC-drug transporters using common databases revealed length variations of these 3′-UTRs (alternative polyadenylation) and conflicting data to reported microRNA binding sites which are partially not located within 3′-UTRs as described. This is also true for commercially distributed vectors containing these 3′-UTRs linked to reporter gene constructs. The fact that the expression level of ABC-transporters contributes to pharmacoresistances or insufficient drug responses necessitates a detailed analysis of the 3′-UTRs of drug transporters and their possible length variations.

Objective: The objective of this study is to identify the exact length and possible sequence variations of the 3′-UTRs of the major drug transporters ABCB1, ABCG2, ABCC1 and ABCC2 and to analyze the existence of different 3′-UTR lengths in various cell-lines as well as drug resistant cancer cells and their dug sensitive counterparts.

Methods: 3′-RACE and various standard PCR experiments were performed using cDNA of different human cell lines and primary human tissues. The abundance of 3′-UTR fragments was analyzed using quantitative RT-PCR.

Results: Five different ABCB1 3′-UTR length variants were identified. Previously reported microRNA binding sites were located only on the three longer fragments. Imatinib resistant leukemia cells expressed predominantly shorter 3′-UTRs, where miRNA binding sites are absent [1]. For ABCC2 also five different 3′-UTR length variants were identified. Reported microRNA binding sites for ABCC2 are completely absent on the shortest fragment. In silico analysis of ABCG2 and ABCC1 revealed six different 3′-UTR lengths for ABCG2 and eight for ABCC1. Experimental verification of the in silico analysis is currently in process.

Conclusions: Shortening of the 3′-UTRs of ABC-drug transporters cause loss of microRNA-dependent post-transcriptional mRNA silencing leading to elevated protein levels and elevated drug insensitivity or drug resistances, respectively.

Reference: 1. Bruhn O, Drerup K, Kaehler M, Haenisch S, Röder C, Cascorbi I. Pharmacogenomics 2016;17(4):327-40.

CYP2D6 allele specific copy number analysis using TaqMan® SNP Genotyping Assays and digital PCR

Dominique Dewolf¹, Toinette Hartshorne², Sunali Patel², Kathleen Hayashibara² & David Keys².

1 ThermoFisher Scientific, 16 avenue du Québec, Villebon-sur-Yvette, France, 91941

2 ThermoFisher Scientific Genetic, Medical & Applied Sciences, Oyster Point boulevard, South San Francisco, CA, USA, 94404

The major drug metabolizing enzyme, CYP2D6, is encoded by a highly polymorphic gene. Over 100 star allele haplotypes are known, which can contain SNP, InDel, and copy number variants (CNVs) and which fall into 3 main functional categories (full, reduced, or none). The diploid star allele content is predictive of CYP2D6 drug metabolizer phenotype (ultrarapid, extensive, intermediate, or poor). We previously described a workflow whereby sample SNP genotype and CNV analysis results, generated using TaqMan® SNP assays and TaqMan® Copy Number assays, respectively, can be translated to star allele diplotypes using AlleleTyper™ software. However, for samples that carry CYP2D6 duplications and are heterozygous for key SNPs, the specific allele that is duplicated cannot always be identified. A phenotype can be assigned if all 3 alleles are of the same functional category, but if alleles are from different functional categories there may be 2 possible phenotypes. To address this issue, we developed a method to detect allele-specific copy number by digital PCR using the QuantStudio™ 3D Digital PCR System.

Potential role of single nucleotide polymorphisms of XRCC1, XRCC3, and RAD51 in predicting acute toxicity in rectal cancer patients treated with preoperative radiochemotherapy

G Gentile¹, M Borro¹, L Nicosia², Osti MF², M Simmaco¹

¹ Department of Neurosciences, Mental Health and Sensory Organs (NESMOS), Sapienza University, 00189 Rome, Italy

² Institute of Radiation Oncology, Sant’ Andrea Hospital, 00189 Rome, Italy

Background: Colorectal cancer is the third most common cancer in the western world and preoperative radiochemotherapy and total mesorectal excision are widely used for the treatment of locally advanced rectal cancer. Sometimes, neoadjuvant radiochemotherapy (N-RCT) is associated with high rates of complications reducing patient’s compliance during treatment.

Genetic factors may influence tissues radiosensitivity and the development of side effects. Radiotherapy (RT) exerts its cytotoxic effects through the damage to cell structures, proteins, and DNA, then individual variation in the mechanisms of DNA repair and xenobiotic metabolism may modify the response of normal tissues.

Some studies showed that polymorphisms of genes involved in the DNA repair pathways could influence cellular sensitivity to radiation and chemotherapeutic agents.

Objective: The aim of this study was to analyzed the association between polymorphisms of DNA repair genes and acute toxicity in rectal cancer patients treated with N-RCT.

Design: Sixty-seven patients were genotyped for the following SNPs: XRCC1 rs25487, XRCC3 rs1799794 and rs861539, RAD51 rs1801320 and GSTP1 rs1695.

Results: RAD51 correlated with acute severe gastrointestinal toxicity in heterozygosity (Aa) and homozygosity (AA) (P=0.036). Grade ≥ 3 abdominal/pelvis pain toxicity was higher in the Aa group (P=0.017) and in the Aa +AA group (P=0.027) compared with homozygous (aa) patients. Acute skin toxicity of any grade occurred in 55.6% of the mutated patients versus 22.8% in the wildtype group (P=0.04) for RAD51. XRCC1 correlated with skin toxicity of any grade in the Aa +AA group (P=0.03) and in the Aa group alone (P=0.044). Grade ≥ 3 urinary frequency/urgency was significantly higher in patients with AA (P=0.01), Aa (P=0.022), and Aa +AA (P=0.031) for XRCC3 compared with aa group.

Conclusions: Our study suggested that RAD51, XRCC1, and XRCC3 polymorphisms may be predictive factors for radiation-induced acute toxicity in rectal cancer patients treated with preoperative combined therapy¹.

Reference: Osti MF et al, Am J Clin Oncol. 2015 Mar 24.

Characterization of a novel single nucleotide polymorphism in thiopurine methyltransferase

Sara Helander¹, Iu Yan Ping Heidi², Anna Zimdahl Kahlin¹, Cheung Chun Wah², Shek Chi Chung², Leung Moon Ho³, Lars-Göran Mårtensson⁴, M. Lindqvist Appell¹

¹ Division of Drug Research, Department of Medical and Health Sciences, Linköping University, SE-581 85 Linköping, Sweden

²Chemical Pathology Laboratory, Department of Pathology, Queen Elizabeth Hospital, Hong Kong SAR, China.

³ Department of Medicine, Queen Elizabeth Hospital, Hong Kong SAR, China.

⁴ Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden.

Background: Thiopurine drug induced toxicity is associated with defects in the thiopurine methyltransferase (TPMT) gene. TPMT is a polymorphic enzyme, with most of the single nucleotide polymorphisms (SNPs) causing an amino acid change, which in most cases results in low enzymatic activity of the TPMT protein. The SNP for TPMT*3C, c.719A>G results in an amino acid substitution at position 240 from Tyr to Cys. Tyr240 is highly conserved and mutation at Tyr240 may alter the protein stability. Cellular studies show that the TPMT co-factor S-adenosylmethionine (SAM) could modulate TPMT activity. Although studies have been performed both with patient material and in cellular assays a limited number of studies have studied the direct effect of SAM on TPMT stability.

Objective: The objective was to characterize the novel patient allele c.719A>C together with the more common variant c.719A>G, resulting in amino acid shift from Tyr to Ser, p.Y240S and Tyr to Cys, p.Y240C respectively. In addition, the wild-type protein TPMT*1 was characterized and both variants were compared against TPMT*1.

Design: TPMT c.719A>C (p.Y240S) was identified using DNA sequencing and pyrosequencing. The enzymatic activity in red blood cells was determined. Recombinant TPMT c.719A>C protein, TPMT*3C and the wild type TPMT*1 were produced in E.coli, purified with standard methods and characterized using biophysical methods such as Circular Dichroism (CD) and Isothermal Titration Calorimetry (ITC). The recombinant enzymatic activity was determined using a fluorescence assay.

Results: The new TPMT SNP, c.719A>C (p.Y240S) have intermediate enzymatic activity in red blood cells. We evaluated the thermal stability and show that TPMT p.Y240S is less stable than both TPMT*1 and TPMT*3C. Furthermore, addition of SAM increases the stability for all protein variants, thus higher molar excess of SAM are needed in order to stabilize TPMT*3C and TPMT p.Y240S. In addition, SAM increases the long-term stability of both TPMT*1 and TPMT*3C. In agreement with thermal stability data, TPMT p.Y240S shows a remarkably decreased enzymatic effect compared to TPMT*1 and TPMT*3C.

Conclusions: Our studies gain further insight to the TPMT enzyme and the possible stabilizing effects of SAM. We show that both TPMT p.Y240S and TPMT*3C have lower enzymatic activity and are less stable compared to TPMT*1. All protein variants used in or study can be stabilized by the addition of SAM.

Acknowledgements: This study was supported with grants from LiU Cancer network, FORSS, the foundation Lars Hiertas memory and the Samariten foundation.

Reference: Karas-Kuzelicki N, Smid A, Tamm R, Metspalu A, Mlinaric-Rascan I. From pharmacogenetics to pharmacometabolomics: SAM modulates TPMT activity. Pharmacogenomics. 2014;15(11):1437-49. Epub 2014/10/11.

Polymorphism within the RYR2 transporter as a potential predictor of statin associated myalgia/myopathy in Czech population

Hubacek J.A.¹, Dlouha D.¹, Adamkova V.¹, Češka R.², Vrablik M.²

¹Center for Experimental Medicine, IKEM, Prague, Czech Republic; ²3^rd Department of Internal Medicine, 1^st Faculty of Medicine, CU, Prague, Czech Republic

Background: Statins are the most common drugs used in both primary and secondary cardiovascular prevention. Genetic variants might play a major role in determination of the adverse statin effects, like myalgia or myopathy.

Objectives: Gene for ryanodine receptor 2 gene (RYR2) belongs to the candidates with potential to influence risk of statin induced muscle problems (myalgia/myopathy). RYR2 codes for tetramer channel, which is major source of calcium required for muscle excitation-contraction and rs2819742 variant within this gene was associated with cerivastatin induced rhabdomyolysis. Design:RYR2 rs2819742 (G>A at chromosome 1, g.237990122) intronic polymorphism was successfully (CR 99.3%) analysed in heterogeneous group of 288 patients with statin induced myopathy (~ 90% on simvastin or atorvastatin, 10 mg or 20 mg per day) and 2, 492 healthy adult controls (none on statin treatment, CR 99.8%). Polymorphism was analysed using PCR-RFLP.

Results: Distribution of the individual RYR2 genotypes in Czech patients with statin induced myalgia/myopathy (GG=30.0%, GA=53.9%, AA=16.1%) was slightly different from the control population (GG=36.0%, GA=48.1%, AA=15.9%; P<0.05). In comparison with GG homozygotes, carriers of at least one A allele were on higher risk of statin associated myalgia/myopathy development (OR [95%CI]=1.31 [1.00 - 1.70]; P=0.048).

Conclusions: Results of the pilot study suggest that the rs2819742 variant within the gene for RYR2 receptor could be associated with statin induced myalgia/myopathy in Czech patients on low doses of common statins.

Supported by project MH CR - IN 00023001, IKEM.

Clinical barriers for the application of pharmacogenetics in the UK: statistical analysis of limitations of pharmacogenetic resources with a focus on warfarin and clopidogrel treatment and impact of the decreasing costs of pharmacogenetic tests

S S Joshi, E A Shephard and M J Bartlett

Division of Biosciences, University College London (UCL), London WC1E 6BT, United Kingdom.

Background and Hypothesis: A large amount of evidence is available to physicians in UK about pharmacogenetic tests for prescription of Warfarin and Clopidogrel. The resources available to support the cardiologists on how to and when to use pharmacogenetic tests need to be more accessible. The lack of clear guidance and high perceived costs of the pharmacogenetics tests act as barriers to adoption of testing before prescription. The principal aims of this study are:

1. To understand the opinion of cardiologists about the available resources that provide guidance on pharmacogenetic testing with respect to Warfarin and Clopidogrel, 2. To determine the limitations of the current resources, 3. To access the awareness about the decreasing cost of the pharmacogenetic tests.

Design: The survey methodology that has been designed for the purpose of this project includes a questionnaire (15-20 questions). This questionnaire will be used to record information about the knowledge gap, limitations of the resources and the decreasing costs of pharmacogenetic testing. As the study focuses on pharmacogenetics pertaining to warfarin and clopidogrel, the sample will mainly consist of cardiologists. The data generated will be analysed using non-parametric tests to identify statistically significant relations in the data. Main Findings and Project Impact: This project will make an important assessment about the impact of the support mechanisms available for the interpretation of pharmacogenetic tests for warfarin and clopidogrel and perceived costs associated with genetic testing.

The results of the study will be used to make some inferences about the improvements that are needed in the interface between the cardiologists and pharmacogenetic testing.

References: Johansen Taber K, Dickinson B. Pharmacogenomic knowledge gaps and educational resource needs among physicians in selected specialties. PGPM. 2014;:145.

Correlation between CYPs 1A2, 2C9, 2C19, 2D6 and 3A4 hydroxylation phenotype and genotype in Ecuadorians

Fernando De Andres¹, Santiago Terán², Francisco Hernández³, Enrique Terán² and Adrian Llerena¹

¹CICAB Clinical Research Centre, Extremadura University Hospital and Medical School, Badajoz, Spain

²Colegio de Ciencias de la Salud, Universidad San Francisco de Quito. Quito, Ecuador

³Facultad de Ciencias Médicas, Universidad Estatal de Guayaquil. Guayaquil, Ecuador.

Background: Genotype analysis of CYP450 enzymes is an essential, but not definitive to determine the interindividual variability observed for drug metabolism, as of the genotype-metabolic phenotype discordance observed. Therefore, the analysis of the CYP450 hydroxylation capacities should help to predict individual’s responses to the administration of more than 75% of prescribed drugs.

Objective: to explore the relationship between the main CYP450 genetic polymorphisms and the actual hydroxylation capacity (metabolic phenotype) in Ecuadorians.

Design: 139 healthy individuals were genotyped and phenotyped by using the CEIBA cocktail approach¹. Plasma concentrations of the probe drugs and metabolites were quantified and metabolic ratios were calculated to determine each individual’s actual CYPs metabolic capacity. The genotype-phenotype relationship was evaluated by correlation analysis between ‘activity score’ and/or genotype, and MR_log.

Results: No adverse effects were reported and, according to the genotype analyses, no poor metabolizers were found for CYP2C9, whereas 0.752% and 3.10% of the subjects were classified as poor metabolizers for CYP2C19 and CYP2D6, respectively. Additionally, a prevalence of ultrarapid metabolizers (gUMs) of 15.79% for CYP2C19 and 5.43% for CYP2D6 was found. Metabolic ratios correlated with activity score for CYP2C9, CYP2C19 and CYP2D6 and with CYP1A2 genotype. Nevertheless, the frequency histograms and probit analysis showed no clear distinction among individuals when grouped by genotype or activity score, and all the individuals with lower drug metabolizing capacity carried zero active genes. However, some individuals carrying enhanced activity alleles showed lower drug metabolic capacity than individuals with reduced activity alleles.

Conclusions: This is the first study that simultaneously determined the genotypes, drug metabolizing phenotypes and analyses their correlation for the main CYP450 enzymes on Ecuadorians. A key role of the genetic polymorphisms is observed, though the actual metabolic capacity needs to be confirmed by a phenotyping approach.

Acknowledgements: Supported by AEXCID-Junta Extremadura (13/A001) to SIFF, and ISCIII (CD13/00348). Coordinated by the RIBEF network.

1.De Andrés F, Sosa-Macías M, Llerena A. A rapid and simple LC–MS/MS method for the simultaneous evaluation of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 hydroxylation capacity. Bioanalysis. 2014; 6:683-696.

Interethnic CYP2D6, CYP2C9 and CYP2C19 variability across Native American populations and the relevancy of ancestry

A LLerena¹, MEG Naranjo¹, F Rodrigues-Soares², R Barrantes³, LR Calzadilla⁴, FE Estévez-Carrizo⁵, M Grazina⁶, M López-López⁷, GE Moya⁸, R Ortiz-López⁹, R Ramírez-Roa¹⁰, I Rodeiro¹¹, AP Sarmiento¹², MG Sosa-Macías¹³, E Tarazona-Santos², E Terán¹⁴, & CEIBA-Consortium of the Iberoamerican Network of Pharmacogenetics and Pharmacogenomics RIBEF.

¹CICAB Clinical Research Centre, UEX, Spain. ²UFMG, Brazil. ³UCR, Costa Rica. ⁴CCSM La Habana Vieja, Cuba. ⁵UM, Uruguay. ⁶UC.PT, Portugal. ⁷UAM, México. ⁸PUC and GENOS, Argentina. ⁹UANL, México. ¹⁰UNAN, Nicaragua. ¹¹CEBIMAR, Cuba. ¹²PUJ, Colombia. ¹³IPN-CIIDIR, México. ¹⁴USFQ, Ecuador.

Background:The genetic polymorphisms of CYPs (CYP2D6, CYP2C9, and CYP2C19) are one of the major determinants of the interethnic variability in the drug response. Pharmacogenetics studies have been widely developed in Caucasians, but further studies are required in other populations, such as Latin-Americans (resulting of interethnic crosses among Amerindians, Europeans and Africans), and specifically in the understudied Native Americans.

Objetives:The CEIBA-RIBEF Network Consortium (European Ibero-American for Population Pharmacogenetics) aims to evaluate the CYP2D6, CYP2C9 and CYP2C19 genotype and phenotype in different Ibero-American populations.

Design:A total of 6060 individuals from Ibero-America were classified according to their self-reported ancestry in Natives (n=1395), Blacks (n=96), Caucasians (n=1824), Ashkenazi Jews (n=174) and Admixed Americans (n=2571). Moreover, Native-Americans were divided in three groups: North-, Central- and South-Native Americans (from Mexico, Costa Rica and Peru, respectively). All subjects were studied for the most relevant CYP2D6, CYP2C9 and CYP2C19 allelic variants, and grouped into poor/ultrarapid metabolizers (gPMs and gUMs, respectively). Differences among groups were evaluated usinga χ² test followed by the Marascuilo procedure.

Results:Interethnic differences were detected among Native Americans and other ethnic groups: the frequency of CYP2C9*2 was lower in Native Americans (1.14%) than in other ethnic groups (7.96-16.61%; p<0.05), except for Black Americans. Moreover, the frequency of CYP2C19*17 was lower in Native Americans (1.59%) than in the rest (12.33-21.34%; p<0.05), and, consequently, CYP2C19 gUMs was less frequent among Native Americans (2.74%) compared with other ethnic groups (20.71-32.93%; p<0.05). Likewise, important differences within the Native Americans have been found: CYP2D6 gPMs were more frequent in Central-Native Americans (10.20%) than in the rest of Native Americans (0.00-0.31%; p<0.05), whereas CYP2D6 gUMs showed a higher frequency in North-Native Americans (9.52%) than in South- and Central Native Americans (0.41-3.57%; p<0.05).

Conclusions:A large variability on CYPs polymorphisms has been detected in different ethnic groups from Ibero-America, especially for Native Americans. The interethnic differences described could result in unexpected side-effects or therapeutic failures when these individuals are treated with CYP2D6, CYP2C19 or CYP2C9 substrates.

Acknowledgments:Supported by Junta Extremadura-AEXCID (13/A001) MESTIFAR to the RIBEF Network IberoAmerican Society of Pharmacogenetics SIFF (www.ribef.com).

Personalized cancer radiotherapy: a role for DNA repair SNPs as radiogenomic biomarkers?

AM Marques1, LS Santos2.

1Instituto Universitário de Ciências da Saúde, CESPU, 4585-116 Gandra, Portugal; 2Universidade

Católica Portuguesa, Instituto de Ciências da Saúde - Viseu, 3504-505 Viseu, Portugal.

Background: Ionizing radiation has the ability to induce DNA damage and, therefore, to activate apoptotic cell death. This ability is the basis for cancer radiotherapy but normal cells may also be affected alongside with tumour cells. Since DNA repair mechanisms exist to counteract radiationinduced DNA damage, it is possible that variants in coding or regulatory regions of DNA repair genes may influence the efficacy and safety of radiotherapy and, ultimately, affect the clinical outcome of cancer patients submitted to such therapy.

Objective: Through systematic review of the clinical evidence available, we aimed to identify DNA repair SNPs that may correlate with the clinical outcome of cancer patients submitted to adiotherapy. Such SNPs have the potential to be used as radiogenomic biomarkers in personalized cancer therapy.

Design: The PUBMED database was searched using the following MeSH terms: neoplasms AND Polymorphism, Single Nucleotide AND DNA repair AND radiotherapy. 104 articles, published up to March 1, 2016, were retrieved. On applying predefined inclusion/exclusion criteria, 79 articles were excluded. 25 articles were thus eligible for further data extraction.

Results: A high number of significant associations were observed between multiple clinical outcome endpoints (e.g. overall or disease-specific survival) and SNPs across different DNA repair pathways (direct damage reversal, base excision repair, nucleotide excision repair, homologous recombination), DNA damage response genes (e.g. ATM) and other genes with suspected DNA repair function (e.g. RECQL). The XRCC1 Gln399Arg substitution (rs25487) was the DNA repair SNP most frequently associated with response to radiotherapy, the variant allele being consistently associated with decreased survival. Associations between the XRCC2 Arg188His (rs3218536) variant allele and decreased survival and between the ERCC2 Lys751Gln (rs13181) variant allele and increased survival are also suggested by different studies. Despite less consistent, evidence also exists regarding the synonymous ERCC1 substitution Asn118Asn (rs11615).

Conclusions: DNA repair SNPs across different DNA repair pathways appear to modulate the individual clinical outcome in response to radiotherapy. Evidence is stronger for the XRCC1

Gln399Arg substitution (rs25487). Integration of such radiogenomic biomarkers into the clinical decision process may, in a near future, allow for the optimization of the therapeutic approach to several types of cancer, maximizing the benefit while minimizing the risk.

The most prevalent variant allele of CYP1A2 does not affect clozapine and olanzapine pharmacokinetics

M Nijenhuis¹, M van Rhenen¹ and the Dutch Pharmacogenetics Working Group²

¹Royal Dutch Pharmacists Association, The Hague, the Netherlands, ²VHM Deneer, St. Antonius Hospital, Nieuwegein; AH Maitland-van der Zee and A de Boer, Department of Pharmaceutical Sciences, University of Utrecht, Utrecht; J Mulder, Pharmacy of the Wilhelmina Hospital, Assen; GAPJM Rongen, Department of Pharmacology – Toxicology, University Medical Center St Radboud, Nijmegen; RHN van Schaik, Department of Clinical Chemistry, Erasmus Medical Center, Rotterdam; JJ Swen and HJ Guchelaar, Pharmacy of the Leiden University Medical Center, Leiden; DJ Touw, Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Groningen; J van der Weide, Clinical chemistry laboratory, Hospital St. Jansdal, Harderwijk; B Wilffert, Department of Pharmacy, University of Groningen, Groningen; EJF Houwink, General Practice Maastricht Wolfs, Schreurs and Houwink, Maastricht; the Netherlands.

Background: The inactivation of clozapine and olanzapine is increased by smoking, which induces CYP1A2. The most prevalent allele of CYP1A2 in the Netherlands (*1F) has been reported to increase this induction compared to the second most frequent allele (*1A). An infrequent allele (*1C) results in diminished CYP1A2 activity.

Objective: To provide a national guideline on whether and how clozapine and olanzapine treatment should be adjusted in patients with CYP1A2 variant genotypes.

Design: All available information on the effect of variant CYP1A2 alleles on clozapine and olanzapine was collected from literature.

Results: Pharmacokinetics of clozapine and olanzapine did not differ between CYP1A2 *1A and *1F. This was also true in smokers.

For clozapine, there were indications that *1C increased the risk of adverse effects. For this reason, plasma concentration guided dose adjustment is recommended. This recommendation was incorporated in the decision support system of all Dutch pharmacies, general practices and hospitals and appears when clozapine is prescribed for patients labeled as *1C/*1C or *1C-heterozygote.

For olanzapine no effect of *1C on pharmacokinetics or clinical effects was found.

Conclusions: No difference in pharmacokinetics of clozapine and olanzapine was observed for CYP1A2 *1A compared to CYP1A2 *1F. For clozapine, CYP1A2 *1C might increase the risk of adverse effects.

Acknowledgements: This work was funded by the Royal Dutch Pharmacists Association and through the Horizon2020 project ‘Ubiquitous Pharmacogenomics (U-PGx): Making actionable pharmaco-genomic data and effective treatment optimization accessible to every European citizen’ (update and implementation in other European countries).

Reference: Earlier work:

1.Swen JJ et al. Clin Pharmacol Ther 2011;89:662-73.

Iatrogenically induced opioid dependence, pharmacogenetics and pain

Planelles B^1,2, Almenara S², Muriel J ^4,5, Núñez A⁶, Inda MM^3,4, Puga C^4,5, Morales D⁷, Margarit C^1,3, Peiró AM^{1, 2, 3}.

(1) Pain Unit, Department of health of Alicante - General Hospital (DHA-GH), Spain

(2) Neuropharmacology on Pain and Functional Diversity (NED), Research Unit, Department of health of Alicante - General Hospital, ISABIAL, Spain

(3) Pharmacology Unit, Department of health of Alicante - General Hospital (DHA-GH), Spain

(4) Foundation for the Promotion of Health and Biomedical Research of the Valencian Community (FISABIO), Alicante, Spain

(5) Occupational Observatory, University Miguel Hernandez (UMH), Elche, Spain

(6) Pharmacology Unit, Virgen del Rocío Hospital - Sevilla, Spain

(7) Operative Research Center (CIO), UMH, Elche, Spain

Aim of Investigation: Opioids have high therapeutic interest as analgesics but in a low percentage of patients, could associate aberrant drug-related behaviour. This is scarce studied in non-cancer chronic pain patients.

Methods: A cohort study was performed with outpatients (n=70) diagnosed with opioid iatrogenic dependence. They followed a structured and progressive opioid reduction dose program witth conversion to buprenorphine or tramadol, along 6 months. Study was focused on analgesic efficacy, abstinence (Opioid Withdrawal Scale, OWS), adverse side effects, functional status and aberrant drug-related behaviour. Genotyping Single Nucleotide Polymorphism of the OPRM1 (rs1799971), OPRD1 (rs4985), COMT (rs4680), ABCB1 (rs1045642) and ARRB2 (rs409) genes was performed by real-time PCR. Ethical Committee approved the study. Statistical analysis was performed using R.3.2.0.

Results: A significant reduction of the total daily dose (TDD) converted to morphine, was achieved (247.25±163.72 vs. 115.03±100.14, p= 0.033) without abstinence, presenting a moderate pain intensity. Quality of life tended to improve, as well as, the number of adverse reactions reported by the patients throughout the visits. Genotype 118-AA for OPRM1 gene required significantly lower TDD (codominant model, AA=90.83±15.36; AG=134.05±27.05; GG=308.40± 0.00 mg/day; p=0.037).

Conclusions: Our program showed effectiveness in reducing TDD with a good conversion to buprenorphine, significantly associated to 118-AA OPRM1 genotype.

Impact of ABC gene variants on plasma lipid traits and response to atorvastatin in Chilean individuals

Prado Y, Rosales A, Salazar LA.

Center of Molecular Biology and Pharmacogenetics, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile.

Background: Statins constitute the first line treatment to reduce high cholesterol levels. However, there is great interindividual variability in response to these drugs.

Objective: The goal of this study was to investigate the influence of four single nucleotide polymorphisms in genes of the ABC transporter family (ABCC2 rs717620, ABCG2 rs2231142, ABCB1 rs1128503, and ABCB1 rs3842) in the response to short-term low-dose atorvastatin medication in Chilean hypercholesterolemic patients.

Design: We included 127 Chilean hypercholesterolemic patients treated with 10 mg/day atorvastatin for one month. Lipid profile was determined before and after drug administration. Genotyping of the variants rs1128503, rs3842, rs717620 and rs2231142 was performed using TaqMan^® Drug Metabolism Genotyping Assays.

Results: Following statin therapy, there was a reduction of TC, LDL-C and TG concentrations (p<0.05). Also, HDL-C levels increased (p<0.05). Minor allele frequencies for rs1128503, rs3842, rs717620, rs2231142 variants were 0.453, 0.154, 0.232 and 0.075, respectively. LDL-C response to atorvastatin was not associated with none of the studied polymorphisms (p>0.05). However, the rs3842 was associated with TG variability after atorvastatin medication in both men and women (p=0.0035), while rs717620 was associated with TG variability after atorvastatin medication in men only (p=0.0039).

Conclusion: This study indicates that LDL-C reduction following atorvastatin therapy is not influenced by the SNPs evaluated. However, the ABCB1 rs3842 and ABCC2 rs717620 polymorphisms showed an association with TG concentration in response to atorvastatin medication in Chilean hypercholesterolemic subjects.

Acknowledgements: Fondecyt-Chile (1130675) and CONICYT Scholarship.

Reference: Dean M, Hamon Y, Chimini G. The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res. 2001; 42(7):1007-17.

Evaluation of epigenetic mechanisms in THP-1 cells treated with statins

J Lagos¹, RDC Hirata², MH Hirata², LA Salazar¹

¹Center of Molecular Biology & Pharmacogenetics, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile; ²Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

Background: Statins are HMG-CoA reductase inhibitors. Alongside enzymatic inhibition, statins induce intracellular activation of transcription factors, leading to an increase in low-density lipoprotein receptors (LDLRs) coupled to the plasma membrane of hepatocytes, enhancing clearance of plasma cholesterol. Several proteins with key functions, which are regulated by statins, are involved in this pathway. However, to date, there are no reports describing epigenetic mechanisms contributing to their regulation.

Objective: To evaluate the effect of DNA methylation, histone modification and miRNAs expression in the modulation of intracellular cholesterol metabolism genes in monocytic cells treated with statins.

Design: THP-1 cells in the absence and presence of 10 µM of atorvastatin or simvastatin were cultured. Gene expression of HMGCR, LDLR, SCAP, INSIG1, INSIG2, SREBF1, SREBF2, MBTPS1, KPNB1 and MBTPS2 was evaluated by real-time PCR. DNA methylation in specific areas of promoter regions of the LDLR, SREBF2 and HMGCR genes was evaluated by sequencing after modification with sodium bisulfite. The modifications in H3 and H4 histones was determined by colorimetric assays and miR-29a, miR-29b, miR-33a, miR-33b, miR-300 and miR-454 expression was evaluated by real-time PCR.

Results: Both statins induced overexpression of LDLR, HMGCR, SREBF2 and INSIG1 genes, while simvastatin additionally overexpressed SCAP, MBTPS1 and MBTPS2. No changes in methylation status were observed in the promoter regions of LDLR, SREBF2 and HMGCR. Only atorvastatin induced an increase in H3K4 di- and trimethylation, H3K9 monomethylation and H3K36 di- and trimethylation. Both statins increased monomethylation of H3K36 and H3K79 di- and trimethylation, along with an increase in H3K14 and H3K9 acetylation and H3Ser28 phosphorylation. Simvastatin increased monomethylation of H4K20 and H4Ser1 phosphorylation. Both treatments were associated with H4K5 acetylation and only atorvastatin was associated with a decreased H4K20 trimethylation. MiR-29b and miR-454 showed increased expression after treatment with both statins, while only atorvastatin induced miR-33b overexpression.

Conclusions: Both statins investigated differentially overexpressed genes involved in intracellular metabolism of cholesterol, probably due to greater transcriptional activity as a consequence of the modifications observed in histones H3 and H4 and by miRs modulation. Our results constitute the first report evaluating simultaneously three epigenetic mechanisms.

Acknowledgements: FONDECYT – Chile (1130675); CONICYT Scholarship (21130987)

Reference: Brown MS, Goldstein JL. Cell 1997; 89(3):331–340.

Cytochrome P450 2 C19 polymorphism does not contribute to in-stent restenosis in Chilean patients who underwent percutaneous coronary intervention receiving clopidogrel

J Ruedlinger¹, Y Prado¹, N Saavedra¹, F Lanas^{1, 2}, B Bobadilla², M Potthoff², L Pérez³, T Zambrano¹, LA Salazar¹

¹Center of Molecular Biology and Pharmacogenetics, Scientific and Technological Bioresource Nucleus (BIOREN); ²Faculty of Medicine, Universidad de La Frontera, Temuco, Chile; ³Faculty of Medicine, Universidad de Concepción, Concepción, Chile.

Background: Several data proved the significance of CYP2C19*2 polymorphism in the development of in-stent restenosis in vascular disease after endovascular treatment and stent implantation. In addition, it has been reported that single nucleotide variants within the CYP2C19 gene, the hepatic enzyme involved in clopidogrel biotransformation to its active metabolite, result in significant interindividual variations regarding therapeutic efficacy of the drug. Clopidogrel is a widely used antiplatelet drug for secondary prevention of atherosclerotic events in high risk patients, especially those undergoing percutaneous coronary interventions (PCI), who receive it in combination with aspirin as a dual antiplatelet therapy.

Objective: The aim of this study was to evaluate the impact of the loss of function variant CYP2C19*2 in the occurrence of in-stent restenosis in Chilean coronary artery disease patients who underwent PCI and received clopidogrel.

Design: A total of 144 patients were included. Patients with stenosis>50% in the angioplasty site were defined as cases and those with<50% as controls. Clinical and demographic variables were registered. CYP2C19*2 rs4244285 SNP was genotyped using TaqMan^® Assays.

Results: The study included 68 cases (mean age 62.6 ± 9.9; 79.4% male) and 76 controls (mean age 64.9 ± 10; 71.1% male). Diabetes and bare metal stents (BMS) were observed more often in cases than controls (p=0.03 and p=0.002, respectively). Genotypic frequencies for the rs4244285 variant did not differ significantly between the groups (p= 0.12). Nonetheless, the minor allele was observed in greater proportion in patients who did not develop in-stent restenosis (0.05 vs 0.12, p= 0.04). There was no significant association between the loss of function rs4244285 variant and the occurrence of in-stent restenosis after PCI (OR= 0.4; 95% CI: 0.163 - 0.999; p= 0.05).

Conclusions: We found no association between the CYP2C19* 2 variant and coronary in-stent restenosis development in patients treated with clopidogrel. Our results should be interpreted with caution, since clinical and angiographic factors contribute to this condition, particularly diabetes, which is also associated with a low response to clopidogrel.

Acknowledgements: Fondecyt-Chile (1141292) and CONICYT Scholarship.

Reference: Kracskó B. Anatol J Cardiol 2015; 15(5):354.

Gender differences in genes polymorphisms of cytochrome P450 (CYP2C9, CYP4F2) and its impact on the phenindion dosage in patients with valvular atrial fibrillation

V. Shakhidzhanova¹, D.Sychev², A.Tretyakov³

1.I.M.Sechenov First Moscow State Medical University, Moscow, Russia, 119048; 2 Russian Medical Academy of Postgraduate Education, Moscow, Russia, 125993; 3.Belgorod National Research University, Belgorod, Russia, 308000

Background: Gender differences are important for personalized medicine.

Objective: To obtain the differences in phenindion dosing in men’s and women’s population, depending on polymorphisms of CYP2C9, CYP4F2.

Design: The study included 22 women (63.1 years), and 20 men ( 62.5 ), with valvular atrial fibrillation. The frequency of alleles and genotypes of CYP2C9 and CYP4F2 genes were performed by the method PCR-RFLP (restriction fragment length polymorphism). Statistical analysis was performed by using χ2 Pearson and Student’s t-test.

Results: The target levels of INR (TLINR) (2.0-3.0) for women were achieved by the dose of phenindion 72,3 ± 5,4 mg; for men - 88,5 ± 6,7 mg (t=1,84, p>0.05). It was noticed out that the frequency of successful achieving TLINR differed due to the sex. So only 3 women (13.6%) haven’t achieved TLINR (wINR-), on phenindion dose - 110 ± 22,1 mg. 8 men (40%) haven’t achieved the TLINR (mINR-) - (x2=17,3, p <0,0001), phenindion dose - 116,25 ± 4,0 mg. 19 female patients who have achieved TLINR (wINR +) – on phenindion dose 66,3 ± 3,5mg.12 male patients (mINR +), the dose of phenindion was 70,0 ± 4,7; (P> 0,05). At the same time all patients were comparable for age, body mass index (30.1 for women and 30.7 for men). The following gene polymorphisms were found out in the group (wINR-) :* 1 * 1, CYP2C9 – (n=3), CC CYP4F2 – (n=3). The distribution of genes in the group (mINR-) was following: * 1 * 1 CYP2C9 - 5 patients, * 1 * CYP2C9 - 3, CC CYP4F2 - 6, CT - 2 patients. In general the frequency of genes polymorphism *1* 1CYP2C9 (wild) in the group of (wINR-) was 100% vs 62% for men (mINR-) (x2=44,5, p<0,001).

Conclusions: The male patients with WAF that didn’t achieve TLINR on the adequate therapy of phenindion were found out for the three times often than women and in the men’s group cytochrome P450 polymorphisms were presented more diverse.

References: Shakhidzhanova V, Sychev D. C3435T polymorphism and its influence on the dose of phenindion, Como Eurotrombosis 2015:92.

The Effects of Glipizide and Acarbose on Obesity and Glucose Metabolism in 3T3-L1, AML12 cell lines and their co-cultures

Meliha KOLDEMİR GÜNDÜZ¹, Mehtap ÇEVİK¹, Penbe ÇAĞATAY², Belgin SÜSLEYİCİ DUMAN¹

¹Department of Biology, Marmara University, Faculty of Science and Arts, İstanbul, Turkey.

²Department of Biostatistics and Medical Science, İstanbul University İstanbul Faculty of Medicine, İstanbul, Turkey

Background: The unipotent preadipocytes 3T3-L1, are ideal for studying the molecular events responsible for the conversion of preadipocytes into adipocytes since they can either remain as preadipocytes or undergo conversion to mature adipose tissue. Hepatocytes, are commonly used in gene regulation studies.

Objective: The aim of the study is to evaluate how The active substances of oral-anti-diabetic drugs sush as glipizide and acarbose, effect the cells reproduction activities and how they change the expressions of FTO, CD68, NIBAN, and RAN genes which could change insulin signalization, also be effective in the adipogenetic process, have unknown functions and related to obesity in 3T3-L1 adipocytes, AML12 hepatocytes and adipocyte-hepatocyte co-cultures.

Design: Cell proliferation was examined with iCELLigence system. The time and amount of active substances of the oral-anti-diabetic drugs which were applied to cells were determined real-time according to IC50 value. FTO, CD68 NIBAN and RAN gene expressions were determined with qPCR.

Results: When single and multiple doses of glipizide and acarbose in co-culture’s were compared respectively, the 24 hour IC50 values were determined as 180 µM and 17 mg/ml in adipocytes; 72 µM and 23 mg/ml in hepatocytes, 41.5 µM and 5 mg/ml in co-culture cells. FTO gene expression was found to be decreased in the glipizide administered cells, except for co-cultured hepatocytes. CD68 gene expression was found to be decreased in the glipizide administered cells, except for co-cultured hepatocytes. As a response to glipizide application, NIBAN gene expression was found to increase in adipocytes, whereas decreased in the co-cultured adipocytes. As a result of glipizide application, while RAN gene expression in co-cultured hepatocyte was silenced in other study groups it was increased. In the acarbose applied cells FTO and CD68 gene expressions were decreased in all groups. In the acarbose applied cells while NIBAN gene expression was decreased in adipocytes, it was fund to increase in co-cultured adipocytes. In the acarbose applied cells RAN gene expression was found to increase in all groups.

Conclusion: When compared to single cultures, the low drug doses in co-cultures shows that the doses that are determined, as proper doses less harmful, by determining specially personalized dose can be used in treatment instead of using high doses. The decreasing effects of glipizide and acarbose on CD68 and FTO gene expressions may show the protective effect of drug on inflammation and obesity. In glipizide and acarbose applied insulin resistant mature adipocytes, NIBAN gene expression’s high levels may show antiapoptotic effect and as to RAN gene expression’s high levels may show it’s regulatory effect in glucose homeostasis.

Acknowledgment: This study was supported by Marmara University (Project No: FEN-C-DRP-131113-0433).

Modelling the Structural and Functional effects of nucleotide polymorphisms in the human NAT1 drug metabolising enzyme

R Cloete¹, W Akurugu², C Werely³, P van Helden³, and A Christoffels¹

¹South African MRC Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Private Bag X17, Bellville, Cape Town 7535, South Africa.

²Noguchi Memorial Institute for Medical Research, University of Ghana, P. O. Box LG 581 Accra, Ghana.

³SA MRC Centre for Molecular and Cellular Biology, and DST-NRF Centre of Excellence for Biomedical TB Research, Dept. of Biomedical Sciences, Faculty of Medicine & Health Sciences, Stellenbosch University, P. O. Box 241, Cape Town 8000, South Africa

The human drug metabolising enzymes (DME), arylamine N-acetyltransferases (NAT1 and NAT2) determine the duration of action of amine-containing drugs by impacting the metabolic balance between the activation and detoxification of these compounds. Single nucleotide polymorphisms (SNPs) in the NATs have been implicated in both the inter-ethnic and inter-individual variation in the phenotypic treatment profiles of patients. We investigated the effect of several novel non-synonymous SNPs on the structure and function of the NAT1 enzyme by simulation modelling studies. Interestingly, these SNPs were not found in nor associated with the catalytic pocket of the enzyme.

The in silico prediction and simulation analyses utilised tools such as GROMACS, mCMS, SIFT and POLYPHEN-2 to investigate the effects of the SNPs on the human NAT1 enzyme. For the purpose of the analysis we used the wild type NAT1 crystal structure (PDBID: 2IJA), containing the acetylated cysteine-68 residue complexed with coenzyme A. In addition, the substrate para-aminobenzoic acid (PAS) was also complexed to the structrure. The NAT1 SNPs were identified in a South African population cohort of mixed ancestry.

The simulation results showed that two of the investigated novel SNPs, resulting in changes at amino acids 231 (V>G) and 245 (N>I) respectively, affected the function of the NAT1 protein, whilst two others, at 242 (R>M) and 264 (E>K) gave contradictory results, and could not be interpreted. Three of the altered amino acids, 231G, 242I and 264K showed stabilisation energies between -0.58 and -5.09 kcal/mol which is indicative of protein destabilisation. In addition the similation studies indicated that the amino acid substitutions at V231G and E264K, also occupied energy minima in support of a predictive protein destabilisation.

NAT1 SNPs have generally not been validate in vitro, to our knowledge. We propose that the use of simulation studies could provide an appropriate alternative to qualify and quantify unknown SNP effects on protein structure and function, particularly as a selective screening methodology. The observations from this study could inform a strategy for incorporating genotypic data (i.e, functional SNP alleles) with phenotypic information (slow or fast acetylators) to prescribe more effective treatment regimens.

Characterization of the single nucleotide polymorphism 497A>G in the drug metabolizing enzyme thiopurine methyltransferase

A. Zimdahl Kahlin¹, S. Helander¹, L-G Mårtensson², M. Lindqvist Appell¹

¹Department of Medical and Health Sciences, Division of Drug Research, Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden ²Department of Physics, Chemistry, and Biology, Linköping University, 581 83 Linköping, Sweden

Background: Thiopurine methyltransferase (TPMT) is a polymorphic enzyme that converts cytotoxic thiopurine drugs into both active and inactive metabolites. In Caucasians 10 % carries a single nucleotide polymorphisms (SNP) causing a less functional enzyme leading to an increased risk of severe adverse reactions during treatment with normal thiopurine doses. Today about 40 SNPs are known and named by the TPMT nomenclature committee (www.imh.liu.se/tpmtalleles).

Recently, we discovered a SNP in the drug metabolizing enzyme TPMT, in a young female patient with acute lymphoblastic leukemia. It had earlier been reported from the genomic ClinSeq Project but has not been further characterized.

Objective: The objective of this study was to characterize the TPMT SNP 497A>G, to describe the properties and function of the resulting protein in comparison to the wild type TPMT.

Design: In our study the TPMT 497A>G was characterized in context of enzyme activity in RBC and WBC, cDNA sequencing and heredity. We also produced recombinant human TPMT 497A>G in Escherichia Coli bacteria and investigated the stability and structural changes of the protein structure with biophysical methods (1).

Results: The SNP consists of a 497A>G mutation which cause an amino acid shift of Y166C. Our results shows that 497A>G is a heritable variant of TPMT that decrease the patient´s enzyme activity dramatically. The overall structure of the enzyme is normal but the amino acid shift causes a pronounced less stable enzyme.

Conclusions: We present a full characterization of the nonfunctional TPMT SNP 497A>G, a SNP that influence the metabolism of thiopurine drugs, widely used during treatment of childhood leukaemia.

Acknowledgements: The authors express their gratitude to Britt Sigfridsson, Monica Häger, Annette Molbaeck and Åsa Schippert for excellent technical assistance. This study was supported with grants from Stiftelsen Linköpings Läkaresällskaps Forskningsfond and Östgötaregionens Cancerfond.

Reference: 1. Wennerstrand P, Dametto P, Hennig J, Klingstedt T, Skoglund K, Lindqvist Appell M, Mårtensson L-G. Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*5. Biochemistry. 2012;51:5912-20.