6th Santorini Conference Biologie Prospective 2012 ...

DOI 10.1515/dmdi-2012-0025

Drug Metab Drug Interact 2012; 27(3): A1–A48

Congress Abstracts*)

6th Santorini Conference Biologie Prospective 2012

Systems Biology and Personalized Health Science and Translation Santorini Island, Greece, September 30 – October 02, 2012

Scientific Committee Bontoux Nathalie (Massy, France), Bühlmann Roland P. (Schönenbuch, Switzerland), Domdey Horst (Martinsried, Germany), Fitzgerald Peter (Crumlin, Co Antrim, UK), Froguel Philippe (Lille, France), Henney Adriano (Macclesfield, UK), Humphries Steven E. (London, UK), Kepes François (Evry, France), Kussmann Martin (Lausanne, Switzerland), Llerena Adrian (Budajoz, Spain), Manolopoulos Vangelis G. (Alexandroupolis, Greece), Marc Janja, (Ljubljana, Slovenia), Michel Gerd (Geneva, Switzerland), Noyer-Weidner Mario (Berlin, Germany), Pallaud Céline, Roche, (Basel, Switzerland), Patrinos George (Patros, Greece), Roses Allen (Durham, USA), Seifert Martin (Munchen, Germany), Siest Gérard (Nancy, France), Superti-Furga Giulio (Vienna, Austria), Van Schaik Ron (Rotterdam, NL), Sophie Siest (Nancy, France)

Organizing Committee Biologie Prospective, (Head: Pr Gérard Siest) & Research Unit “Cardiovascular Genetics”, (Head: Dr Sophie Siest), Université de Lorraine, Nancy, France. Abdelsalam Saleh, Azimi Nezhad Mohsen, Ben Rezig Laurence, Bonnefond Amélie, El Shamieh Said, Godjo Thibaut, Herbeth Bernard, Hiegel Brigitte, Lambert Daniel, Masson Christine, Ndiaye Ndeye Coumba, Rancier Marc, Shahabi Payman, Stathopoulou Maria, Siest Gérard, Sophie Siest & the participation of all the other members of the research team.

*) These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax, and usage are the authors.

Authenticated | [email protected] Download Date | 9/20/12 7:38 PM



CONFERENCES & WORKSHOPS 30 SEPTEMBER 2012 - MORNING

SESSION: FROM SYSTEMS BIOLOGY TO SYSTEMS MEDICINE Interactome Networks and Human Disease M Vidal Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology Dana-Farber Cancer Institute & Department of Genetics, Harvard Medical School, Boston, MA 02115 For over half a century it has been conjectured that macromolecules form complex networks of functionally interacting components, and that the molecular mechanisms underlying most biological processes correspond to particular steady states adopted by such cellular networks. However, until a decade ago, systems-level theoretical conjectures remained largely unappreciated, mainly because of lack of supporting experimental data. To generate the information necessary to eventually address how complex cellular networks relate to biology, we initiated, at the scale of the whole proteome, an integrated approach for modeling proteinprotein interaction or “interactome” networks. Our main questions are: How are interactome networks organized at the scale of the whole cell? How can we uncover local and global features underlying this organization, and how are interactome networks modified in human disease, such as cancer?

Ageing populations, complex diseases and unmet medical need: The need for a systems approach to 21st Century Medicine A Henney Macclesfield, United Kingdom The world population is increasing at a rate of about 80 million people per year and is estimated to reach around 8 billion by the year 2025. By that time, those over the age of 65 is expected to represent 10% of the population; in Europe it is calculated to be closer to 25%. This presents a significant problem for healthcare budgets as there is a disproportionately greater prevalence in the elderly of chronic, complex diseases that are debilitating and difficult to treat. These are areas of major focus for novel therapies, but pharmaceutical industry figures show the success of delivery of new medicine to market has declined significantly, whilst the full cost of doing has increased massively. One reason for the continued lack of success is likely to arise from the focus on reductionist, cell and molecular screens that capitalise on detailed target information emerging from advances in molecular and cellular biology, but which lack information on the physiological context in which the compound is ultimately intended to operate. Failure of medicines in phase II or phase III is extremely costly and is now largely related to a failure in efficacy: even though we think we know a lot about the building blocks of biological systems, we actually know very little about how they operate in the physiological networks that underpin diseases. We need to understand how targets operate in physiological networks and, as importantly, within the context of the elderly who will present at clinic with co-morbidities, taking a

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number of medications. Integrative systems approaches that address the dynamics of networks operating in disease offer promise that this can be achieved, and their potential to have an impact on 21st century medical practice is not in doubt. What is lacking is sufficient evidence of application in the real world and reduction to practice. The challenge is how to evaluate the added value that these advances in science bring to existing medical practice, and to find ways of exploiting them routinely in the clinic. This task is dependent more than ever on the need to bring exploratory, blue skies science and busy, pragmatic, practicing physicians together to test the potential of, and troubleshoot, unproven technologies. Advocates of using mathematics, engineering and physics to understand biological network behaviour are increasingly vocal, and modelling and simulation is now seen as an inevitable evolution in biology post genome. Indeed, analysts suggest it is an essential component of the changes that need to take place in the pharma business model if it is to survive. This is now even more important, with the increased interest in personalisation, segmentation or stratification of treatment, demanding an improvement in our understanding of the connection between molecular function and emerging phenotype.

Systems approach to decipher the logic of life interlacing all fields of biomedicine D Ghosh, J Sengupta Department of Physiology, All India Institute of Medical Sciences, New Delhi The human endeavour to explore the logic of life has a history of long journey; it began about three thousand years before Christ was born. The unresolved issue of understanding of the logic of life has been taken a centre stage nevertheless, for the fact that it is the primal point of all aspects of health and its maintenance, disease and its cure and management. The long journey of the quest has substantiated this understanding at all levels of biomedical research. There is absolutely no doubt about the great achievements of reductionists’ approach in dissecting mechanistic basis of multicellular life process over teleonomists’ approach in analyzing life process during last a few centuries since William Harvey (1578-1657). During the last century, however, there has been a galloping awareness that a systems approach using theories and practices to undertake analysis and synthesis in a holistic way is imperative to crack the logic of life, more for the reason that such systems approach shall allow for interlacing among various aspects of health, disease and their management in the real life. This issue will be examined in the present paper.

Drug bioproduction and global optimization of the transcriptional scheme in microorganisms F. Képès 1,2, C. Bouyioukos 1, B.C. Jester 1, T. Lepage 1 Epigenomics Project / institute of Systems and Synthetic Biology, Genopole®, CNRS, University of Evry, France; 2 Department of BioEngineering, Imperial College, London, UK Background: Protein drugs and vaccines are typically produced by microorganisms in a single step involving the expression of a single foreign gene encoding this protein. Paradoxically, the production of a simple —non-protein— drug is a more complex task that involves the optimized expression of several foreign genes, encoding altogether 1


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the metabolic pathway required to synthetize the non-protein drug. State of the art today is to design and construct short pathways which involve, at most, a dozen enzymes encoded by as many genes stimulated by as many stimuli. Objective: The goal is two-fold, to break the ceiling of a dozen-gene co-regulation, and to intensely trigger all genes in the pathway with a single stimulus. Design: Bench and computational studies are currently combined to decipher the threefold interdependence between gene expression, chromosome conformation and genome layout. Results: Co-regulated genes tend to be located at periodical or proximal positions along the bacterial chromosome (1). This specific genome layout is interpreted as a means to cluster co-functional genes in space through the appropriate folding and conformation of chromosomes. This point was demonstrated using a thermodynamic model of DNA folding under the influence of localized pairwise interactions such as those mediated by transcription factors. Conclusions: This periodical genome layout is found, not only in eubacteria, but also in eukaryotic yeast, meaning that the workhorses of drug bioproduction share this feature. It will be exploited and re-designed to reach the above double goal. Along this way, we envisage to construct generic strains that allow the coordinated and efficient expression of many genes, to streamline and rationalize the production of drugs from engineered microorganisms. Acknowledgements: This work was supported by the Sixth European Research Framework (GENNETEC project number 034952), by the Seventh European Research Framework (ST-FLOW project number 289326), and by CNRS, CRIF and Genopole®. Reference: Junier I, Hérisson J, Képès F. Genomic organization of evolutionarily correlated genes in bacteria: limits and strategies. J Mol Biol 2012, 419, 5, 369–386.

Genetic risk factors and a mitochondrial hypothesis for late-onset Alzheimer’s Disease AD Roses Zinfandel Pharmaceuticals, Inc., Durham, NC Using phylogenetic mapping techniques, we examined variable length polyT length repeat clustering at the rs10526523 locus [523] and the genotype of APOE polymorphisms connected within this region of linkage disequilibrium [LD] on chromosome 19. Haplotypes of the cis-connected polymorphisms differentiated multiple age of onset curves for Caucasian individuals in the population between 62 and 83 years of age. This LD region studied contained three known expressed genes: TOMM40, APOE and APOC1, but the rationale for phylogenetic mapping of this region was to determine if the highly replicated association of APOE genotypes with age of onset of AD were solely attributable to APOE or whether another cis-acting locus could better define the AD association and be informative in more individuals at risk for AD. More than a dozen genome-wide association studies [GWAS] using platforms that contained single nucleotide polymorphisms [SNPs] from TOMM40 and APOC1 had clearly found that TOMM40 SNPs were highly significantly associated with AD as well, but virtually all the conclusions were interpreted as a confirmation of the APOE association leading to a search for an additional risk region elsewhere in the human genome.

It was now possible to explain why APOE4 had been consistently found to be associated with AD and how the APOE3-connected 523 sequences were variable, leading to heterogeneity in age of onset for APOE 3/4 and APOE 3/3 individuals. The association of AD with the variants at the 523 locus provided the opportunity to resolve age of onset curves much more accurately for APOE3 and APOE2 carriers than simply mapping to the APOE genotypes. For example, the original data published in 1993 and confirmed over the years demonstrated that the so-called APOE3/4 genotypes resulted in a mean age of onset of 75 years. Now, with 523 polyT repeat data, two distinct APOE3/4 age of onset curves for the two 523 species found cis- to APOE could be resolved. The long-very long (L-VL) data, representing an E4 L allele is paired to either an E3 short(S) or VL polyT repeat at the 523 locus, demonstrated two distinct age of onset curves with a 7-8 year difference in the mean age of onset [70 years versus 78 years]. Similarly APOE3/3 and APOE2/3 genotypes were resolved into three different curves using the 523 genotypes: S-S, S-VL, or VL-VL. Tested for APOE2 allows distinct risk curves to be identified amongst APOE3/3 and APOE2/3 subjects. Onset risk for cognitive symptoms for the next 5-7 years could be stratified, and led to design of a prospective biomarker qualification study with a simultaneous primary prevention clinical trial. An algorithm of TOMM40-523 genotype, APOE genotype, and age with define cognitively normal subjects estimated to have a high or low risk for onset of cognitive symptoms for their particular age at normal neuropsychological testing, Their TOMM40-523 genotypes, and whether carriage of an APOE4 allele allows to differentiation of risk for APOE4-negative subjects who possess APOE3/3, APOE2/3, or APOE2/2 genotypes. A series of experiments dating from 1998 contributes to a decreased mitochrondrial dynamics that, in the neuronal cells, magnifies the deficit because each neuron receives its mitochondrial content at conception. Neurons cannot die and be replaced as other tissues. This mechanism also may account for the pathogenesis of other, much less common, maternally transmitted mitochondrial diseases.

Maps of open chromatin from association signals to function P Deloukas Hinxton, United Kingdom

The public health genomics challenge: translating systems medicine into healthcare systems A Brand Institute for Public Health Genomics, Cluster Genetics & Cell Biology; Maastricht University, 6229 ER Maastricht, The Netherlands Rapid scientific advances and tools in genomics such as in the light of epigenomics, microbiomics and systems biology supported by new ICT solutions not only contribute to the understanding of disease mechanisms, but also provide the option of new promising applications in human health management during the whole life-course. What was little time ago a vision for a new era of public health, in which advances from the -omic sciences would be integrated into strategies aiming at benefiting population health, is now responding to the very pressing need for the development of effective personalized healthcare going even beyond personalized medicine based



on a systems medicine approach. While the utility of most genetic tests and biomarkers is still not evidence-based, the real take home message stops here and is a different one. In the personalized medicine setting the traditional assessment and evaluation tools just do not work anymore. Thus, we clearly face the need for a new paradigm moving from population health to personal health. The paradigm shift depends on the willingness to restructure policies, and there is a clear urgency to prepare health care systems and policy makers in time. So far, all stakeholders including policy-makers and the private sector are struggling to translate the emerging knowledge into public health. Public Health Genomics (PHG) is the area of public health ensuring that scientific advances in genomics (“from cell...”) triggered by innovative technologies are timely, effectively and responsibly translated into health policies and practice for the benefit of population health (“...to society”). The implementation of PHG requires increased concerted activities. The Institute for Public Health Genomics (IPHG) at Maastricht University aims to fulfil this task in all European Member States by hosting the European Centre for Public Health Genomics (ECPHG) and coordinating the Public Health Genomics European Network (PHGEN). Furthermore, it closely collaborates with the European Flagship Pilot ITFoM on the future of medicine being one of the most ambitious worldwide largescale, science-driven, research initiatives that aim to achieve the visionary goal of the “virtual human”. Cesuroglu T, van Ommen B, Malats N., Sudbrak R, Lehrach H, Brand A. Public health perspective: from personalized medicine to personal health. Personalized Medicine 2012;9(2):115-119.

Identifying distinct patient subpopulations based on underlying molecular mechanisms: an inflammatory bowl disease case study D Laifenfeld, D Drubin, J Park, AVan Hooser, B Frushour, R Deehan Selventa, One Alewife Center, Cambridge MA 01420 Background: It has long been recognized that some patients may respond well to a particular intervention, whereas others may gain little or no benefit. As diseases are classically characterized by their phenotype and not always sub-categorized by the specific mechanisms or genotypes contributing to the phenotype, applying a focused molecular targeted therapy may not be effective in most patients and can obscure the benefit to a responder sub-population. Objective: The objective of the study was to identify predictive biomarkers for infliximab response in inflammatory bowl disease (IBD) based on TNF signaling levels (TNF Molecular Footprint) in individual patients. Design: To detect TNF signaling in colon, a 256 gene Molecular Footprint was generated from our causal knowledgebase and applied to colon samples from a training set of 43 patients with IBD (24 patients with UC and 19 with Crohn’s colitis). Six healthy control subjects along with the training set patients were stratified by their individual levels of TNF pathway activation. Standard biomarker development methods were applied on data from patients with the highest 20% and lowest 20% TNF activation level to develop a 20-gene biomarker panel differentiating high and low TNF patients. Results: The healthy controls had the lowest levels of TNF pathway activation, and low levels of activation in treated patients correlated with response (p = 3e-8), confirming our hypothesis. The TNF

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pathway activation biomarker, using detection of TNF pathway amplitude to predict response (low, “normal-like” TNF pathway activation expected to lead to higher response), performed with a 70% responder predictive value and a 100% non-responder predictive value in a independent test set of 23 UC patients where outcomes to infliximab were known. Conclusion: This example with infliximab in UC provides initial validation that our approach for patient stratification by disease-driving mechanisms can be used to predict response to a targeted therapy. Once patient populations are identified and stratified by levels of different disease-driving mechanisms, biomarkers can be generated for each subset driven by a distinct mechanism, as we did here with TNF.

Metabolomics: Bridging gaps in systems biology O Yanes Centre for Omic Sciences, Rovira i Virgili University, Tarragona, Spain (http://www.yaneslab.com/) Metabolomics is a newly emerging field focused on the profiling of small, naturally occurring molecules collectively known as the ‘metabolome’. While the genome and proteome represent upstream biochemical events, the functions of which are subject to epigenetic regulation and post-translational modifications, respectively, metabolites are the functional output of cellular reactions and therefore more closely correlate with phenotype. Here a mass spectrometry (MS)- and nuclear magnetic resonance (NMR)-based metabolomic platform will be detailed that provides a method for identifying cellular pathways that are perturbed during disease. In addition, the integration of metabolomic technologies with untargeted and targeted experimental approaches in proteomics and genomics may offer meaningful data for the correlation of biochemical changes with phenotype.

30 SEPTEMBER 2012 – AFTERNOON WORKSHOP PROTAGEN Patient stratification in clinical trials using high quality immunoproteome profiling: Examples from autoimmune diseases and cancer Chair: Vangelis E. Manolopoulos, Alexandroupolis, Greece Moderator: Peter Schulz Knappe, Dortmund, Germany

WORKSHOP GENOMATIX Next generation sequencing tools Chair: George P. Patrinos, Patras, Greece Moderator: Martin Seifert, Genomatix, Munich, Germany

WORKSHOP RANDOX Biochip Array Technology applications Chair: Steve Humphries, London, United Kingdom Moderator: John Lamont, Co Antrim, United Kingdom


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Functional epistatic interactions and blood pressure levels S Siest EA 4373, University of Lorraine, Nancy, France Epistatic interactions, even important in the knowledge and understanding of the genetic mechanisms involved in blood pressure phenotypic variance are until now very poorly elucidated. In the present study, we in-depth characterized these G*G interactions for 10 genetic variants previously reported to be associated with blood pressure levels, in a large population using a multilocus assay and a pre-planned two-phase approach. We selected rs1799752Ins>del in ACE, rs5882A>G in CETP, rs1801133C>T in MTHFR, rs662A>G in PON1, rs1800629G>A in TNF, rs5355C>T in SELE, rs6046>A in F7, rs1800790G>A in FGB, rs328C>G in LPL and rs3025058T>Ins in MMP3 in order to establish a new specific multilocus essay for Hypertension the “HTN-Evidence InvestigatorTM biochip” designed by Randox Laboratories, Antrim, UK. We first tested the associations of the above SNPs with blood pressure levels in 3,600 French individuals (discovery cohort) and after replication of the results in 4,620 additional European individuals we searched their association with the expression of different genes putatively implicated in cardiovascular pathways in peripheral blood mononuclear cells from a subsample of 90 individuals. We further assessed G*G interactions between SNPs highlighting significant associations. In the discovery cohort we found 2 SNPs associated with decreased blood pressure levels (P≤3.7 x10-3 and P=5x10-3). Both variants interacted in order to decrease blood pressure levels (P≤0.048). This interaction was replicated in the 4,620 additional individuals (P=0.03). Furthermore, by the transcriptomic investigation in peripheral blood mononuclear cells, we proposed the molecular mechanism involved explaining 4% of BP variation. These findings reveal the importance of epistatic interactions in blood pressure genetics.

SESSION: OMICS FOR SYSTEMS MEDICINE APPLICATION IN ONCOLOGY On the clinical use of genome wide association studies: breast cancer DG Cox Cancer Research Center of Lyon, INSERM U1052, Lyon, France Background: Genome wide association studies (GWAS) are a powerful tool in the arsenal of the molecular genetic epidemiologist. While there was great promise of GWAS with respect to disease etiology, few such studies have had measurable impact on disease prevention (i.e. screening) or treatment, particularly pertaining to breast cancer. We will explore the place of GWAS in the clinical setting using the example of breast cancer. Objectives: We will review the history of GWAS in the context of breast cancer, and define potential future directions for applying genomic analyses to clinical aspects of this disease of great importance to public health. Results: While a number of GWAS on breast cancer risk have been published, the majority of the variants described, while common in the populations studied, have had very moderate effects on disease

risk. As a consequence, these variants have little impact on screening practices. Furthermore, despite the first breast cancer GWAS being published nearly 5 years ago, little is known about how these variants, or those they are markers for, influence disease risk. Despite the relative lack of influential results of GWAS of breast cancer incidence, genetic variants may still be of interest with respect to clinical outcomes of breast cancer. Here, the difficulty lies in the heterogeneity of breast cancer types, and need for sufficient (both in terms of detail and length) follow-up to achieve numbers necessary to carry out effective GWAS studies. Conclusions: GWAS remain a powerful tool to study the influence of genomics on diseases such as breast cancer. Properly conducted GWAS studies among breast cancer patients may provide insights into mechanisms of disease progression, as well as potential prognostic markers. Markers with prognostic value can furthermore be used as randomization variables in clinical trials, potentially increasing the effectiveness of treatment regimes.

The mevalonate pathway in cancer cells: getting control on apoptosis C Le Jossic-Corcos*, Y Dréano*, S Commet*, C Gouin*, M Pesson*, K Trillet*, Y Amet*, D Lucas*, B Simon*, E Pécou-Gambaudo**, L Corcos*. *INSERM U1078-ECLA, Faculty of medicine, SFR-ScInBioS, University of Brest, 22 avenue Camille Desmoulins, 29200 Brest, FRANCE; ** Bioinformatics modeling and simulation, SOBIOS SA, 2000 route des Lucioles, Aristote A, 06901 Sophia Antipolis, FRANCE; Email address: [email protected] Background: The mevalonate biochemical pathway leads to the synthesis of cholesterol, but also to the production of C15 (farnesyl pyrophosphate, FPP) and C20 (geranylgeranyl pyrophosphate, GGPP) intermediates that prenylate, and thereby activate, small G proteins from the Ras, Rho, Rac etc. families. Collectively, these proteins stimulate cancer cell growth and motility when they carry activating mutations. Statins inhibit the HMG-CoA reductase enzyme, which converts HMG-CoA into mevalonate, lower cholesterol in humans and efficiently trigger cancer cell apoptosis. Objective: Our aim is to circumvent the activity of the mevalonate pathway, as it is believed that apoptosis induction by statins may be due, in part, to their ability to prevent prenylation of small G proteins. Design: We have undertaken an integrated analysis aimed at determining the activity of the pathway in response to statins through surveying several endpoints by analytical and cellular approaches: Enzyme activities (HMG-CoA reductase, Mevalonate Kinase, FPP Synthase and Squalene Synthase), FPP and GGPP amounts, protein prenylation (Ras, RhoA,…) and apoptosis levels. Results: Lovastatin blunted the mevalonate pathway, as shown by the strong decrease in both FPP and GGPP, and triggered apoptosis in cancer cell lines. Supplementation with FPP or GGPP reduced apoptosis induction, even more so than cholesterol. The suppression of Rho prenylation – but not that of Ras – was associated with the effect of lovastatin, indicating that Rho proteins participate in the control of cell apoptosis in response to the drug. A bioinformatics model is being built to integrate the biochemical parameters and apoptosis data. Conclusion: This approach will provide a systems’ biology view of the cancer cell killing effects brought about by mevalonate shortage.



This should lead to a better understanding of the alterations in cancer cell metabolism and allow efficient cell death induction upon using the predictions from the model. Acknowledgements: This study was supported by the BioIntelligence program, the INSERM, the University of Brest, the “Ligue Contre le Cancer”, the “Association pour la Recherche sur le Cancer” (ARC) and the “Structure Fédérative de Recherche” (SFR) ScInBioS.

Cancer-genetics guided discovery of blood biomarkers - a systems biology approach R Schiess Zurich, Switzerland A key barrier to the realization of personalized medicine for cancer is the identification of biomarkers. A two-stage strategy for the discovery of serum biomarker signatures corresponding to specific cancer-causing mutations and its application to prostate and ovarian cancer in the context of the commonly occurring phosphatase and tensin homolog (PTEN) tumor-suppressor gene inactivation will be presented. PTEN is one of the most commonly inactivated genes in human cancer and has been identified as lost or mutated in several sporadic cancers, including endometrial carcinoma, glioblastoma, breast, and prostate cancer. It is expected that signaling-pathway-activating mutations such as PTEN loss produce changes in the proteomes of affected tissue, and these changes should be detectable as discrete biomarker signatures in the serum.

Companion Diagnostics and targeted immunotherapies J-Y Bonnefoy VP R&D Transgene SA, Illkirch, France Inflammation has long been thought to be associated as a cause or a consequence of many acute or chronic diseases. Same holds true for cancer and chronic infectious diseases. Yet little is known on drugs which act by inducing or inhibiting inflammation and their benefit on the cause of those latter diseases. This may indeed also depend on the ongoing inflammatory status of the patient when such inflammatory modulating drugs are administered. In this context, Transgene is developing viral-based therapeutic vaccines for the treatment of cancer and infectious diseases. Those products are based on live MVA viruses which likewise any other viruses do provoke some inflammation upon injection. In a recent Phase IIb clinical study, we observed that cancer patients who at baseline did present an inflammatory status, actually suffered a safety signal whereas conversely patients with a non-inflammatory status at baseline did very significantly benefit from the product as measured by overall survival. This indicates an important equilibrium between inflammation and immune response to viral-based therapeutic vaccines.

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Mathematical modelling of proliferation in cell populations with applications to therapeutic optimisation in oncology F Billya, J Clairambaulta, O Fercoqb INRIA Paris-Rocquencourt, LJLL, Université Pierre et Marie Curie, 4, place Jussieu, 75005 Paris, France; bINRIA Saclay-Île-de-France, CMAPX, Ecole Polytechnique, 91128 Palaiseau Cedex, France a

Background: Decrease of cell proliferation in proliferating tissues is the result of the action of anticancer drugs, searched for in tumours, avoided in healthy tissues. In cancer chronotherapies, differences between healthy and cancer proliferating cell populations w.r.t. control by circadian clocks on cell cycle progression at phase transitions are exploited to enhance anticancer effects while minimising unwanted toxic side effects. Objectives: To identify a mathematical model of the cell division cycle in proliferating cell populations, with investigation of desynchronisation of cancer cell populations w.r.t. cell cycle timing, compared to healthy ones. To obtain optimal control algorithms of drug delivery to define best chronotherapeutic strategies. Design: The proliferation of cell populations, cancer or healthy, is described by a population dynamics model of the Von FörsterMcKendrick type, in which targets, for both external drugs and internal clocks, are checkpoints, gating at phase transitions. The phase transition gating rates, are identified in individual cells in cultures using a fluorescence-based technique (FUCCI). The circadian clock control is a fixed cosine-like gate opener at transitions, assumed to be looser in cancer (less synchronised) than in healthy cell populations. Drug control is a free time-varying modulation of the gating. Uzawa’s algorithm is used to optimise the drug delivery function, with evaluation of proliferation coefficients in the two cell populations. Results: Optimal drug delivery schedules were defined, successfully reducing the cancer population growth rate with limited damage to its equivalent for healthy cells. Conclusion: This methodology, combining a theoretical approach, which allows to predict growth rate in proliferating cell populations, with experimental measurements of the mathematical model parameters, may be used to propose optimised delivery schemes for cytotoxic drugs in cultured cell populations, prior to clinical trials. Acknowledgments: Supported by a grant from the European Research Area in Systems Biology (ERASysBio+) to the French National Research Agency (ANR) #ANR-09-SYSB-002-004 for the research network Circadian and Cell Cycle Clock Systems in Cancer (C5Sys) coordinated by Francis Lévi (INSERM U776, Villejuif, France). Reference: Billy F, Clairambault J, Fercoq O, Gaubert S, Lepoutre T, Ouillon T, Saito S. Synchronisation and control of proliferation in cycling cell population models with age structure. To appear, 2012; downloadable from http://hal.archives-ouvertes.fr/hal-00662885.

01 OCTOBER 2012 - MORNING SESSION: SYSTEMS BIOLOGY AND DRUG TARGETS Applying Systems Biology to Drug Discovery and Development

Petri net modelization: a new tool for dissecting cell signal in cancer patients

A Bril Institut de Recherches Internationales Servier, Suresnes, France

P Braquet Paris, France

The discovery of a novel therapeutic solution requires (1) the identification of a patient subpopulation expected to benefit from the therapy,


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(2) the understanding of the underlying biology describing the pathophysiology of the disease, and (3) the characterization of how an exogenous entity aimed at manipulating the disease pathway could demonstrate benefit to the patient over existing therapies. Today, with the possibility to gain information concerning the biological processes at a molecular level, multi-scale integration and simulation strategies are required to translate any hypothesis into a benefit to the patient. The drug discovery process is therefore a dynamic interdisciplinary field integrating traditional experimental techniques with systems analysis. In brief, drug discovery starts at the patient level with careful detailed analysis of both the phenotype and the molecular network with the aim to identify an innovative hypothesis. As an example, following a careful selection of patients with subtype of breast cancer, it has been possible, based on a detailed analysis of the transcriptome and of the proteome to identify potential drug discovery hypotheses. Such a novel hypothesis (in this case a molecular target) is then tested by using exogenous modulators agents (new chemical entities, biologics) that will need to fulfill all the criteria, in terms of efficacy, safety, druggability, required to benefit the patient. Validating the hypothesis at the patient level is the aim of current translational medicine strategies. From this, it is obvious that every drug discovery project can be characterized by the complexity of the systems, the vast quantity of data and the scattered pieces of knowledge. Therefore, creating an integrating platform allowing each and every scientist involved into a drug discovery process remains needed to facilitate and improve drug discovery and development. Such a platform should facilitate the use of both software and data resources available not only in a specific department but in the scientific community at large.

Genome based assessment of animal models for biomedical research U Certa Non-clinical Safety, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland Background: Drug-safety testing in animal models is part of the regulatory requirements for novel drugs before entry into humans. Refining, replacing and reducing animal studies (3R-principles) is a global commitment supported by all major pharma-companies. Objective: Genome research based improvement of animal experiments. Results: Genomics allows a significant improvement and more rational design of animal experiments and mechanistic studies. In particular the translational value of data for humans can be assessed with high confidence. Data based on the genome of the primate Macaca fascicularis will be presented. Conclusion: The genomes of all major animal models used in biomedical research will lead to a significant improvement of drugsafety and- efficacy. Reference: Ebeling M, et al. Genome Res 2011;21:1746–56.

Gene-gene and drug-drug interactions in pharmacogenetic studies: the case of leukotrienes in asthma M Via Laboratory of Anthropology, Dept. Animal Biology, University of Barcelona, Barcelona, Spain

Background: Advances in functional genomics and systems biology have increased the awareness of the importance of interactions between genes in the study of complex phenotypes. Moreover, it is well known that some drugs can modulate the physiological effect of other drugs with relevant clinical consequences. However, it is still not well understood the role that complex (i.e. third- or higherorder interactions) may play in complex outcomes. Leukotrienes are a family of molecules with an important role in allergic and inflammatory diseases. Several genes are involved in their metabolism and different leukotriene modifier (LM) drugs have been approved for the treatment of asthma. Objectives: i) To detect interactions between leukotriene genes in the risk for asthma, and ii) to quantify the impact of LM drugs and genegene-LM interactions on the response to bronchodilator treatment. Design: 687 parent-child trios from the Genetics of Asthma in Latino Americans (GALA) study where genotyped for SNPs in the LTA4H and ALOX5AP genes in the leukotriene pathway. Pulmonary function was evaluated before and after the administration of albuterol, a shortacting β2-adrenergic receptor agonist. Results: A gene-gene interaction was identified between SNPs at LTA4H and ALOX5AP that conferred a decreased risk for asthma in Latino asthmatics (p=0.003). Moreover, the use of LM was associated with a clinically significant increase in bronchodilator responsiveness in Puerto Rican children (p < 0.001). Analyzing together the genetic and LM effects on the response to albuterol, we found that among carriers of certain LTA4H and ALOX5AP alleles (and their gene-gene interactions) the use of LM was associated with significant increases (ranging from 7 to 10%) in forced expiratory volume (FEV1) after bronchodilator use. Conclusions: Genetic markers and drugs act at different orders of interaction (from individual alleles/drugs to gene-gene-drug-drug interactions) to modulate complex outcomes, such as disease status or drug response. Here we presented results on the leukotriene pathway in asthma, but it is likely that this model can be applied to other complex phenotypes. Acknowledgement: The author was supported by the Beatriu de Pinos Program (2006 BP-A 10144 and 2009 BP-B 00274). The GALA1 study was supported by the following grants: the NIH (HL078885, AI077439, HL088133), the Flight Attendant Medical Research Institute (FAMRI), American Asthma Foundation and the Sandler Foundation).

Contribution of microRNA to drug response I Cascorbi Institute of Experimental and Clinical Pharmacology, Christian Albrechts University, Kiel, Germany Chemoresistance of tumors is often reported to be due to overexpression of efflux-transporters or genetic alterations of signaling pathways. More recently, there is increasing evidence that epigenetic modification contributes to the phenomenon of drug resistance. Despite alteration of DNA methylation or histone-acetylation, deregulated microRNA expression patterns of tumor cells have been identified to interfere with drug response. Attempts to modify the expression of selected microRNAs have partly led to intriguing improvements of chemotherapy response. Recently we could show that the induction of ABCC2 protein by rifampicin is counteracted by microRNA 379 in HepG2 cells,



contribution to the explanation of discrepancies between mRNA and protein expression following external stimuli though PXR ligands. In contrast, down-regulation of certain microRNAs could abolish the suppression of gene expression as exemplified by imatinib resistance of BCR/ABL-overexpressing K-562 cells through up-regulation of ABCG2 (BCRP). The use of microRNA as biomarkers is currently not established, however epigenetic factors may be considered to better understand inter-and intraindividual variation of drug response.

Human lymphoblastoid cell lines: tools for drug response biomarkers discovery D Gurwitz Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel Human lymphoblastoid cell lines (LCLs) are commonly used in genomic studies as the best representation of donors. They are often employed for searching disease risk alleles. In addition, LCLs are valuable tools for drug response biomarkers discovery, using the genome-wide hypothesis free approach for detecting genes whose expression levels may indicate favorable or poor drug response. The example of studying biomarkers for individual response to selective serotonin reuptake inhibitors (SSRIs) will be presented in detail. SSRIs are the most commonly used class of antidepressants for treating major depression. However, >30% of patients do not respond sufficiently to their first-line antidepressant drug treatment and require alternative therapeutics. Genome-wide studies searching for SSRI response DNA biomarkers or studies of candidate serotonin-related genes so far gave inconclusive or contradictory results. As a first step toward studies with patients’ lymphocytes, we used the alternative approach of genome-wide transcriptome-based search for SSRI response biomarkers in human lymphoblastoid cell lines (LCLs). We first screened 80 LCLs from healthy adult female individuals for growth inhibition by paroxetine. Fourteen LCLs with extremely high and low sensitivities to paroxetine were chosen for genome-wide expression profiling with Affymetrix microarrays. Genome-wide transcriptomic analysis has identified 158 genes whose levels of expression differed by >1.5-fold and p4). In pediatric patients, VKAs are mainly used to prevent thromboembolism in cardiac diseases. In a cohort of 118 children (median age 9 years, 3 months-18 years), we evaluated i/the relative contribution of non-genetic factors and VKORC1/CYP2C9/CYP4F2 genotypes on warfarin response (n=83); ii/the influence of these factors on the time spent within/above/below the range. Height, target INR, VKORC1 and CYP2C9 genotypes were the main determinants of warfarin dose requirement, explaining 69.7% of the variability. None of the covariates were associated with the time spent above or below the INR range. In these special populations, VKORC1 and CYP2C9 variants significantly influenced the warfarin response. The potential usefulness of pharmacogenetic-based versus empiric warfarin therapy on clinical outcomes remains to be assessed. Further work is needed to determine whether pharmacogenetics could minimize the risk of over- and under-anticoagulation, especially at the start of treatment and could improve the management of these patients. Reference: Moreau C, Bajolle F, Siguret V, Lasne D, Golmard JL, Elie C, Beaune P, Cheurfi R, Bonnet D, Loriot MA. Vitamin K antagonists in children with heart diseases: Height and VKORC1 genotype are the main determinants of the warfarin dose requirement. Blood 2012; 119(3):861–7.

Genetic variation in GATA-4 might affect the coumarin maintenance dose RMF van Schie1, JAM Wessels2, LGJ van Hoorn1, TI Verhoef1, T Schalekamp1, S le Cessie3,4, FJM van der Meer5,6, FR Rosendaal4,5, LE Visser7, M Teichert7, A Hofman7, PN Burhe7, A de Boer1, AH Maitland-van der Zee1 1 Pharmaco-epidemiology and Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; 2Clinical Pharmacy & Toxicology, Leiden University Medical Center, Leiden, The Netherlands; 3Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands; 4Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands; 5Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands, 6Medial, medical-diagnostic laboratories, Hoofddorp, The Netherlands; 7 Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands

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of the best explaining SNP for acenocoumarol were validated in the Rotterdam Study. This cohort study among approximately 15,000 persons was designed to investigate different diseases in a population aged over 45 years. Complete data was available for 1265 acenocoumarol users. For phenprocoumon no replication study was available. Results: Significant effects on the acenocoumarol maintenance dose were found for haplotypes GG and AG in haploblock 4 and for haplotype GG in haploblock 5&6. These effects were also observed for 3 SNPs that were part of these haplotypes. The largest dose differences were found for rs3735814 in patients being wild type for CYP2C9. The dosages decreased from 2.92 mg/day for the GATA-4 wild type patients to 2.65 mg/day for the patients carrying 1 GATA-4 variant allele to 2.37 mg/day for patients carrying 2 GATA-4 variant alleles (p=0.004). Results for rs3735814 could not be replicated in the Rotterdam Study. For phenprocoumon, no significant effects were observed. Conclusion: Genetic variations in GATA-4 appeared to influence the acenocoumarol maintenance dose in the Pre-EU-PACT study. However this could not be replicated in the Rotterdam Study. No significant association was found for phenprocoumon.

01 OCTOBER 2012 – AFTERNOON SESSION: CLINICAL PHARMACOGENETICS AND THERANOSTICS HLA, immunogenetics, pharmacogenetics and personalized medicine M Pirmohamed Liverpool, United Kingdom

ESPT ROUND TABLE Implementation of pharmacogenomics in clinical setting in European public health care system Moderators: A Llerena (Badajoz, Spain) & R Van Schaik (Rotterdam, The Netherlands) General discussion with ESPT board members and representatives of European and International Institutions: I Cascorbi (IUPHAR), P Marquet (IATDMCT), M Papalouca (EMA), A Huriez (EPEMED), V Oeding (EDMA), A Brandt Kirsten Steinhausen (ESF)

Pharmacogenetics in Daily Routine Clinical Practice Background: It has been shown that the transcription factor GATA-4 is involved in the transcriptional regulation of CYP2C9. Therefore, it is hypothesized that genetic variations in GATA-4 play a role in the variability in coumarin dose response. Objectives: To investigate whether the phenprocoumon and acenocoumarol maintenance dose is influenced by genetic variations in GATA-4. Design: The Pre-EU-PACT database was used, which contains information about 624 phenprocoumon users and 471 acenocoumarol users. The influence of variations in GATA-4 on the phenprocoumon and acenocoumarol maintenance dose was investigated by performing an ANOVA trend analysis, stratified for CYP2C9 genotypes. Results

W Siffert Institute of Pharmacogenetics, University Hospital, 45147 Essen, Germany Background: During the past decade an immense effort has been invested in the attempt to identify pharmacogenetic markers which may help to individualize drug therapy with emphasis on both, drug safety and drug efficacy. However, which of these findings has made it from bench to bedside? In my report I summarize which of these tests are routinely done in a typical university hospital setting, i.e. at the University Hospital of Essen, Germany.


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Results: In general, and with one exception, all tests which are routinely performed aim at reducing severe adverse drug affects. Most, but not all of the single nucleotide polymorphisms (SNPs) determined are located in genes whose products play a major role in drug metabolism. TPMT: Thiopurine methyltransferase (TPMT) catalyzes the methylation of thiopurine drugs such as azathioprine and 6-mercaptopurine. Several mutations in the TPMT gene correlate with low enzyme activity and adverse effects such as myelotoxicity. Hence, genotyping TPMT makes it possible to identify patients at high risk for drug toxicity. Azathioprine is frequently used in patients with M.Crohn, colitis ulcerosa, rheumatoid arthritis, to name but a few. Approximately 10 % of the German population carry only one active allele of TPMT, 0.3 % have no TPMT activity. In heterozygous subjects the dose of azathioprine is reduced by 50 %, patients with a complete lack of TPMT activity. Do not receive this drug. UGT1A1: Metastatic colorectal cancer is frequently treated with irinotecan, a topoisomerase-I inhibitor. The UGT1A1 gene encodes for an enzyme that metabolizes irinotecan, and its genetic variants were shown to be associated with increased drug toxicity. Carriers of the variant need an at least 30 % reduction of their drug dose. DPYD: Dihydropyrimidine dehydrogenase (DPD) is the initial ratelimiting enzyme in endogenous pyrimidine catabolism and is responsible for the reduction of the pyrimidine analog 5-fluorouracil (5-FU). DPD deficiency is known to cause potentially lethal toxicity in patients receiving 5-FU. A variety of SNPs located in DPYD are associated with severe toxicity of 5-FU. For that reason, allpatients scheduled for 5-FU are routinely genoyped for SNPs in DPYD. HLA-B*5701: Hypersensitivity to abacavir affects about 4% of patients who receive the drug for HIV-1 infection. Screening for HLA-B*5701 drastically reduces abacavir hypersensitivty and is mandatory for all patients scheduled for this drug. Conclusion: Despite widely spread scepticism some of the pharmacogenetic markers have a proven utility in clinical practice. Reimbursement issues have been resolved. Adverse drug reactions can be reduced following the implementation of pharmacogenetic procedures into routine clinical practice.

02 OCTOBER 2012 – MORNING SESSION: ENVIRONMENTAL EPI- AND METAGENOMICS Systems Approaches to Health Maintenance and Disease Prevention

subjects a priori into case/control groups, we study the same cell/ animal/human over time with repeated environmental/nutritional challenges to capture individual health trajectories and recognize deviations from an individual’s optimal lifetime health course. Methods: The profiling techniques of proteomics [J. Proteomics 2008], metabonomics [Curr. Opin. Biotechnol. 2008], and transcriptomics [Anal. Chem. 2006] are employed to show nutritional efficacy and dietary mechanisms. Genetic methodologies stratify individuals enrolled in nutritional intervention studies. We explore epigenetics to improve our understanding of environmental influences, including nutritional programming of metabolic health [Nutr. Rev. 2010]. Proteomics is a central platform in our –omics set of systems technologies and comes in two flavours [J. Proteome Res. 2010]: as discovery and validation tool for (a) biomarkers and (b) bioactives, such as peptide and protein ingredients. While we had built a robust and automated chips-based gene expression platform [Anal. Chem. 2006], we have now established deep sequencing technologies. Results: Using proteomics, we have investigated: effects of low GI diets on weight maintenance [Mol. Nutr. Food Res. 2011]; development of early life intestinal [Mol. Cell. Proteomics 2011] and systemic [Mol. Immunol. 2011] immune competence; candidate allergy biomarkers [J. Proteome Res. 2011]; milk bioactives [J. Proteomics 2010]; and probiotics [J. Proteomics 2009]. A complex interplay between the microbiota and the host immune system shapes the latter throughout life, but little is known about the microbial effect on key players of the adaptive immune system, the B2 B cells. We evaluated the effect of commensal bacteria on B cell ontogeny and function [Mol. Immunol. 2011]. Combining classical immunology with transcriptomics, we revealed an influence of gut microbiota on function of mucosal B2 B cells. Dynamic postnatal intestinal development is characterized by morphological changes coinciding with functional adaption to the nutritional change from a diet rich in fat (milk) to a diet rich in carbohydrates on from weaning. We analyzed changes of primary intestinal epithelial cells from jejunum during the early and middle suckling, as well as the weaning period in mice, using a labelfree proteomics approach [Mol. Cell. Proteomics 2011]. We provided the first time-resolved proteomics of intestinal epithelial cells along postnatal intestinal development. Key Message: We develop and apply systems biology in longitudinal studies with repeated challenges to better understand metabolic flexibility and healthy ageing.

Developmental Programming of type 2 diabetes and obesity

M Kussmann1,2 1 Nestlé Institute of Health Sciences (NIHS), Proteomics and Metabonomics Core, Lausanne, Switzerland. 2Aarhus University, Faculty of Science, Aarhus, Denmark

S Ozanne Metabolic Research Laboratories, Institute of Metabolic Science, University of Cambridge, Cambridge CB2 2QR, UK

Scope: We deploy systems technologies encompassing –omics and (epi)genetics as a function of diet and lifestyle [Pers. Med. 2008] and address nutritionally actionable health conditions such as immune balance [Mol. Cell. Proteomics 2011], energy metabolism [Mol. Nutr. Food Res. 2011], physical [Physiol. Genomics 2011] and mental performance and digestive health [J. Proteomics 2008]. At the Nestlé Institute of Health Sciences (NIHS) we are building systems biology platforms, natural human cell models, rodent genetic resources, and human clinical trials to better understand healthy ageing and metabolic, cognitive and intestinal health. Instead of separating study

It is now over twenty years since epidemiological studies revealed that there was a relationship between patterns of early growth and subsequent risk of diseases such as type 2 diabetes, cardiovascular disease and the metabolic syndrome. Studies of identical twins, individuals who were in utero during periods of famine and animal models have provided strong evidence that the early environment, including early nutrition, plays an important role in mediating these relationships. The concept of “early life programming” is therefore widely accepted. However the mechanisms by which a phenomenon that occurs in early life can have long-term effects on the function



of a cell and therefore metabolism of an organism many years later are only starting to emerge. A number of potential mechanisms have been suggested including: (1) Permanent structural changes in an organ, such as the endocrine pancreas and brain, resulting from suboptimal levels of an important factor during a critical period of its development. (2) Permanent effects on regulation of cellular ageing through increases in oxidative stress leading to macromolecular damage, including that to DNA causing premature cell senescence and (3) Persistent alterations in epigenetic modifications (e.g. DNA methylation and histone modifications) leading to changes in gene expression. Several transcription factors have been shown to be susceptible to programmed changes in gene expression through such mechanisms. These conceptually are attractive targets of programmed epigenetic regulation, as through regulation of their expression a whole network of other genes will be regulated. Further understanding of the extent and nature of such processes will enable the development of preventative and intervention strategies to combat the burden of common diseases such as type 2 diabetes and obesity.

Epigenetics and environment: a complex relationship MF Fraga1,2 Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid E-28049, Spain; 2 Cancer Epigenetics Laboratory, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain 1

The relationship between epigenetics and environment is an emerging field that promises exciting revelations in the near future. Epigenetic mechanisms, including DNA methylation and histone modification, play an important role in normal development. Besides the developmental ontogeny of epigenetic modifications, there is also considerable stochastic variation, often without apparent biological purpose. These epigenetic variation depend on genetic (intrinsic) and environmental (extrinsic) factors. Some of the epigenetic alterations described during development, as hypermethylation at specific promoters and decrease of global DNA methylation, are also associated with cancer. Future challenge in the field is the determination of the role of these epigenetic alterations in the establishment of specific aging and disease phenotypes.

Perinatal nutrition, epigenetics & later metabolic risk K Godfrey MRC Lifecourse Epidemiology Unit and NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton UK Perinatal nutrition, epigenetics and later metabolic risk Background: Experimental studies in animals indicate that particular maternal exposures during pregnancy can have specific effects on body composition in the offspring, with long-term implications for subsequent metabolic phenotype and cardiovascular risk. In animals the environment during early life induces altered phenotypes in ways which are influenced or mediated by epigenetic mechanisms, but until recently there has been little direct evidence in humans and

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understanding of which developmental influences can alter body composition in the offspring is incomplete. Objective: To define maternal exposures associated with offspring adiposity and elucidate underlying epigenetic mechanisms. Design: Follow up studies within the Southampton Women’s Survey (SWS), in which the pre-pregnant characteristics of 12,583 women were assessed at recruitment; 3,160 of these women have subsequently become pregnant. The babies have been measured at birth and anthropometric measurements made at 6 months, 1, 2, 3, 4, 6 and 8 years of age. Body composition by dual energy X-ray absorptiometry is assessed in samples of the offspring at birth, and at 4, 6 and 8 years. Results: Using DXA-assessed measurements of body composition, we have shown greater adiposity in the offspring in association with higher maternal adiposity, poor quality maternal diets in pregnancy (characterised by frequent consumption of energy-dense, micronutrient-poor foods), low maternal vitamin D status, excess gestational weight gain, and short duration of breastfeeding. Many of the relationships between early experience and adiposity in childhood are most clearly seen when using the DXA-assessed measures. Using Sequenom MassARRAY we have found that greater methylation of a single CpG within the RXRA promoter measured in umbilical cord was strongly associated with greater adiposity in later childhood.1 Perinatal measurements of DNA methylation explained >25% of the variance in childhood adiposity. These findings were replicated in a second independent cohort.1 Conclusions: Our data provide the first human evidence that epigenetic processes in non-imprinted genes have an important role in early growth and later body composition. Understanding the associations with maternal exposures and direct measures of adiposity provides insights into the aetiology of childhood obesity, with implications for the design of intervention studies.

Reference 1. Godfrey KM, et al. Epigenetic gene promoter methylation at birth is associated with child’s later adiposity. Diabetes 2011;60:1528–34.

The Study of the Human Microbiome and Implications for Human Health and Disease KE Nelson The J. Craig Venter Institute, Rockville, Maryland, USA The numbers of microbial cells that inhabit the human body significantly outnumber the number of host somatic cells. Available new sequencing technologies and ‘omics approaches are allowing for an indepth characterization of these species – many of which have been implicated in various health and disease conditions. Our recent studies on the human microbiome highlight a higher degree of microbial diversity within and across individuals than was previously appreciated as well as new microbial species whose roles remain unexplored. Access to human populations, healthy as well as those with diseases ranging from periodontitis and diabetes to gastrointestinal disorders present new possibilities for defining diagnostics, treatments and probiotics for microbial associated human diseases. It is clear that the advent of metagenomics holds significant promise for increasing our understanding of many microbial diseases associated with the human body, inclusive of those that are yet to be characterized.


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SESSION: EPIGENETIC AND PHARMACOGENOMIC APPLICATIONS Epigenetics and microRNAs S Gay Department of Rheumatology University Hospital CH-8091 Zurich Switzerland Our laboratory has over the last 10 years turned into an epigenetics research center. By studying the pivotal role of synovial fibroblasts (SF) in rheumatoid arthritis (RA) (1), M Neidhart has reported that RASF derived are expressing a Line-1 element which is only expressed during the stage of hypomethylation and is thereby inducing p38delta and galectin-3. Along with future analysis of methylation of specific promoters such as CXCL12 in RA, we studied also the benefits of methylating the hypomethylated RASF. Since epigenetic modifications are also associated with regulatory mechanisms mediated through sumoylation/ubiquination, H Maciejewska could report that the apoptosis regulating SUMO-1 degrading enzyme SENP1 regulates the acetylation at the distal end of the MMP-1 promoter in RASF. J Stanczyk in our laboratory found first by hybridization miR-145 and 155 to be expressed in RASF and monocytes, and later by RT-PCR using specific primers for all differentially expressed in RASF. In analyzing subsequently the functional role of individual miRs, she has reported that mir-203 is a strong regulator of IL-6. Most interesting was that IL-6 was neither regulated by TLRsignaling nor proinflammatory cytokines such as IL-1 and TNFa, but by methylation in the promoter of the miR-203. This finding illustrates that the expression of certain microRNAs is regulated by methylation. In related work within the FP7 Masterswitch project we could not induce arthritis in miR 155 ko mice (2). M Brock in his PhD thesis showed that IL-6 regulates the microRNA cluster 17/92 in the down regulation of bone morphogenic protein receptor 2 (BMPR2) in vascular cells during the development of pulmonary hypertension and that microRNA-18a enhances the interleukin-6-mediated production of the acute-phase proteins fibrinogen and haptoglobin in human hepatocytes (3). 1. Lefèvre S et al Synovial fibroblasts spread rheumatoid arthritis to unaffected joints. Nat Med 12:1414-20, 2009 2. Kurowska-Stolarska M et al. MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis. Proc Natl Acad Sci USA 2011;108:11193-8. 3. Brock M et al MicroRNA-18a enhances the IL-6 mediated production of the acute-phase proteins fibrinogen and haptoglobin in human hepatocytes. J Biol Chem 286:40142-50, 2011

Clinical outcomes databases F Frueh Bethesda, USA

Pharmacogenomics of statin related side effects Lars Kuepfer Systems Biology and Computational Solutions, Bayer Technology Services GmbH Interindividual variability in clinical endpoints and occurrence of potentially severe adverse effects may hamper drug development at

all phases of (pre-) clinical research. Hence, a comprehensive understanding of the processes governing both pharmacokinetics and pharmacodynamics is of utmost importance. Physiology-based pharmacokinetic (PBPK) models enable the mechanistic investigation of drug distribution and drug action at a mechanistic level of detail. Most notably, the computational models can be used to incorporate heterogeneous experimental data ranging from gene expression profiles at the cellular scale to physiological parameters at the wholebody level into one integrative modeling framework. Likewise, PBPK models allow differentiating between specific physiological properties of the patient on the one hand and drug physicochemistry on the other. Using the example of statin pharmacogenomics, we show here how targeted experimental information can support the model-based investigation of patient-specific pharmacokinetics and moreover help to explain occurrence of adverse drug reactions. PBPK models of different statins will be presented which include various proteinmediated processes governing a particular distribution or metabolisation behavior, respectively. We will then show how inter-individual variability in pharmacokinetics emerges as a consequence of a patient’s genetic and phenotypic predisposition. The performance of the PBPK models will be demonstrated by comparison of simulated time profiles and experimentally measured plasma concentration curves. Based on such comprehensive models, occurrence of specific side effects will be analysed and compared to clinical observations. The methods presented outline how computational models together with targeted experimental data allow a mechanistic investigation of drug efficacy and toxicity. Such integrative approaches may have significant implications for the development of individualized therapeutic strategies with a favorable risk-benefit profile in the future.

CYP2D6 and the severity of suicide attempts Peñas-Lledó EM1, Blasco-Fontecilla H2, Dorado P1, Vaquero-Lorenzo C2, Baca-García E2, Llerena A1 1 CICAB Clinical Research Centre, Extremadura University Hospital and Medical School, Badajoz, Spain; 2Department of Psychiatry, Jiménez Díaz Foundation, Autónoma University, IIS, Madrid, Spain Background & Aim: Among people who die by suicide, an increased frequency of CYP2D6 active gene multiplication has been described. Therefore, the present study analyzed the relationship between the severity of the suicidal intent and CYP2D6 number of active genes among survivors. Methods: A group of 342 individuals were evaluated with Beck Suicide Intent Scale within 24 h of the failed attempt. ‘Severe’ suicide attempters were classified as those scoring above percentile 75 in the objective circumstances section of the Suicide Intent Scale Scale. A group of 377 healthy controls were also genotyped. Results: A higher number of ‘severe’ suicide attempters carrying ≥2 active CYP2D6 genes as compared with the rest of the patients population (p < 0.01) or the healthy control group (p < 0.01) was found. Conclusion: Considering that ‘severe’ suicide attempters are more likely to die by suicide, CYP2D6 genetic polymorphism might be of use as a biomarker of death by suicide, which is in agreement with previous findings. Acknowledgments: Supported by the Institute of Health Carlos III-FIS and the European Union (FEDER) Grants PI10/02758, PI10/02010, and CP06/00030 (PD), JCI-2011-11050 (CVL), CIBERSAM;



Gobierno de Extremadura, and the European Union (FEDER) Grant BS10023. Reference: Pharmacogenomics. 2012;13(2):179–84.

02 OCTOBER 2012 – AFTERNOON SYSTEMS BIOLOGY, GWAS IN MULTIFACTORIAL DISEASES Mirror aetiologies of severe obesity and very low body mass index S. Jacquemont1, F. Zufferey1, C. Golzio2, E.H. Sherr3, N.D. Beckmann1, E. Hanson4, A. Maillard1, L. Hippolyte1, A. Mace5, C. Ferrari6, Z. Kutalik5, J. Andrieux7, R. Bernier8, A.I.F. Blakemore9, S. Bouquillon7, B. Delobel10, W. Andrew-Faucett11, R.P. Goin-Kochel12, L. Harewood13, S. Lebon14, D.H. Ledbetter11, C. Lese-Martin15, K. Mannick13, D. Martinet1, M.B. Ramocki16, S.J. Spence17, K. Steinmann18, J. Tjernagel19, R.G. Walters9, J.E. Spiro19, P. Froguel9, N. Katsanis2, A. Reymond13, W. Chung20, J.S. Beckmann1,5, on behalf of the Simons VIP and the 16p11.2 European Consortia 1 Service de Génétique Médicale, Centre Hospitalier Universitaire Vaudois, Lausanne; 2Dept of Cell biology, Duke University, Durham, North Carolina; 3Dept of Neurology, University of California, San Francisco; 4Dept of Psychiatry, Children’s Hospital Boston, Harvard Medical School, Boston; 5Dept of Medical Genetics, University of Lausanne; 6Dept of Psychiatry, Centre Hospitalier Universitaire Vaudois, Lausanne; 7Institut de Génétique Médicale. Hopital Jeanne de Flandre, CHRU de Lille, Lille; 8Dept of Psychology, Center on Human Development and Disability, University of Washington, Seattle; 9Dept of Genomics of Common Disease, Imperial College London, London; 10Centre de Génétique Chromosomique. Hôpital Saint-Vincent de Paul, GHICL, Lille; 11Genomic Medicine Institute, Geisinger Clinic, Danville; 12Dept of Pediatrics, Psychology Section, Baylor College of Medicine, Houston; 13Center for Integrative Genomics, University of Lausanne; 14Dept of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne; 15Dept of Human Genetics, Emory University, Atlanta; 16Dept of Pediatrics, Section of Pediatric Neurology and Developmental Neuroscience, Baylor College of Medicine, Houston; 17Dept of Neurology, Children’s Hospital Boston, Harvard Medical School, Boston; 18Dept of Neurology, Children’s Research Institute & University of Washington, Seattle; 19Simons Foundation, New York; 20Dept of Pediatrics and Medicine, Columbia University, New York Extensive GWAS identified numerous loci associated with obesity, but these account for a small percentage of its heritability. We previously reported a highly penetrant form of obesity resulting from a heterozygous deletion at the 16p11.2 locus, accounting for 0.7% of morbidly obese cases and absent in healthy non-obese European individuals. This recurrent ~600kb 16p11.2 BP4-BP5 deletion is also among the most frequent known genetic etiologies of autism spectrum disorder (ASD) and related neurodevelopmental disorders. Carriers of the reciprocal duplication show significantly reduced postnatal weight and BMI, this duplication being one of the first identified genetic variants underlying clinical conditions associated with being underweight. The reciprocal impacts of gene dosage variation on (1) gene expression, (2) head circumference, (3) eating behaviors and (4) BMI indicate that the mirror etiologies of severe obesity and being underweight might stem from contrasting effects on energy balance.

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Analyses of zebrafish and mouse embryos suggest KCTD13 as a major driver for the neurodevelopmental phenotypes associated with this rearrangement. Our study highlights a promising strategy for identifying missing heritability in complex traits. To systematically assess the impact of this deletion, we collected clinical data on 285 deletion carriers and performed detailed evaluations on 72 carriers and 68 intrafamilial controls. These data show that the 16p11.2 deletion impacts in a quantitative and independent manner on FSIQ, behavior and BMI, possibly through direct influences on neural circuitry. Although nonspecific, these features are clinically significant and reproducible, suggesting that BMI can be considered as a neurological disease. These studies demonstrate the burden of rare variants with strong effects in common disease, and highlight the necessity for large patient cohorts to characterize their clinical consequences.

Next generation association studies for complex traits E Zeggini Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1HH, United Kingdom Genome-wide association studies of complex traits have been successful in identifying common variant associations, but a substantial heritability gap remains. The field of complex trait genetics is shifting towards the study of low-frequency and rare variants, which are hypothesised to have larger effects. This next generation of association studies makes use of sequencing technologies and imputation approaches to access rare variants. The field of statistical genetics has been actively developing and evaluating tests tailored to the joint analysis of multiple rare variants. In addition, the study of these variants can be empowered by focusing on isolated populations, in which they may have increased in frequency and linkage disequilibrium tends to be extended. To narrow down causal variants, substantial effort has also been invested in trans-ethnic mapping genetic studies.

Integrative approach for VEGF genetic study through genome- wide methodology S Siest, EA 4373, University of Lorraine, Nancy, France Vascular endothelial growth factor (VEGF) is implicated in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. It is associated with numerous pathologies including cardiovascular diseases, diabetes, several types of cancer, cognitive decline and dementia, and reproductive and immune-inflammatory disorders. Also, VEGF inhibitors are used in cancer, macular degeneration and rheumatoid arthritis treatment, showing however significant toxicity. Given the high heritability of this trait, 60-80%, pharmagenomic studies could assist in the prediction of better response and/or lower toxicity. However, the genetic component of VEGF is still not well described. We will present the identification, by a Genome Wide Association Study (GWAS), of VEGF genetic variants and interconnexions of these biomarkers with other disease-associated molecules in healthy populations. The GWAS was performed in 3,527 healthy participants (Framingham Heart Study) and the most significant results (P G and FASL -844C>T. Statistical analysis was done using SPSS 18.0 for Windows. Results: After a median follow-up time of 48 months radiation induced severe late side effects were valued. In a Kaplan-Meier analysis evaluated by log rank test, the -844C>T polymorphism was correlated with a decreased risk for the development of severe rectal and/or urinary side effects RTOG ≥2. In a univariate Cox proportional hazard analysis carriers of the -844C>T variant showed significant associations with a lower risk for the occurrence of late high-grade toxicity (HR= 0.661, 95% CI 0.510 to 0.857; p=0.001). In a following multivariate Cox regression analysis the significance for the analyzed FASLG polymorphism remained a predictive factor for lower risk regarding the development of severe late rectal and/or urinary side effects (HR=0.637, 95% CI 0.434 to 0.934; p=0.002). For the remaining analyzed polymorphisms no significant results could be achieved. Conclusions: We conclude that genetic variants in the FASL gene may influence the risk of high-grade late rectal toxicity after radiotherapy in prostate cancer patients. The detection of genetic factors predictive for the occurrence of late toxicities will contribute to the identification of patients at high risk of late toxicity leading to an individualization of radiation treatment.

A14 - LiverAtlas: A unique integrated knowledge database for systems-level research of liver and hepatic disease Y Zhang1,2, C Yang1,2, T Chen1, M Li1, X Wang1, F He1,2, Y Zhu1 Beijing Institute of Radiation Medicine, Beijing, China 102206; 2 Institute of Basic Medical Sciences, CAMS, Beijing, China 100730 ([email protected]) 1

Background: A large amount of liver-related physiological and pathological data exist in publicly available biological and bibliographic databases, which are usually far from comprehensive and not integrated. Data collection, integration, and mining processes pose great challenges to scientific researchers and clinicians interested in the liver. Design: To address the above problems, we constructed LiverAtlas (http://liveratlas.hupo.org.cn). Results: In this version, LiverAtlas covers data on liver-related genomics, transcriptomics, proteomics, metabolomics and hepatic diseases. Additionally, LiverAtlas provides a wealth of manually curated information, relevant literature citations, and cross-references to other databases. Importantly, an expert-confirmed Human Liver Disease Ontology, including relevant information for 224 types of hepatic disease, has been constructed and is used to annotate LiverAtlas data. Furthermore, we demonstrated an example of applying LiverAtlas data to identify candidate markers for hepatocellular carcinoma at systems level.

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Conclusion: LiverAtlas is the most comprehensive liver and hepatic disease resource, which facilitates scientists and clinicians without a computational background to analysis their data at systems-level, and offers great contributions to biomarker discovery and drug target selection in liver diseases. Acknowledgements: This work was funded by the Chinese National Key Program of Basic Research (2010CB912700, 2011CB910601). Reference: Arias I, Wolkoff A, Boyer J, Shafritz D, Fausto N, Alter H, et al. The liver: biology and pathobiology: Wiley 2009.

B1 - Anti-inflammatory effects of cholesterol-lowering drugs in TNFα-induced endothelial cells A Cerda, MH Hirata, RDC Hirata School of Pharmaceutical Sciences, University of Sao Paulo Background: Dyslipidemia is a key factor in the pathogenesisof the atherosclerosis through the direct relationship of cholesterol excess with the lipid-composition of the plaque. Moreover, cholesterol contents have been largely associated to the inflammatory process, an initial step in atherosclerosis by modulating adhesion molecules and inflammatory cytokines. Statins (inhibitors of cholesterol synthesis) have been previously reported to reduce pro-inflammatory biomarkers, however there is little information about molecular mechanisms controlling this process and whether other cholesterol-lowering drugs, such as theezetimibe (inhibitor of cholesterol absorption), can modulate theexpression ofadhesion molecules or inflammatory cytokines. Objective: To evaluate the effects of statins (atorvastatin and simvastatin) and ezetimibe on expression ofthe genes Intercellular adhesion molecule 1 (ICAM1), Nuclear Factor kappa B (NFKB), Interleukin 8 (IL8), Interleukin 6 (IL6), Interleukin 1 beta (IL1B) and Matrix Metalloproteinase 9 (MMP9) in cultured human endothelial cells. Design: Human umbilical vein endothelial cells (HUVEC) were exposed to various concentrations ofatorvastatin, simvastatin or ezetimibe (0 to 5.0 μM) for 12h or 24h. First, membrane integrity and DNA fragmentation tests were performed using flux cytometry to evaluate the cell viability after treatments. HUVEC were activated with human TNFα (10ng/ml) during 6h and then cells were submitted to total RNA extraction using Trizol reagent. Relative quantification of mRNA was performed using predesigned Taqman assays (LifeTechnologies, USA). Results: Cytotoxicity tests showed that the three drugs did not affect cell viability up to 5.0 μM. The expression of all genes was increased after 6h induction by TNFα in different extents (2 to 18 fold, pA) and *4 (6009 C>T) allelic variants by PCR-RFLP assay and direct sequencing. Results: The variant allele frequencies of CYP2C9*2, CYP2C9*3, VKORC1 -1639G>A, 9041 G>A and 6009 C>T were 15.4%, 5.6%, 34.6%, 41.9% and 23.5%, respectively. Among the CYP2C9 variants the intermediate metabolizer genotypes (*1/*2, *1/*3) decreased the dose at least (5%), however, among the poor metabolizer genotypes the *2/*2 decreased the daily dose with 12% and the *3/*3 with 29%. In addition to this, the *2/*3 genotype had the most impact on dose reduction (59%). Significant association was found between the acenocoumarol mean daily dose and the VKORC1 genotype of the patients (p0.05) nor 33b (P>0.05) microRNAs for 12 and 24 hours treatment; however a tendency was observed to increased values of MIR-33a when HepG2 were exposed to 10μM during 24h (90% increased compared to vehicle control, p=0.065). Conclusions: Atorvastatin treatment does not influence significantly the expression of the mir33 family in hepatocellular carcinoma cells. More functional studies are necessary to investigate the role of statins in the regulation of cholesterol metabolism mediated by microRNAs.

C1 - Factor VII levels, Factor VII genetic variants and the risk of acute coronary syndrome among Arab-African-Tunisians S Ben-Hadj-Khalifa1, B Lakhal2, B Nsiri1, W Y Almawi3, T Mahjoub1 Research Unit of Biology and genetics of Hematologic and autoimmune diseases, Faculty of Pahrmacy, Monastir, Tunisia; 2 Cytogenetics and Biology Department, Farhat Hached University Teaching Hospital, Sousse, Tunisia; 3 College of Medicine and Medical Sciences, Arabian Gulf. University, Manama, Bahrain 1

Background: The importance of the extrinsic haemostatic system, of which factor VII/VIIa (FVII/FVIIa) is a key constituent, in acute coronary syndrome (ACS) is well recognized, and the superimposed thrombus on eroded or ruptured atherosclerotic plaques is a key event in ASC pathogenesis. Elevated FVIIa levels were demonstrated to enhance and accelerates vessel occlusion, resulting in the coronary event. The production of FVII is genetically controlled, and several FVII gene polymorphisms, associated with altered FVII production, were reported. Theses include the functional exon 8 single nucleotide polymorphism (SNP) R353Q (rs36208070), and the promoter insertion/deletion -323P0/10 (rs6046). The association of FVII variants with ACS appears to be influenced by racial/ethnic background (Asians versus Europeans) [Mo X, 2011], however no data on this association in North African communities. Objective: Accordingly, we investigated the contribution of the FVII gene variants R353G and -323P0/10, and altered FVII plasma levels to the risk of ASC in a North African Tunisian Arabs. Design: Cohort consists of 308 ASC cases and 312 age-, gender- and ethnically-matched control subjects; FVII antigen levels were determined by ELISA. Regression analysis was used in assessing the association of FVII variants and changes in FVII levels to the overall risk of ASC. Results: Significantly higher FVII antigen levels were seen in ACS patients (P A, c*84T>C) and one novel variant (c.*3C>A). Homozygous mutations occurred in 22% and compound heterozygote in 11,3% of probands, i.e. in 56 of individuals the genetic cause of hearing loss was confirmed. The proportion of negative subjects, without finding pathogenic allele reached 55%. Most frequent GJB2 mutations detected in individuals were c.35delG, c.1-3201G>A, c.71G>A (37%, 4,8%, 3,6% respectively). The large GJB6 deletion (delD13S1830) simultaneously with c.35delG GJB2 mutation was found in one family. Conclusions: DNA analysis of patients with SNHL brought mutational spectrum of the GJB2 and GJB6 genes in our cohort and also


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confirmed the genetic cause of hearing loss in 56 patients as key information for genetic counseling and clinical prognosis mainly for cochlear implants candidates. Supported: by the APVV 0148-10, VEGA 1/0465/11 and UK/7/2011 projects and TRANSMED 2 (ITMS:26240120030).

C15 - Searching for Multiple sclerosis genomic candidate regions by genome-wide synthesis of heterogeneous data sources A Maver, B Peterlin Clinical institute of Medical Genetics, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia Background: Multiple sclerosis (MS) is a debilitating autoimmune neurologic condition characterized by demyelination in central nervous system (CNS), leading to symptoms of severe motor and sensory neurological disturbances. In recent decade, development of high-throughput parallel technology has enabled the possibility of scrutinizing complete profile of molecular alterations. Inherent statistical issues of multiple testing and high false-positive rates have, however, hampered the attempts to entirely elucidate molecular background. Objectives: To propagate the discovery and increase detection specificity these studies, we performed an integrative synthesis of data originating from heterogeneous sources of global molecular alterations in MS. Design: Data for inclusion was collected from 39 studies or bioinformatic sources. Altogether, 158.520 distinct significant results discovered on 16 different biological levels were included in the dataset prior to statistical analyses. A custom rank product prioritization approach based on genomic position of included results was utilized for data synthesis and nested permutational cycling was then employed to determine significant accumulation of most significant results discovered on most diverse biological levels. Results: In total, 381 genomic regions were characterized with significant accumulation of results, reaching local permutation p-value minima below 0.001. Follow-up characterization of selected regions revealed them to contain 409 genes of which 87 (21.3%) overlapped with those tracked by the HuGENet database of disease-gene associations, while a large proportion of genes located in discovered regions have not yet been investigated in MS. Conclusions: Our results suggest that there exists a substantial body of genes, whose significant involvement in MS is suggested by evidence from heterogeneous and complex body of data from multiple ‘omic’ studies, but focused studies of their direct role in MS have yet to be performed and they thus present as plausible targets in further validation studies. References: Maver A, Peterlin B. Bioinformatics 2011;27:1971-8.

C16 - Optimization of methods for isolation and quantification of plasma mRNA HMOX-1 in patients with stable angina B Mirjanić-Azarić1, D Stojanović2, S Avram3, D Černe4 University Clinical Centre Banja Luka, 2Health Center Laktaši, BiH. 1,4 Faculty of Pharmacy, University of Ljubljana, Slovenia

1,3

Background: Circulating (cell-free) nucleic acids are present in small amounts in the plasma of healthy individuals, but increased levels of nucleic acids of plasma have been reported in a number of clinical disorders. The discovery of circulating nucleic acids has opened up new possibilities for non-invasive detection and monitoring of various disease conditions. Objective: Our intention was to evaluate and optimize methods for RNA isolation, mRNA transcription and quantitative PCR and to achieve reliable mRNA quantification. Design: Peripheral blood was taken into EDTA tubes (Becton Dickinson, Plymouth, UK) after at least 12 hours of fasting; sample processing took place within 1 h of collection and double-spin procedure was used in all subjects to obtain plasma nucleic acids. Plasma is stored in liquid nitrogen if not used immediately. RNA was isolated from 3 ml plasma using QIAamp Circulating nucleic acid kit and the QIAvac 24 Plus, according to the manufacturer’s instruction, but with certain modifications: the incubation time for proteolysis was 5 min., RNA was eluted from the column with 47μL RNase –free water and the collected eluate used for the second elution of RNA from the same column. Eluted mRNA was transcribed to cDNA with Super Script VILO cDNA Synthesis Kit (Invitrogen.Carlsbad.CA.USA). cDNA was then quantified by real –time polymerase chain reaction ( RT-PCT, ABI PrismTM SDS) for HMOX-1 gene ( TaqMan Gene Expression Assays, Applied Biosystems). Relative quantification was based on the expression levels of target gene versus a housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-housekeeping gene was determined by reagents TaqMan Human Endogenous Control Plate (Applied Biosystems, ZDA). All reactions were run in triplicates and the average threshold cycle (Ct) calculated. Results: Ct values of three mRNA quantifications for HMOX-1 for example, were 32.12; 32.08 and 32.24; StdDev Ct is 0.082. The results were very good. Analytical between-run imprecision of average threshold cycle, expressed as coefficient of variation was below 2%. Conclusion: Introduction of new methods, such as fully automated systems for nucleic acid purification, together with rapid PCR systems, further plasma mRNA analysis and it is reasonable to access its potential usefulness in non-invasive in vivo monitoring of gene expression profile in vascular beds.

C17 - A Genome-Wide Association Study Identifies rs2000999 as a Strong Genetic Determinant of Circulating Haptoglobin Levels NC Ndiaye 1*, P Froguel 2,3*, A Bonnefond 1,2, N Bouatia-Naji 2, A Dechaume 2, G Siest 1, B Herbeth 1, M Falchi 3, L Bottolo 3, RM Guéant-Rodriguez4, C Lecoeur 2, MR Langlois, Y Labrune 2, A Ruokonen, S El Shamieh 1, MG Stathopoulou 1, A Morandi 2, C Maffeis, D Meyre 2, JR Delanghe, P Jacobson, L Sjöström, LMS Carlsson, A Walley, P Elliott, MR Jarvelin, GV Dedoussis 1, Sophie Siest 1,4. PloS ONE 2012;7(3)e32327. * co-first authors 1 EA4373 – ‘Cardio-vascular Genetics’ Research Unit, Université de Lorraine, Nancy, France; 2 Centre National de la Recherche Scientifique (CNRS) 8199 - Institute of Biology, Pasteur Institute, Lille 2 University, Lille, France; 3 Genomic Medicine, Imperial College London, Hammersmith Hospital, London, England; 4 Department of Internal Medicine and Geriatrics, ‘Centre Hospitalier Universitaire de Nancy’, Nancy, France



Haptoglobin is an acute phase inflammatory marker. Its main function is to bind hemoglobin released from erythrocytes to aid its elimination, and thereby haptoglobin prevents the generation of reactive oxygen species in the blood. Haptoglobin levels have been repeatedly associated with a variety of inflammation-linked infectious and non-infectious diseases, including malaria, tuberculosis, human immunodeficiency virus, hepatitis C, diabetes, carotid atherosclerosis, and acute myocardial infarction. However, a comprehensive genetic assessment of the inter-individual variability of circulating haptoglobin levels has not been conducted so far. We used a genomewide association study initially conducted in 631 French children followed by a replication in three additional European sample sets and we identified a common single nucleotide polymorphism (SNP), rs2000999 located in the Haptoglobin gene (HP) as a strong genetic predictor of circulating Haptoglobin levels (Poverall=8.16102 59), explaining 45.4% of its genetic variability (11.8% of Hp global variance). The functional relevance of rs2000999 was further demonstrated by its specific association with HP mRNA levels (β=0.2360.08, P=0.007). Finally, rs2000999 was associated with decreased total and low-density lipoprotein cholesterol in 8,789 European children (Ptotal cholesterol=0.002 and PLDL=0.0008). Given the central position of haptoglobin in many inflammation-related metabolic pathways, the relevance of rs2000999 genotyping when evaluating haptoglobin concentration should be further investigated in order to improve its diagnostic/therapeutic and/or prevention impact.

C18 - Relationship between CYP2D6 ultrarapid metabolism and eating disorder symptoms Peñas-LLedó EM1, González I2*, Dorado P, Calzadilla LR2*, Pérez B4, Alvárez M4, González-Naranjo ME1, LLerena A1 1 CICAB Clinical Research Centre, Extremadura University Hospital and Medical School, Badajoz, Spain; 2Havana Psychiatric Hospital, Cuba (*Present address: Llerena Hospital, Servicio Extremeño de Salud SES, Spain); 3**Centro Comunitario de Salud Mental La Habana Vieja, La Habana, Cuba; 4Calixto García Medical School, Instituto Superior de Ciencias Médicas de La Habana, Cuba and CEIBA-RIBEF Consortium Background and Objective: CYP2D6 is involved in the metabolism of antidepressant drugs and of endogenous serotonin. We have shown an increased number of CYP2D6 active alleles, indicative of higher enzyme hydroxylation capacity, among patients with eating disorders. Thus, we studied eating symptoms in healthy volunteers and analysed whether symptomatic vs. asymptomatic females presented differences in CYP2D6 activity. Methods: Cuban university females (N=159) with no history of psychopathology or psychotropic treatment completed the Eating Disorder Inventory (EDI). CYP2D6 genotypes and hydroxylation capacity determined by debrisoquine metabolic ratio (MR) were already analyzed. Results: All women were at low-risk for “EDI-global” (>42), “EDIdrive for thinness” (>14) or “EDI-body dissatisfaction” (>17), but 47.7% were at moderate-risk for “EDI-bulimia” (≥5). This risky group vs. low-risk “Bulimia” included a higher number of phenotypically ultrarapid (MR12.6) (pT (H155Y) and loss of function SNPs: 1405A>G, 496G>A, 1513A>C, and 1729T>A were analyzed, in addition to SNPs of IL-6 (-174), TNF-α (-308), LTA (+252), and the anti-inflammatory NQO-1 (+609) (NAD(H) quinone-reductase-1, using pyrosequencing. Data were processed by mu-stat.

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Results: Individual SNP signatures discriminating cases from controls ranged between p=0.91 and 0.067. The analysis of SNP diplotypes in P2X7 revealed that the SNP areas between SNP496G>A and SNP1405A>G were more relevant (p=0.0255) than SNPs in the signalling part of P2X7 corresponding to region 1405A>G and 1729T>A (p= 0,251). The IL-6 SNP (-174) contributed to a better defined risk genotype (p=0.0028), however, the most important increase of probability was obtained by including the NQO-1SNP (+609). NQO-1 neutralizes hydroquinone-based oxidative stress occurring by environmental toxins and by mitochondrial respiration. In summary, IL-6 (SNP -174), P2RX7 SNP area 489-1405 and NQO-1 defined the best SNP combination to discriminate cases from controls (p=0.00076). muStat automatically guards against overfitting. Combinations of more than three SNPs had lower levels of significance. Conclusion: Multivariate analysis of various genes contributing to ATP-induced inflammation, cytokine responsiveness, and oxidative stress is valid to analyse individual SNPs as well regions between SNPs in single genes. The genetic analysis may efficiently guide the more costly analysis of biomarkers in complex inflammatory diseases. Reference: Geistlinger J, Du W, Groll J, Liu F, Hoegel J, Foehr KJ, Pasquarelli A, Schneider EM. Clin Chim Acta. 2012;413:39–47.

C24 - Influence of cadmium and lead on structural changes of bone tissue: μ-FTIR analysis MS Stanković, RS Nikolić, JM Jovanović, NS Krstić Deaprtment of Chemistry, Faculty of Sciences and Mathematics, University of Niš, Serbia Background: Heavy metals as pollutants in environment are very serious health and ecological problem. They are toxic, not biodegradable and have a very long period of half life in soil.1 In this study have been analyzed thigh bone samples of Wistar rats in order to recognize eventual changes in bone mineral tissue structure as a consequence of an intoxication by cadmium and lead with and without supplements (lipoic acid, LA and glutathione, GSH). Design: Female Wistar rats, 6 weeks of age, were bred in laboratory conditions and normal dietary regime in the vivarium of Faculty of Medicine (University of Niš) and intoxicating with heavy metals CdCl2 and (Pb(CH3COO)2. Experimental animals were euthanized after the final exposure by anesthesia with ketalar. Freshly dissected bones biopsy cleaned of soft tissue were washed by physiological solution and air-dried on sterile gauze. μ-FTIR analyses were performed at bone samples of experimental rats without any former preparation in reflection mode using Bruker Tensor 27 spectrometer equipped with He-Ne laser and coupled to a Hyperion 1000 IR microscope equipped with a 15× objective. Results: In 4000-600 cm-1 region there are no significant differences among FTIR spectra, except for specimens treated with Pb and Pb+GSH and Cd in ν3PO4 absorption domain (1085 cm-1). These bands are shifted to higher frequencies. μ-FTIR spectroscopic investigation in 600-400 cm-1 region indicates changes of the ν2PO4 vibrations (479 cm-1). The absorptions for specimens treated with Pb and Cd are shifted to lower frequencies which is correlated to higher values of reduces mass. This implies a partial replacement of Ca in hydroxyapatite with Pb and Cd. Conclusion: This technique proves to be useful in monitoring the type and degree of structural changes of mineral tissues in conditions


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of acute or chronically intoxicity with heavy metals. μ-FTIR analysis showed that the heavy metal intoxication leads to changes in the structure of bone tissue, as result of partial replacement of Ca in hydroxyapatite with Pb and Cd. Acknowledgement: This work was supported by the Ministry of Education and Science of the Republic of Serbia, Project No. TR31060. References: E. Merian, Metals and their compounds in the environment. Occurrence, analysis and biological relevance, Weinheim, New York, 1991, p. 469-479

C25 - Smoking denial in cardiovascular disease studies. SJ Wallner-Liebmann1, TB Grammer2, H Mangge3, W März2,3, W Renner3 1 Center of Molecular Medicine, Institute of Pathophysiology & Immunology, Medical University Graz, Austria. 2Synlab Center of Laboratory Diagnostics, Wasserturmstraße 71, 69214 Eppelheim, Germany. 3Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University, Graz, Austria Background: Assessment of self-reported smoking behavior in cardiovascular studies may lead to inaccurate measures of nicotine exposure. A more objective measurement of nicotine exposure can be done by measurement of plasma cotinine levels. Objective: Aim of the present study was to define the rate of discordance between self-reported smoking behavior and biochemically defined smoking status. Design: Data from 3.316 patients hospitalized for coronary angiography, who completed a questionnaire on smoking behaviour, were analysed. As a biochemical assessment of smoking status we used a cut off serum cotinine level of 15 μg/l. Results: Smoking denial, defined as a discrepance between high cotinin levels and self-reported never- or ex-smoking status, was observed 3.7% of the study participants. In a logistic regression analysis with step-wise inclusion of sex, age, CAD, previous MI and educational level, only male sex (odds ratio male/female: 2.00, 95% CI 1.22 - 3.33; p = 0.007) and age (odds ratio per year: 0.79, 95% confidence interval 0.66 - 0.94, p = 0.008) were associated with smoking denial. Conclusions: A mis-classification rate of 3.7% in the evaluation of such an important risk factor may lead to blurred effects and favor false negative results. The results of the present study substantiate the importance of biochemical markers for smoking assessment in cardiovascular studies.

C26 - The role of natural products in modulating the epigenetic pathway response during carcinogenesis MI Panagiotidis Laboratory of Pathology, University of Ioannina, Medical School, University Campus, Ioannina 45110, Greece Epigenetic changes have been implicated during tumor development and progression. For example, various studies have elucidated the connection between DNA methylation and cancer development primarily by indicating that hypermethylation of GC-rich DNA regions plays a major role in cancer development by silencing tumor suppressor genes. However, in higher eukaryotes, modifications in

DNA methylation patterns alone are not sufficient to induce cellular transformation to malignancy. Recent evidence suggests that there is an apparent interdependence between DNA methylation and histone modifications (i.e. methylation, acetylation, deacetylation, phosphorylation, ubiquitination, etc.) that regulates genome-wide changes in chromatin structure and consequently transcriptional regulation. For example, histone methylation can influence transcriptional activity in a way where methylation of histone H3at lysine 4 (H3-K4) is used as a marker for transcriptional active genes whereas histone H3 methylation at lysine 9 (H3-K9) as a marker for gene silencing. On the other hand, botanical extracts are rich sources of agents capable of modulating the epigenome and thus attenuate progression to cancer development. In fact, studies have indicated that such extracts may contain activities with the potential to modulate gene expression, maintain the epigenome and thus serving as promising therapeutic agents. For example, extracts of Artemisia dracunculus L (i.e. Russian tarragon) and Artemisia terbosum L (i.e. Chinese chives/garlic chives) have both been shown to have anticancer effects via their ability to down-regulate DNA methyltransferases (DNMTs) activity and specifically DNMT1 and DNMT3 [Kim et al., 2007]. In addition, a variety of other natural products have also been implicated in modulating the epigenetic pathway and thus show potential in cancer chemoprevention. For example, Psammaplin A (isolated from the sponge Pseudoceratina purpurea) has been observed to act as potent inhibitor of DNMT1 and histone deacetyltransferases (HDACs) [Pina et al., 2003]. In addition, the main polyphenol from green tea, (−)-epigallocatechin-3-gallate (EGCG), has been shown to inhibit DNMTs and consequently reactivate the expression of previously silenced genes like p16INK4a and hMLH1 in tumor cells [Fang et al., 2003]. Two other polyphenols (caffeic and chlorogenic acids derived from coffee) have also been reported to act as DNMTs inhibitors and particularly of DNMT1 [Lee et al., 2006]. In addition, isoflavones from soybean like genistein has been shown to inhibit DNA methylation and lead to the subsequent re-expression of methylation-silenced genes (i.e. RARβ, p16INK4a, MGMT) [Fang et al., 2005]. Finally, our group is currently investigating into the role of natural products in cancer chemoprevention (e.g. essential oils from Satureja thymbra and Satureja parnassica) as well as in modulating the epigenetic pathway response during prostate and hepatocellular carcinomas (e.g. silibinin, etc.).

C27 - Clinical Necessity of Partitioning of Human Plasma Haptoglobin Reference Intervals by Recently-discovered rs2000999 P Shahabi, G Siest, B Herbeth, N-C Ndiaye, S Visvikis-Siest Université de Lorraine, Cardiovascular Genetics Research Unit, EA 4373, Nancy, F-54000, France Background: Very recently, we identified a novel polymorphism, rs2000999, located in haptoglobin gene (HP) as a strong genetic determinant of the haptoglobin levels (Hp). We aim to determine the reference values of Hp on the basis of its main sources of biological variation including the rs2000999 in a large French origin population, the STANISLAS Family Study (SFS). Methods: Through a stepwise regression analysis, the main biological variables of Hp levels were identified in 3,129 “apparently” disease-free individuals of the SFS. Hp reference ranges were



subsequently established in a subgroup of 2,923 selected healthy subjects, as the reference population. Results: The plasma reference values of Hp ranged 0.08-1.97 g/L in males and 0.08-2.19 g/L in females. Gender, age, smoking, plasma levels of hemoglobin and the newly-discovered HP polymorphism, rs2000999, were found to be the strongest biological predictors of the Hp concentrations in human plasma. Hp levels, in both genders and in all age groups, were negatively associated with the presence and

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number of rs2000999 minor allele. The median (50th percentile) of the Hp plasma level in males with RR genotype was 270% of those with rr genotype and in females, median of Hp level in plasma in subjects with RR genotype was 168% of those with rr genotype. Conclusion: To be reliably interpretable in daily medical practice, the HP polymorphism, rs2000999, should be considered for partitioning its reference values. This polymorphism may also help for setting decision limits for medical interpretation of Hp concentrations.


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Author Index

Abad-Santos, F. 26 Absenger, G. 22, 39 Adámková, V. 26, 38 Almawi, W.Y. 34 Almazán, J. 42 Alonso, E. 28, 29 Alvárez, M. 41 Amet, Y. 6 André, N. 31 Andrew-Faucett, W. 15 Andrieux, J. 15 Asadinejad, M. 18, 19 Aseev, M.V. 38 Avram, S. 40 Aydin, A.E. 30 Azevedo, A.P. 21 Baca-García, E. 14, 35 Bahamondes, C. 42 Bakkaloglu, H. 30 Balogová, M. 39 Bapishev, K. 27 Barouki, R. 10 Beaune, P. 10 Beckmann, J.S. 15 Beckmann, N.D. 15 Beltman, P.A. 34 Beltrán, L. 24 Ben-Hadj-Khalifa, S. 34 Benachour, H. 37 Benetos, A. 37 Bernier, R. 15 Billy, F. 7 Blakemore, A.I.F. 15 Blasco-Fontecilla, H. 14, 35 Bonnefond, A. 40 Bonnefoy, J.-Y. 7 Bottolo, L. 40 Bouatia-Naji, N. 40 Bouquillon, S. 15 Bourbon, M. 35 Bouyioukos, C. 3 Brand, A. 4 Braquet, P. 7 Bril, A. 7 Brion, M. 28 Burhe, P.N. 11 Caamaño, J.M. 30 Calıskan, Y.K. 30 Calzadilla, L.R. 41 Camacho, V. 21 Canetti, D. 18 Carcas-Sansuan, A. 26

Cariani, E. 18, 27 Carlsson, L.M.S. 40 Carracedo, A. 28 Cascorbi, I. 8 Cerda, A. 23, 24, 26, 34 Certa, U. 8 Černe, D. 40 Češka, R. 26 Chatzimichail, A. 27 Chen, T. 23 Chung, W. 15 Ciccolini, J. 31 Civcic, M. 35 Clairambault, J. 7 Commet, S. 6 Corcos, L. 6 Cox, D.G. 6 Cuevas, A. 30 Culafic, D.j. 36 Cvetković, V. 41

Falchi, M. 40 Fercoq, O. 7 Ferrari, C. 15, 18 Ferreira, T.C. 21 Fraga, M.F. 13 Froguel, P. 15, 17, 40 Frueh, F. 14 Frushour, B. 5

Dahan, L. 31 Daskalaki, A. 20 De Andres, F. 24, 25 de Andrés, F. 25 de Boer, A. 11, 34 De la Rubia, S. 25 Dechaume, A. 40 Dedousis, G. 17 Dedoussis, G.V. 40 Deehan, R. 5 Delanghe, J.R. 40 Delobel, B. 15 Deloukas, P. 4 Desnos, M. 33 Dimitrijevic-Sreckovic, V. 35, 36 Djordjević, L.j. 41 Djordjevic, P. 35, 36 Dlouhá, D. 26 Dorado, P. 14, 24, 25, 26, 28, 29, 35, 39, 41 Dorea, E.L. 26 Dréano, Y. 6 Drubin, D. 5 Du, W. 42 Duga, B. 31

Gacic, J. 35 Galaviz-Hernández, C. 39 Gallego, A. 28 Gaspar, J. 41 Gaspar, J.F. 21 Gašperíková, D. 32, 39 Gaussem, P. 33 Genvigir, F.D.V. 26 Gerger, A. 22, 39 Germano, J.F. 28 Gerunda, G.E. 27 Ghosh, D. 3 Giannakopoulou, E. 37 Gil, O.M. 21 Gjorgoski, I. 42 Glotov, A.S. 38 Glotov, O.S. 38 Godfrey, K. 13 Goin-Kochel, R.P. 15 Golzio, C. 15 Gomes, B.C. 21 González, I. 41 Gonzalez-Naranjo, M.E. 24, 25, 28 González-Naranjo, M.E. 24, 25, 29, 39, 41 González-Vacarezza, N. 26 Gostiljac, D. 35 Gouin, C. 6 Gouveia, R. 21 Grammer, T.B. 44 Guéant-Rodriguez, R.M. 40 Guimarães, E. 26 Gulyaev, A. 27 Guney, I. 30 Gurtekin, M. 30 Gurwitz, D. 9 Gusukuma, M.C. 26

Ehret, G. 17 El Shamieh, S. 17, 37, 40 Elliott, P. 40 Elloul, I. 37 Esmaeili, H. 22 Estévez-Carrizo, F. 26 Evrard, A. 31

Habibi, M. 19 Hache, H. 20 Hanson, E. 15 Hao, Y. 18 Harewood, L. 15 He, F. 23 Henney, A. 3


Herbeth, B. 37, 40, 44 Hippolyte, L. 15 Hirata, M.H. 23, 24, 26, 28, 34 Hirata, R.D. 28 Hirata, R.D.C. 23, 24, 26, 34 Hirata, T.D.C. 28 Hofman, A. 11 Hoseini Bereshneh, A. 18, 19, 20 Hubáček, J.A. 26, 38 Hučková, M. 32, 39 Humphries, S. 5, 17 Iemmolo, R.M. 27 Ilić, B. 41 Ille, T. 36 Iordanidou, M. 27 Ivashchenko, T.E. 38 Jacobson, P. 40 Jacquemont, S. 15 Jakubíková, J. 39 Jančová, E. 32 Janeski, H. 35, 36 Janicsek, I. 31 Jarvelin, M.R. 40 Jester, B.C. 3 Jiang, Y. 18 Joković, N. 41 Jovanović, J.M. 38, 43 Jović, J. 41 Jung, H. 29 Jungić, S. 20 Kandel, B. 9 Kapp, K.S. 22 Karas Kuzelicki, N. 32 Katsanis, N. 15 Kenessary, A. 27 Képès, F. 3 Kheyrmand Parizi, P. 20 Kilicaslan-Ayna, T. 30 Klein, K. 9 Klimeš, I. 32, 39 Knappe, P.S. 5 Kocić, G.M. 38 Koenig, W. 43 Krenn-Pilko, S. 22 Krsmanović, M.M. 38 Krstić, N.S. 38, 43 Kuepfer, L. 14 Kühn, A. 20 Kurilov, R.V. 38 Kushugulova, A. 27


Kussmann, M. 12 Kutalik, Z. 15 Ľ Barák 32 Labat, C. 37 Labrune, Y. 40 Lacarelle, B. 31 Lagos, J. 28 Laifenfeld, D. 5 Lakhal, B. 34 Lamont, J. 5 Langlois, M.R. 40 Langsenlehner, T. 22 Langsenlehner, U. 22 Lazalde-Ramos, B. 39 le Cessie, S. 11 Le Jossic-Corcos, C. 6 Lebon, S. 15 Lecoeur, C. 40 Ledbetter, D.H. 15 Lehrach, H. 20 Lepage, T. 3 Lese-Martin, C. 15 Li, J. 20 Li, L. 18 Li, M. 23 Li, N. 18 Lima, P.H.O. 28 Limbert, E. 21 LLerena, A. 11, 14, 24, 25, 26, 28, 29, 35, 39, 41 López, M. 28 López-López, M. 29 Loriot, M.A. 10, 33 Lucas, D. 6 Luchessi, A.D. 24, 28 Lukac Bajalo, J. 21 Mace, A. 15 Machín, E. 24, 258 Maffeis, C. 40 Mahjoub, T. 34 Maillard, A. 15 Maitland-van der Zee, A.H. 11, 34 Makchrova, I.А. 38 Mangge, H. 44 Manita, I. 21 Mannick, K. 15 Manolopoulos, V.E. 5 Manolopoulos, V.G. 27, 37 Marc, J. 21, 29 Marino, M. 27 Marochkina, E.U. 38 Maroussi, S. 37 Martinet, D. 15 Marx-Neuhold, E.A. 39 März, W. 44 Maschke-Dutz, E. 20 Mašindová, I. 32, 39

Masson, C. 37 Matejić, J. 41 Maver, A. 40 Meceska-Jovcevska, J. 42 Melegh, B. 31 Melegh, B.I. 31 Mercier, C. 31 Mesanyová, J. 38 Metspalu, A. 32 Meyre, D. 40 Mihajilov-Krstev, T. 41 Milano, G. 31 Milek, M. 32 Mirjanić-Azarić, B. 40 Missale, G. 18 Mlinaric-Rascan, I. 32 Montalti, R. 27 Morandi, A. 40 Moreau, C. 10, 33 Nagizbek-kyzy, E. 27 Nane, I. 30 Ndiaye, C.N. 17 Ndiaye, N.C. 37, 40, 44 Nelson, K.E. 13 Nikolić, R.S. 38, 43 Nsiri, B. 34 Nurgozhin, T. 27 Nzietchueng, R. 37 Olivani, A. 18 Ortega, A. 28, 29 Ostanek, B. 29 Ouafik, L. 31 Ozanne, S. 12 Panagiotidis, M.I. 44 Paraskakis, E. 27 Paré, G. 9 Park, J. 5 Patrinos, G.P. 5 Pavlovčinová, G. 39 Pécou-Gambaudo, E. 6 Pejovic, M. 35 Peñas-LLedó, E.M. 24 Peñas-Lledó, E.M. 14, 25, 35 Peñas-LLedó, E.M. 24, 25, 26, 29, 39, 41 Peoc’h, K. 10 Pereira, T. 41 Pérez, B. 41 Pesson, M. 6 Peterlin, B. 40 Peycheva, S. 20 Pilli, M. 18 Pinto, G.A. 26 Pirmohamed, M. 11 Piťha, J. 38 Popovic, S. 35, 36 Prodan Zitnik, I. 21, 29

Profant, M. 39 Pugacheva, I.V. 38 Quaranta, S. 31 Ragia, G. 27, 37 Rajković, J. 41 Ramocki, M.B. 15 Redekop, W.K. 34 Renner, W. 22, 39, 44 Reymond, A. 15 Ribeiro, A.L. 41 Ribeiro, V. 41 Rosales, A. 28, 30 Rosendaal, F.R. 11 Roses, A.D. 4 Rota, C. 18, 27 Rueff, J. 21 Rumpianesi, G.L. 27 Ruokonen, A. 40 Saavedra, N. 30 Sabaghian, E. 22 Sadr-Nabavi, A. 18, 19, 20 Saiz-Ruiz, J. 35 Salazar, L.A. 28, 30, 34, 42 Salovska, J. 42 Samonigg, H. 22 Sampaio, M.F. 28 Sandoval, M. 30 Šandriková, V. 32 Sanhueza, J.A. 42 Santos, L.S. 21 Schalekamp, T. 11 Schiess, R. 7 Schippinger, W. 22, 39 Schneider, E.M. 42 Seifert, M. 5 Seitz, J.F. 31 Sengupta, J. 3 Senturk-Ciftci, H. 30 Sepúlveda, S. 30 Serdjebi, C. 31 Seybold, M. 42 Seybold, M.P. 16 Shahabi, P. 44 Shakeri, M.T. 22 Sharman, A. 27 Sherr, E.H. 15 Siest, G. 40, 44 Siest, S. 6, 15, 37, 40, 44 Siffert, W. 11 Siguret, V. 10 Silbiger, V.N. 28 Silva, S.N. 21 Simon, B. 6 Sipeky, C. 31 Sjöström, L. 40 Skerjanec, M. 29 Smadja, D.M. 33


A47

Smid, A. 32 Soldatovic, I. 35, 36 Sonakya, V. 16 Sosa-Macias, M.G. 39 Sousa, A.G. 28 Spence, S.J. 15 Spiro, J.E. 15 Sreckovic, B. 35, 36 Sredojević-Dugalić, G. 38 Stamkova, M. 42 Staněk, V. 38 Staník, J. 32 Staníková, D. 32 Stanković, M.S. 43 Stathopoulou, M.G. 40 Steinmann, K. 15 Stojanović, D. 40 Stotz, M. 22 Supiyev, A. 27 Szabo, I. 31 Szkandera, J. 22, 39 Szymezak, J. 33 Talos, G. 31 Tamm, R. 32 Tanajura, L.F. 28 Tarkovskaya, I.V. 38 Tavridou, A. 27, 37 Teichert, M. 11 Teo, Y.-Y. 16 Terán, E. 24 Thomas, M. 9 Thurner, E.M. 22 Tjernagel, J. 15 Trejo, H.D. 29 Trenti, T. 27 Trillet, K. 6 Tubić, B. 20 Turkmen, A. 30 Tzvetkov, M. 33 Valentínová, L. 32 van der Meer, F.J.M. 11 van Hoorn, L.G.J. 11 Van Hooser, A. 5 Van Schaik, R. 11 van Schie, R.M.F. 11, 34 Van Viet, H. 33 Vaquero-Lorenzo, C. 14, 35 Varga, L. 39 Varnai, R. 31 Vashukova, E.S. 38 Verhoef, T.I. 11, 34 Via, M. 8 Vidal, M. 3 Vieillefond, V. 33 Visser, L.E. 11 Vrablík, M. 26 Walley, A. 40

A48


Wallner-Liebmann, S.J. 44 Walters, R.G. 15 Wang, X. 23 Weissmueller, M. 22 Wessels, J.A.M. 11 Wierling, C. 20 Winder, T. 22

Wittkowski, K.M. 16, 42 Wu, P. 18 Wu, S. 18 Yanes, O. 5 Yang, C. 23 Yermekbayeva, B. 27

Zambrano, T. 34 Zanelli, P. 18 Zanetti, A. 18 Zanger, U.M. 9 Zeggini, E. 15 Zerbini, A. 18 Zhang, Y. 23


Zholdybaeva, E. 27 Zhu, Y. 23 Zhumadilov, Z. 27 Zlatković, B. 41 Zufferey, F. 15 Zulus, B. 22, 39