EUROESPES MEDICINE PERSONALIZED MEDICAL TREATMENT
PERSONALIZED, GENOMIC
MEDICINE
AT THE FO R E FRON T IN G EN OM IC M EDICIN E A N D R E S E ARCH WE´RE LEA DERS IN PERSON A LIZ ED M E D IC IN E
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The EuroEspes Journal
DECEMBER - 12-13 - 2008 www.euroespesannualconference.org
Ist Meeting of the World Association of Genomic Medicine
Genomic Medicine and Pharmacogenomics. Future Challenges for Personalized Medicine in the European Union
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Index of Contents Gen-T 2 | December 2008 International Edition
05 Welcome Message
The Abyssal Waters of Personalized Medicine
08 Programme
ENGLISH
10 Programa ESPAÑOL 12 Programa
Sponsor
EuroEspes Foundation
Editor-in-Chief Ramón Cacabelos
Administrative Management
Gladys Bahamonde administracion@gen-t.es
GALEGO
15 Honorary Committee 18 Organizing Committee 20 Speakers
Publisher
EuroEspes Publishing Co.
In Order of Presentation
59 Opening and Closing Ceremony Addresses 65 Poster Presentation Abstracts 92 Index of Authors In Alphabetical Order 97 Sponsors
Head of Production
Javier Masoliver publi@euroespes.com
Graphics
Antonio Bermo Ramón Vázquez
Gen-T
Santa Marta de Babío s/n 15165 Bergondo Coruña, Spain [T] +34 981 780 505 ISSN 1888-7937 LEGAL DEPOSIT C 2084-2008 © Copyright 2008 Gen-T takes no responsibility for the opinions and/or criteria expressed by the authors, and reserves the property rights of the works published.
Total or partial reproduction, literary or iconographic, is expressly prohibited without the prior consent of the editor.
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WELCOME MESSAGE The Abyssal Waters of Personalized Medicine
T
he past century brought about substantial novelties in our lives, including changes in politics and economics, education, science and technology. Progressive modifications in the population pyramid and the emergence of a magnified aged population, as the result of the duplication of life expectancy over a time interval of 100 years, caused an increased rate of chronic disorders (heart disease, cancer, dementia) which have become the greatest problems of health, cause of death and disability in our society. New nutritional habits have contributed to increasing the manifestation of health problems such as hypercholesterolemia, arteriosclerosis, diabetes, obesity or the metabolic syndrome. The tragic increase in traffic accidents sowed death, brain trauma and disability in millions of families. The industrialization of the developed world has paid an enormous price in terms of air pollution and environmental contamination. Changes in our life style are not always equivalent to improvements in our quality of life. The progress of science and technology, as well as telecommunications, introduced the concept of the information society, and the power of money was gradually replaced by the power of knowledge. In this context, at the turn of the century, the Human Genome Project and subsequent “animal and plant genome projects” capitalized the interest of our society and revolutionized many fossilized concepts in medicine and biology. We are now facing a new era in which the molecular world has to explain physiology and anatomy, and in which classical descriptive medicine must be replaced by a molecular medicine capable of explaining the pathogenic mechanisms of disease. At the same time, from a public health perspective, the medicine of protocols must be replaced by the medicine of persons with an individual identity. This personalized medicine will take advantage of the knowledge provided by structural genomics, functional genomics, transcriptomics, proteomics, and metabolomics, to give us an insight into novel concepts of etiology, pathogenesis, predictive diagnosis, and molecular therapeutics. At present, more than 90% of common diseases are of
unknown etiology. Most medical syndromes and specific disorders are explained by diverse pathogenic hypotheses which might not be true. The interactions between the interior milieu (endogenous factors) and the exterior milieu (exogenous factors) to elicit a particular disease are practically illusory o imaginative, except perhaps in the case of infectious disorders. Epidemiological studies have to rely on biological (objective) markers rather than on speculative (subjective), biased questionnaires. More than 80% of chronic disorders are complex and multifactorial, in which both genomic and environmental factors interact to induce a pathogenic cascade leading to a particular phenotype; and the pathogenesis of the vast majority of these phenotypes (clinical pictures) is highly hypothetical and changeable depending upon fashionable interests. The final consequence of this maze is that most therapeutic interventions in human disease are of poor value. In most pathologies, pharmacological treatments are useful in 20-40% of the cases, but a final cure is obtained in less than 5% of the cases. Most of our treatments are symptomatic or palliative, but not curative in nature. This discouraging picture must be changed to enter into a scientific medicine based on molecular evidence aimed to prevent rather than to treat (to predict rather than to repair).
Ramón Cacabelos President of the EuroEspes Foundation Chairman of the Organizing Committee
We believe that the genomic era is a favourable time to jump into a brighter future in medical sciences. Genomic medicine is still in its infancy and the most keenly anticipated “personalized medicine” is still in an embryonic stage. From a technical point of view there are still many black holes in the understanding of our genome. There are thousands of dark islands in the abyssal mantle of the human genome. Our genome is the recapitulation of our genetic history, and it is likely that large segments of our genomic junk are in functional dialog with the 25.000-30.000 genes integrating the functional part of the human genome. Epigenetics is probably a fundamental mechanism in the heterogeneity of complex disorders. RNA interference (RNAi) and gene silencing are intriguing mechanisms of health and disease. Mitochondrial DNA-related pathology remains cryptic and enigmatic. The interaction between mitochondrial DNA and genomic (nuclear) DNA demands further elucidation. The impact of nutrition on genomic function is an appealing idea still devoid of convincing documentation. Genomic medicine will evolve in parallel with technological and scientific progress, but a personalized medicine requires a profound change in the mentality of administrators, physicians, the pharmaceutical industry, and society. Ultraconservative positions of those who are afraid of novelty will slow down the progress of science and the implementation of new procedures in medical practice to improve the welfare of the population. Education should run in parallel with technological and scientific developments. Physicians must become familiar with genomics and bioinformatic tools to optimize diagnosis and therapeutics. December 2008
7
Programa IIIMESSAGE WELCOME Conferencia Anual EuroEspes
Many other aspects of the genomic era need a profound revision, such as ethical, legal, and social issues. Genomic medicine will not advance if in many developed societies there is not the appropriate education and regulation of procedures to perform standard genotyping. Without genotyping as a routine in clinical practice there is not real genomics. We have to shorten the periods lagging between a scientific breakthrough and its practical application in medicine. Good regulators are those who facilitate the application of novel procedures following an efficient law. Bureaucrats are redundant interpreters of circular rules. If time is money, approximately 60% of the costs of drugs are the consequence of inefficient regulations and ineffective procedures in drug development. The stage fright of genomics is founded in many instances on statements of ignorance; other fears are quite reasonable, especially when important educational issues are not posed appropriately by the players or when the incorporation of a new technology implies the existence of winners and losers. The cost of new technologies is another important issue to take into account. The health and disease inequalities between rich and poor societies cannot be ignored so as not to increase the gap between developed and developing countries. The obscene appetites of some dumping powers in the industry sector should be controlled by responsible governments. Genomic Medicine is the new umbrella under which all medical specialities and health disciplines should be protected. Mendelian disorders represent less than 5% of human genetic disorders; in contrast, more than 80% of complex, multifactorial disorders (cardiovascular disease, cancer, hypertension, atherosclerosis, dementia, movement disorders, neuropsychiatric disorders, etc) exhibit a clear genomic component in which multiple genes distributed across the human genome might be involved. The interaction of genomic factors with environmental factors is at the basis of most pathogenic mechanisms associated with complex disorders (the most common pathology in humans). The genomic approach to medical practice is not a threat to conventional procedures, but a novel tool to optimize diagnostics and therapeutics. Genomic procedures have to be particularly useful in drug development and therapeutics. With the present methodologies, only one drug out of 6000 compounds in development reaches phase II clinical trials, with enormous money waste during 10-12 years of pharmacological research. Our current trial-and-error prescriptive methods are responsible for 60-80% of unnecessary drug adverse events and 40-60% of cases of wrong prescription. Pharmacogenetics and Pharmacogenomics are very attractive disciplines to help in drug development from early stages of development to (i) define disease-specific drug targets, (ii) to recruit the appropriate patients for clinical trials, (iii) to reduce costs in drug development, (iv) to reduce side-effects, (v) to reduce the pharmacological cost of most disease treatments, and, in general terms, to improve efficacy and safety issues. 8
December 2008 - international edition
The IIIrd EuroEspes Annual Conference The IIIrd EuroEspes Annual Conference (2008) is devoted to Genomic Medicine and Pharmacogenomics, with special emphasis on the Future Challenges for Personalized Medicine in the European Union. This is an initiative of the EuroEspes Foundation, a Spanish non-profit organization dedicated to the advancement of science and dissemination of knowledge, in cooperation with the R&D Directorate-General of the European Commission and the Autonomous Government of Galicia (Xunta de Galicia), represented by the Regional Ministry of Innovation and Industry (Dirección Xeral de I+D+i) and the Regional Ministry of Economy and Treasury (IGAPE, Instituto Galego de Promoción Económica). The organizers are very proud of and thankful to Their Royal Majesties King Juan Carlos and Queen Sofia of Spain, who have agreed to hold the Honorary Presidency of the IIIrd EuroEspes Annual Conference. Our Honorary Committee, integrated by Emilio Pérez Touriño (President of the Autonomous Government of Galicia), Bernat Soria Escoms (Minister of Health and Consumer Affairs), Dolores Vilarino Santiago (President of the Galician Parliament), Manuel Ameijeiras Vales (Government Delegate in Galicia), Alejandra Pérez Márquez (Mayoress of Bergondo), Fernando Xavier Blanco Álvarez (Regional Minister of Innovation and Industry), Salustiano Mato (R&D Director General, Regional Ministry of Innovation and Industry), Salvador Fernández Moreda (President of the Provincial Council of A Coruña), Isacio Sigüero Zurdo (President of the Organization of Professional Medical Associations), José Luís Baltar Pumar (President of the Provincial Council of Ourense), Antonio Garrigues Walker (President of Garrigues Asesores), José Manuel Silva (R&D Director General, European Commission), Alain Vanvossel (Research Directorate General, European Commission), Manuel Hallen (Head of the Medical and Public Health Research, Research Directorate General, European Commission), Christian A. Scerri (in representation of the Minister of Health of Malta), Vasco María (Chairman of the Board, Authority of Medicine and Health Products, Ministry of Health of Portugal), Gerardo Jiménez Sánchez (Director of the Genomic Medicine Institute of Mexico, Ministry of Health of Mexico), Antonio Leaño Reyes (Rector of the Autonomic University of Guadalajara, Mexico), Juan Sánchez Aldana Ramírez (President of the Municipality of Zapopan, Mexico), Víctor Manuel Ramírez Aguiano (Rector of the University Centre of Health Sciences, University of Guadalajara, Mexico), Rafael López (President of Parggon Pharmaceuticals, Mexico), Eugenio Luigi Iorio (President, International Observatory of Oxidative Stress, Italy), the Rectors of 40 public and private Spanish Universities, the Presidents of 20 Official Medical Associations, the Presidents of 15 Official Pharmaceutical Associations, the Presidents of 8 Royal Medical Academies, and the Presidents of 28 Medical Societies also deserve our deepest gratitude for giving personal and institutional support to this Conference. All these distinguished persons and the institutions that they represent are admirable examples of compromise and
WELCOME MESSAGE
solidarity with the idea that Genomic Medicine needs to be propelled in the European Union to reach maturity in the coming years. In this endeavour, public and private institutions, universities, medical centres, research centres, medical and pharmaceutical associations, educational centres of any level, citizens´ associations, and state administrations have to be involved. In this early stage of development of Genomic Medicine, the mastery of pioneering experts in the field is essential to delineate guidelines, recommendations, and practical issues in order to lay the foundations for an efficient Genomic Medicine in European society and in other countries around the world. In this regard, we are very grateful to the invited speakers who, coming from Europe, USA, Japan, and Latin America, have agreed to joint together in this Conference to open an intellectual debate on the future of Genomic Medicine. Urs A. Meyer (Switzerland), John R. Cockcroft (UK), Filippo De Braud (Italy), José Manuel Silva (Belgium), Gerardo Jiménez (México), Masatoshi Takeda (Japan), Carmen Vigo (USA), Marvin Edeas (France), Francesco Marotta (Italy), Allen D. Roses (USA), Munir Pirmohamed (UK), Ian P. Hall (UK), Valter Lombardi (Spain), Amalio Telenti (Switzerland), Alain Vanvossel (Belgium), and Stefan Prause (Germany) are distinguished personalities in different fields of Genomic Medicine from whom all of us have much to learn. In fact, all of us are beginners in Genomic Medicine.
The World Association of Genomic Medicine (WAGEM) With the opportunity of the celebration of the IIIrd EuroEspes Annual Conference, the recently created World Association of Genomic Medicine (WAGEM) will hold its first constitutional meeting. The WAGEM is intended to be an open forum to all health professionals who wish to create, construct and use Genomic Medicine as an instrument to optimize their medical practice and research activities. All medical specialities have a place within the structure of the WAGEM. Suggestions and recommendations from any sector of medical, pharmaceutical, biological, biotechnological, and health sciences are welcome to appropriately serve our scientific community and our society.
The World Guide for Drug Use and Pharmacogenomics (WG-PGX) During the IIIrd EuroEspes Annual Conference, The World Guide for Drug Use and Pharmacogenetics (WGPGX) will be announced. The WG-PGX is the first multiauthored Guide in the world which covers over 1000 drugs of current use, with their pharmacogenomic profile, brand names in Europe, USA, Canada, Japan, and Latin America, hundreds of genes associated with the most prevalent human diseases, the classification of drugs, the international classification of human diseases, and genotype-phenotype correlations in the Caucasian population. The WG-PGX is intended to be a practical tool for physicians and specialists of any
medical field, pharmacologists and pharmacists, biologists, geneticists, and other health professionals. We hope that this new tool will help physicians in their daily practice to optimize therapeutics.
Welcome to Coruña In my condition as President of the EuroEspes Foundation and Chairman of the Organizing Committee of the IIIrd EuroEspes Annual Conference, I want to thank all of you for attending this Conference, and wish all of you a pleasant stay in the beautiful and cosmopolitan environment of Coruña city. This is a very ancient town of Celtic origin where the Atlantic Ocean taught people to live in tribal harmony for centuries. Progress means change. Inevitably, the new has to replace the old under the guidance of respect and common sense in order to preserve all that which has proved to be timeless.
Bibliography Aziz H, Zaas A, Ginsburg GS. Peripheral blood gene expression profiling for cardiovascular disease assessment. Genomic Med 2007; 1:105-112. Cacabelos R (Ed). The World Guide for Drug Use and Pharmacogenomics. EuroEspes Publishing, Coruña, 2009 (in press). Cacabelos R, Fernández-Novoa L, Lombardi V, Kubota Y, Takeda M. Molecular Genetics of Alzheimer’s disease and aging. Meth Find Exper Clin Pharmacol 2005; 27(Supll 1):1-573. Cacabelos R, Takeda M. Pharmacogenomics, nutrigenomics and future therapeutics in Alzheimer’s disease. Drugs Future 2006; 31(Suppl B):5-146. Cohen N (Ed). Pharmacogenomics and Personalized Medicine. Humana Press, New Jersey, 2008. Hall IP, Pirmohamed M (Eds). Pharmacogenetics. Taylor & Francis, New York, 2006. Kalow W, Meyer UA, Tyndale RF (Eds). Pharmacogenomics. Drugs and the Pharmaceutical Sciences Vol 113. Marcel Dekker, New York, 2001. Kumar D. Genomic medicine: a new frontier of medicine in the twenty-first century. Genomic Med 2007; 1:3-7. Roses AD. Pharmacogenetics and drug development: the path to safer and more effective drugs. Nature Rev Genet 2004; 5:645-656. Yan Q (Ed). Pharmacogenomics in Drug Discovery and Development. Methods in Molecular Biology 448.Humana Press, New Jersey, 2008. www.euroespesannualconference.org www.fundacioneuroespes.org www.wagem.org December 2008
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Programme IIIrd EuroEspes Annual Conference
PROGRAMME
FRIDAY 12
10
9:00
Reception of Delegates
9:30
Opening Ceremony
• Honorary Members • European Union Representatives • Spanish Authorities • Autonomic Authorities • Local Authorities • Chairman
10:30
Plenary Lecture-1 Genomic Medicine: Challenges and Opportunities
Urs A. Meyer (Switzerland)
11:15
Genomic Medicine and Pharmacogenomics of Cardiovascular Disorders
John R. Cockcroft (United Kingdom)
11:45
Coffee Break
12:00
Genomic Medicine and Pharmacogenomics of Cancer
Filippo De Braud (Italy)
12:30
Genomic Medicine and Pharmacogenomics of Neuropsychiatric Disorders
Ramón Cacabelos (Spain)
13:30
Lunch
16:00
Genomic Medicine and Pharmacogenomics in the European Union
José Manuel Silva (European Commission, R&D Directorate-General, Belgium)
16:30
Genomic Medicine in Mexico
Gerardo Jiménez (Mexico)
17:00
Development of Genomic Medicine in CNS Disorders in Japan
Masatoshi Takeda (Japan)
17:30
Pharmacogenomics in Drug Discovery in USA
Carmen Vigo (USA)
18:00
Coffee Break
18:15
Nutrigenomics in Europe
Marvin Edeas (France)
18:45
Human Nutrition in Genomic Medicine: Future Age-Management Strategies
Francesco Marotta (Italy)
December 2008 - international edition
Programme
IIIrd EuroEspes Annual Conference
9:00
Plenary Lecture-2
The Future of Pharmacogenomics in Medical Practice: A New Model of Pipeline Pharmacogenetics
Allen D. Roses (USA)
9:45
Pharmacogenetics and Metabolic Disease
Munir Pirmohamed (United Kingdom)
10:15
Genomic Medicine and Pharmacogenomics of Respiratory Diseases
Ian P. Hall (United Kingdom)
10:45
Coffee Break
11:00
Genomic Medicine and Pharmacogenomics of Immunological Disorders and Vaccines
Valter Lombardi (Spain)
11:30
Genomic Medicine and Pharmacogenomics of Infectious Diseases
Amalio Telenti (Switzerland)
12:00
Genomic Medicine and Pharmacogenomics in the EU Research Programmes
Alain Vanvossel (European Commission, Belgium)
12:30
Novel technologies for the Implementation of Pharmacogenomics in Medical Practice
Stefan Prause (Germany)
13:00
General Discussion and Conclusions
13:30
1st Meeting of the World Association of Genomic Medicine
14:00
Closing Ceremony
Best Poster Presentation Prize Presentation of the EuroEspes Awards 2008
21:00
EuroEspes Dinner and Farewell Party
Formal Dress
PROGRAMME
SATURDAY 13
December 2008
11
Programa III Conferencia Anual EuroEspes
PROGRAMA
VIERNES 12
12
9:00
Recepción y Entrega de Documentación y Credenciales
9:30
Ceremonia de Inauguración
• Miembros del Comité de Honor • Autoridades Extranjeras • Autoridades Nacionales • Autoridades Autonómicas • Autoridades Locales • Presidente del Comité Organizador
10:30
Conferencia Plenaria - 1 Medicina Genómica: Desafíos y Oportunidades
Urs A. Meyer (Suiza)
11:15
Medicina Genómica y Farmacogenómica de los Trastornos Cardiovasculares
John R. Cockcroft (Reino Unido)
11:45
Descanso
12:00
Medicina Genómica y Farmacogenómica del Cáncer
Filippo De Braud (Italia)
12:30
Medicina Genómica y Farmacogenómica de los Trastornos Neuropsiquiátricos
Ramón Cacabelos (España)
13:30
Comida
16:00
Medicina Genómica y Farmacogenómica en la Unión Europea
José Manuel Silva (Comisión Europea, Bélgica)
16:30
Medicina Genómica en México
Gerardo Jiménez (México)
17:00
Desarrollo de la Medicina Genómica de los Transtornos del Sistema Nervioso en Japón
Masatoshi Takeda (Japón)
17:30
Farmacogenómica en el Descubrimiento de Fármacos en Estados Unidos
Carmen Vigo (EEUU)
18:00
Descanso
18:15
Nutrigenómica en Europa
Marvin Edeas (Francia)
18:45
Nutrición Humana en Medicina Genómica: Estrategia para el Manejo Futuro del Envejecimiento
Francesco Marotta (Italia)
December 2008 - international edition
Programa IIIrd Conferencia Anual EuroEspes
9:00
Conferencia Plenaria - 2
Futuro de la Farmacogenómica en la Práctica Médica: Un Nuevo Modelo de Desarrollo Farmacogenético
Allen D. Roses (EEUU)
9:45
Farmacogenética y Transtornos Metabólicos
Munir Pirmohamed (Reino Unido)
10:15
Medicina Genómica y Farmacogenómica en las Enfermedades Respiratorias
Ian P. Hall (Reino Unido)
10:45
Descanso
11:00
Medicina Genómica y Farmacogenómica en Trastornos Inmunológicos y Vacunas
Valter Lombardi (España)
11:30
Medicina Genómica y Farmacogenómica en Enfermedades Infecciosas
Amalio Telenti (Suiza)
12:00
Medicina Genómica y Farmacogenómica en los Programas de Investigación de la Unión Europea
Alain Vanvossel (Bélgica)
12:30
Nuevas Tecnologías para la Implementación de la Farmacogenómica en la Práctica Médica
Stefan Prause (Alemania)
13:00
Discusión General y Conclusiones
13:30
Primera Reunión de la Asociación Mundial de Medicina Genómica
14:00
Ceremonia de Clausura
Entrega de los Premios EuroEspes 2008 Entrega del Premio al Mejor Póster
21:00
Cena de Gala EuroEspes y Fiesta de Despedida
Etiqueta
PROGRAMA
SÁBADO 13
December 2008
13
Programa III Conferencia Anual EuroEspes
PROGRAMA
VENRES 12
14
9:00
Acreditación e recepcion de Delegados
9:30
Ceremonia de Inauguración:
• Membros do Comité de Honra • Representantes da Unión Europea • Autoridades Nacionais. • Autoridades Autonómicas • Autoridades Locais • Presidente do Comité Organizador
10:30
Urs A. Meyer (Suiza)
11:15
Mediciña Xenómica e Farmacoxenómica en Trastornos Cardiovasculares
John R. Cockcroft (United Kingdom)
Conferencia Plenaria -1 Mediciña Xenómica: Desafíos e Oportunidades
11:45
Descanso
12:00
Mediciña Xenómica e Farmacoxenómica do Cancro
Filippo De Braud (Italy)
12:30
Mediciña Xenómica e Farmacoxenómica de Trastornos Neurosiquiátricos
Ramón Cacabelos (Spain)
13:30
Xantar
16:00
Mediciña Xenómica e Farmacoxenómica na Unión Europea
José Manuel Silva (Comisión Europea, Bélxica))
16:30
Mediciña Xenómica en México.
Gerardo Jiménez (Mexico)
17:00
Desenvolvemento da Medicina Xenómica dos Transtornos do Sistema Nervioso en Xapón
Masatoshi Takeda (Xapón)
17:30
Farmacoxenómica e Desenvolvemento de Fármacos en USA
Carmen Vigo (USA)
18:00
Descanso
18:15
Nutrixenómica en Europa
Marvin Edeas (Francia)
18:45
Nutrición Humana na Mediciña Xenómica: Estratexia para o Manexo Futuro do Envexecemento
Francesco Marotta (Italia)
December 2008 - international edition
Programa III Conferencia Anual EuroEspes
9:00
Conferencia Plenaria - 2
O futuro da Farmacoxenómica na Práctica Médica: Un Novo Modelo de Desenvolvemento Farmacoxenético
Allen D. Roses (EEUU)
9:45
Farmacoxenética e Trastornos do Metabolismo
Munir Pirmohamed (Reino Unido)
10:15
Mediciña Xenómica e Farmacoxenómica en Enfermedades do Aparato Respiratorio
Ian P. Hall (Reino Unido)
10:45
Descanso
11:00
Mediciña Xenómica e Farmacoxenómica en Trastornos Inmunolóxicos e Vacinas.
Valter Lombardi (España)
11:30
Mediciña Xenómica e Farmacoxenómica en enfermedades infecciosas.
Amalio Telenti (Suiza)
12:00
Xenómica e Farmacoxenómica en “Innovative Medicine Initiative” (IMI)
Alain Vanvossel (Director Executivo IMI. Comision Europea. Bélxica).
12:30
Novidades tecnolóxicas para a implementación da farmacoxenómica na práctica médica
Stefan Prause (Alemania)
13:00
Debate Xeral e Conclusións
13:30
Primeiro Encontro da Asociación Mundial de Mediciña Xenómica (WAGeM)
14:00
Cerimonia de Clausura
Entrega do Premio ó Mellor Póster Presentación dos Premios EuroEspes 2008
21:00
Cea de Gala EuroEspes e Festa de Despedida
Etiqueta
PROGRAMA
SÁBADO 13
December 2008
15
Committees
Honorary Committee
Presidency
Their Royal Majesties King Juan Carlos and Queen Sofía of Spain
Spanish and Local Authorities
Emilio Pérez Touriño President of the Autonomous Government of Galicia
Bernat Soria Escoms Spanish Minister of Health and Consumer Affairs
Dolores Villarino Santiago President of the Galician Parliament
Manuel Ameijeiras Vales Government Delegate in Galicia
Alejandra Pérez Márquez Mayoress of Bergondo
Fernando Xavier Blanco Álvarez Regional Minister of Innovation and Industry, Autonomous Government of Galicia
Salustiano Mato R&D Director General, Regional Ministry of Innovation and Industry, Autonomous Government of Galicia
Salvador Fernández Moreda President of the Provincial Council of A Coruña
Isacio Sigüero Zurdo President of the Organization of Professional Medical Associations
José Luis Baltar Pumar President of the Provincial Council of Ourense
Antonio Garrigues Walker President of Garrigues Asesores
International Authorities
José Manuel Silva
University Authorities
Francisco González Lodeiro
R&D General Directorate, European
Rector Magnificus of the University of
Commission, Brussels
Granada
Alain Vanvossel
Jaime Oraá Oraá
European Commission, Research
Rector Magnificus of the University of
Directorate General, Belgium
Deusto
Manuel Hallen
Francesc Xavier Grau Vidal
Head of Unit “Medical and Public Health
Rector Magnificus of the Rovira i Virgili
Research’ at the Research Directorate General
University
of the European Commission in Brussels
Christian A. Scerri Clinical and Molecular Geneticist, in representation of the Minister of Health of Malta
Vasco Maria Chairman of the Board, Authority of Medicine and Health Products, in
Edelmiro Mateos Rector Magnificus of the Royal University Centre “María Cristina”, Escorial
Juan Juliá Igual Rector Magnificus of the Polytechnic University of Valencia
Juan Manuel Suárez Japón
representation of the Minister of Health of
Rector Magnificus of the International
Portugal
University of Andalucía, Seville
Gerardo Jiménez Sánchez
Lluís Ferrer i Caubet
Director of the Genomic Medicine Institute
Rector Magnificus of the Autonomous
of Mexico, in representation of the Minister
University of Barcelona
of Health of Mexico
Antonio Leaño Reyes Rector of the Autonomic University of Guadalajara, Mexico
Juan Sánchez Aldana Ramírez President of the Municipality of Zapopán, Mexico
Víctor Manuel Ramírez Anguiano Rector of the University Centre of Health Sciences, University of Guadalajara, Mexico
Rafael López President of Parggon Pharmaceuticals, Mexico
Eugenio Luigi Iorio President, International Observatory of Oxidative Stress, Italy
Esther Giménez-Salinas Colomer Rector Magnificus of Ramón Llull University, Barcelona
Ángel Gabilondo Pujol Rector Magnificus of the Autonomous University of Madrid
Francisco José Martínez López Rector Magnificus of the University of Huelva
Félix Faura Matéu Rector Magnificus of the Polytechnic University of Cartagena
Joan Viñas Salas Rector Magnificus of the University of Lleida
Manuel Parras Rosa Rector Magnificus of the University of Jaen December 2008
17
Committees
Javier Uceda Antolín
Carlos Berzosa Alonso-Martínez Rector Magnificus of the Complutense University of Madrid
Rector Magnificus of the Polytechnic University of Madrid
Julio Lafuente López
Eduardo Doménech Martínez Rector Magnificus of the University of La Laguna (Santa Cruz de Tenerife)
Rector Magnificus of the Public University of Navarra
Manuel J. López Pérez
Josep Argemí Renom Rector Magnificus of the International University of Catalunya (Barcelona)
Rector Magnificus of the University of Zaragoza
Vicente Gotor
Montserrat Casas Ametller Rector Magnificus of the University of the Balearic Islands
Rector Magnificus of the University of Oviedo
Adelaida de la Calle Martín
Virgilio Zapatero Gómez Rector Magnificus of the University of Alcalá (Madrid)
Rector Magnificus of the University of Málaga
Evaristo J. Abril Domingo Rector Magnificus of the University of Valladolid
Ernesto Martínez Ataz
Maria del Rosario Sáez Yuguero Rector Magnificus of the Catholic University of Ávila
Daniel Peña Sánchez de Rivera Rector Magnificus of the Carlos III University of Madrid
Rector Magnificus of the University of Castilla-La Mancha
Juan Antonio Cobacho Gómez
Imma Tubella i Casadevall Rector Magnificus of the Open University of Catalunya
Rector Magnificus of the University of Murcia
Josep Samitier i Martí Rector Magnificus of the University of Barcelona
Rafael Cortés Elvira Rector Magnificus of Camilo José Cela University, Madrid
Ignacio Jiménez Raneda Rector Magnificus of the University of Alicante
Alfonso Murillo Villar Rector Magnificus of the University of Burgos
Francisco Tomás Vert Rector Magnificus of the University of Valencia
Alberto Gago Rodríguez Rector Magnificus of the University of Vigo
Diego Sales Márquez
Luis Enrique de la Villa Gil Rector Magnificus of the Open University of Madrid
Rector Magnificus of the University of Cádiz
Jesús Rodríguez Marín Rector Magnificus of Miguel Hernández University, Elche
José Alberto Parejo Gámir Rector Magnificus of the Cardenal Herrera-Ceu University, Valencia
Joaquín Luque Rodríguez Rector Magnificus of the University of Seville 18
December 2008 - international edition
Kepa Urigoitia Saudino President of the Official Medical Association of Álava
Pedro Trillo Parejo President of the Official Medical Association of Orense
Ricardo Miranda Muñoz-Ortíz President of the Official Medical Association of Cádiz
Miquel Bruguera i Cortada President of the Official Medical Association of Barcelona
Enrique de la Figuera Von Wichmann President of the Official Medical Association of Zaragoza
Manuel Gómez Benito President of the Official Medical Association of Salamanca
Rodrigo Martín Hernández President of the Official Medical Association of Tenerife
Rosa M. Pérez Pérez President of the Official Medical Association of Lleida
Antonio Arroyo Guijarro President of the Official Medical Association of Alicante
José Luís Díaz Villarig President of the Official Medical Association of León
Ismael Sánchez Hernández President of the Official Medical Association of Teruel
Joan M. Gual Julià President of the Official Medical Association of Balearic Islands
Alfredo Milazzo Estefanía President of the Official Medical Association of La Rioja
José Ramón Huerta Blanco
Official Medical Associations
Carlos Javier González-Villardell Urbano President of the Official Medical Association of Seville
Cosme Naveda Pomposo President of the Official Medical Association of Vizcaya
President of the Official Medical Association of Soria
Antonio Luna Fantony President of the Official Medical Association of Jaén
Luis Campos Villarino President of the Official Medical Association of Pontevedra
Committees
José Mª Rodríguez Vicente
Javier Tudela von Schmiterlöw
President of the Official Medical Association
President of the Official Pharmaceutical
of Zamora
Association of Málaga
Rodolfo Castillo Wandossell
Manuel Pérez Fernández
President of the Official Medical Association
President of the Official Pharmaceutical
of Murcia
Association of Seville
Official Pharmaceutical Associations
Francisco Asís de la Maza Ruiz
President of the Official Pharmaceutical Association of Pontevedra
President of the Official Pharmaceutical
Association of Cantabria
Association of the Balearic Islands
Alberto García Romero
President of the Official Pharmaceutical
President of the Official Pharmaceutical
Association of Valencia
Association of Madrid
Mª Pilar Garcia Ruiz President of the Official Pharmaceutical Association of Navarra
Rosa María Lendoiro Otero
edical Academies
Alfonso Ballesteros Fernández President of the Royal Medical Academy of e Balearic Islands
Fernando Solsona Motrel
President of the Official Pharmaceutical
President of the Royal Medical Academy of
Association of A Coruña
Zaragoza
Prudencio Rosique Robles
J.A. García-Rodríguez
President of the Official Pharmaceutical
President of the Royal Medical Academy of
Association of Murcia
Salamanca
Juan Carlos Gimeno Barranco
José López Sastre María José Moreno President, Spanish Society of Pain
Isabel Vallejo Díaz President, Business Federation of Spanish Pharmacists
Ramón Pujol Farriols
Antoni Real Ramis
President of the Official Pharmaceutical
Javier Climent Grau
Medical Societies President, Spanish Society of Neonatology
Luis Alberto Amaro Cendón
Máximo Poza y Poza
President of the Spanish Society of Internal Medicine
Luis Aguilera García President of the Spanish Society of Family and Community Medicine
José Manuel Bajo Arenas President of the Spanish Society of Gynaecology and Obstetrics
Julio Ancochea Bermúdez President of the Spanish Society of Pneumology and Thoracic Surgery
Manuel Aguilar Diosdado President of the Spanish Society of Diabetes
Jesús Román Martínez Álvarez
President of the Official Pharmaceutical
President of the Royal Medical Academy of
President of the Spanish Society of
Association of Zaragoza
Murcia
Dietetics and Nutritional Sciences
Lourdes Bergillos Moretón
José Carro Otero
President of the Official Pharmaceutical
President of the Royal Medical Academy of
Association of Asturias
Galicia
Carlos Muñoz Sanz
Jacint Corbella i Corbella
Miguel Carrero López President of National Healthcare Provision
José L. Pérez Sáenz President of the Spanish Society
President of the Official Pharmaceutical
President of the Royal Medical Academy of
of Infectious Diseases and Clinical
Association of “Las Palmas de Gran Canaria”
Catalunya
Microbiology
Francisco Peinado Martínez
María del Carmen Maroto Vela
Juan Pedro López Siguero
President of the Official Pharmaceutical
President of the Royal Medical Academy of
President of the Spanish Society of
Association of Huelva
Granada
Paediatric Endocrinology
Cecilio J. Venegas Fito
Benjamín Narbona Arnau
Miguel García Montes
President of the Official Pharmaceutical
President of the Royal Medical Academy of
President of the Spanish Association of
Association of Badajoz
Valencia
Medical Biopathology December 2008
19
Committees
Esther de la Viuda
Pedro Gil Gregorio
Inmaculada García Montes
President of the Spanish Society of
President of the Spanish Society of
President of the Spanish Society of
Contraception
Geriatrics and Gerontology
Rehabilitation and Physical Medicine
Ismael Herruzo Cabrera
Santiago Martínez del Olmo
Feliciano J. Ramos Fuentes
President of the Spanish Association of
President of the Spanish Association of
President of the Spanish Association
Radiotherapy and Oncology
Pharmacists and Analysts
of Human Genetics
Fermín Mearin
Juan Enrique Domínguez Muñoz
President of the Spanish Society of
President of the Spanish Society of
Gastroenterology
Digestive Pathology
Luis Aguilera García
Carlos Sánchez Marchori
President of the Spanish Society of Family
President of the Spanish Society of Sports
and Community Medicine
Traumatology
Luis Martí-Bonmatí
Luis Jiménez Murillo
F. Gilsanz Rodríguez President of the Spanish Society of Anaesthesiology, Reanimation and Pain Therapy
Francisco Vicente Álvarez Menéndez President of the Spanish Society of Clinical Biochemistry and Molecular Pathology
Damiano Galimberti
President of the Spanish Society of Medical
President of the Spanish Society of
President, Association of Italian Anti-Aging
Radiology
Emergency Medicine
Physicians
Organizing Committee
Chairman
Ramón Cacabelos MD, PhD, DMSc
Committee Members
Adolfo Sánchez (Spain) Ana Isabel Vallejo (Spain) Antón Álvarez (Spain) Arturo Romero (Spain) Carlos Miramontes (Mexico) Carmen Vigo (USA) Enrique Fernández (Spain) Francesco Marotta (Italy) Francisco Bernárdez (Mexico) Francisco Javier Jiménez (Mexico) Gildardo Zafra (Mexico) Isabel Hoffman Miles (Canada) Jerzy Leszek (Poland) José Antonio Carrizosa (Spain) 20
December 2008 - international edition
José García-Morales (Spain) José Iglesias (Mexico) José Miguel Sempere (Spain) Juan Carlos Carril (Spain) Juan Manuel Vieites Baptista (Spain) Lola Corzo (Spain) Lucía Fernández-Novoa (Spain) Luis Álvarez (Spain) Marta Rodríguez (Spain) Marvin Edeas (France) Masatoshi Takeda (Japan) Pablo Álvarez de Linera (Spain) Ricardo Palacios (Spain) Rodolfo Rodríguez (Spain) Ruth Llovo (Spain) Salvador Harguindey (Spain) Stefan Prause (Germany) Toshihisa Tanaka (Japan) Valter Lombardi (Spain)
Secretariat
Adam McKay Amanda Bello Antonio Bermo Ciprián Rivas Esther Barros Gladys Bahamonde Javier Masoliver Juan Antonio Mesonero Ramón Alejo Ricardo Palleiro Rocío Martínez
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Speakers In Order of Presentation Speakers
Urs A. Meyer M.D.
Abstract
GENOMIC MEDICINE: CHALLENGES AND OPPORTUNITIES Urs A. Meyer urs-a.meyer@unibas.ch
Biozentrum, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland
Friday 12 - 10:30
The interindividual variation of the human genome sequence explains pharmacogenetic observations and is an important component of personalized medicine. Personalized medicine is a strategy to improve clinical outcome by precise diagnosis ( e.g. subphenotypes of cancers ) by optimizing drug choice and drug dose to the problem and need of the individual patient, by taking into account the individual’s genome sequence and environmental (e.g. smoking, other drugs) and host factors (e.g. age, sex, previous diseases). The reasonable hope is that this strategy will decrease the number of adverse drug reactions (ADRs) and increase the efficacy of drug therapy.
22
Earlier comparisons of haploid human genome sequences suggested that two individuals differ by only 3 million basepairs or 0.1 % of the total sequence. More recent studies have revealed additional structural diversity, including variations in gene copy number and large insertions and deletions. Moreover, sequences of several diploid genomes show considerable sequence differences (~ 4 million basepairs) between the chromosomes of a chromosomal pair. Therefore, the sequence difference between two unrelated individuals may be 1 or 2 % of total sequence, or 30 to 60 million basepairs. Recent developments in genomics include the International HapMap project (http://snp.cshl.org) in four different populations, faster and less expensive sequencing technologies and dense genotyping chips or beads for analysis of 0.5 to 1 million of SNPs (single nucleotide polymorphisms).These developments have led to 1) further studies of human sequence diversity, e.g. by the plan to sequence 1000 human genomes (http://www.1000genomes.org) or even the genomes of 100,000 volunteers (PGP, personal genome project (http://www.personalgenomes.org) and posting the genomes and corresponding medical histories on the web. 2) genome-wide association studies with the goal of identifying the genetic risk factors leading to common diseases (http://genome.gov/WGAstudies), 3) studies to identify genetic risks of severe adverse drug reactions, 4) the Cancer Genome Atlas(TCGA), an effort to identify all common mutations in human cancers. 5) Another recent development is “consumer genomics”, i.e. the emergence of commercial companies offering to genotype >500’000 SNPs for individuals at a prize of less than 400 US $. The search for pharmacogenomic biomarkers involved in ADRs or efficacy problems has focused on variations of genes for drug metabolizing enzymes in particular cytochromes P450 or MHC Class I genes for immune-mediated toxicities. These biomarkers include genes for enzymes important in the metabolism of anticancer drugs such as CYP2D6 for tamoxifen. Other drugs for which variants in CYPs are of clinical importance are codeine, antidepressants and neuroleptics, and warfarin. Warfarin is difficult to dose due to its narrow therapeutic range and wide interpatient variability. This variability is caused by the interaction of genetic, environmental and host factors. Variants of two genes, CYP2C9 and VKORC1, contribute ~ 35 %, -age, gender, body surface area, type of disease or concurrent drug treatment another ~30 % to the individual variation in warfarin dose. Because overdosing and underdosing is frequent and leads to severe ADRs (hemorrhage) or insufficient anticoagulation, dosing algorithm based on genotypes of CYP2C9 and VKORC1 and individual clinical factors have been developed and are increasingly used to predict the dose of warfarin. Meyer, U.A. Genes and the individual response to treatment. In: The LANCET Treating Individuals (P.M. Rothwell, editor) Elsevier Limited, 2007, pp. 151-168. Kalow, W., Meyer, U.A., Tyndale, R.F. (eds). Pharmacogenomics. Taylor & Francis, Boca Raton, 2005. Meyer, U.A. Pharmacogenetics-five decades of therapeutic lessons from genetic diversity. Nature Reviews Genetics 5:669-676, 2004.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Urs A. Meyer received B.A. and M.D. degrees from the Universities of Geneva and Zürich, with additional training in biochemistry and metabolic diseases for 2 years at the University of Zurich (thesis). After internship and residency training in Internal Medicine at the University of California, San Francisco (UCSF), he was a Postdoctoral Fellow in Clinical Pharmacology at UCSF and Fellow in Hepatology at the University of Texas Southwestern Medical School, Dallas, Texas. In 1971, Urs A. Meyer was recruited as Assistant Professor of Medicine and Pharmacology to UCSF where he established his own research group and in 1974 returned to Switzerland as Associate Professor and Chief of the Division of Clinical Pharmacology at the University of Zürich Medical School. Since 1983 Urs A. Meyer is Professor of Pharmacology at the Biozentrum of the University of Basel, where he also was Acting Chairman of the Biozentrum from 1993 to 1995. In 1992 - 1993, Urs A. Meyer was a visiting professor in the Department of Molecular Pharmacology at Stanford University, Palo Alto, USA, and in 2008 a Sabbatical Professor at the Center for Drug Evaluation and Research (CDER) at the Food and Drug Administration (FDA) in Silver Springs, MD, USA. Urs A. Meyer’s research has focused on interindividual variation of drug response throughout his career, from studying the pharmacogenetic disease porphyria to the pharmacogenetics of drug metabolism and more recently transcriptional regulation of drug metabolizing enzymes by nuclear receptors and xeno-endobiotic crosstalk. He has authored over 350 publications and is listed on ISI’s Highly Cited Researcher’s database. Urs A. Meyer has served in various WHO and NIH functions, most recently as member of the External Scientific Panel for the NIH Pharmacogenetics Research Network. He was president of the clinical section of the Swiss National Science Foundation, is an elected member of the Swiss Academy of Medical Sciences, and was Vicepresident of the Academia Europaea. Urs A. Meyer has been the recipient of numerous awards and honors including the Cloetta Award for Medical Research, the Rawls-Palmer Award fro Progress in Medicine, the Robert Pfleger Research Award and most recently the RT Williams Distinguished Scientific Achievement Award of the International Society for the Study of Xenobiotics - among many others.
December 2008
23
Speakers In Order of Presentation Speakers
Prof.
John R Cockcroft Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS OF CARDIOVASCULAR DISORDERS Professor John R Cockcroft cockcroftjr@Cardiff.ac.uk
Professor of Cardiology, Wales Heart Research Institute, Cardiff CF14 4XN Improved understanding of the human genome has stimulated researchers to study the effects of genetic polymorphisms on the prediction of disease and the actions of drugs in the hope of developing improved therapies, targeted at individual patients, based on their genetic makeup.
Friday 12 - 11:15
Since cardiovascular disease is a major cause of death and morbidity, it has become a major focus of research into the interaction between the genome and cardiovascular drugs. Genetic differences influence response to cardiovascular drugs in 3 major ways. Pharmacokinetic interactions, especially drug metabolism, as relevant to the cytochrome P450 system. Pharmacodynamic interactions such as exemplified by polymorphisms of the beta adrenoceptors. Finally, genes in the causal pathway of disease can modify the effects of drugs; here ApoE has been widely studied.
24
A number of major systems are involved in the genesis of cardiovascular disease including the renin angiotensin system, the sympathetic nervous system, the lipid, inflammation and coagulation systems. Polymorphisms within these systems have been shown to influence the effect of drugs such as ACE inhibitors, beta-blockers, statins and anticoagulants. However, results of such studies have often been conflicting and as yet have not resulted in major therapeutic changes. Considerable heterogeneity in ethnicity, clinical domains and outcome have made meta analysis of accumulated data difficult to perform and there is now an urgent need for high quality randomised control trials. Perhaps the most exciting and clinically relevant application of pharmacogenomics in the management of cardiovascular disease is the prediction of adverse responses to widely used drugs. Indeed, polymorphisms in the SCLO1B1 gene can predict the development of statin-induced myopathy. In addition response to warfarin is also influenced by genetic variation in both the cytochrome P450 2C9 (CYP2C9) gene and the Vitamin K apoxide reductase gene (VKORC1). These findings have already led researchers and clinicians to advocate screening for these polymorphisms in clinical practice to reduce the incidence of warfarin associated bleeding. Finally, polymorphism in genes coding for cardiac ion channels have been shown to predispose to the development of life threatening cardiac arrhythmias in response to certain drugs. Again suggesting that screening for such polymorphisms may reduce the incidence of drug-induced torsade de points or ventricular fibrillation. Although cardiovascular pharmacogenomics is a promising field, as yet, there are no tests that appear close to clinical utility. The literature is characterised by many exploratory findings that have not been replicated. Strong and consistent associations between particular genotypes and drug response will be required for pharmacogenomic findings to be translated into clinical practice. Furthermore, clinical trials will be necessary to determine whether patient outcomes are actually improved by treatment directed by genetic information.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Professor Cockcroft is Professor of Cardiology at the Wales Heart Research Institute in Cardiff. He is also Special Professor of Cardiovascular Disease Prevention at the University of Nottingham and visiting Professor in the Graduate School of Biomedical Engineering, the University of New South Wales, Sydney, Australia. Most recently Professor Cockcroft is visiting Professor at Columbia Presbyterian Hospital New York. His major research interests focus on endothelial function and arterial stiffness in health and disease. Recently he has become interested in the mechanisms of vascular calcification especially in patients with renal disease. He is currently researching the relationship between osteoporosis and vascular calcification. He has published over 150 peer reviewed articles and has co-authored books on hypertension and coronary heart disease. He is a founding member of the Association for Research into Arterial Structure and Physiology (ARTERY) and is co-organiser of the Association’s conferences. Currently he is the Secretary of The European Association of Clinical Pharmacology and Therapeutics (EACPT) Professor Cockcroft’s clinical interests focus on hypertension and cardiovascular disease prevention and he was a member of the committee which produced the Welsh National Service Framework for cardiovascular disease. He is especially interested in patient empowerment and promoting more informed involvement with their care and treatment, and has lectured widely to patient groups on hypertension and cardiovascular disease. Indeed, 8 years ago he established the first patient self referral clinic in the UK, which has proved extremely popular with patients interested in establishing their own cardiovascular risk and learning more about cardiovascular risk factors in general. He has also run mobile cardiac vascular risk factor screening clinics Wales wide and also in England. Currently he runs an open access metabolic syndrome clinic. He is currently advising the CDC in Washington on including measurements of arterial stiffness in the NHANES Study. Professor Cockcroft is a member of the British, European, American and International societies of Hypertension the British Cardiac Society and also the European Association for the Study of Diabetes. He is a member of the editorial board of the Journal of hypertension and Hypertension. Most recently, he has been re-lected Secretary of The European Association of Clinical Pharmacology and Therapeutics (EACPT). He is President of The Association for Research into Arterial Structure and Physiology (ARTERY) and is Editor in Chief of the associations journal Artery Research.
December 2008
25
Speakers In Order of Presentation Speakers
Filippo Guglielmo de Braud M.D.
Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS OF CANCER 1
Filippo de Braud MD, 2 Marco Foiani PhD, 1 Francesca Toffalorio MD PhD, 3 Elisa Giovannetti MD PhD
New Drug Development Unit at European Institute of Oncology Milan 2 Prof Molecular Biology University of Milan, Director IFOM, FIRC Institute of Molecular Oncology 3 Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands 1
filippo.debraud@ieo.it
Friday 12 - 12:00
Genomic biomarkers are the foundation of “personalized medicine” . They can be used either to select population of patients suitable to receive proper treatments avoiding toxicity either to select the optimal drug for specific cancer cells population. Nevertheless, methodology of clinical trials and drug development trials must be improved to allow pharmacogenetics to ameliorate treatment outcome by permitting the selection of patients most suitable to benefit from the proposed treatment and/ or to exclude those tumours unlike to be sensitive. Few recent clinical studies have addressed the use of predictive molecular markers of activity and toxicity for cancer treatment, but the results must be interpreted with care as it will be presented.
26
In past years, many compounds were found to be active on tumor cells, without any exact knowledge of their targets and of their molecular mechanisms of working. We now can take advantage of a set of new tools, namely pharmacogenomics, to better define how small biologically active compounds can exert their function and, more importantly, identify mechanisms of resistance/sensitivity to these drugs. We intend to use yeast as a model of pharmacogenomic approach for new drugs development. The screening of an yeast knock out library with the experimental compounds will performed contemporary to phase I studies in order to identify mutants that exhibit resistance or hypersensitivity to drug treatments. Mechanistic studies will be carried out when applicable. Yeast has proven to be a powerful instrument to this purpose: its small, compact genome has completely been sequenced (Goffeau et al., 1996; Winzeler et al., 1999), showing that many yeast proteins share similarity to human ones. Several of these are involved in human diseases (Hartwell et al., 1997), making this simple organism very appealing for anticancer drug discovery. We intend to use yeast as a model system to identify drug responsive genes that will be then validated in human cells and tissues. From 2006 at IFOM a pharmacogenomic platform is operative and devoted to the identification of the chemical genetic profiles of several anticancer drugs. Several drugs have been tested, including conventional cytotoxic drugs and “biological drugs”. Mechanistic studies are also ongoing aimed at elucidating the mechanistic aspects of drug activity.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Filippo Guglielmo de Braud, MD obtained his Medical Degree in 1984 and the Specialization in Oncology in 1988 from the University of Milan. Since December 2000 he has been Director of Clinical Pharmacology and New Drugs Development Unit at the European Institute of Oncology, Milano Italy and a member of the Technical Scientific Commission (CTS) for drug approval of the Italian Medicine Agency since February 2001. He is also a member of several professional societies. During his training and earlier work experiences he was involved in basic and clinical research at National Cancer Institute of Milan (Italy) Royal Free Hospital School of Medicine in London, Wayne State University of Detroit and Institute Gustave Roussy of Paris. He is author and co-author of more than 120 publications on peer reviewed journals and 20 book chapters. He is also clinical Editor of the START Oncology project, on the state of oncology in Europe, supported by “Allenaza Contro il Cancro”. www.startoncology.net Member of American Society for Clinical Oncology (ASCO) Member of American Association for Cancer Research (AACR) Member of European Society for Medical Oncology (ESMO) Member of Società Italiana di Oncologia Medica (AIOM) Member of Connective Tissue Oncology Society (CTOS) Member of Società Italiana di Farmacologia (SIF)
December 2008
27
Speakers In Order of Presentation Speakers
Ramón Cacabelos M.D., Ph.D., D.M.Sc.
Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS OF NEUROPSYCHIATRIC DISORDERS Ramón Cacabelos rcacabelos@euroespes.com
EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, EuroEspes Chair of Biotechnology and Genomics, Camilo José Cela University, 15165-Bergondo, Coruña, Spain
Friday 12 - 12:30
Neuropsychiatric disorders and dementia represent a major cause of disability and high cost in developed societies. Central nervous system (CNS) disorders are the third greatest health problem in developed countries, representing 10-15% of deaths, after cardiovascular disorders (25-30%) and cancer (20-25%). Approximately 127 million Europeans suffer brain disorders. The total annual cost of brain disorders in Europe is about €386 billion, with €135 billion of direct medical expenditures (€78 billion, inpatients; €45 billion, outpatients; €13 billion, pharmacological treatment), €179 billion of indirect costs (lost workdays, productivity loss, permanent disability), and €72 billion of direct nonmedical costs. Mental disorders represent €240 billion (62% of the total cost, excluding dementia), followed by neurological diseases (€84 billion, 22%).
28
Most disorders of the central nervous system (CNS) share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways, poor therapeutic outcomes, and chronic disability. The lack of accurate diagnostic markers for early prediction and an effective therapy of CNS disorders are the two most important problems to efficiently diagnose and halt disease progression. The pharmacological treatment of CNS disorders, in general, accounts for 10-20% of direct costs, and less than 30-40% of the patients are moderate responders to conventional drugs, some of which may cause important adverse drug reactions (ADRs). In the case of dementia, less than 20% of the patients may benefit from current drugs (donepezil, rivastigmine, galantamine, memantine), with doubtful cost-effectiveness. The pathogenic mechanisms of most CNS disorders (e.g., psychosis, depression, anxiety, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis, etc) are poorly understood. This circumstance complicates the implantation of a molecular intervention to neutralize causative factors. In fact, more than 80% of the 25.000 genes integrating the human genome are expressed in the CNS at different periods of the life span, and only a few neurotransmitters (e.g., noradrenaline, dopamine, acetylcholine, GABA, histamine, and less than 10 neuropeptides) are the actual targets of conventional psychopharmacology. Common features in CNS disorders include the following: (a) polygenic/complex disorders in which genomic and environmental factors are involved; (b) deterioration of higher activities of the CNS; (c) multifactorial dysfunctions in several brain circuits; and (d) accumulation of toxic proteins in the nervous tissue in cases of neurodegeneration. Extensive molecular genetics studies carried out in the past two decades have shown that most CNS disorders are multifactorial and polygenic. For example, 256 genes have been associated with dementia, 151 with schizophrenia, 230 with depression, 87 with bipolar disorder, 123 with anxiety, 51 with personality disorders, 90 with language disorders, 90 with sleep disorders, 21 with drug addiction, 184 with stroke, 32 with cerebrovascular disorders, 83 with migraine, 713 with different types of ataxia, 422 with epilepsia, 152 with movement disorders, 70 with Parkinson’s disease, 46 with demyelinating disorders, 113 with motor neuron disease, 89 with neuromuscular disorders, and 466 with brain tumors. Another paradigmatic example of heterogeneity and complexity is dementia, one of the most heterogeneous disorders of the CNS. The genetic defects identified in AD over the past 25 years can be classified into 3 main categories: (a) Mendelian or mutational defects in genes directly linked to AD, including (i) 32 mutations in the amyloid beta (Aβ)(ABP) precursor protein (APP) gene (21q21); (ii) 165 mutations in the presenilin 1 (PS1) gene (14q24.3); and (iii) 12 mutations in the presenilin 2 (PS2) gene (1q31-q42). (b) Multiple polymorphic variants of risk characterized in more than 200 different genes distributed across the human genome can increase neuronal vulnerability to premature death. Among these genes of susceptibility, the apolipoprotein E (APOE) gene (19q13.2) is the most
December 2008 - international edition
Speakers
In Order of Presentation
prevalent as a risk factor for AD, especially in those subjects harbouring the APOE-4 allele, whereas carriers of the APOE-2 allele might be protected against dementia. APOE-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of AD. (c) Diverse mutations located in mitochondrial DNA (mtDNA) through heteroplasmic transmission can influence aging and oxidative stress conditions, conferring phenotypic heterogeneity. Many of these genetic associations could not be replicated in different settings and different populations due to many complex (methodological, technological) factors. Furthermore, the same genomic defect can give rise to apparent diverse phenotypes, and different genomic defects can converge in an apparently common phenotype, this increasing the complexity of genomic studies (e.g., patient recruitment, pure controls, concomitant pathology, epigenetic factors, environmental factors). Recent advances in genomic medicine can contribute to accelerating our understanding on the pathogenesis of CNS disorders, improving diagnostic accuracy with the introduction of novel biomarkers, and personalizing therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug development and clinical practice. Pharmacogenetic and pharmacogenomic factors may account for 60-90% of drug variability in drug disposition and pharmacodynamics. The introduction of novel procedures into an integral genomic medicine protocol for CNS disorders is an imperative requirement in drug development and in the clinical practice to improve diagnostic accuracy and to optimize therapeutics. This kind of protocol should integrate the following components: (i) clinical history, (ii) laboratory tests, (iii) neuropsychological assessment, (iv) cardiovascular evaluation, (v) conventional X-ray technology, (vi) structural neuroimaging, (vii) functional neuroimaging, (viii) computerized brain electrophysiology, (ix) cerebrovascular evaluation, (x) structural genomics, (xi) functional genomics, (xii) pharmacogenetics, (xiii) pharmacogenomics, (ix) nutrigenetics, (x) nutrigenomics, (xi) bioinformatics for data management, and (xii) artificial intelligence procedures for diagnostic assignments and probabilistic therapeutic options. All these procedures, under personalized strategies adapted to the complexity of each case, are essential to depict a clinical profile based on specific biomarkers correlating with individual genomic profiles. The natural course of technical events to achieve efficient goals in pharmacogenetics and pharmacogenomics includes the following steps: (a) genetic testing of mutant genes and/or polymorphic variants of risk; (b) genomic screening, and understanding of transcriptomic, proteomic, and metabolomic networks; (c) functional genomics studies and genotype-phenotype correlation analysis; and (d) the development of pharmacogenetics and pharmacogenomics, addressing drug safety and efficacy, respectively. With pharmacogenetics we can understand how genomic factors associated with genes encoding enzymes responsible for drug metabolism regulate pharmacokinetics and pharmacodynamics (mostly safety issues). With pharmacogenomics we can differentiate the specific disease-modifying effects of drugs (efficacy issues) acting on pathogenic mechanisms directly linked to genes whose mutations determine the disease phenotype. The pharmacogenomic outcome depends upon many different determinant factors including (i) genomic profile (family history, ethnic background, disease-related genotype, pharmacogenetic genotype, pharmacogenomic genotype, nutrigenetic genotype, nutrigenomic genotype), (ii) disease phenotype (age at onset, disease severity, clinical symptoms), (iii) concomitant pathology, (iv) genotype-phenotype correlations, (v) nutritional conditions, (vi) age and gender, (vii) pharmacological profile of the drugs, (viii) drug-drug interactions, (ix) gene expression profile, (x) transcriptomic cascade, (xi) proteomic profile, and (xii) metabolomic networking. The dissection and further integration of all these factors is of paramount importance for the assessment of the pharmacogenomic outcome in terms of safety and efficacy. More than 80% of psychotropic drugs are metabolized by enzymes known to be genetically variable, including: (a) esterases: butyrylcholinesterase, paraoxonase/arylesterase; (b) transferases: Nacetyltransferase, sulfotransferase, thiol methyltransferase, thiopurine methyltransferase, catecholO-methyltransferase, glutathione-S-transferases, UDP-glucuronosyltransferases, glucosyltransferase, histamine methyltransferase; (c) Reductases: NADPH:quinine oxidoreductase, glucose-6-phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehydehydrogenase, monoamine oxidase B, catalase, superoxide dismutase, trimethylamine N-oxidase, dihydropyrimidine dehydrogenase; and (e) December 2008
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cytochrome P450 enzymes, such as CYP1A1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A5 and many others. Polymorphic variants in these genes can induce alterations in drug metabolism modifying the efficacy and safety of the prescribed drugs. Drug metabolism includes phase I reactions (i.e., oxidation, reduction, hydrolysis) and phase II conjugation reactions (i.e., acetylation, glucuronidation, sulfation, methylation). The principal enzymes with polymorphic variants involved in phase I reactions are the following: CYP3A4/5/7, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1B1, CYP1A1/2, epoxide hydrolase, esterases, NQO1 (NADPH-quinone oxidoreductase), DPD (dihydropyrimidine dehydrogenase), ADH (alcohol dehydrogenase), and ALDH (aldehyde dehydrogenase). Major enzymes involved in phase II reactions include the following: UGTs (uridine 5’-triphosphate glucuronosyl transferases), TPMT (thiopurine methyltransferase), COMT (catechol-O-methyltransferase), HMT (histamine methyltransferase), STs (sulfotransferases), GST-A (glutathion S-transferase A), GST-P, GST-T, GST-M, NAT2 (N-acetyl transferase), NAT1, and others. Polymorphisms in genes associated with phase II metabolism enzymes, such as GSTM1, GSTT1, NAT2 and TPMT are well understood, and information is also emerging on other GST polymorphisms and on polymorphisms in the UDP-glucuronosyltransferases and sulfotransferases. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. About 60-80% of the psychotropic agents currently used for the treatment of neuropsychiatric disorders are metabolized via enzymes of the CYP family, especially CYP1A2, CYP2B6, CYP2C8/9, CYP2C19, CYP2D6 and CYP3A4. The most frequent CYP2D6 alleles in the European population are the following: CYP2D6*1 (wild-type) (normal), CYP2D6*2 (2850C>T)(normal), CYP2D6*3 (2549A>del)(inactive), CYP2D6*4 (1846G>A) (inactive), CYP2D6*5 (gene deletion)(inactive), CYP2D6*6 (1707T>del)(inactive), CYP2D6*7 (2935A>C)(inactive), CYP2D6*8 (1758G>T)(inactive), CYP2D6*9 (2613-2615 delAGA)(partially active), CYP2D6*10 (100C>T)(partially active), CYP2D6*11 (883G>C)(inactive), CYP2D6*12 (124G>A) (inactive), CYP2D6*17 (1023C>T)(partially active), and CYP2D6 gene duplications (with increased or decreased enzymatic activity depending upon the alleles involved). In the Spanish population, where the mixture of ancestral cultures has occurred for centuries, the distribution of the CYP2D6 genotypes differentiates 4 major categories of CYP2D6-related metabolyzer types: (i) Extensive Metabolizers (EM)(*1/*1, *1/*10); (ii) Intermediate Metabolizers (IM)(*1/*3, *1/*4, *1/*5, *1/*6, *1/*7, *10/*10, *4/*10, *6/*10, *7/*10); (iii) Poor Metabolizers (PM)(*4/*4, *5/*5); and (iv) Ultra-rapid Metabolizers (UM)(*1xN/*1, *1xN/*4, Dupl). In this sample we have found 51.61% EMs, 32.26% IMs, 9.03% PMs, and 7.10% UMs20,74-77. The distribution of all major genotypes is the following: *1/*1, 47.10%; *1/*10, 4.52%; *1/*3, 1.95%; *1/*4, 17.42%; *1/*5, 3.87%; *1/*6, 2.58%; *1/*7, 0.65%; *10/*10, 1.30%; *4/*10, 3.23%; *6/*10, 0.65%; *7/*10, 0.65%; *4/*4, 8.37%; *5/*5, 0.65%; *1xN/*1, 4.52%; *1xN/*4, 1.95%; and Dupl, 0.65%. Approximately 60-80% of CNS drugs are metabolized via enzymes of the CYP gene superfamily; 18% of neuroleptics are major substrates of CYP1A2 enzymes, 40% of CYP2D6, and 23% of CYP3A4; 24% of antidepressants are major substrates of CYP1A2 enzymes, 5% of CYP2B6, 38% of CYP2C19, 85% of CYP2D6, and 38% of CYP3A4; 7% of benzodiazepines are major substrates of CYP2C19 enzymes, 20% of CYP2D6, and 95% of CYP3A4. About 10-20% of Caucasians are carriers of defective CYP2D6 polymorphic variants that alter the metabolism of many psychotropic agents. Another 100 genes participate in the efficacy and safety of psychotropic drugs. The incorporation of pharmacogenetic/pharmacogenomic protocols to CNS research and clinical practice can foster therapeutic optimization by helping to develop cost-effective pharmaceuticals and improving drug efficacy and safety. To achieve this goal several measures have to be taken, including: (a) educating physicians and the public on the use of genetic/genomic screening in daily clinical practice; (b) standardizing genetic testing for major categories of drugs; (c) validating pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulating ethical, social, and economic issues; and (e) incorporating pharmacogenetic and pharmacogenomic procedures to both drugs under development and marketed drugs in order to optimize therapeutics.
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December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Graduate in Medicine and Surgery, Oviedo University Medical School, Oviedo, Spain (1980). International Fellowship, Ministry of Education, Science and Culture of Japan, Department of Psychiatry, Osaka University Medical School, Osaka, Japan (1981-1983). International Fellowship, Ministry of Education, Science and Culture of Japan, Postgraduate I-IV, Department of Psychiatry, Osaka University Medical School, Osaka, Japan (1983-1987). Ph.D. (Medicine & Surgery), Santiago University Medical School, Santiago de Compostela, Spain (1985). Ph.D., D.M. Sci. (Doctor in Medical Science/Internal Medicine/Psychiatry), Osaka University Medical School, Osaka, Japan (1987). Associate Professor of Psychiatry, Department of Psychiatry, Santiago University Medical School, Santiago de Compostela, Spain (1987-1989). Visiting Professor, Department of Psychiatry, Navarra University Medical School, Pamplona, Spain (1990). Visiting Professor, Department of Pathology, New York University Medical Center, New York, USA (1991). Tenure Professor, Department of Human Physiology, Complutense University Medical School, Madrid, Spain (1989-1995). International Master in Hospital Management, International School for Hospital Management, Madrid, Spain (1996). President of EuroEspes Corp. (1991-). Director, Institute for Central Nervous System Disorders, La Coruña, Spain (1991-). Coordinator, European Dementia Network (EUDENET)(1992-2000). President, EuroEspes Foundation (1992-). President, Spanish Association of Neurogerontology and Neurogeriatrics (1994-2005). Director General, EuroEspes Biomedical Research Center, La Coruña, Spain (1995-). Director, Department of Clinical Neurosciences, CIBE, La Coruña, Spain (1995-). President, EuroEspes Biotechnology (EBIOTEC), La Coruña, Spain, (2001-). President, Ebiotec Foundation, La Coruña, Spain (2003-2008). Professor & Chairman, EuroEspes Chair of Biotechnology and Genomics, Camilo José Cela University, Madrid, Spain (2003-).
Research Projects: National Projects (56), International Projects (52)
Publications: Original papers (318), chapters and reviews (160), Abstracts and Congress Communications (645), Books (17), Other publications (155) Member of 30 National and International Scientific Societies and Associations Referee of 26 National and International Journals Member of 25 International Advisory Boards Patents: 6 Lectures, Seminars, Conferences and Courses: 201 Awards and Scientific Honors: National (9), International (4) Editor-in-Chief of: ANNALS OF PSYCHIATRY/BASIC AND CLINICAL NEUROSCIENCES (1990-2004), and NEUROGERONTOLOGY & NEUROGERIATRICS (1993-2004) Editor-in-Chief, The EuroEspes Journal, Gen-T (2007-)
December 2008
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José Manuel Silva Rodríguez EUROPEAN COMMISSION, R&D DIRECTORATE-GENERAL
Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS IN THE EUROPEAN UNION José Manuel Silva Rodríguez rtd-secretariat-dg@ec.europa.eu
Director General, European Commission, Directorate-General for Research
Friday 12 - 16:00
We have witnessed unprecedented advances in life sciences during recent years. The sequencing of the human genome and other genomes, together with the development of new large-scale analysis techniques have revolutionised biomedical research and provided new insights into the function of genes in normal and pathological situations. Although the attempts to integrate genomic knowledge into clinical practice are still in the early stages, genomic sciences are expected to have a profound impact on clinical medicine in the era of genomic medicine.
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The first EU-funded projects addressing human genetics date back to FP2 (1987-1991). Since then, the EU has played a cohesive role in addressing the fragmentation of the genomics and post-genomics research community in Europe by funding collaborative research projects bringing together top-level researchers from different countries in the Union. In FP6 (2002-2006) the coordination and integration aspects were strengthened to contribute towards the creation of the European Research Area (ERA) for the European “internal market” for research. Still today the vast bulk of research in Europe is implemented independently by national or regional programmes, which leads both to unwanted duplications and to opportunities missed, to achieve critical mass. Only 15% of European publicly financed civil R&D is financed in a cross-border collaborative manner. In recent years, the EU has launched many new initiatives, such as the ERA-NET scheme, to decrease fragmentation and to boost the impact and efficiency of public research. In 2007, the Commission published the Green Paper on the European Research Area which was followed by a large public consultation. Based on this, new ERA initiatives are being launched in 2008. The new initiatives address researchers’ careers and mobility, research infrastructures, knowledge sharing, joint programming, and international science and technology cooperation. They aim at establishing durable partnerships with Member States and stakeholders, including business, universities and research organisations to move forward with the implementation of the ERA. The Health Theme of the current Framework Programme, FP7 (2007-2013), provides an unparalleled opportunity to advance genomic medicine. The focus is on translational research turning discoveries of basic research into the development of new preventive, diagnostic and therapeutic methods to be applied in medicine. The overall funding earmarked for Health research is €6100 million. This represents a significant boost to European science and technology for the prevention, diagnosis, treatment and control of diseases, ultimately improving the health of European citizens. In 2007, the EU took a major step toward strengthening Europe’s position as a centre of excellence in biopharmaceutical R&D by launching the Innovative Medicines Initiative (IMI). The aim of IMI is to support faster discovery and development of better medicines for patients and enhance Europe’s competitiveness by ensuring that its biopharmaceutical sector remains a dynamic high-technology sector. IMI will ensure that Europe’s biomedical sciences receive targeted strategic support for the benefit of patients, as well as the scientists and citizens of Europe.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Agricultural engineer (ETSI AGRONOMOS – MADRID) Degree in External trade (CEU-MADRID) Diploma in European Communities (CEPADE-MADRID) Since 1971 until his incorporation into SOIVRE, while an inspector in the Ministry of Economy and Trade, he was a professor at the School of Agricultural Engineers in Madrid and worked in the private sector in export companies as well as on animal food projects.
Ministry of Economy and Trade (Madrid) 1979-1982 Employed in the Directorate general of Internal Trade and in the Directorate General of Trade Policy
Secretaria de Estado (Madrid) 1983-1986 Counsellor in agricultural affairs and member of the delegation for the Spanish accession negotiations.
European Commission Since 1986, he worked in the following posts: 1986 Member of the Cabinet of the Vice-President Manuel Marin. 1987-1990 Head of unit for processed fruit and vegetables division. 1990 Head of unit, Tobacco division. 1991-1993 Head of unit, wine, spirits and derived products division. 1993-1997 Chief advisor of the Directorate General of Agriculture and Director of the Common Market Organisation for Vegetable Products. 1997 Deputy Director General of Agriculture responsible for rural development. Dec. 1999-2005 Director General for Agriculture and Rural Development. Jan. 2006 Director General for Research.
December 2008
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Gerardo Jiménez-Sánchez M.D., Ph.D.
Abstract
GENOMIC MEDICINE IN MEXICO Gerardo Jiménez-Sánchez, MD, PhD. mmora@inmegen.gob.mx
National Institute of Genomic Medicine, Mexico. Periferico Sur No. 4124, Torre Zafiro II, 6to. Piso, Col. Jardines del Pedregal, Mexico D.F. 01900, Mexico.
Friday 12 - 16:30
Mexico faces important demographic and epidemiological transitions with significant implications to patterns of disease, disability, and death. On the one hand, there are problems of underdevelopment and, on the other, the emerging challenges of the chronic and degenerative diseases of the industrialized world. For these diseases, prevention becomes a key strategy for alleviating a major burden to the economy and health of the population. Genomic medicine has become a priority to the Mexican government as a means of finding new strategies to tackle common diseases (Jimenez-Sanchez G. Science 2003. 300(5617): 295). In 2000, strategic planning for genomic medicine began, from a feasibility study and a multi-institutional consortium effort, to the creation of a National Institute of Genomic Medicine (INMEGEN) by the Mexican congress in 2004 (Séguin B, Nat Rev Genet. 2008 Oct;9 Suppl 1:S5).
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INMEGEN is designed to develop world class translational research focused on national health problems. Most Mexicans are Mestizos resulting from admixture of Amerindian, Spaniard and African populations. The admixture process has led to particular genomic ancestry structure. To optimize the use of human genome information to improve healthcare in Mexicans, we are systematically evaluating genomic variability of the Mexican population (http://diversity.inmegen.gob.mx). We are including additional Mexican Amerindians and increasing SNP density to better understand the admixture process in Mexicans, and develop more suitable tools to analyze the genetic bases of complex diseases in this population. In addition to the construction of a haplotype map of the Mexican population, INMEGEN is developing several genome-wide association studies for common diseases, such as diabetes, obesity, cardiovascular disease, and cancer, as well as other translational medicine projects that include biomarkers discovery for several kinds of cancer, pharmacogenomics, and nutrigenomics (JimenezSanchez, G. Genome Res. 2008; 18 (8): 1191-1198). The design of INMEGEN includes an intellectual property unit and a business incubator to develop goods and services to improve healthcare for the Mexican population. Thus far, this strategy has been successful, however there are challenges that still need to be addressed, including increased investment in science and technology to stimulate a more vigorous and competitive research environment, development of more effective basic and clinical scientific synergies, recruitment and training of more human resources in genomic medicine, developing mechanisms to stimulate translational research, and developing a more modern regulatory framework to ensure that genomic medicine will successfully contribute to improve healthcare in the Mexican population (Hardy BJ, Nat Rev Genet. 2008 Oct;9 Suppl 1:S23).
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Dr. Gerardo Jimenez-Sanchez was born in Mexico City in 1965. He obtained his Medical Doctor degree from the National Autonomous University of Mexico (UNAM). He did his residency in Pediatrics at the National Institute of Pediatrics and earned his Ph.D. degree in Human Genetics and Molecular Biology from the Johns Hopkins University in Baltimore, MD, USA. He received his diploma in business administration from the IPADE Business School. Dr. Jimenez-Sanchez is Director General of the National Institute of Genomic Medicine of Mexico, Professor of Genomic Medicine at the National Autonomous University of Mexico and Investigator of the Mexican Health Foundation. He is affiliate member to the Institute of McKusick-Nathans Institute of Genetic Medicine at the Johns Hopkins University. In August 2003, he was elected Founder President for the Mexican Society of Genomic Medicine and served as President of the I and II National Congresses of Genomic Medicine in 2004 and 2006. Dr. Jimenez-Sanchez is a certified pediatrician and a member of the National Academy of Medicine, the American Society of Human Genetics, the American Society of Gene Therapy, the Society for Inherited Metabolic Disease, the European Society of Inborn Errors of Metabolism and the Human Genome Organization (HUGO). He is a founder member of the National Commission for the Human Genome in Mexico and member of the Mexican Academy of Pediatrics, the Mexican Society of Pediatrics, the Mexican Association of Human Genetics, and the Mexican Society of Biochemistry. In 2007, became member of the Board of Directors, P3G (Public Population Projects in Genetics) International Consortium, Canada. In 2007, he was elected Chairman of the Working Party on Biotechnology at the Organization for Economic Co-operation and Development (OECD). He is the leading investigator in the Mexican Genomic Diversity Project and the Mexican Medical Resequencing Initiative. His current research focuses on the study of human disease causing genes, production of animal models for the study of human diseases and the development of genomic medicine in Mexico. He is Course Director in following graduate courses: “Introduction to Genomic Medicine”, “Genomic Applications to Clinical Pediatrics” and “Genomics in Internal Medicine”, first of its kind in Latin America. Dr. Jimenez-Sanchez’ work has resulted in the publication of articles and chapters in specialized journals and books. He received the Research in Pediatrics Award of the Society for Pediatric Research in 1999. Along with his colleagues David Valle and Barton Childs, he produced the first medical analysis of the human genome, published with the first draft of the human genome in Nature in 2001. He received the National Award in Clinical Investigation “Dr. Miguel Otero” from the Government of Mexico. In April of 2003, he was appointed Silanes Professor in Genomic Medicine. In 2004, he received the Golden Masters Award from the International Forum of Business Administration.
December 2008
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Masatoshi Takeda M.D., Ph.D., D.M.Sc.
Abstract
DEVELOPMENT OF GENOMIC MEDICINE IN CNS DISORDERS IN JAPAN mtakeda@psy.med.osaka-u.ac.jp
Masatoshi Takeda, Takashi Morihara, Kojin Kamino, Leonides Canuet, Masayasu Okochi, Toshihisa Tanaka, Takashi Kudo Department of Psychiatry, Osaka University Graduate School of Medicine, Japan
Friday 12 - 17:00
Alzheimer’s disease (AD) is the most common form of dementia. In developed countries the prevalence of AD is estimated at 3-10% in 65-year-olds and is expected to double every decade thereafter. After age, family history is the second greatest risk factor for AD. Genetic studies have dramatically accelerated AD research, not only for the understanding of the disease but also for the development of treatment and diagnosis.
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Genetic analysis has shown that amyloid precursor protein (APP), presenilin 1 and 2 mutations cause familial AD. Most of these mutations increase the production of Ab42 protein and display a typical AD phenotype, except at an early age of onset. These facts have greatly helped to understand AD, including sporadic cases, this being an extremely complex disease, and have been the major evidence to support the amyloid cascade hypothesis. New treatment and diagnosis methods based on this hypothesis are currently undergoing clinical trials. The majority of AD cases occur sporadically. The APOE-Îľ4 allele of the apolipoprotein (ApoE), closely connected to lipid metabolism, is the strong genetic risk factor for late onset AD. More than 500 genes have been examined as AD risk candidate genes and 10 or more AD association studies are published monthly. No gene except ApoE, however, has been established as an AD risk gene so far, because of poor replications. In this presentation, the expanding of genetic information is overviewed and the application of genetic information to treatment will be discussed.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Masatoshi Takeda has been Chairman Professor, Department of Psychiatry, Osaka University Graduate School of Medicine since April 1996. He graduated from Dartmouth College, New Hampshire (United States) in 1972, Osaka University Medical School (Japan) with an MD in 1979 and Graduate School of Osaka University with a PhD in 1983. Professor Takeda started his research career with the work on functional impairment of cytoskeletal proteins in dementia brain. He spent two years as a post doc in University of Florida (USA), and Baylor College of Medicine (USA) during 1985-1987 and then continued neurochemistry and molecular biology of cytoskeletal proteins in Department of Neuropsychiatry, Osaka University Medical School, which has been one of the leading centers of psychogeriatrics in Japan. His team is now working on the mechanism of neurodegeneration in aged and dementia brains. His department also carries out clinical research projects, searching for biological markers for degenerative dementia, functional neuroimaging of dementia, neuropsychology, psychophysiology, psychopathology, and drug development in psychogeriatrics. The Japanese now enjoy the longest life span in the world (78 years for males and 84 years for females). Japanese society has experienced the world’s fastest transition from an aging society (above 7% elderly population) into an aged society (above 14% elderly in 1995) in 24 years. One out of four Japanese will be older than 65 years in the 21st century. Professor Takeda believes this unique situation requires the Japanese Psychogeriatric Association to produce more scientific data and clinical experience to other international societies. Professor Takeda, as President-elect of IPA, is keen to stimulate research and personal exchanges between developing countries and Japan.
December 2008
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Carmen Vigo M.D., Ph.D.
Abstract
PHARMACOGENOMICS IN DRUG DISCOVERY IN USA Carmen VIgo cvigo@atlaspharmaceuticals.com
Director of Transplant Diagnostics and Applied Markets, Invitrogen Corporation
Friday 12 - 17:30
Pharmacogenetics has greatly benefited from the rapid advances associated with the Human Genome Project and its derivative the HapMap. More than 1.4 million single nucleotide polymorphisms (SNP) were identified in the initial sequencing of the human genome, with over 60,000 of them in the coding regions of genes. Some of these SNP have already been associated with substantial changes in the metabolism or effects of medications and are now being used to predict clinical response. Nowadays we can assess more than 1,000,000 polymorphisms or the expression of more than 25,000 genes in each participant of a clinical study at affordable costs.
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Serious adverse drug reactions have been shown to cause or contribute to 6 to 7 % of all hospitalizations, a 2-day increase in the average length of hospitalization, and 100,000 deaths annually in the United States alone, and may cost as much as the drug treatment itself. Adverse drug reactions are also a major problem during the development of a drug. In total, approximately 4% of all new medical agents are withdrawn from the market owing to adverse drug reactions. During the period 1995 to 2005 at least 34 drugs were withdrawn mainly as a result of hepatotoxic or cardiotoxic effects notably, cerivastatin, nefazodone, rofecoxib (vioxx), terfenadine, and troglitazone. The search for pharmacogenomic biomarkers that could be used to identify patients at an increased risk for drug-related toxic effects has often focused on variation within genes encoding drugmetabolizing enzymes, transporters, and receptors. For immune-mediated toxic effects, much focus has been placed on the MHC class I genes and on the human leukocyte antigens (HLA), which are involved in the regulation of immune response to infection and in malignant transformation. Several HLA alleles have already been unequivocally associated with toxicity in response to drug treatment. As examples of immune-mediated toxic effects is abacavir, a guanosine reverse transcriptase inhibitor and an antiretroviral treatment against human immunodeficiency virus (HIV), which is widely prescribed and has an 8% association with serious toxicity. The FDA has approved to screen all the HIV patients receiving this drug. Elimination of patients with HLA-B*5701 positive has eradicated toxicity in 100% of the White population, however it doesn’t reduce toxicity in the Black population and this allele is rare in Asians. Despite its usefulness, the drug industry is very hesitant to release drugs requiring pharmacogenetic testing because the basis for prescription is more complex than for drugs not requiring testing. However HLA-B*5701 genotyping appears to be an effective pharmacogenomic test in White populations allowing clinicians to avert a specific toxic effect of a drug. Transport proteins play an important role in regulating the absorption, distribution, and excretion of many medications. Members of the ATP–binding cassette family of membrane transporters are among the most extensively studied transporters involved in drug disposition and effects. An example is a member of the ATP-binding cassette family, P-glycoprotein, encoded by the human ABCB1 gene (also called MDR1). A principal function of P-glycoprotein is the energy-dependent cellular efflux of substrates, including bilirubin, several anticancer drugs, cardiac glycosides, immunosuppressive agents, glucocorticoids, HIV type 1 protease inhibitors, and many other medications. The expression of P-glycoprotein in many normal tissues suggests that it has a role in the excretion of xenobiotics and metabolites into urine, bile, and the intestinal lumen. At the blood–brain barrier, P-glycoprotein in the choroid plexus limits the accumulation of many drugs in the brain including digoxin, ivermectin, vinblastine, dexamethasone, cyclosporine, domperidone, and loperamide. Genetic variation in drug targets (e.g., receptors) can have a profound effect on drug efficacy with over 25 examples already identified, including angiotensin-converting enzyme (ACE), which affects the renoprotective actions of ACE inhibitors; Arachidonate 5-lipoxygenase (ALOX5), affecting the response to ALOX5 inhibitors; and sequence variants with a direct effect on response occur in the
December 2008 - international edition
Speakers
In Order of Presentation
genes for the 2-adrenoreceptor, affecting the response to 2-agonists. Others have been identified in Dopamine receptors, affecting antipsychotic drugs, estrogen receptors affecting hormone replacing therapy, glycoprotein subunits affecting platelet aggregation, and serotonin transporter affecting the action of antidepressants. Pharmacogenetics has changed the practices and requirements in preclinical and clinical drug research; large clinical trials with pharmacogenomic add-ons appear to have become more common in pharmaceutical companies and academic institutions. Over 150 clinical trials are being carried out in the USA with pharmacogenomics as one of the components of the study. In summary, the convergence of pharmacogenomics and human genomics means that physicians can now individualize therapy in the case of a few drugs. As we expand our knowledge the selection of the best drug for each patient should also increase. However, there are a number of critical issues that must be considered as strategies are developed to elucidate the inherited determinants of drug effects. A formidable one is that the inherited component of the response to drugs is often polygenic. Approaches for elucidating polygenic determinants of drug response include the use of anonymous SNPs maps to perform genome-wide searches for polymorphisms associated with drug effects, and candidate-gene strategies based on existing knowledge of a medication’s mechanisms of action and pathways of metabolism and disposition. Both these strategies have potential values and limitations. However, the candidate-gene strategy has the advantage of focusing resources on a manageable number of genes and polymorphisms that are likely to be important, and it has produced encouraging results in a number of studies. The limitations of this approach are the incompleteness of knowledge of a medication’s pharmacokinetics and mechanisms of action. Gene-expression profiling and proteomic studies are evolving strategies for identifying genes that may influence drug response. One of the most important challenges in defining pharmacogenetic traits is the need for well-characterized patients who have been uniformly treated and systematically evaluated to make it possible to quantitate drug response objectively. To this end, the norm should be to obtain genomic DNA from all patients enrolled in clinical drug trials, along with appropriate consent to permit pharmacogenetic studies. Because of marked population heterogeneity, a specific genotype may be important in determining the effects of a medication for one population or disease but not for another; therefore, pharmacogenomic relations must be validated for each therapeutic indication and in different racial and ethnic groups. Remaining cognizant of these caveats will help ensure accurate elucidation of genetic determinants of drug response and facilitate the translation of pharmacogenomics into widespread clinical practice.
Curriculum Vitae
Dr. Vigo is a Ph.D. recipient in Biochemistry from The Royal College of Surgeons of England, University of London. She is a business entrepreneur and experienced scientist with over 20-years background in the biotech industry in California. She currently directed the area of transplant diagnostics at Invitrogen, and was the former Chief Scientific Officer, co-founder, and board of directors-member of Atlas Pharmaceuticals developing drugs to treat liver regeneration. Previously, she was the Research Director at Vitagen, a cell-therapy company dedicated to treat fulminant hepatic failure using bioartificial liver devices and studying the chemical and genetic mechanisms that induce hepatic cell lines to express specific liver enzymes via transcription factor regulation and enzyme cloning and transfection. Earlier, Dr. Vigo worked at two CNS companies, Neurocal, Inc., and Athena Neurosciences, now Élan Pharmaceuticals. At Athena Neurosciences she was the first scientist to isolate and characterize beta amyloid peptide in the CSF, showed its release by cells in culture and five-fold over production by fibroblasts from patients with Swedish familial AD mutation. Her contributions to AD were internationally acclaimed and were awarded the Norage-Pharmacia Award for her achievements. Dr. Vigo later founded Neurocal where she pioneered the development of antioxidant and antiapoptotic drugs to treat AD and stroke. Her career spanned the whole range of biopharmaceutical product development from target discovery, process science, to clinical trials. She has extensive experience in large pharmas, Syntex, and small to medium biotech companies. She has been an invited speaker in numerous scientific meetings, has published over 75 peer-reviewed research papers, four books, and is the inventor of 14 patents. December 2008
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Marvin Edeas President of the French Society for Antioxidants
Abstract
NUTRIGENOMICS IN EUROPE Marvin Edeas edeas@orange.fr
Dr Marvin Edeas, Professor of preventive medicine, President of the International Society of Antioxidants in Nutrition & Health (ISANH) The future impact of nutrigenomics in Europe is still poorly understood. Our current challenge is to bring together scientific, industrial and political visions of Nutrigenomics to discuss the ways to reach the ultimate aim which is consumer health improvement.
Friday 12 - 18:15
Nutrigenomics will not only promote our nutrition but will help to prevent diabetes, obesity, neurodegenerative diseases and especially aging.
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However, many strategic questions must be discussed and taken into consideration during this conference: • How can we integrate and interpret Nutrigenomic analysis? • Are the different levels of molecular analysis a limit to Nutrigenomic approach? • How can we combine data from transcriptomics, proteomics and metabolomics to describe the biological effect of nutrients? • How can we bypass the difficulties of data interpretation? • What are the implications of innovations for development and manufacturing? • How can we integrate new technologies in Nutrigenomics? • Nutrigenomic technologies: limitation in personal development food? • How could this emerging concept impact industry? • What is the cost of Nutrigenomic strategy? During my presentation I will discuss personalized nutrition, how it can prevent diseases and provide wellbeing.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Professor Edeas is the Chairman and organizer of the Paris Nutrigenomics conference 2008 and the first conference on superoxide dismutases at Pasteur Institute. Professor Edeas has edited the following books: Latest advances on Anti-ageing and neurodegenerative disease Latest advances on Skin Anti-ageing and antioxidants Latest advances on slimming and obesity Latest advances on antioxidant applications on health Latest advances on SOD and clinical applications Prof. Edeas is president of the Task Force Group on Antioxidants Prof. EDEAS founded the International Society of Antioxidants in Nutrition & Health (ISANH) in 1998 Member of 7 National and International Scientific Societies and Associations Lectures, Seminars, Conferences and Courses: 75 Patents: 3 Professor Edeas’ clinical interests focus on the impact of oxidative stress and antioxidants on chronic diseases such as obesity, cancer, viral infection and diabetes. He has studied how free radicals and ROS can activate the HIV and how supplementation by Antioxidants can decrease the oxidative stress observed in HIV infected patients and increase CD4. He has shown that Antioxidants can play a role not only due to their antioxidant activities, but can also affect the redox status of the cells, and this can affect gene expression and regulation. He has demonstrated why many antioxidants failed in many clinical studies. He worked on glycation of antioxidants such as SOD and its inactivation in diabetic patients. The mitochondrial paradox is one of his current areas of interest.
December 2008
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Speakers In Order of Presentation Speakers
Prof.
Francesco Marotta Abstract
HUMAN NUTRITION IN GENOMIC MEDICINE: FUTURE AGE-MANAGEMENT STRATEGIES Prof. F. Marotta, MD, PhD WHO-cnt for Biotech. & Natural Med., University of Milano and 2 G.A.I.A. Age-management & Research Foundation.
1,2
Friday 12 - 18:45
fmarchimede@libero.it
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“Functional Foods” represent an emerging opportunity and they will certainly play a consistent and important role in future. Such a novel nutraceutical perspective entirely depends on the development of new innovating solutions aiming at acting on organic systems. A biochemistry- and molecular biology-specific development together with biotechnological methods support the hypothesis that some nutrients can modulate body functions. This would play a definite role in health conditions as well as in the reduction of disease risk and synergistic-integrated approach in established therapeutic regimens and, ideally, slowing the aging process itself. Indeed, efforts at unfolding the causes of aging are limited by the complexity of the problem. Aging changes take places from the molecular to the organismic level, moreover environmental factors affect experimental observations, secondary effects complicate the understanding of primary mechanisms and precisely defined, easily measurable “biomarkers” are lacking. One difficulty has to do with the overlap between aging and disease. Both the aging process and diseases can cause changes in the body, which affect life span. The idea of biomarkers is to measure the aging process, but it has been cumbersome to separate this out from the effects of a disease if one is present, in other words, if a marker reflects aging or a pre-morbid condition more common in the aged. As a matter of fact, no one unifying theory exists, since the mechanisms of aging could be quite distinct in different organisms, tissues, and cells. A fundamental question remains whether aging is a single process with several different results, or are separate processes going on in different body systems, such as the immune system, the bones, the muscles. Moreover, the post-genomic research line represents an ideal match with Functional Foods, thus leading to an integrated nutrigenomic strategy. Nutrigenomics mainly aims at studying genetic and epigenetic interactions with a nutrient as to lead to a phenotype change and therefore to the cell metabolism, differentiation or apoptosis. As for new generation studies, however, it is too early yet and still many are the interactions to be assessed between nutrients and host and between nutrients themselves, and possibly many mechanisms will play an important role all together. Nonetheless, such new scenario requiring a multi-speciality team approach has to be strongly promoted with the aim to test how and whether a behavior (such as exercise), drug, or dietary additive slow down the aging process which is the ultimate goal of interventions.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
1981 Graduation from Medical School, Catania University, Italy with highest marks cum Laude. 1981 - 1982 University of Chicago, Visiting Fellow in Gastroenterology 1983-1986 University of Cape-Town, Groote Schuur Hospital, Clinical and Research Registrar in Gastroenterology 1986 Completing the Post-Graduate School in Gastroenterology, University of Catania, Italy with highest marks cum Laude. 1986-1988 University of Hirosaki, Japan, Dept of Surgery. Ph.D. course granted by the Japanese Ministry of Education and following a Japanese Language Proficiency course at Tohoku University-Sendai, Japan. At the end of the experimental studies (Superoxide anion generation and role of macrophage oxidative burst in several models of acute pancreatitis) granted a PhD as the only one obtained in the medical field in Japan by an italian after the post-war period. 1988 - 1990 National Cancer Center Hospital, Tokyo, Japan. Fellowship granted by the Japanese Ministry of Science & Technology. Studies on pancreatic cancer. 1991-1998 Chief Consultant in Gastroenterology, S. Anna Hospital, Como, Italy 1998 - Consultant Hepato-GI Unit, S. Giuseppe Hosp., Milano, Italy 2007 - Full-immersion stage at Goodgene genomic lab., Seoul, S. Korea
Appointments: Consulting Professor, WHO-affiliated Center for Biotechnology, Traditional Medicine Post-Graduate School, Dept. of Anatomy, University of Milano, Italy. Visiting Professor at major Japanese Institutions Editor-in-Chief of International Journal of Probiotics & Prebiotics Consultant-in-charge for Lombardia region of Chronic Pancreatitis Project Group. Referee and Editorial Board Member of a number of European, Asian and Middle-East journals, several of them of outstanding value. Advisor for Eastern Countries of JOP, the first electronic peer-reviewed journal of Pancreatology (www.joplink.net). Organizing President of the 25th Annual meeting of the Italian Association of Pancreatology 2001. Consultant in charge of the Italian counterpart of the Italy-Japan Exchange Research Project in Pancreatology (in progress) Member of several foundation and research institutes of aging Member of the European Federation of Biotechnology and of the International Society Study of Xenobiotics. Member of the Genetic Association Database of National Institute of Aging, USA Scientific Director of Italian Association Age-Management Pharmacists
Publications: 96 full papers (most of which as leading author), over 370 scientific communications and lectures and a few book sections dealing with experimental and clinical gastroenterology, nutraceutics, oxidative stress and aging. He is co-inventor of some patents in nutraceuticals in USA. He is the co-editor of Sperling-Kupfer book “Il Manifesto della Lunga Vita (The Manifesto of Long Life), a successful mediahit 500-page and over 50-world authorities endorsed book on aging intervention.
December 2008
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Clinical Genetics and Genomics of Complex CNS Disorders Health Biotechnology Human Identification Genetics
Pharmacogenetics Food Intolerance Nutraceuticals Drug Development
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www.ebiotecmexico.com
Speakers In Order of Presentation Speakers
Allen D. Roses M.D., FRCP
Abstract
THE FUTURE OF PHARMACOGENOMICS IN MEDICAL PRACTICE: A NEW MODEL OF PIPELINE PHARMACOGENETICS Allen D. Roses, MD mindy.mcpeak@duke.edu
Saturday 13 - 09:00
Duke University, Jefferson-Pilot Professor of Neurobiology and Genetics, Professor of Medicine (Neurology), Director, Deane Drug Discovery Institute, Senior Scholar, Fuqua School of Business
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President & CEO, Cabernet Pharmaceuticals, Inc. In order for pharmacogenetics to be integrated into medical practice, the pharmaceutical business model must evolve. Rigorous science must be applied during drug development to collect meaningful data that can be used to demonstrate positive and negative predictive value for important unmet medical needs. This is, however, an economic component that must also be considered for achieving success in the market-place. Industry frequently decries the lack of incentives, but fails to evolve away from historic comfortable structures. The current rage in the pharmaceutical industry is to decrease R&D, specifically the Discovery component and move towards a functional Development company by purchasing drug candidates from smaller companies or academic institutions. This can frequently be successful, but is only an opportunistic structure with many protagonist companies competing for few assets. I will discuss a new model for academic-industry synergies which provides for commercialization-sharing opportunities for academic institutions, an integration of PGX technologies and project planning to produce pipeline data, and can be managed as a series of small virtual companies with laser-like functional organization across the pharmaceutical pipeline. Three Phase III clinical trials for rosiglitazone for the treatment of AD will complete over the next six months, testing a pharmacogenetic hypothesis. These studies built on previous work of a 24 week clinical trial, AD patients who did not carry an APOE4 allele demonstrated significance clinical improvement compared to placebo patients. Following a VXDS discussion with the FDA, the 48 week Phase III Program was initiated in 2005. One set of facts are apparent and well known: it takes over a decade to complete the development of a drug. There are also two apparent outcomes for the Phase III program, there will be a new drug for Alzheimer’s disease - or not. Should the clinical results be positive, planning for a prevention trial using a PPARγ agonist, similar to rosiglitazone, would represent the next logical extension of medical practice significance. This is precisely the strategy of the Deane Drug Discovery Institute at Duke University. By creating a holding company for intellectual property related to newly discovered genetic variants that are predictive of age of onset, [called Shiraz Pharmaceuticals, Inc.], it becomes practical to predict those normal individuals between 65-85 years of age who are most likely to develop AD over the next five years.This provides an opportunity to increase the medical significance with a therapeutic strategy to test a preventative therapy and simultaneously, in the placebo population, validate the diagnostic variants. The outcome is that, rather than commercialize the diagnostic as a predictor of disease, there is the possibility of using that diagnostic to advise age of treatment of individuals who are at greatest risk of AD. When should normal individuals initiate a pharmaceutical treatment is a more powerful medical rationale for the exclusive commercial application of this intellectual property. There is a real difference in strategies.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Allen D. Roses, MD, FRCP (Hon) was appointed as Senior VP, Pharmacogenetics for GlaxoSmithKline, July 2006. Previously, he held the position of Senior VP, Genetics Research for GlaxoSmithKline. In 1997, Dr. Roses joined Glaxo Wellcome in 1997 and was charged with organizing genetic strategies for susceptibility gene discovery, pharmacogenetics strategy and implementation, and integration of genetics into medicine discovery and development. In the GSK R&D structure, genetics, genomics, proteomics and bioinformatics were part of Genetics Research and supported the entire R&D pipeline. His group recently performed the proof of principle experiments for using linkage disequilibrium mapping to identify susceptibility loci for drug adverse events. With respect to hypersensitivity to the drug, abacavir, GSK identified the HLA_B5701 locus in 2002 with candidate gene analyses, and then prospectively established the sensitivity (97%) and specificity (>99%) – leading to a re-labelling of the drug expected in late 2008. In 1997, when Dr. Roses left Duke University Medical Center, he was the Jefferson Pilot Professor of Neurobiology and Neurology, Founding Director of the Joseph and Kathleen Bryan Alzheimer’s Disease Research Center, Chief of the Division of Neurology, and Director of the Center for Human Genetics. Dr. Roses was one of the first clinical neurologists to apply molecular genetic strategies to neurological diseases. His laboratory at Duke reported the chromosomal location for more than 15 diseases, including several muscular dystrophies and Lou Gehrig’s disease. He led the team that identified APOE as a major, widely-confirmed susceptibility gene in common late-onset Alzheimer’s disease in 1992. Translation of these findings to metabolic pathway analyses, drug discovery and development continued in GSK with three Phase III trials of rosiglitazone for the treatment of AD. In October 2007, Dr. Roses retired from GSK to return to Duke University where he will direct the Deane Drug Discovery Institute. He has also started a new company, Cabernet Pharmaceuticals Inc, which will initially operate as a pharmacogenetics consulting company, with growth expected with the initiation of PGX project design and management for the Pharmaceutical, biotech, HMO and CRO industries. Dr. Roses is a member of the FDA Science Board since 2003, where his expertise is in clinical neuroscience, pharmacogenetics, and exploratory drug discovery.
December 2008
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Speakers In Order of Presentation Speakers
Munir Pirmohamed M.D., Ph.D., D.M.Sc.
Abstract
PHARMACOGENETICS AND METABOLIC DISEASE Munir Pirmohamed munirp@liv.ac.uk
Saturday 13 - 09:45
Department of Pharmacology, The University of Liverpool, Ashton Street, Liverpool, L69 3GE
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Metabolic disease is a generic term which encompasses a number of different pathological entities, which individually and collectively, represent major public health issues in the developed, and now also, in the developing countries. There are effective drugs for the treatment of most of the metabolic diseases; however, all of these drugs are associated with variable efficacy, and in some cases toxicity. It is also important to note that metabolic disease can sometimes be induced by drugs, i.e. it is an adverse drug reaction, and this may also have a genetic basis. The presentation will focus on some key areas where appropriate drug use can have major beneficial effects (diabetes and hyperlipidemia), and one scenario, the use of highly active antiretroviral therapy (HAART) in HIV disease, where drug use leads to different metabolic disorders. Type II diabetes is an important public health problem. A number of drugs are available for treatment including metformin, sulphonylureas and glitazones. Metformin is the drug of first choice, and there are new compounds coming through (e.g. glipitins) where very little work has been done in relation to pharmacogenetics. For the established compounds, there is now extensive evidence that disposition of the compounds can be affected by variation in drug metabolising enzyme and transporter genes. However, how this translates in terms of dosing has not been evaluated clinically. The most interesting and clinically relevant findings have been in (a) neonatal diabetes in children with heterozygous activating mutations in KCNJ11, encoding the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, who can be switched from insulin to sulphonylureas; and (b) maturity onset diabetes of the young with hepatic nuclear factor-1alpha (HNF1alpha) mutations, who can be treated with low doses of sulphonylureas. Various adverse effects can be caused by anti-diabetic agents, but little work has been done to identify predictors of toxicity. With the recent issues with rosiglitazone and thrombotic cardiac adverse events, this may be a fruitful area for further research. Statins are widely used for the treatment of patients with hyperlipidemia, and will in the future, be increasingly used for primary prevention. They are highly effective both for lowering lipids and for secondary prevention of vascular disease. Nevertheless, there is variability in response, and this has stimulated research on identifying genetic predictors of response. This has focused on pharmacokinetic and pharmacodynamic targets, but most studies have either been negative or produced contradictory findings. The target for statin action, the HMG-CoA reductase gene, has been evaluated, and the most significant association relates to a splice variant. Further studies utilising whole genome association approaches are on-going. Statins are associated with myopathy and occasionally rhabdomyolysis; recent data have suggested that variation in SLCO1B1, the transporter responsible for statin influx, may be responsible for at least 60% of cases of simvastatin-associated myopathy. HAART has been hugely successful in converting HIV from a rapidly lethal disease to one which had a chronic indolent course. However, this has been at the expense of some common and life-threatening adverse reactions. The most prominent amongst these has been lipodystrophy syndrome, which is characterised by fat re-distribution, hyperlipidemia, insulin resistance and occasionally diabetes mellitus. These patients also have a higher risk of cardiovascular morbidity and mortality. Protease inhibitors, which are a component of HAART, have been particularly associated with lipodystrophy, although other agents such as efavirenz can also cause isolated hyperlipidemia. Genetic factors, for example in the TNF-alpha locus, have been shown to predispose to HAART-associated lipodystrophy, while a combination of genes predisposes to hyperlipidemia. These areas will be covered in the presentation.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Munir Pirmohamed qualified in Medicine in 1985, undertook a PhD in Pharmacology in 1993, and was appointed Consultant Physician at the Royal Liverpool University Hospital in 1996. He was awarded a Personal Chair in Clinical Pharmacology at The University of Liverpool in 2001, and in 2007, was appointed to the NHS Chair of Pharmacogenetics. He is also Deputy Director of the MRC Centre for Drug Safety Sciences in Liverpool. Professor Pirmohamed is a Member of the Commission on Human Medicines and Chair of its Pharmacovigilance Expert Advisory Group. His main area of research is in pharmacogenetics and drug safety. Adverse reactions to drugs are a major cause of illness in the population. The research aims to maximise the benefits of drugs and minimize their harms. This is being achieved through the use of different strategies ranging from improvements in prescribing to the development of genetic and other tests for predicting and monitoring individual susceptibility to toxicity.
December 2008
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Speakers In Order of Presentation Speakers
Prof.
Ian P. Hall Abstract
PHARMACOGENETICS AND PHARMACOGENOMICS OF RESPIRATORY DISEASE Ian P Hall
ian.hall@nottingham.ac.uk
Division of Therapeutics and Molecular Medicine, University Hospital of Nottingham, Nottingham NG7 2UH, UK.
Saturday 13 - 10:15
Genetic approaches can potentially be of value in respiratory medicine by either defining novel markers for predicting efficacy or adverse drug responses, or by identifying novel potential targets for drug development.
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Pharmacogenetic approaches to date have identified a small number of specific genetic markers which predict side effect profiles of drugs used in the treatment of respiratory diseases with a reasonable degree of accuracy. Two examples are the use of TPMT phenotyping/genotyping to predict myelosuppression following use of azathioprine in interstitial lung disease; and alpha 1 antitrypsin genotyping to define subsets of patients with early onset COPD requiring altered clinical management. However, in general, to date, the predictive value of genetic markers for assessing efficacy of commonly used drugs in conditions such as asthma has been less good. There has for example been extensive work on ADRB2 polymorphisms and response to β2 agonists, but whilst there are consistent effects in clinical studies using short acting agents (SABAs) large scale retrospective studies have failed to identify clinically important effects in clinical trials with long acting agents such as salmeterol and formoterol used as recommended by national guidelines. This subject however remains important with the development of novel longer acting agents with full agonist properties. Genomic approaches have resulted in the identification of a number of genes which contribute to disease risk in respiratory diseases. The best examples are for rarer diseases such as cystic fibrosis and primary pulmonary hypertension, where single gene defects explain all or much of the disease phenotype. In common diseases such as asthma it is becoming clearer that multiple genes of relatively small effect contribute to disease risk. Interestingly, the genes which have been identified in asthma including ADAM33, PHF11, DPP10 and GPRA are not in gene families which would intuitively have been thought to be important in disease pathogenesis. Identification of these genes, and clarification of the functional role played by variants in these genes, therefore has the potential to (i) further our understanding of disease pathogenesis and (ii) may lead to novel therapeutic targets in the disease.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Current Positions 1. Head, Division of Therapeutics and Professor of Molecular Medicine. 2. Director of Clinical Research for the Faculty of Medicine and Health Sciences, University of Nottingham.
University Education 1976 - 1979 Lincoln College, Oxford University, BA Physiological Sciences (upper 2nd) 1979 - 1982 Oxford University Medical School, BM, BCh, Oxford
Postgraduate Qualifications May 1996 FRCP (London), October 1985 MRCP (London), March 1991 DM (Nottingham)
Prizes and Awards July 1993-1998 National Asthma Campaign Senior Research Fellowship July 1991-1992 Medical Research Council Travelling Fellowship September 1990-91 Royal Society of Medicine Derrick Dunlop Travelling Fellowship.
Other Positions MRC College of Experts 2005MRC PSCSB and Experimental Medicine review panels 2006-9 Wellcome Trust expert review group 2005RAE 2008 subpanel 4 member
Grants Held I hold the grants listed below which are currently active and in addition was a principal investigator on both the Nottingham MRC Co-operative (1998-2003) Cell Signalling in Infection and Inflammation (£350,000) and Wellcome JIF award Fluorescent correlation spectroscopy (PIs, Institutes of Cell Signalling, Infections and Immunity and Pharmaceutical Science) £900,000, 1999 - 2004.
Current 1. Novartis funded collaboration: functional relevance of beta2 receptor polymorphisms (2003-2008) (£175,000) 2. Department of Health - collaborative grant with Professors RW Dingwall, RJ Murphy, DK Whynes, J Kai, K Cox and G Currie (University of Nottingham): External evaluation of NHS Service Development Initiatives in Genetics (2004-2008) (£752,477) 3. MRC project grant. Defining the mechanisms underlying physiological control of cyclic AMP by phosphodeisterases in human airway smooth muscle. (2005-2008) (£206,000). 4. Asthma UK: AUGOSA (genome wide association study in severe asthma) £170,000 5. MRC Capacity Building studentship: Molecular genetics of histamine H4 receptor expression in human airways. (2007-2010) (£58,000) 6. Research collaboration with Astra Zeneca: biomarkers in interstitial lung disease (2007-8) (£30,000) 7. Research collaboration with GSK (coapplicants Prof R Spiller, Prof Y Mahida) Cytokine polymorphisms and the Genetics of irritable bowel syndrome, £250,000 8. MRC grant: Calcium homeostasis in airway smooth muscle (2008-11) (£450,000) 9. EPSRC: hyperpolarized Xe lung imaging (coapplicants Morris, Hall, Barlow, Wolber) (2008-2011) (£1,200,000)
December 2008
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Speakers In Order of Presentation Speakers
Valter Ruggero Maria Lombardi Terlizzi M.D., Ph.D.
Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS OF IMMUNOLOGICAL DISORDERS AND VACCINES Dr. Valter Ruggero M. Lombardi biotecnologia@ebiotec.com
Saturday 13 - 11:00
Molecular Immunology Department, Euroespes Biotechnology, Bergondo Industrial Estate, La Coruña, Spain.
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The immune system is composed of a network of cells producing various humoral factors that function together to maintain a state of homeostasis. In the normal host, presentation of antigen for a specific protective immune response elicits a complex series of events that result in a mixed cellular and humoral protective response. Extracellular parasites (i.e., bacteria) incite primarily a humoral response, while intracellular parasites (i.e., virus, fungi, mycobacteria, etc.) elicit a cell-mediated response. The host immune system has a variety of mechanisms to direct the immune response into the humoral versus cellular direction-nature of antigen presenting cells (APC), major histocompatibility complex (MHC) restriction, and availability of specific T- and B- cell components. However, the central control of the cellular versus humoral response to an antigenic challenge appears to be via production of specific cytokine milieu. A central source if these cytokines comes from CD4+ helper T-cell subpopulations, often referred as Th1 and Th2 cells. Human Th1 cells secrete a specific cytokine profile including interferon-gamma (IFN-γ) and tumor necrosis factor-beta (TNF-β). These cytokines are important helper factors in the generation of cellular immune responses, including antigen-specific cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Additionally, IFN-γ in particular has an antagonistic activity against Th2 cytokines.. Interleukin-12 (IL-12), produce primarily by activated macrophages, plays a central role in inducing IFN-γ production. In contrast, Th2 cells secrete IL-4, IL-5, IL-9, IL-10, and IL-13 which are involved in isotype switching of B-cells as well as proliferation and differentiation into antibody-secreting plasma cells. In particular, IL-4 and IL-13 are involved in the isotype switch from IgM to IgE, the antibody responsible for classical allergic disease. IL-4 and IL-10 are also regulatory cytokines, antagonizing the activity of Th1 cytokines. In humans, helper T-cells, can have a third cytokine secretory pattern. Th0 cells are capable of secreting all of the above-mentioned cytokines and are often thought of as precursors. Whether this represents a differentiation pathway for Th cells remains to be determined. Thus, it can be said that the nature, the intensity, and duration of a specific immune response depends upon the delicate balance between Th1 and Th2 numbers and/or activities. Since it is now appreciated that many other cell types besides the Th cells produce IFN-γ, IL-4, IL-5, IL-9, and IL-10, many scientists now refer to these as type 1 and type 2 cytokines, supporting cellular and humoral immune responses, respectively. More recently, specific regulatory pathways have been described to control the overall immune balance and thus effector responses. The best described involve regulatory T-cells that are variably characterized by phenotype as well as cytokine production. T-cells that are CD4+/CD25+ and produce varying amounts of transforming growth factor-β (TGF-β) and/or IL-10 are able to regulate Th1/Th2 activity as well as effector cell function including T-cell proliferation, cytotoxic T-cell killing, and helper T-cell dependent antibody production. Indeed, Treg deficiency (either in number or function) has been implicated in a variety of immunopathological states including multiple sclerosis, systemic lupus erythematosus, and various allergic states. Advances in molecular genetics have provided tools for unravelling some of the mysteries regarding inheritance of risk factors for immune diseases. Two different strategies have been used to identify susceptibility genes for immunological disorders, including positional cloning and the candidate gene approach. Once narrowed, genetic linkage studies and genetic association studies have detected genetic variations utilizing variable number of tandem repeats (VNTRs) and single-nucleotide polymorphism (SNPs) in high probability loci that promote atopy, asthma, mulptiple sclerosis and systemic lupus erythematosus. More than 1.4 million single-nucleotide polymorphisms have been identified in the initial sequencing of the human genome, with over 60,000 of them in the coding region of genes. Some of these singlenucleotide polymorphisms have already been associated with substantial changes in the metabolism or effects of specific currently used drugs, and some are now being used to predict clinical response. Because most drug effects are determined by the interplay of several gene products that influence the pharmacokinetics and pharmacodynamics of pharmacological compounds, including inherited diffe-
December 2008 - international edition
Speakers
In Order of Presentation
rences in drug targets (e.g., receptors) and drug disposition (e.g., metabolizing enzymes and transporters), polygenic determinants of drug effects have become increasingly important in pharmacogenomics. Loci on chromosomes 5, 6, 8, 11, 12, 13, 16, and 20 have so fare been identified to potentially have significant effects on immune associated disorders development. Genetic variants in Th2 immune signaling pathways such as IL-13 (chromosome 5), IL-4Rα (chromosome 16), and STAT-6 (chromosome 12), as well as variants of a metalloprotease and disintegrin ADAM33 (chromosome 20), which could be involved in airway remodelling after allergic inflammation, have been found. Also, loci controlling nitric oxide (NO) synthesis and metabolism as well as TNF and other pro-inflammatory cytokines and chemokines have been looked at as possible candidates influencing the associated risk to the development of different immune disorders. Preventive strategies during the past decade have offered new approaches to neuroprotective therapy, and in the case of Alzheimer’s disease (AD) both active and passive immunizations have been developed. Although cognitive functions were less declined in patients with high antibody titers against beta amyloid, the first clinical trial using the active vaccine AN-1792 was unsuccessful due to the occurrence of meningoencephalitis in some of the vaccinated patients. More recently, both passive and active vaccines to beta-amyloid have been successfully tested in transgenic mice and seem to be a real alternative to the AN-1792 vaccine. A careful and accurate interpretation of the involvement of the immune system during the course of AD, taking into account the type of immunization (passive, active, or a combination of both), choice of adjuvant, immunization schedule, can be expected to lead to more effective results in the interest of new treatments. In order to establish valuable correlations, we suggest that the rule should be to obtain, store and analyze genomic DNA from all patients enrolled in clinical drug trials, along with appropriate consent to perform gene-expression profiling and proteomic studies for identifying genes that may play an important role in drug response in diseases affecting the immune system.
Curriculum Vitae
Obtained his degree in Biology in 1985 and his Ph.D. in Medical Science in 1988 at the University of La Sapienza in Rome. From 1985 to 1987 he was a research fellow at the Pediatrics Department, Faculty of Medicine, University of La Sapienza. Rome. From 1987 to 1991 he was a research fellow at the department of Immunology, Faculty of Mmedicine, at Karolinska Institute in Stockholm and he worked in the field of HIV research. During this time he has been involved in many projects related to the mechanisms of HIV infection, mother-to-child HIV transmission, epidemiology of HIV in African countries, molecular aspects of HIV-receptor interactions, molecular mechanisms of virus entry and replication, and in vitro inhibition of HIV replication by pharmacological compounds. In 1991 he became a researcher at the Department of Pediatrics, Faculty of Medicine, University of Tor Vergata, Rome. In 1992 he obtained a Master degree in Immunology at the Department of Immunology, Karolinska Institute, Stockholm: “Transmission mechanisms of HIV-1 virus during pregnancy”. Since 1996, he is the Director of Biotechnology Division at Euroespes Biotechnology, La Coruña, Spain. His main research fields concern, assay development and high throughput screening methods for drug discovery, assay development for primary cell culture screening, assay development for new immunological tests, assay development for human neurodegenerative diseases, assay development for isolation and identification of new natural compounds. He is author of more than 60 scientific publications in international journals.
Publications (selected): Lombardi, V.R.M., Fernández-Novoa L., Etcheverría, I., Seoane, S., Cacabelos, R. Title: Studies on the immunological, biochemical, haematological and growth regulation by Scomber scombrus fish protein extract supplementation in young pigs. Animal Science Journal, 2005; 76:159-170. Lombardi, V.R.M., Fernández-Novoa L., Etcheverría, I., Seoane, S., Cacabelos, R. Effects of fish derived lipoprotein extracts on activation markers, Fas expression and apoptosis in peripheral blood lymphocytes. International Immunopharmacology, 2005; 5:253-262. Lombardi, V.R.M., Etcheverría, I., Fernández-Novoa L., Díaz J., Seoane, S., Cacabelos, R. In vitro response of rat microglia and human polymorphonuclear cells (PMN) to immunoactive compounds. Oriental Pharmacy Exp Medicine, 2005; 5:216-230. Lombardi, V.R.M., Pereira J., Etcheverría, I., Fernández-Novoa L., Seoane, S., Cacabelos, R. Improvement of immune function by means of Conger Conger extract in an in vivo rat model of cold stress. Food and Agricult Immunology, 2006; 17:1-13. December 2008
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Speakers In Order of Presentation Speakers
Amalio Telenti M.D., Ph.D.
Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS OF INFECTIOUS DISEASES Amalio Telenti amalio.telenti@chuv.ch
Institute of Microbiology, University of Lausanne
Saturday 13 - 11:30
Pathogens have been a constant force in the evolution of humans. Selective pressures have modeled the human genome contributing to genetic variation, which in turn, may contribute to modern diversity on susceptibility to infection.
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Humans display great differences in susceptibility to pathogens such as tuberculosis, malaria and HIV (e.g. some individuals are not infected despite repeated exposure), and in disease progression (e.g. some individuals are infected but will never develop disease). We have participated in the first two genome-wide analysis of genomic determinants of susceptibility to AIDS, and are we are currently studying the expression pattern in CD4 T cells from infected individuals for whom genome-wide genotype exists. This information is now placed in the context of signals of evolutionary pressure in the primate and human genomes. The final goal of this work is in the understanding of pathogenesis. The laboratory also explores the field of pharmacogenomics at three levels: marker discovery (development of ADME pathway SNP arrays), marker evaluation (in the context of population pharmacokinetic/pharmacogenetic models and drug metabolism), and translational studies (predictive value for the clinics). The final goal of this work is personalization of care.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
A. Telenti, MD (Infectious Diseases) and PhD (Microbiology) is professor of medical microbiology and director of the Institute of Microbiology at the University Hospital Center of the University of Lausanne, Switzerland. A graduate in medicine from the University of Oviedo, Spain, he trained in internal medicine and infectious diseases at the Mayo Clinic, Rochester, Minnesota; and in microbiology research at the university of Berne, Switzerland, and at the Albert Einstein College of Medicine, New York. From 1994 to 1997, Dr. Telenti was on staff at the Institute of Microbiology at the University of Bern, Switzerland. From 1997 until 2003, he was director of the HIV Unit and Laboratory at the University Hospital in Lausanne, Switzerland. Dr. Telenti has worked on the molecular basis of mycobacterial resistance to anti-tuberculous drugs, and in the field of antiretroviral resistance and viral fitness. Dr. Telenti’s primary scientific interest is in the field of host genetics and pharmacogenetics in HIV disease. These projects are supported by the Swiss National Science Foundation. He is a member of the Swiss HIV Cohort Study, an associate editor of Antiviral Therapy; he is editor of www.HIV-pharmacogenomics.org www.hiv-pharmacogenomics.org, and a member of the scientific executive board of SystemsX.ch, the Swiss national program for Systems Biology.
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Speakers In Order of Presentation Speakers
Alain Vanvossel European Commission, Research Directorate General
Abstract
GENOMIC MEDICINE AND PHARMACOGENOMICS IN THE EU RESEARCH PROGRAMMES Alain Vanvossel Alain.Van-Vossel@ec.europa.eu
Saturday 13 - 12:00
Interim Executive Director of Innovative Medicines Initiative (IMI) European Commission, Directorate-General for Research, Directorate for Health
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The European Union has committed €6100 million to the Health research in the 7th Framework Programme for Research (FP7) between 2007 and 2013. The objectives of the Health Theme are to improve the health of European citizens and to increase the competitiveness of the European health-related industries, while addressing global health issues. Emphasis is on translational research turning discoveries of basic research into the development of new preventive, diagnostic and therapeutic methods. This provides an unprecedented opportunity to integrate recent advances in genomic sciences into clinical management of common diseases and into better prediction of therapeutic efficiency of medicines. In several European countries, a multitude of national and regional population and disease-oriented biobanks have been systematically collected for decades via the national healthcare systems, representing a unique European strength. Recognising the power of population-based approaches in the study of genetic susceptibility for multifactorial diseases, the EU has funded a number of collaborative research projects in population genetics in the Research Framework Programmes. Altogether the EU has provided support of more than €60 million for collaborative research in this area between 2002 and 2008. Continuing support in the FP7 Health Theme will allow Europe to develop and maintain a leading global position in genetic epidemiology and population genetics research. In the FP7 Health Theme, translational research is supported in priority areas tackling major diseases such as cancer, cardiovascular diseases, neurological conditions, diabetes, rare diseases and other chronic diseases. Specific sections have also been devoted to research confronting global threats including HIV/AIDS, malaria, tuberculosis, anti-microbial drug resistance, emerging epidemics and neglected infectious diseases. Furthermore, health biotechnology research includes the development of more precise and reliable diagnostic tools, innovative therapeutic approaches to cure diseases, and pharmacogenomics approaches for prediction of therapeutic efficiency and adverse effects of medicines. By launching the Innovative Medicines Initiative (IMI) in 2007, the EU started a new initiative for strengthening Europe’s position as a centre of excellence in biopharmaceutical R&D. IMI is a unique pan-European public and private sector collaboration aiming to support faster discovery and development of better medicines, at the same time ensuring that its biopharmaceutical sector remains a dynamic high-technology sector. The strategic research agenda for IMI includes e.g. the development of novel methods of risk prediction and benefit–risk assessment using pharmacogenomics approaches, and the development of pharmacogenetic markers to divide patients to disease subtype groups of responders and nonresponders for certain treatments. Potential focus areas for knowledge management include personalised medicine, pharmacogenomics, and the integration of genomic testing data into diagnostic and therapeutic decision support tools. More information on the current and past EU Framework Programmes for Research can be obtained on the Cordis website (cordis.europa.eu).
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
Alain Vanvossel studied medicine at the Vrije Universiteit Brussel (VUB) and obtained his diploma as Doctor in medicine in 1978. After a passage as assistant in the Health Centre and the Public Health School of the Vrije Universiteit Brussel (VUB) and as health inspector in the Belgian Ministry of Labour, he entered the European Commission 1986 in the Division “Health Protection (Euratom) & “Public Health” in Luxembourg, where he was co-responsible for actions relative to public health, drug addiction, AIDS/ HIV prevention and implementing the actions proposed by the Commission concerning radiation protection of the public. In 1988, he turned to the newly set up Programme “Europe against cancer” in Brussels and was in charge of the initiatives on primary prevention (tobacco, screening, cancer registration, carcinogenic agents). In 1994, he became, in the Directorate-General for Health and Consumer Protection of the Commission (SANCO), the secretary of the High Level Committee on Public Health. Since 14 years now, Dr Vanvossel is active at the Research Directorate-General of the European Commission. As Scientific Officer, he dealt with diverse area such ageing research, public health research, cancer research and research on environment and health, within the 4th and 5th Framework Programmes. In 2001, he became Head of the Unit “Control of infectious diseases” and in 2002, in the context of the 6th Framework programme, Alain Vanvossel headed the unit “Major diseases”. From 2006 till 2008 Alain Vanvossel was head of the Unit : “Coordination of national research programmes – Relations with European research organisations” in DG Research . Begin 2008, he was appointed as interim Executive Director of the Innovative Medicines Initiative Joint Undertaking (IMI JU), responsible for the preparatory actions to set up the IMI JU, and as head of the Unit F3 “Infectious diseases”.
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Speakers In Order of Presentation Speakers
Stefan Prause PharmGenomics GmbH
stefan.prause@pharmgenomics.de
Abstract
NOVEL TECHNOLOGIES FOR THE IMPLEMENTATION OF PHARMACOGENOMICS IN MEDICAL PRACTICE 3
Eidens M., 3 Prause S., 1 Pfützner E., 2,3 Weise A.,3 Klemm M., 1 Weber M.M., 2 Forst T., 2 Musholt P., Pfützner A
2,3
Clinic of the Johannes Gutenberg-University, Mainz; Germany, 2 Institute for Clinical Research and Development, ikfe GmbH, Mainz; Germany, 3 PharmGenomics GmbH, Mainz; Germany
Saturday 13 - 12:30
1
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Background: The implementation of pharmacogenetics into daily medicine is hindered by time consuming and expensive technologies, which still require sophisticated equipment. Thus, pharmacogenetic testing remains reserved to centralized laboratories. In addition, cost-effective testing is only achieved for analyses of 1 to 5 markers per patient sample (real-time PCR etc.) or from 1,000 to 500,000 markers (microarrays) per sample. However, since pharmacogenetic testing frequently requires assessment of 5 – 200 markers per patient analysis, a diagnostic gap exists, which is not served by common technologies on the market. We here describe the first pharmacogenetics test which addresses this diagnostic gap with a rapid, cost effective method utilizing inexpensive equipment. We use a macroarray technology, which further does not use fluorescence. The reaction chip carrying the marker detection spots is integrated into a common 1.5 ml reaction tube. This feature enables technicians to handle the chip with equipment found in common laboratories. Moreover, the detection scheme is based on a robust precipitation reaction, which also reduces costs of the reading device enormously. Methods: The macroarray layout has 15x15 spots having a distance of 160 µm from each other. Each marker spot was duplicated to ensure highest accuracy. Probe design was performed according to the Santa Lucia TM calculation algorithm. The probes were designed in a way that the respective variant is located in the center of the probe. One perfect match and one mismatch probe was designed in order to cover both possible alleles. Allele discrimination was obtained by the difference in the melting behavior of the two probes. Final visualisation of the bound targets to the probes occurred by applying an enzyme coupled detection scheme resulting in a probe specific precipitation pattern. Results: Genotyping with respect to allele discrimination was successfully established for the DPYD alleles *2A, *3, *4, *5, *6, *8, *12, *13, A551T, and M166V, which all are associated with at least a partial DPD deficiency. A tailor-adjusted multiplex PCR amplification in two 0.2 ml PCR tubes facilitates the amplification of the targets enormously compared to current approaches. Discussion: With the described method it is possible to use one single reaction to detect 10 different DPYD alleles affecting the human 5-FU metabolism in a negative way. Using the developed assay may help to save costs for genotyping while delivering a higher information content compared to current assays such as RT-PCR or sequencing. Conclusion: As the described macroarray technology is easily adapted for other pharmacogenetic or other genetic applications (e.g., a CYP2D6 ultra rapid and poor metabolizer assay), the method is suitable to introduce pharmacogenetics into medical practice and routine diagnostic applications.
December 2008 - international edition
Speakers
In Order of Presentation
Curriculum Vitae
2003 – 2006. Study of the Bachelor program “Applied Biology” at the University of Applied Sciences Bonn-Rhein-Sieg (Germany) August 2006. Degree of Bachelor of Science with the thesis “Establishment of High-Throughput Screening Methods for Pharmacogenetic relevant Polymorphisms in the Dopamine Receptor D2 (DRD2) Gene on the LightCycler®-System” 2006 – 2008. Study of the Master program “Applied Biology with Biomedical Sciences” at the University of Applied Sciences Bonn-Rhein-Sieg (Germany) with the special topic “Genetics” May 2007. Nomination for the “NUK Businessplan” contest with PharmGenomics in Cologne (Germany) Okt. 2007. Lecturer and manager of the practical course “Pharmacogenetics” at the University of Applied Sciences Bonn-Rhein-Sieg (Germany) April 2008. Degree of Master of Science with the thesis “Development of a Novel DNA Microarray Chip for Genotyping the Human Cytochrome P450 2D6 Gene” May 2008. Beginning the PhD program at the University of Mainz (Germany) May 2008. Winner of the “Exist” grant for PharmGenomics (supported by the German Federal Ministry of Economic Affairs and Technology as well as the European Social Fund of the European Union) July 2008. Cofounder and Managing Director of the company PharmGenomics GmbH in Mainz (Germany)
Publications Prause S., Eidens M., Weise A., Weiher H., Forst T., Pfützner A. Development of a High Throughput Method for Screening the Dopamin(DRD2) Receptor Gene Polymorphisms based on the LightCycler System. Clin. Lab. (submitted) Eidens M., Weise A., Prause S., Weiher H., Dahmen N., Manderscheid N., Forst T., Pfützner A. Development and Validation of a High Troughput Method for two Novel Mutations in the 5-HydroxyTryptamine (Serotonine) Receptor Gene Am J Pharmacogenomics (submitted) Weise A., Prause S., Eidens M., Musholt P.B., Forst T., Pfützner A. Prevalence of CYP450 Genes in Patients with Type 2 Diabetes Diab Technol Therap. (submitted)
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Totally Convinced and with One Clear Vision About the Importance and Impact of Genomic Medicine Spearhead in Genomic Medicine in the Latin American Market
PIONEER IN MARKETING GENOMIC MEDICINE AND NUTRACEUTICAL PRODUCTS IN MEXICO
Parggon Laboratorios Av. Vallarta No. 7000, Col San Juan de Ocotan Zapopan 45019, Jal. Mexico Tel. 52-33-3682-0377 | www.parggon.com
PROUDLY MEXICAN COMPANY
Opening and Closing Ceremony Addresses
Miguel García Montes President, Spanish Association of Medical Biopathology (AEBM) The technological breakthroughs achieved over the last years are allowing us to unravel the molecular mechanisms pertaining to the etiopathogeny of various groups of diseases and the genetic dispositions which condition these mechanisms. We are beginning to understand the reason for the different ways of reacting, by different individuals, to the same physical, chemical or biological stimulus, the reason why some people fall ill and others do not when faced with some of these; the basis for the differences in the evolution of the same disease, and what conditions the fact that a patient may or may not respond to the same treatment. This knowledge, channelled by various experimental strategies, is increasingly being applied in human clinical medicine, permitting the diagnosis, the type and the stage of different diseases, the definition of factors or markers of individual predisposition to the same, the search for methods to forestall them, or the indication of which therapeutic alternative will be the most efficient for a particular patient. At laboratories devoted to welfare we are incorporating, at a rapid pace, various applications of genomics into clinical diagnosis: the study of alleles of genes whose products intervene directly in the appearance or the evolution of many diseases; or in the field of Microbiology, those which allow us to detect, typify and quantify various micro-organisms. These studies are requested in order to confirm a diagnosis when faced by a clinical surmise or subsequent to the knowledge of positiveness of a biochemical marker; in order to assess individual predisposition when faced by a familiar case, or for preventive or epidemiological reasons. An illustrative example of what we can expect from genomic medicine may be found in the works of Professor Cacabelos concerning neurodegenerative diseases, in particular Alzheimer’s Disease. Over 20 years ago, he sensed and proved the presence of molecules which caused inflammation, these being responsible for cellular lesions, in the brain tissue and in the cerebrospinal fluid. His observations have also shown that the increase in production, and the local aggregation of beta-amyloid, in conjunction with the inflammatory process, are responsible for the oxidative aggression toward the neurones and the synaptic loss between them and therefore for dementia, and that the conditioning factors of local inflammation, the vulnerability of the neurones and the accumulation of beta-amyloid are genetically regulated by alleles which become ever better known. His many pharmacogenomic studies have shown that therapeutic response in Alzheimer’s is also genotype-specific, and that many cases which present problems of efficacy or safety are associated with characterised changes in the genome. In addition to his contribution to functional genomics applied to the search for effective medication and for preventive strategies for Alzheimer’s, Professor Cacabelos has shown that environmental factors also take part in its appearance and evolution; among these, diet plays an ever more deciding role. Several of his messages insist that the choice of a diet which is in keeping with the requirements, the nutritional situation and the genotype of the patient, that is to say, individualised nutrition, is an effective weapon for forestalling, mitigating or curing certain diseases, and also for improving the quality of life in old age. In the case of patients with dementia, the interaction of nutrients with certain genes may have an influence on lipid metabolism, on cerebral functions, on cerebrovascular and metabolic risk and on the longevity of the patients. Professor Cacabelos’ research, conclusions and contributions in the field of “nutrigenomics” and of “nutraceutical” food and drugs have contributed to the fact that the medical community is aware of their importance. Thanks to this work we now have at our disposal wide-spectrum drugs with protective properties and no toxic side-effects, which are applied increasingly not only in neurodegenerative diseases, but in many others which affect humanity.
Damiano Galimberti President of A.M.I.A.– Italian Association of Anti-Aging Physicians.
Gaining Health It is possible to significantly reduce the burden of premature death, disease and disability through comprehensive action on the leading causes and conditions. Investing in prevention would improve the quality of life and well-being of people and societies. There are five key messages to guide action: • Prevention throughout life is effective and must be regarded as an investment in health and development. • Society should create health-supporting environments, thereby also making healthy choices easier choices. • Health and medical services should be fit for purpose, responding to the present disease burden and increasing opportunities for health promotion. • People should be empowered to promote their own health, interact effectively with health services and be active partners in managing disease. • Universal access to health promotion, disease prevention and health services is central to achieving equity in health. Most chronic diseases have a multifactorial etiology and result from complex interactions between individuals and their environment, including their opportunities for promoting health and countering their vulnerability to risks. A person’s genetic make-up is likely to be important in the probability of developing certain diseases, such as diabetes, cardiovascular disease, cancers, schizophrenia and Alzheimer’s disease. Although patterns of inheritance are not clear-cut, gene-environment interactions may play a major role. In fact the personal risk of developing disease can be dependent on the interaction between the individual, his or her personal susceptibility, and the wider environment. While the reduction and control of the more modifiable risk factors and wider determinants remain the cornerstone of action in the prevention and control of many chronic diseases, it is likely that in the next 5–10 years an increasing December 2008
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Opening and Closing Ceremony Addresses
number of tests will be developed for single-gene disorders and for genetic predisposition to common disease. It will be possible to test asymptomatic individuals for genetic disorders and to identify those at higher genetic risk, thus enabling earlier, more targeted interventions to be made before symptoms appear; it will also be possible to test symptomatic individuals to confirm diagnosis and offer novel treatments. The scale of the potential for health gain from these scientific advances is as yet unknown but is likely to have significant consequences for the organization, staffing and delivery of health services, as well as to raise issues of ethics and equity of access. For example, to prevent or significantly (several years) delay age associated cognitive impairment and dementia are a very important goal in the contest of a preventive strategy. About Alzheimer disease mutated alleles in several pro-inflammatory and metabolic genes have been included in the risk profile, since they have resulted to be associated with an increased risk of AD from case control studies and/or with an accelerated rate of cognitive decline during the clinical progression of the disease. Gene variants in several genes influence the risk of developing AD and the cognitive deterioration during the progression of the disease. These gene variants form the genetic risk profile predictive of age–related cognitive decline and AD. This first genetic risk profile can be used to identify healthy subjects with increased risk of developing cognitive decline and AD. The profile also gives useful information for a personalized early therapy approach. Pro-inflammatory genotype profiles can be used to identify healthy individuals with high risk of developing cognitive decline and dementia. This means a real prevention and a reduction of health costs for the governments.
Anna Soler Vice-president of the Spanish Association of Human Genetics (AEGH) The Spanish Association of Human Genetics (AEGH) was founded in 1974 as a non-profit scientific organization, to which the great majority of the Spanish geneticists belong. At present, it has more than 650 members, who come from Biology, Medicine, Pharmacy, Chemistry and/or Biochemistry, and who are devoted to the different branches of Human Genetics: clinical, educational or research areas in hospitals, universities, private or public centres. Their professional fields include Dysmorphology, Genetic Counselling, Cytogenetics, Biochemical Genetics, Cancer Genetics and Molecular Genetics. The AEGH members have witnessed the evolution of Human Genetics over the last 35 years: the first chromosome studies with banding techniques, the development of fluorescent in situ techniques (FISH), the discovery of the polymerase chain reaction (PCR), the exponential identification of involved genes and causative mutations of mendelian disorders, the Human Genome Project, the more recent technical advances in microarrays and sequencing analysis, the approach to common and complex diseases based on the knowledge of human genome polymorphisms... These developments in Human Genetics have been reflected in our Association: among the youngest members there is an increasing number of professionals in the genomic area, working in hospitals, universities or specialized research centres. Our website (www.aegh.org) offers information about Spanish centres which perform molecular studies and describes the genetic pathologies diagnosed by each centre. The impact of genomic medicine is also reflected in the subjects of the annual congress organized by the Association; likewise, the number of scientific communications related to genomic studies has been increasing in the last years. Translating the new emerging genomic knowledge into public health and medical care is one of the major challenges for the coming years. The new genomic-based medicine will deal with susceptibility risks and predictive tests for common diseases as well as therapeutics based on pharmacogenomics. There will be a need for trained professionals to deal with the different aspects of diagnosis and its transmission to the patients, and a proper genetic counseling will be of crucial importance. Since its foundation, our Association has been a platform to share and exchange scientific knowledge, and this will still be one of our main goals in the new era of genomic medicine.
Jorge Teijeiro Vidal Vice-president of the Royal Academy of Medicine and Surgery of Galicia.
Genomic Medicine in the European Union Since its inception, EuroEspes has established a noteworthy collaboration with institutions from La Coruña and Galicia for the dissemination of knowledge generated by means of its basic, social-healthcare and applied clinical research. The breakthroughs achieved in these fields of attention to patients are most significant, and they will be highlighted during this important world-level conference. The Royal Academy of Medicine and Surgery of Galicia, founded in 1831 by King Fernando VII of Spain initially in Santiago, was established in La Coruña in 1833. Obviously, its functions have changed over these 177 years. The Official Medical Associations, created in 1898, have progressively undertaken some of the functions of the Royal Academies. This has also happened with the structures of official healthcare during the 20th century. The Royal Academy is associated with the Institute of Spain, and with the Galician Autonomous Government’s Regional Ministry of Health since October 2003, due to the transfer of Healthcare from the Spanish State to the Autonomous Community of Galicia. 62
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Opening and Closing Ceremony Addresses
Among the current tasks of the Royal Academies the following may be mentioned: issuing the forensic and occupational medical reports which may be required by the competent authorities, issuing reports to private persons on matters of their competence, promoting and stimulating medical studies and research. This last task is carried out in scientific updating sessions. In addition to the distinguished academicians, noteworthy doctors and basic researchers are invited, for the dissemination of biomedical knowledge. Research is also stimulated by means of the annual awards for the best scientific works. Three awards are of interest for the research carried out in this IIIrd EuroEspes Annual Conference: the Caixanova Award for the best work, on a free theme, on a social-healthcare study of an area of Galicia; the “Pedro Barrié de la Maza, Conde de Fenosa” Award for for the best work presented, on a free theme, on medical sciences, and the Jiménez Herrero Award for a work on Geriatrics within a clinical framework or its social aspect. These may be viewed on the Royal Academy’s website http://www.ramycga.org/ The Royal Academy is made up of 300 Corresponding Members who are resident in Galicia, the rest of Spain or abroad. They are nominated for having received an Award or by election, due to works of excellence. The management and administration of the Royal Academy corresponds to the Numerary Academicians, currently 34 of these, who represent the various basic and clinical medical specialities. The Numerary Academicians elect the Governing Committee, consisting of six members, who undertake the tasks of management for a period of four years. The Royal Academy, as a veteran Galician organisation, is gratified by the holding of the First Meeting of the World Association of Genomic Medicine, and the constitution of the same, in our city. We wish it success in the years to come, and hope that European citizens will be able to benefit from this new personalised medicine.
Christian Scerri Clinical and Molecular Geneticist, Mater Dei Hospital, Malta. The phenomenal leaps that life sciences has made in the past 10 years, especially in human genomics and in health information technology, leave no doubt that a revolution is about to occur in the health care sector in particular personalized health care through pharmacogenomics. With its central position in the Mediterranean, Malta’s history has been characterized by migratory waves, in particular from the Sicilian Island interspersed by periods of mass exodus. Studies on various genetic disorders have identified a number of founder effects within the Maltese population, the result of the latest migratory influx starting around 1530. The presence of these founder effects, the small geographical area and the tightly knit and relatively large family structure, offer an excellent opportunity to study complex disorders. Apart from formulating a basis upon which future personalized health care can evolve, the Maltese government has recognised the need to tap this opportunity so as to bolster the strong pharmaceutical industry on the Island. During the past 10 years or so, a number of strategic steps and investments have been taken so as to attract research opportunities in the field of pharmacogenomics. Mater Dei Hospital, Malta’s state of the art University Hospital opened its doors to the public for the first time on 29 June 2007. The 250,000 sqm complex includes 825 beds and 25 operating theatres. Catering for the major medical specialties and in close proximity to the University Campus (in fact it is practically an extension of the campus) it is equipped with the state of the art diagnostic and treatment equipment, including an Integrated Health Information System. The easy access to medical records and tests makes it an ideal place to carry out genetics research. The government has also embarked on a modernization exercise within the University and in particular it has just availed of a €4.5M grant from the European Regional Development Fund (ERDF) to upgrade the existing health biotechnology research facility of the University, a project that is planned to be completed by 2010. In addition, Health Biotechnology has been identified as one of the 4 main areas of research within the National Strategy for Research and Innovation (2007-2010). This has enabled local scientists to apply for and obtain research grants to study complex disorders such as coeliac disease, diabetes mellitus, epilepsy, atherosclerosis and myocardial infarction and osteoporosis. The initiation of the SmartCity Project as well as the recently accepted ERDF funded Biopark project shall augment these facilities and offer an opportunity for further collaboration between industry and the academia. In addition, European Social Funds (ESF) are being tapped to offer opportunities for Maltese graduates to specialize in relevant health biotechnology and informatics fields as well as entice Maltese scientists working abroad to return. Such an investment cannot succeed if the necessary legal and financial framework is not in place. The government, through its Medicines Authority, has laid down the necessary legislation for clinical trials whilst the Health Ministry’s Bioethics Consultative Committee is in the process of formulating guidelines on genetics testing. The third pillar in the government’s endeavour towards a thriving pharmacogenomics research base is the incentives package offered towards the setting up of investments projects in the fields of health biotechnology. These packages, administered by MaltaEnterprise include access to finance, tax credits, training programmes and R&D and Innovation programmes. Membership within the European Union, is offering new opportunities for our scientists and health workers to participate within the EU’s research programmes and be able to collaborate with prominent scientists in the field, as well as to attract investment in innovative fields such as pharmacogenomics. December 2008
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Opening and Closing Ceremony Addresses
Rafael Cortés Elvira Rector, Camilo José University (Madrid) Considerable challenges have been set for Universities with the appearance of the European Space for Higher Education, not only in the field of teaching, but also in other areas with a greater direct impact on the regional and national economy. Recently, the traditional missions of Universities, teaching and research, have been joined by a third: the transfer of the results of this research, that is to say, to give back to society what the latter has granted to the University, in the form of economic value and social development. A model based on the generation of new knowledge generated by means of basic research gives way to new possibilities where basic and applied research go hand-in-hand, and where the economic yield of the intellectual or industrial products becomes indispensable. Research at traditional Universities has been directed towards a model of Open Science, aimed at scientific publications regardless of the value or economic return, by means of the retaining of the intellectual or industrial property rights. The classic research model, where the researcher concentrated on his or her own lines of research now gives way to another where the socio-economic relevance of research, and the express demand of the social agents reign supreme. Here, social, industrial and economic progress is not in contradiction with market values. The process of protection of intellectual and industrial property, its valuation and subsequent marketing are not just elements for the enhancement of regional enrichment, but are also markers of the wealth of the country itself. The EuroEspes Biomedical Research Centre is a good example of the surmounting of these alleged contradictions which face the Spanish University model, combining the traditional aspects of scientific publications with support for innovation, the valuation of the knowledge generated and the transmission of the results to society, thus maximising social, business and economic development within its field of knowledge. In this way, the implementation of six product patents and two technological ones is added to the over 1,500 scientific contributions on the etiopathogeny and treatment of CNS degenerative diseases (which has placed EuroEspes among the most productive scientific groups in the European Union). And this is the commitment undertaken by Camilo José Cela University together with the EuroEspes Biomedical Research Centre in the development and consolidation of the EuroEspes Chair of Biotechnology and Genomics, directed by Dr. Cacabelos. By means of this Chair an internationalisation process has begun with the signing of an agreement with the Autonomous University of Guadalajara (Mexico) in order to facilitate and develop the exchange of medical, teaching and research personnel in programmes of varying scientific content. Likewise, for the next academic year a Master’s degree in Health Biotechnology will be developed, which will mean the commencement of a doctorate programme in biotechnology and genomic medicine, with a clearly international calling. The IIIrd edition of the EuroEspes Annual Conference represents yet another element in the consolidation of EuroEspes as a leading centre in Genomic Medicine, with worldwide recognition, as may be seen from the international category of the speakers, and it is a stimulus in the Spanish scientific field. The promise, part of which is already a reality, of the possibility of disease prevention and individualised treatment according to the human genome sequence and its genetic variations, is one of the greatest challenges which this Congress and medicine in general faces. And all this without neglecting the goals expounded above: generating economic and industrial value, and in particular, improving people’s quality of life. Finally, I would like to congratulate the organisers through Dr. Cacabelos, the real driving force of this Congress, and to wish all the participants the greatest of successes and a pleasant stay in Galicia, one of the most privileged places in Spain.
Víctor Manuel Ramírez Anguiano Rector of the University Centre of Health Sciences, Guadalajara University (Mexico) Genomic medicine represents a powerful armamentarium to tackle down most chronic diseases, and currently several acute diseases, which have not, so far, been defeated. Thus, this new and powerful biotechnological set of weapons enable us to make use of molecular diagnostics to detect silent diseases, otherwise undetectable by conventional analysis. Moreover, elucidation of the complete and final human genome code will allow us the design of specific pharmacogenomic treatments for patients on the basis of their individual genetic code. Regarding new medical treatments, gene therapy has emerged as a true hope for treatment for many chronic diseases. Up to November 2008, 1472 approved clinical protocols are currently undergoing and sooner than later we will be witness of the results. At the University of Guadalajara, specifically at the Institute of Molecular Biology and Gene Medicine of the Centre for Human Health Sciences, a team led by Dr. Juan Armendariz-Borunda has developed two strategies to induce regression of hepatic cirrhosis using approaches of human gene therapy. One of them consisted in the use of an adenoviral vector bearing a modified cDNA coding for a non-secreted form of human urokinase Plasminogen Activator (Ad huPA) to deliver the gene in vivo. This cDNA was chosen to diminish the risk of bleeding, which would be particularly problematic in cirrhotic animals that may have preexisting coagulopathy. AdhuPA administration led to almost complete restoration of periportal and centrilobular fibrosis compared to 64
December 2008 - international edition
Opening and Closing Ceremony Addresses
a progressive fibrosis in CCl4-cirrhotic animals without adenovector injection. Similar observations were obtained when we used a cDNA coding for a matrix metalloproteinase-8 (MMP-8) targeted to cirrhotic rat livers by means of a replicative-deficient Adenoviral vector. In both cases, we have conducted extensive studies of bio-distribution, bio-availability, toxicity, etc. In other words, we know exactly the pharmacology of our gene therapy approaches for a further use in human beings affected by fibrotic diseases caused by Hepatitis C or B viruses chronic infection, alcohol, secondary biliary cirrhosis, primary biliary cirrhosis. Needless to say, all biotech developments are properly registered and fulfill quality assurance and control standard operating procedures. In addition to resolution of fibrosis, liver regeneration was also observed in cirrhotic rats treated with both therapeutical adenovectors. Gene treatment in both models correlated with improvement in acitis, functional hepatic tests and gastric varices, indicating a diminished intrahepatic blood pressure in animals injected with the adenovectors. There is a great deal of data we need to recollect before we attempt to use these biotech strategies in human patients. We are aware of that. However, these and other equally innovative and cutting-edge technologies give us the notion that, we are certainly on the right path.
Eugenio Luigi Iorio International Observatory of Oxidative Stress, Salerno, Italy. eugenioluigi.iorio@oxidativestressobservatory.org
Future challenges for personalized medicine in the European Union. The contribution of international observatory of oxidative stress The International Observatory of Oxidative Stress (IOOS) is a no profit organisation having the mission to promote, to develop, to diffuse and to translate, when possible, the basic knowledge on oxidant species and antioxidants into clinical practice either in Human or in Veterinary Medicine, for a better quality of life (www.oxidativestress.observatory.org). The IOOS was founded in 2003 and its location has been recently moved from Parma (Italy) to Salerno, the city where the oldest European Medical School (Schola Medica Salernitana) was born. The IOOS cooperates with many academic institutions worldwide, especially in Europe, Asia and America, with the aim of promoting experimental and clinical studies on free radicals and antioxidants. In particular, every year, starting in 2003, in Tokyo (Japan), the president of IOOS is an invited speaker at the “FREE RADICAL SEMINAR” organised by the Wismerll Foundation with the aim to spread oxidative stress evaluation in Japan and East Asia. This year a special chapter on oxidative stress, written by IOOS, will be published in Spain for the Spanish College of Pharmacists (“El laboratorio en el estudio del estrés oxidativo”) (1–6). Recently IOOS developed a special software (WIN OS MANAGERTM) that allows clinicians to evaluate oxidative stress in clinical practice by means of two screening tests (i. e. d-ROMs test and BAP test), both invented by the Italian researcher Mauro Carratelli (7). Every clinician who performs these tests can send the results – via INTERNET – to the IOOS for statistic purposes. By means of this software a virtual medical network is now being created worldwide, with the main aim to personalize – by means of genomics – any medical action (see REDOXOMICS project). In this context IOOS appreciates the efforts of EUROSPES in this very important scientific meeting and it wishes a successful continuation of the ANNUAL CONFERENCE in the next years.
References: 1. Iorio EL. Oxidative stress and nutrition. Proceedings VI Macedonian Nutrition Congress. November 19-21, 2004. Thessaloniki, Greece. 2004. Pp. 143–144. 2. Iorio EL. Thermal medicine and oxidative stress. Rev Latinoamer Quim. 2005. 33 (suplemento especial): 63. 3. Iorio EL, Ferreri C, Cozzolino R, Manara S, Ferreri R. Plasma total oxidant status and erythrocyte plasmamembrane lipidomics. A preomising sinergy in the molecular diagnostics of anti-aging. Proceedings 9th International Congress of Aesthetic Medicine. 2007, October 11th –13th Milan, Italy. 2007. Pp. 145. 4. Pasquini A, Luchetti E, Marchetti V, Cardini G, Iorio EL. Analytical performances of d-ROMs test and BAP test in canine plasma. Definition of the normal range in healthy Labrador dogs. Vet Res Commun. 2008. 32 (2): 137–143. 5. Mandas A, Congiu MG, Balestrieri C, Mereu A, Iorio EL. Nutritional status and oxidative stress in an elderly Sardinian population. Mediterr J Nutr Metab. 2008. DOI 10.1007/s12349-008-0016-1. 6. Banfi G, Iorio EL, Corsi MM. Minireview: oxidative stress, free radicals and bone remodeling. Clin Chem Lab Med. 2008. Oct 10. [Epub ahead of print]. PMID: 18847368. 7. Iorio EL, Cinquanta L, Pisano R. A diagnostic algorithm to manage oxidative stress. Australasian J Cosmet Surg. 2006. 2 (1) : 26-30.
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P1
Redoxomics. An integrated and practical approach to genomics, metabolomics and lipidomics to manage oxidative stress 1
Eugenio Luigi Iorio, MD, PhD, 2 Maria Grazia Marin, BD, PhD 1 International Observatory of Oxidative Stress, 2 Salerno, Italy, OXIGENLAB s. p. a., Brescia, Italy eugenioluigi.iorio@oxidativestressobservatory.org
In all living organisms a delicate equilibrium between the production and the elimination of oxidant chemical species (OCS) takes place (1). The production of OCS – which include either radical or non-radical oxygen-, nitrogen-, carbon-, chlorine-, sulphur-centred species – can depend on both exogenous agents (e. g. radiations, chemicals, bacteria) and endogenous phenomena (e. g. activation of specific enzymatic or non-enzymatic reactions) (2). The level/activity of antioxidants systems – which include either endogenous (e. g. antioxidant enzymes, like catalases and peroxidases) or exogenous agents (e. g. vitamins and vitamin-like compounds) devoted against OCS – is related to genetic and environmental factors (lifestyle mainly) (3). An increased production of OCS and/or a decreased efficacy of antioxidant systems can lead to the breakdown of oxidative balance thus generating the so-called oxidative stress (OS) (4). Oxidative stress is generally recognised to play a pathogenic role either in early ageing or in several inflammatory and/or degenerative diseases, including atherosclerosis and hypertension (and their consequences, such as stroke, myocardial infarction, etc.), Alzheimer’s disease, Parkinson’s disease, cancer and so on (5). Unfortunately, OS is not a “disease”, according to the traditional sense of this word. Indeed, OS is the unwanted effect of the breakdown of a biochemical equilibrium. Therefore it can impact, often deceitfully, upon the onset and/or the course of several basic diseases. As it is not a classical disease, OS does not exhibit a specific clinical picture, but it hides itself behind the symptoms and the signs of the basic disease. Therefore, OS can be found only if the clinician submits the patient to specific biochemical tests. At long last, our laboratory (OXIGENLAB, Brescia, Italy) in cooperation with the International Observatory of Oxidative Stress (Salerno, Italy) now offers clinicians the opportunity to manage the oxidative stress by means of REDOXOMICS, an integrated approach where genomics and metabolomics (mainly lipidomics) interact with a innovative panel of oxidative balance biomarkers in order to offer to the patients a personalised analysis aimed to prevent oxidative damage, to diagnose and to monitor oxidative stress and, finally to evaluate the indication and the effectiveness of antioxidant supplementations and/or therapeutic interventions (6, 7). 1. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. 2nd Edn, Clarendon Press, Oxford. 1989. 2. Gardes-Albert M. Physico-chemical aspects of reactive oxygen species. Ann Pharm Fr. 2006. 64(6):365–372. 3. Valko M, Leibfritz D, Moncol J, et Al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007. 39(1):44–84. 4. Delattre J. Introduction: from molecular oxygen to oxidative stress and radical biochemistry. Ann Pharm Fr. 2006. 64(6):363. 5. Favier A. Oxidative stress in human diseases. Ann Pharm Fr. 2006. 64 (6): 390–396. 6. Ridker PM, Brown NJ, Vaughan DE, et Al. Established and emerging plasma biomarkers in the prediction of first atherothrombotic events. Circulation. 2004. 109 (Suppl. IV): IV-6 –IV-19. 7. Iorio EL, Cinquanta L, Pisano R. A diagnostic algorithm to manage oxidative stress. Australasian J Cosmet Surg. 2006. 2 (1) : 26-30.
P2
Different gene expression of protein precursor, PENK, POMC and PDYN in human spermatozoa 1,2
N Subirán, 2 ML Candenas, 3 CG Ravina, 3 M. Fernández-Sánchez, 1 J Irazusta, 2 FM Pinto
Department of Physiology, University of the Basque Country. UPV/EHU. 48940 Bizkaia. Spain, 2 Chemical Research Institute, CSIC-University of Sevilla. 41092, Sevilla, Spain, 3 Valencian Infertility Institute. 41011 Sevilla, Spain 1
Two-thirds of infertile men do not present spermatogenetic defects and have apparently normal semen quality. In this context, recent findings suggest that most idiopathic male infertilities are caused by genetic factors. Mature spermatozoa have a stable population of mRNAs that are synthesised during spermatogenesis and selectively maintained in mature sperm cells. So, ejaculated spermatozoa can be used as a non-invasive proxy for investigations of testis specific infertility. Proenkephalin (PENK), proopiomelanocortin (POMC) and prodynorphin (PDYN), are the genes that encode the precursor proteins of opioid peptides. All of them are expressed in the testis. So, the aim of this study was to investigate the presence of PENK, POMC and PDYN transcripts in human normal fertile men. RT-PCR was used to detect the transcripts for PENK, POMC and PDYN on human December 2008
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spermatozoa isolated on a discontinuous Percoll gradient (40%-80%). We detected the presence of PENK and PDYN transcripts but not POMC transcript in mature sperm cells from normozoospermic men. These findings confirm the existence of a stable and selective mRNA population in mature sperm. In addition these results support the idea that opiod system may be a useful biochemical tool for the diagnosis and treatment of male infertility. This work has been supported by a grant from the Spanish Ministry of Education and Science (BFU2006-07779), Basque Government (GIC07/84)and IVI Foundaction S.L. (Contrato Instituto Sevillano de Infertilidad-CSIC cod.20071101) N. Subirán holds a fellowship from the University of the Basque Country.
P3
Human neurodegenerative diseases (hndds) and cancer: an integral approach to the etiopathogenesis and treatment of hndds within the frame of the trophic factor withdrawal syndrome (TFWS) 1 4
Salvador Harguindey, 2 Ramón Díaz de Otazu, 3 Jon Arlucea, 1 José Catalán, 1 Jose Luis Arranz, 3 Gorka Orive, Stephan J. Reshkin, 5 Ramón Cacabelos Institute of Clinical Biology and Metabolism, 2 Hospital Txagorritxu, 3 University of the Basque Country, Vitoria, Spain; 4 University of Bari, Italy; EuroEspes Biomedical Research Center, Coruña, Spain. 1 salvaszh@telefonica.net
1 5
A proton [H+]-related integral approach to the etiopathogenesis of malignancy underlying the initiation, local progression and the metastatic process of cancer has been described by us recently (S. Reshkin et a., FASEB J 2000; 14: 2185-2197; S. Harguindey et al., Bioch. Biopyhs. Acta Revs. Cancer 2005; 1756: 1-24). This is mainly mediated by the genetically-controlled Na+/H+ antiporter at the cell membrane, but also by other proton transporter systems (PTs). The results afforded by this new and all-embracing paradigmatic approach has allowed us to advance a new and integral approach to cancer etiopathogenesis and therapeutics aiming to trigger selective cancer cell death through the induction of low pHi-mediated apoptosis. Selective apoptosis of malignant cells is achieved through the inhibition of the Na+/H+ antiporter leading to low pHi-mediated apoptosis (I. Rich et al., Blood 2000; 95: 1427-1434; S. Harguindey et al., Bioch. Biopyhs. Acta Revs. Cancer 2005; 1756: 1-24; S. Harguindey et al, Curr Alzheimer Res 2007; 4(1): 53-65; S. Harguindey et al., Neuropsych Dis & Treatment, 2008 (in press). The failure of tumor cells to undergo apoptosis following chemotherapeutic treatment also appears to be highly dependent on their resistance to undergo selective intracellular acidification. Contrariwise, low pHi-mediated spontaneous apoptosis in different HNDDs seems to be the main mediated terminal mechanism of metabolic collapse and cell death in many cases of neurodegeneration (AM Vincent et al, J. Neurobiol. 1999; 40(2): 171- 184). These observations lead to view cancer and HNDDs as as opposite pathological processes of chronic cellular homeostasis (spontaneous low pHi-mediated apoptosis in HNDDs versus the specific and pathological high pHi-mediated antiapoptosis in cancer). This integrated approach leads to propose opposite forms of treatment for HNDDs and malignant diseases. In different HNDDs, the Na+/H+ antiporter can be downregulated, either genetically or via a decreased microenvironmental stimulation, by an endogenous lack of platelet and non-platelet derived growth factors, and this could be of paramount importance in the etiopathogenesis and progression of low pHi-mediated apoptosis in neurodegeneration. Finally, growth and trophic factor (GFs) withdrawal (TFWS) in HNDDs, either from neural origin or otherwise, indicate the need to further investigate the potential and concerted utilization of certain GFs in the treatment of Alzheimer’s disease (AD) and other HNDDs. In a preliminary study of our group, platelets from AD patients studied under electron microscopy appear as normal platelets. In spite of this, potential functional platelet abnormalities in HNDDs, like a decrease in their output of platelet-derived growth factors (PDGF), would suggest a therapeutic potential for PDGF in these pathologies, either through young platelet transfusions or other ongoing clinical methods, like VEGF-containing slow release microcapsules (see also, abstract by A. Portillo, et al. Microencapsulated VEGF-expressing cells as as a potential treatment for Alzheimer´s Disease).
P4
Altered mRNA levels of angiotensin-converting enzymes (ACE, ACE-2 and APA) in renal tumors 1
Itxaro Pérez, 1 Lorena Blanco, 1 Luis Casis, 1 Adolfo Varona, 2 Jose I López, 3 Aitziber Ugalde, 4 Gorka Larrinaga Dept of Physiology, Basque Country Univ, Leioa, Bizkaia, Spain. 2 Dept of Anatomic Pathology, Hospital de Cruces-Osakidetza, Basque Country Univ, Barakaldo, Bizkaia, Spain. 3 Dept of Anatomic Pathology, Hospital de Basurto, Bilbao, Bizkaia, Spain. 4 Nursing School, Basque Country Univ, Leioa, Bizkaia, Spain. Corresponding author: iperez105@ikasle.ehu.es 1
Renin angiotensin system (RAS) is, in its classic concept, an endocrine peptidergic system involved in blood pressure regulation and hydroelectrolitic balance. However, in recent years it has been demonstrated that kidney and other tissues possess local RAS which have been implicated in several long-term processes such as cell growth, differentiation, angiogenesis and proliferative disorders. Ang II levels are controlled through 68
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Angiotensin Converting Enzymes (ACE-1 and ACE-2 and APA). These ectopeptidases are highly expressed in renal tubules and play important roles in the regulation of renal function by the intrarenal RAS (iRAS). Moreover, dysregulation of these cell surface peptidases has been associated to renal injury. However, these studies have been mainly focused on non-neoplastic kidney diseases. The goal of this study was to evaluate if profiles of ACE, ACE 2 and APA mRNA levels are altered in a subset of 16 clear cell (CCRCC) and 4 Chromophobe (ChRCC) renal cell carcinomas, and 4 renal oncocytoma (RO). These renal entities are of different histogenetical origins (CCRCC proximal tubules; ChRCC and RO distal) and used to behave differently with regard to prognosis and aggressiveness. mRNA levels given in relative units of expression were measured by qRT-PCR using TBP, PPLA, ACTB, and SDHA as genes of reference. The mRNA levels of ACE, ACE-2 and APA were altered in a tumor type dependent manner. Thus, the expression of these peptidases strongly decreased in ChRCC (ACE decreased 10-fold, ACE-2 99-fold and APA 77-fold) and RO (ACE decreased 4-fold, ACE-2, 4-fold and APA 21-fold). Studied peptidase expressions did not vary significantly in CCRCC. These results suggest that losses of expression which these peptidases suffer appear to be dependent of the histogenetic arising and/or aggressive behavior of the different tumor-types we analyzed. All co-authors are aware of the content of the abstract. Supported by grants from the Jesús Gangoiti-Barrera Foundation and Basque Government (GIC07/84).
P5
Protein expression of CD10 and CD26 markers in renal tumors 1
Lorena Blanco, 1 Itxaro Pérez, 1 Javier Gil, 2 Luz Candenas, 2 Francisco Pinto, 1 Adolfo Varona 1 Dept of Physiology, Basque Country Univ, Leioa, Bizkaia, Spain. 2 Institute for Chemical Research, CSIC, Sevilla, Spain. Corresponding author: bcbblcrl@ehu.es
Several cell surface proteins have been proposed to be useful markers in tumor diagnosis and prognosis. Among them CD10 and CD26 appear to be of clinical interest for renal neoplasia. These two peptidases play a key role in the control of growth and differentiation in renal tissue. The aim of this study is to delineate the profile of expression of CD10 and CD26 in clear cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas and in a benign entity such as renal oncocytoma (RO). CD10 and CD26 mRNA and cell surface expression have been investigated in a series of 16 CCRCC, 6 ChRCC and 4 RO. Tumor and non-tumor tissues were evaluated in every case. Results were stratified by grade (Fuhrman) and stage (2002 TNM) in the CCRCC group. Conventional immunohistochemistry and mRNA levels given in relative units of expression with TBP, PPLA, ACTB, and SDHA as genes of reference were performed in all cases. Males predominated in the series (20M/6F) with an average age of 62.5 years (range, 37-78). Tumor Grade in the CCRCC group was Low grade: G1-G2, n=12 vs High grade: G3-G4, n=14. Stage was Low stage: T1-T2, n=16 vs High stage: T3-T4, n=10. The mRNA levels of both peptidases were decreased in all tumor types compared with non-tumor tissue. The decreases were especially pronounced in ChRCC, for both peptidases (CD10 = 46-fold; CD26 = 67-fold), and in RO for CD10 (30-fold); however, these decreases only were statistically significant in ChRCC [0.01 0.008 vs. 0.675 0.332 (CD10)/0.025 0.009 vs. 1.142 0.616 (CD26), Student’s T test p<0.05], with non significant results in CCRCC and RO. The expressions of the two peptidases in the CCRCC group after stratification by Grade and Stage were not significantly different. Immunohistochemistry data supported the mRNA profile. Thus CD10 was positive in all the tumor types, with intense and diffuse staining in CCRCC and mild and focal immunoreaction in both ChRCC and RO. In contrast, CD26 immunostaining was positive in CCRCC (moderate and diffuse) and negative in both ChRCC and RO. Conclusion: CD10 and CD26 are strongly down-regulated in CCRCC, ChRCC and RO. These results support the role of both peptidases in the malignant transformation of proximal and distal renal tubules and confirm its potential use in routine practice as diagnostic and prognostic tools. All co-authors are aware of the content of the abstract. Supported by grants from the Jesús Gangoiti-Barrera Foundation and Basque Government (GIC07/84).
P6
CB1 and CB2 endocannabinoid receptor expression in human renal parenchyma and in the Clear Cell Renal Cell Carcinoma 1
Adolfo Varona, 1 Itxaro Pérez, 1 Jaime Zubero, 2 Jose I López, 1 Jon Irazusta, 3 Gorka Larrinaga Dept of Physiology, Basque Country Univ, Leioa, Bizkaia, Spain. 2 Dept of Anatomic Pathology, Hospital de Cruces-Osakidetza, Basque Country Univ, Barakaldo, Bizkaia, Spain. 3 Nursing School, Basque Country Univ, Leioa, Bizkaia, Spain. Corresponding author: adolfo.varona@ehu.es 1
The detection of endocannabinoids and corresponding receptors (CB1 and CB2) in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities. Cannabinoids have emerged as crucial mediators in a variety of pathophysiological conditions, including cancer. Thus, there is increasing evidence that endocannabinoids have antitumor action, and they are now emerDecember 2008
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ging as suppressors of angiogenesis and tumor spreading in different types of cancer. These evidences point to a potential role of this system as a target for a therapeutic approach of tumors, although, they are still a controversial topic in oncological praxis. Besides a strong expression in the CNS, CB1 is also present in testis, sperm, liver, ovary, muscle, heart, spleen, and pituitary. CB2 distribution remains unclear, although this receptor is broadly associated with the cells of the immune system. However, little is known about the distribution of CB1 and CB2 receptors in human kidney. Overall, Clear Cell Renal Cell Carcinoma (CCRCC) has a worse prognosis than the other types of renal carcinomas. The search for more efficient therapeutics is a priority in the CCRCC. In this work we tested the expression of CB1/CB2 receptors in the human kidney, and analyzed if the expression pattern of CB1/CB2 receptors is altered in the CCRCC. We performed qRT-PCR, western-blot and immunohistochemistry assays of 6 samples from CCRCC patients. Non-tumor tissue surrounding the CCRCC (normal tissue) was used to detect if CB1 and/ or CB2 receptors were expressed in renal parenchyma (n=6). Only CB1 receptor was marked in the normal renal parenchyma, while CB2 was absent from the kidney. Both, mRNA and protein expression for CB1 receptor, dissapeared from CCRCC tissues. These data describe for the first time CB1 expression in human renal normal parenchyma and suggest a role for this receptor in renal carcinoma. All co-authors are aware of the content of the abstract. Supported by grants from the Jesús Gangoiti-Barrera Foundation and Basque Government (GIC07/84).
P7
Cytotoxicity effects induced by FR-91 on human tumor cell lines 1
Martínez E, 1 Chacón R, 2 Etcheverría I, 2 Cacabelos R, 2 Lombardi VRM 1
Georgian Alternative Medicine, Madrid, Spain, 2 EBIOTEC, Biotechnology Division, La Coruña, Spain
Introduction: Scientific attempts for the in vitro evaluation of natural compounds with biological activity have increased in the last two decades, due mainly to the development of highly automated bioassay screening based on colorimetric methods that can measure the inhibition of proliferation of cell cultures. The main objective of the present work was to evaluate the antiproliferative activity of FR91, a standardized lysate of microbial cells of the Bacillus genus, on human tumor cell lines. Material and Methods: HL-60, H.S. 281.T, H.S. 274.T, H.S. 313.T, SW 982, SW 872, H2126, TOV-21G, and WM115 human cell lines were used in the study. All cell cultures were maintained grown as a monolayer, except for HL-60, in DMEM, L-15 and EMEM supplemented with 10% fetal bovine serum. The cultures were maintained at 37ºC in a humified 5% CO2 atmosphere. For tetrazolium-dye (MTT) cytotoxicity assay adherent cell monolayers were trypsinized, washed and plated at 15,000 cells/well in 96 flat bottomed plates. FR91 was added at corresponding wells in duplicate at the concentration of 10 μL/w, 25 μL/w, and 50 μL/w, respectively. Untreated cells were used as control. Plates were incubated for 24 hours at 37ºC. After incubation supernatants were removed from all wells and 25 μL of MTT solution was added to each well. After 2 hours incubation, 125 μL of DMSO was added to the wells and absorbancy was read at 450 on a multiwell spectrophotometer. The percentage of cytotoxicity was calculated as (A-B)/A x 100, where A is the mean optical density of untreated wells and B is the optical density of wells treated with FR91. Results: The results of FR91 activity at the three different concentrations (10 μL/w, 25 μL/w, and 50 μL/w ) showed a clear increase of growth inhibition, mainly in the HL-60, SW 872, SW 982, H.S. 313.T, and TOV-21G cell lines. Conclusions: According to the results obtained in the study it can be concluded that the FR91 microbial cell lysate shows in vitro antiproliferative activities and that this extract is a good candidate for further activity-monitored fractionation to identify activity principles.
P8
Effects of FR-91 on immune cells from healthy individuals and patients with non-hodgkin lymphoma 1
Martínez E, 1 Chacon R, 2 Cacabelos R, 2 Etcheverría I, 2 Lombardi VRM 1
Georgian Alternative Medicine, Madrid, Spain 2 EBIOTEC, Biotechnology Division, La Coruña, Spain
Introduction: Because of the importance of identifying cell immunophenotypes, considerable effort has gone into developing rapid identification methods for cell surface antigen involved in the modulation of immune system. The main objective of the present work was to evaluate the activity of FR91, a standardized lysate of microbial cells of the Bacillus genus, on peripheral blood lymphocytes from healthy individuals (HI) and patients with non-Hodgkin lymphoma (NHL) by following sequential changes in the immunophenotype of mixed-cell populations, as well as in the cytokine production. Material and Methods: Peripheral blood lymphocytes were obtained from 15 HI and from 10 NHL patients. 500 µL of whole blood was diluted 1:1 with RPMI 1640 containing 10% fetal bovine serum, and FR91 was added at corresponding tubes at the concentration of 10 μL/mL, 25 μL/mL, and 50 μL/mL, respectively, and incubated 24 hours at 37ºC in a humified 5% CO2 atmosphere. After incubation 100 µL of blood was added to individual tubes and stained with anti-human CD3-FITC, anti-human CD25-FITC, anti-human CD69-FITC, anti-human HLADR-PE, anti-human CD4-PE, and anti-human CD8-PE. After 70
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incubation and washing, tubes were passed on a flow cytometer and percentages of positive cells were calculated. For cytokine quantification, a Multiplex bead array assays, which permit simultaneous cytometric quantitation of IL-1β, IL-2, IL-6, IL-8, IL-10 p70, IL-12, IFN-γ, TNF-α in solution by capturing these to spectrally distinct beads, has been used. Results: The results of FR91 activity at the three different concentrations show a significant increase with respect to untreated controls of CD3 (p=0.02) and CD8 (p=0.02) antigen expression in the HI group, while a higher increase in CD3 (p=0.002) and CD4 (0.002) was observed in the NHL group. A significant increase in IL-2 (p=0.014), IL-6 (p=0.001), Il-12 (p=0.003), IFN-γ (p<0.0001) and TNF-α (p=0.002) cytokine expression was observed in the HI group. Similar results were observed in the NHL group: IL-1β (p=0.03), IL-6 (p=0.008), Il-12 (p<0.0001), IFN-γ (p<0.017) and TNF-α (p=0.012). Conclusions: The significant increase of CD3+ lymphocytes in both studied groups suggests a possible use of FR91 in patients with altered function of the immune system. In addition, the increase in IL-2, IL-6, IL-12 and TNF-α cytokine production, can be one of the underlying mechanisms by which FR91 control cell proliferation.
P9
Caspase 3 activity in PBMCs from Alzheimer´s Sisease patients: When and why do cells die? Lombardi VRM, Etcheverría I, Fernández-Novoa L, Seoane S, Cacabelos R EBIOTEC, Biotechnology Division, La Coruña, Spain Introduction: Over the past 20 years there has been a near-exponential increase in publications about apoptosis. Around 60 new molecules have been discovered whose known functions are exclusively to do with the initiation or regulation of apoptosis. A further 40 molecules at least, although already associated with important roles in signalling or DNA replication, transcription or repair, have been recognized as affecting the regulation of apoptosis. Material and Methods: 10 mL of blood was collected in EDTA tubes. Peripheral blood mononuclear cells were isolated from 10 healthy subjects (HS) and 10 patients with Alzheimer´s disease (AD). Short term cell cultures have been maintained in RPMI 1640 medium containing antibiotics and 10% fetal calf serum, at 37ºC in a 5% humified atmosphere, during a period of 24 days. Every fourth day cells were analyzed for the presence of spontaneous and induced apoptosis. A fluorometric immunosorbent enzyme assay was used to test quantitative detection of caspase 3 activity. Results: Caspase 3 activity was clearly detectable in lysate of 106 PBMCs with 5% apoptotic cells. A preliminary caspase activity assay has been used to detect caspase activation in short term cell cultures exposed (positive controls) to different concentrations of the apoptosis inducing agent camptothecin. In this model system, the induction of apoptosis was obtained with 1μg/mL of camptothecin. A significant difference between HS and AD caspase 3 activity was observed in both spontaneous and camptothecin induced apoptosis starting from 8 days in culture. Caspase 3 activity in HS was 3% at day 4, 7% at day 8, 22% at day 12, 55% at day 16, 78% at day 20, and 100% at day 24, whereas in the AD group caspase 3 activity was 4% at day 4, 19% at day 8, 35% at day 12, 75% at day 16, and 100% at day 20. Conclusions: PBMCs from normal individuals seem to be able to better survive during time to adverse conditions, such as those represented by short term cultures without the presence of growth factors. In contrast, PBMCs from patients with AD seem to be more vulnerable to stressed growth conditions. These observations suggest that caspase 3 activity is one of the most important mediators of very early stages of apoptosis and that it can work in combination with other effectors, such as TNF and CD95, which have been already demonstrated to be overexpressed during the progression of AD.
P10
ACE-related blood pressure values in the Spanish population 1,2
J. Des, 2 R. Perez, 2 M. Alcaraz, 2 L. Febril, 2 L. Fernandez-Novoa, 2 R. Cacabelos
1 Ophthalmology Department. Ourense Hospital Center. Spain, 2 EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain
Introduction: The angiotensin converting enzyme is a part of the angiotensin-renin system associated with vascular pathology. ACE promotes the conversion of angiotensin I to angiotensin II, a potent vascular constrictor and an inhibitor of acetylcholine liberation. ACE is present in different organs and tissues, including lungs, kidneys, vascular endothelium, heart, and brain. A polymorphism for the gene codes ACE has been described, characterized by the presence (I) or absence (D) of a DNA fragment containing 287 base pairs in the long arm of chromosome 17 (17q23). Individuals carrying the DD genotype show higher serum and tissue levels of ACE than those carrying II. Allele D has been related to cardiovascular diseases, hypertension, cerebrovascular pathology, and cognitive disorders. Aim: To analyze if the ACE genotype and blood pressure parameter values are related in a sample of the Spanish population. Methods: Blood pressure parameters to be studied included systolic arterial pressure (SBP) and diastolic arterial pressure (DBP). Sample members were grouped according to their ACE genotype and comparisons among groups were made for each of the parameters. The ANOVA test was used to compare the groups (P<0.05). Results: One thousand nine hundred and ninety-six adult people constituted the dataset. Seven hundred December 2008
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and seven individuals (707/1996, 35%) carried the DD genotype, 980 (980/1996, 49%) carried ID, and 309 (309/1996, 16%) carried the II genotype. No significant differences among groups for the analyzed parameters were found. Conclusion: ACE genotype is not related to SBP and DBP values in the Spanish population.
P11
ACE-related brain hemodynamic parameters in the Spanish population 1,2
J. Des, 2 R. Perez, 2 M. Alcaraz, 2 L. Febril, 2 L. Fernandez-Novoa, 2 R. Cacabelos
Ophthalmology Department. Ourense Hospital Center. Spain, 2 EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain 1
Introduction: The angiotensin converting enzyme is a part of the angiotensin-renin system associated with vascular pathology. ACE promotes the conversion of angiotensin I to angiotensin II, a potent vascular constrictor and an inhibitor of acetylcholine liberation. ACE is present in different organs and tissues, including lungs, kidneys, vascular endothelium, heart, and brain. A polymorphism for the gene codes ACE has been described, characterized by the presence (I) or absence (D) of a DNA fragment containing 287 base pairs in the long arm of chromosome 17 (17q23). Individuals carrying the DD genotype show higher serum and tissue levels of ACE than those carrying II. Allele D has been related to cardiovascular diseases, hypertension, cerebrovascular pathology, and cognitive disorders. Aim: To analyze if the ACE genotype and haemodynamic parameters are related in a sample of the Spanish population. Methods: Brain haemodynamics were assessed by non-invasive transcranial Doppler ultrasonography and included mean velocity, systolic velocity, diastolic velocity, Gosling’s pulsatility index (PI), and Pourcelot’s resistance index, determined in the left (L) and right (R) middle cerebral arteries, in the L and R anterior cerebral arteries (ACA), and in the vertebrobasilar system. Sample members were grouped according to their ACE genotype and comparisons among groups were made for each of the parameters. The ANOVA test was used to compare the groups (P<0.05). Results: One thousand nine hundred and ninety-six adult people constituted the dataset. Seven hundred and seven individuals (707/1996, 35%) carried the DD genotype, 980 (980/1996, 49%) carried ID, and 309 (309/1996, 16%) carried the II genotype. With regard to RACA, individuals carrying DD showed the highest VM, while they showed a higher VD than those carrying II. With regard to LACA, individuals carrying the II genotype showed the highest PI. No other significant differences among groups for the analyzed parameters were found. Conclusions: the ACE genotype can predict the values of some brain haemodynamics parameters in ACAs. This relationship is laterality-dependent.
P12
FOS-related blood biochemical parameters and brain haemodynamics in the Spanish population 1
R. Perez, 2,3 J. Des, 1 C. Fraile, 1 L. Fernandez-Novoa, 1 L. Corzo, 1 R. Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain. 2 Ophthalmology Department. Ourense Hospital Center. Spain. 1
C-Fos is a cellular proto-oncogene belonging to the gene family of transcription factors. It is thought to have an important role in signal transduction, cell proliferation and differentiation. C-fos expression increases due to a variety of stimuli, including growth factors, cytokines, neurotransmitters, polypeptide hormones, stress and cell injury, and it is considered to have a critical function in regulating the development of cells in forming and maintaining the skeleton. Due to its enhanced expression while neurons discharge, it has been used as an indirect marker of neuronal activity because there are at least two different versions (alleles) of the c-FOS gene, called A and B. This work analyzes a series of ordinary blood parameters and brain haemodynamics in the Spanish population in order to evaluate if the c-FOS genotype predicts them. The blood parameters studied were serum levels of glucose, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, triglycerides, urea, creatinine, uric acid, total proteins, albumin, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, gamma-glutamyl transpeptidase, alkaline phosphatase, bilirubin, lactate dehydrogenase, calcium, phosphorus, chloride, sodium and potassium. Brain haemodynamics were assessed by non-invasive transcranial Doppler ultrasonography and included mean, systolic, and diastolic velocities, Gosling’s pulsatility index (PI), and Pourcelot’s resistance index, determined in the left (L) and right (R) middle cerebral arteries, in the L and R anterior cerebral arteries (ACA), and in the vertebrobasilar system. Sample members were grouped according to their c-Fos genotype and comparisons were made among groups for each of the parameters considered. The ANOVA test was used to compare the groups (P<0.05). Seven hundred and ninety-four adult people were included in the study. Five hundred and fifty-six individuals (556/794, 70%) carried the AA genotype, 198 (198/794, 25%) carried AB, and 40 (40/794, 5%) carried the BB genotype. With regard to serum total proteins, individuals carrying the AA genotype showed higher levels than those carrying AB. With regard to serum calcium, individuals carrying the AA genotype showed higher levels than those carrying BB. With regard to LACA, individuals carrying the BB genotype showed a higher PI than those carrying AA. No other significant differences were found. According to this, the c-FOS genotype is not a useful predictor for ordinary blood parameters and brain haemodynamics. 72
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P13
APOE-related blood biochemical parameters and brain haemodynamics in the Spanish population 1
R. Perez, 2,3 J. Des, 1 C. Fraile, 1 L. Fernandez-Novoa, 1 L. Corzo, 1 R. Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain. 2 Ophthalmology Department. Ourense Hospital Center. Spain
1
The APOE gene encodes apolipoprotein E. Lipoproteins are responsible for packaging cholesterol and other fats, carrying them through the bloodstream to adequate locations for processing. Maintaining normal levels of cholesterol is essential for the prevention of vascular disorders. There are at least three different versions (alleles) of the APOE gene, called e2, e3 (the most common), and e4. This work analyzes a series of ordinary blood parameters and brain haemodynamics in the Spanish population in order to know if the APOE genotype predicts them. Blood parameters included serum levels of glucose, total cholesterol (Ct), high-density lipoprotein-cholesterol (C), low-density lipoprotein (LDL)C, triglycerides (TG), urea, creatinine, uric acid, total proteins, albumin, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, gamma-glutamyl transpeptidase, alkaline phosphatase, bilirubin, lactate dehydrogenase, calcium, phosphorus, chloride, sodium and potassium. Brain haemodynamics, assessed by non-invasive transcranial Doppler ultrasonography, included mean (MV), systolic (SV) and diastolic (DV) velocities, Gosling’s pulsatility index, and Pourcelot’s resistance index (RI), determined in the left (L) and right (R) middle (MCA) and anterior (ACA) cerebral arteries, and in the vertebrobasilar system (BT). Individuals were grouped according to their APOE genotype and comparisons were made among groups for each of the variables. The ANOVA test was used for comparisons (P<0.05). Blood parameters were studied in 2324 adults. Three individuals (3/2324, 0.1%) carried the e22 genotype, 186 (186/2324, 8%) carried e23, 30 (30/2324, 1.3%) carried e24, 1565 (1565/2324, 67.4%) carried e33, 488 (488/2324, 21%) carried e34, and 52 (52/2324, 2.2%) carried the e44 genotype. With regard to serum Ct, individuals carrying the e23 genotype showed lower levels than those carrying e33, e34, or e44 genotypes, while individuals carrying e33 or e34 showed lower levels than those carrying e44. With regard to serum LDL-C, individuals carrying the e23 genotype showed the lowest levels, while those carrying e44 showed the highest. With regard to serum TG, individuals carrying the e23 genotype showed higher levels than those carrying e33 or e44, while individuals carrying the e34 genotype showed higher levels than those carrying e33 or e44. With regard to serum calcium, individuals carrying the e33 genotype showed higher levels than those carrying e34. With regard to serum phosphorus, individuals carrying the e23 genotype showed higher levels than those carrying e34. With regard to serum bilirubin, individuals carrying the e23 genotype showed higher levels than those carrying e34. Brain haemodynamics was studied in 794 adults. Two individuals (2/794, 0.3%) carried the e22 genotype, 66 (66/794, 8.3%) carried e23, 8 (8/794, 1%), 498 (498/794, 62.7%) carried e33, 196 (196/794, 24.7%) carried e34, and 24 (24/794, 3%) carried the 44 genotype. With regard to L- and RMCA, individuals carrying the e24 genotype showed the highest VM, VS and VD, while those carrying the e44 genotype showed the highest RI in LMCA. With regard to LACA, individuals carrying the e24 genotype showed the highest VM, VS, and VD, while individuals carrying e33 showed a higher VM than those carrying the e34 genotype. With regard to TB, individuals carrying the e44 genotype showed a higher RI than those carrying e33. No other significant differences were found. In summary, Ct, LDL-C and TG, and haemodynamics from L/RMCA and LACA are related to the APOE genotype.
P14
Significance of family history in the drug refractory epilepsies 1 2
Fernández Millares, V, 1 Alonso Cerezo, C., 2 Herrera Peco,I, 3 Pastor Gómez, J, 4 Hernando Requejo, V, García de Sola, R Service of Clinical Chemistry, Genetics Unit., 2 Neurosurgery, Epilepsy Unit, 3 Clinical Neurophysiology, Epilepsy Unit, 4 Neurologist, Epilepsy Unit. “La Princesa” University Unit, Madrid. 1
Epilepsies are a group of diseases characterized by recurrent and usually unpredictable seizures. They affect ~ 3% of the population at some time in life, with the peak impacts in childhood and the elderly, making Prevalent epilepsies the most serious chronic neurological disorder, affecting an estimated 42 million people worldwide. It is known that epilepsy can be caused by both genetic and acquired factors. Drug treatment of epilepsy is characterized by unpredictability of efficacy, adverse drug reactions and optimal doses in individual patients. Moreover, a substantial fraction of patients, ~ 33%, develop drug-refractory epilepsy despite optimal treatment. Objective: Assessment of family history in drug refractory epilepsies. Subjects and Methods: A prospective cross-sectional descriptive study of 76 families who have a patient diagnosed with drug refractory epilepsy and referred to the Neurosurgery Service of the University Hospital de la Princesa. We obtained the informed consent and the agreement of the hospital’s ethics committee. We used a structured questionnaire that included the completion of a genealogical tree, detailing the family for three generations. The data indicate the age, if alive or not, and the medical history of each family member. It was not possible to obtain medical reports from relatives of the testing case. We measured the frequency and percentages for relatives of each degree. December 2008
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Results: In our sample of 76 patients, 38 were male (50%) and other 38 women (50%). 56 of them have no family precedents of epilepsy (73.7%), 14 (18.4%) have a relative of first or second degree with epilepsy and 6 (7.9%) show two or more relatives with epilepsy. The analysis of family trees showed a family in which the epilepsy transmitted in an autosomal dominant manner. In other families with one or more members affected, first or second-degree distribution suggests a polygenic inheritance. Conclusions: There is ample evidence that response to AEDs is influenced by genetic factors. A higher proportion of cases with affected relatives compared with other studies is shown in our results. This may be due to the special characteristics of the patients referred to our Genetics Unit (long drug refractory epileptic history with surgery as the only therapeutic option). The present study supports the existence of family aggregation in Drug Refractory epilepsy and supports the presence of genetic bases in drug resistance. Nevertheless, the knowledge resulting from epilepsy pharmacogenetics is expected to be useful in the clinical treatment of epilepsy, in development of new AEDs and in the carrying out of AED trials.
P15
Clozapine-induced adverse effects in schizophrenic patients: Role of polymorphisms in P450 1A2 gene Ferrari M., Bortolaso P., Bolla E., Ceccon F., Marino F., Lecchini S., Vender S., Cosentino M Department of Clinical Medicine, University of Insubria – Varese, Italy Clozapine (CLO) is an atypical antipsychotic drug with a response rate of about 60% in schizophrenic patients with partial or no response to classic neuroleptics, however the risks of adverse reactions (ADR) such as sedation, hypersalivation, tachycardia, dizziness, constipation, orthostatic hypotension, comitial seizure, and weight gain are increased by CLO concentrations above 1000 ng/mL. Several lines of evidence suggest that interindividual variability in the theraputic response to CLO as well as CLO-induced ADR is affected by genetic factors. Cytochrome P4501A2 (CYP1A2) is the major enzyme involved in the metabolism of CLO (about 70%) and its activity is a very good predictor of CLO steady state concentrations. Several polymorphisms have been identified so far in the CYP1A2 gene (http://www.imm.ki.se/CYPalleles/cyp1a2.htm). Of particular interest, two polymorphisms in the promoter region of CYP1A2 gene: CYP1A2*1C (allelic frequency 2-5%) and CYP1A2*1F (allelic frequency 40%) have been associated with decreased enzymatic activity and higher inducibility (compared with CYP1A2*1 alleles), respectively. The aim of the present study was to evaluate the relationship between CLO-induced ADR and the haplotypes formed by CYP1A2*1F and CYP1A2*1C alleles in a population of schizophrenic patients diagnosed according to the classification of DSM IV-TR. Haplotypes were defined as extensive metabolizers (EM; *1/*1, *F/*F; *1/*1, *1/*F; *1/*C, *F/*F); intermediate metabolizers (IM; *1/*C, *1/*F; *C/*C, *F/*F); poor metabolizers (PM; *C/*C, *1/*1; *1/*C, *1/*1; *C/*C, *1/*F). A 3 mL blood sample from each patient was collected and genomic DNA was isolated by Perfect DNA Blood Minikit (Quiagen). Genotyping analysis was performed by Real-Time-PCR. Correlation between ADR and allelic frequency of polymorphisms in CYP1A2 gene was performed by Chi-square test with confidence interval of 95%. Value of P less than 0.05 was considered significant. The study was approved by the ethics committee of the “Ospedale di Circolo-Fondazione Macchi”, Varese, Italy. So far, 7 patients with CLO-induced ADR which led to therapy discontinuation and 8 patients without ADR have been included in the study. Causal relationship between CLO and ADR was estimated by the Naranjo’s algorithm confirmed by WHO-Uppsala criteria. Preliminary results show that no statistically significant difference was found between the allelic frequency of polymorphisms CYP1A2*C and CYP1A2*F in patients with ADR compared with patients without ADR (P= 0.172 and P=0.192 respectively). By contrast in patients with ADR the frequency of PM haplotype was significantly higher compared to patients without ADR (PM 100% ADR; 0% without ADR - IM / EM spirit ADR 33%, 66% without ADR. P = 0,048). These results suggest that CYP1A2 PM genotype is associated with the occurrence of CLO-induced ADR in schizophrenic subjects. Were the association confirmed in a larger sample, CYP1A2 genotyping should be further evaluated as an approach to reduce the risk of ADR occurrence during treatment with CLO.
P16
Difference in beta-amyloid deposition by genetic background Leonides Canuet, Takashi Morihara, Mikiko Yokokoji, Noriyuki Hayashi, Eriko Noshi, Koji Mori, Shinji Tagami, Masayasu Okochi, Toshihisa Tanaka, Takashi Kudo, Kojin Kamino, Masatoshi Takeda Department of Psychiatry, Osaka University Graduate School of Medicine,Japan leonides canuet [leocanon2002@gmail.com] Though efforts have been made in searching for Alzheimer’s disease (AD) risk genes, there is yet no risk gene identified other than apolipoprotein E, indicating methodological limitation of human genotyping. Taking advantage of amyloid pathology and strict control of environmental factors in the evaluation, we have compared amyloid deposition in the brains of mice with different genetic backgrounds. APP Tg mice (Tg2576) usually maintained by B6/SJL hybrid mice were crossed onto C57BL6/J, SJL/J and DBA2/J inbred strains to evaluate the amyloid pathology in the brain. A shortened life span was observed in APP Tg mice with B6 rich genetic background (B6 75%, SJL 25%). No significant life span change, however, 74
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was observed in the mice with either SJL or DBA rich genetic background. The levels of Abeta40 and Abeta42 in Triton and GuHCl fraction from cortex were measured by ELISA. Analysis of 59 APP Tg mice at the age of 12 months showed lower Abeta accumulation in mice with DBA rich genetic background compared with SJL or B6 rich genetic background. Significant differences (p<0.01, Turkey-Kramer) were observed in Abeta40 (-82%) and Abeta42 (-68%) levels in Triton fraction between SJL 84% B6 16% mice and DBA 75% B6 16% SJL 9% mice. Drastic differences (p<0.01, Turkey-Kramer) were observed in Abeta40 and Abeta42 levels from GuHCl fraction between SJL 69% B6 31% and DBA 75% B6 16% SJL 9% mice (-85% and -68%) and between SJL 69% B6 31% and DBA 50% B6 31% SJL 19% mice (-75% and -50%). We focus on SJL and DBA, because the difference in Abeta accumulation was the most drastic and both genetic backgrounds have no impact on life span. More animals are being analyzed and amyloid pathology modifier candidate genes will be investigated using arrays.
P17
MTX pathway polymorphisms useful as predictors of toxicity and outcome in childhood acute lymphoblastic leukemia 1
López-López E., 1 Martin-Guerrero I., 2 Ballesteros J., 3 Piñán MA., 4 Navajas A., 1 and García-Orad A Department of Genetics, Physical Anthropology and Animal Physiology, 2 Department of Neurosciences of the Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, Spain, 3 Department of Haematology and Haemotherapy, 4 Department of Paediatric Oncology of the Hospital de Cruces, Barakaldo, Spain.
1
Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer, and still an important cause of cancer-related death in children. Although the introduction of treatment protocols has improved survival, interindividual differences in drug responses are an important cause of resistance to treatment and adverse drug reactions. Pharmacogenetic studies are providing a rational base for further treatment efficacy and reduction of complications. The aim of the present study was to determine if there was a correlation between genetic polymorphisms and toxicity and/or outcome during therapy in paediatric ALL patients. We analyzed 13 polymorphisms of 10 genes in 55 paediatric ALL patients: 4 genes of the MTX pathway (MTHFR, TYMS, RFC1 and ABCB1), 1 gene of the 6-MP pathway (TPMT) and 5 genes involved in xenobiotic detoxification (CYP1A1, NQO1 and the GSTs GSTM1, GSTT1 and GSTP1). Data were analyzed by using the Fischer exact test. Several associations were found between polymorphisms and clinical parameters, such as that of the MTHFR C677T and A1298C polymorphisms and hepatotoxicity and renal toxicity respectively. Our results indicate that the polymorphisms in the MTX pathway may be useful as predictors of toxicity and outcome in ALL patients.
P18
Is the mechanism of the neuroprotection related to the restoration of the deviated balance between H1R and H2R expression? 1
J.Leszek, 2 T.Basinski, 1 A.Kiejna, 2 M.Jutel Department of Psychiatry,Wroclaw Medical University, Wroclaw, Poland, 2 Department of Internal Medicine and Allergy, Wroclaw Medical University, Wroclaw, Poland
1
Background:Beta-amyloid is responsible for cerebral damage contributing to the development of neurodegenerative disease.Donepezil is a most effective medication in the treatment of this pathology.Its exact mechanism of action is still unknown. The aim of this study: the investigation of the effect of Beta-amyloid on the viability of the cerebral tissue cells along with the investigation of mechanisms of protection by donepezil. Material and Methods: Monocytes were used as a model of microglial cells. Peripheral blood mononuclear cells(PBMC) were isolated by denstity gradient centrifugation. Monocytes were isolated by adhesion on the solid phase and by magnet activated cells sorting(MACS).The cells were stimulated with Betaamyloid (1-42)(0,1mM,1mM),(25-35)(0,1mM,1mM,10mM) and reverse sequence (35-25) (0,1uM,1uM,10uM) as a control. After culturing of the cells for 24h (37,0C, 5%CO2) cell viability was determined by trypan blue staining and cell apoptotic features were determined by annexin binding to phosphatidylserine (PS) and 7-aminoactinomycine (7-AAD) for DNA guanine- cytosine base pair and cytofluorimetric measurement. In addition, total histamine receptor expression and H1R or H2R was measured in fluorescence microscope by staining with histamine-fluorescein alone or after blocking of either histamine 1 receptors(H1R) or H2R with tripelenamine or ranitidine respectively. The effect of donepezil (o,o1uM,o,1uM, 1uM) was also investigated in the same cultures. Results: Beta-amyloid induced monocytes death (death rate 74%) was due to apoptosis.Donepezil (1uM) significantly protected the cells by increasing the viability (death 95%).The mechanism of monocyte death was necrosis and apoptosis as demonstrated by positive staining with both early (annexin) and late (7AAD) apoptotic markers. Total histamine receptor expression was decreased by Abeta (1-42) stimulation from 25,5% to 6,5% of stained cells. Stimulation of monocytes with Beta-amyloid (1-42) increased H1R expression from 4% to 18,5%. Preincubation of the cells with donepezil abrogated this effect. On the contrary, H2R expression was decreased by Beta-amyloid stimulation from 13% to 3%. Donepezil increased the Beta-amyloid supressed expression of H2R up to 8,5%. Conclusion: We conclude that Beta-amyloid induced cell death was due to apoptosis which can be hampered by donepezil. The mechanism of the protection may be related to the restoration of the deviated balance between HR and H2R expression. December 2008
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P19
Comparative study of APOE polymorphisms in the Mexican population: Alzheimer’s disease vs. Indians and mestizos ME Aguilar Aldrete, 2 MC Moran Moguel 2 L Sandoval-Ramírez, 2 RC Rosales-Gómez, 2 SA Gutiérrez-Rubio, 1 MC RodríguezVega, 1 MC Parra Bernal, 3 R Cacabelos
1
Department Of Public Health. University Centre of Health Sciences. University of Guadalajara, Mexico. 2 Biomedical Research Center. Mexican Institute of Social Security (IMSS). 3 EuroEspes, La Coruña, Spain
1
Introduction: The allele E4 of Apolipoprotein E (APOE) has been found to be an important factor in the development of Alzheimer’s Disease (AD), especially in late onset dementia. AD is the fourth cause of death in developed countries; in Mexico it is facing a new epidemic spread, though the APOE genotypes in this population remain unknown. Objective: to determine the APOE genotype in AD patients and in three control groups: Huichols, Tarahumaras and Mestizos, in the Mexican population. Materials and method: DNA was obtained from 40 AD patients, 45 mestizos, 57 Huichols and 24 Tarahumaras. Genotypes were identified by PCR and Hha I digestion in 20% poliacrylamide gel stained with silver nitrate. Results: The allele frequencies were, for the control groups: Huichols: E2=0.05; E3:0.76, E4:0.19; Tarahumaras: E2=0, E3=0.79, E4=0.21; Mestizos: E2=0.04; E3=0.84, E4=0.12, and in AD Patients: E2=0, E3=0.60, E4=0.40. Conclusions: The allelic E4 distribution in AD patients was greater than in other groups. E4 distribution in AD patients or Tarahumaras showed significant differences compared with the mestizo group. The allelic distribution in Tarahumaras or Huichols was different compared with AD patients. The most frequent allele in the control groups is E3 and the E4 allele is more frequent in AD patients. Allelic
Frequencies
GROUPS E2
E3
E4
Mestizos
0.04
0.84
0.12
Huichols
0.05
0.76
0.19
Tarahumaras
0
0.79
0.21
AD Patients
0
0.60
0.40
P20
A pro-inflammatory multi-gene polymorphism profile to predict the risk of Alzheimer’s disease: A working tool for early intervention in healthy subjects 1
Damiano Galimberti, 2 Federico Licastro, 2 Martina Chiappelli, 3 Vittorio Lucchini and 3 Flavio Garoi President of A.M.I.A. – Italian Association of Antiaging Physicians, 2 Department of Experimental Pathology, School of Medicine, University of Bologna, NGB Genetics, University of Ferrara, Italy
1 3
A number of studies have associated risk with certain polymorphisms in genes encoding inflammatory mediators, since alleles that favor increased expression of immunological mediators such as cytokines or acute phase proteins, are more frequent in patients with Alzheimer’s disease (AD) than controls. However, few attempts to concomitantly evaluate allele association from different genes with AD and its clinical progression have been systematically performed. A comprehensive assessment of several inherited variations of immune related genes upon AD risk, and their relative importance compared to APOE has been previously reported (Licastro F., et al Neurobiol Aging, 2006). An extended risk profile including several genes with immune regulatory functions or lipid metabolism influence has been determined in a large case control study (Licastro F., et al Neurobiol Aging, 2006). 76
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A multi-gene risk profile has emerged by case control investigations where gene polymorphism association with AD has been tested. Only polymorphism with a positive significant association with AD has been included in the multigene risk profile. One of the alleles of each polymorphism has resulted associated with an increased risk of developing AD and their relative relevance to the disease and the rate of cognitive decline has been previously assessed in several case control studies (Chiappelli M., et al. Expert Rev. Neurotherapeutics, 2006). Moreover, the concomitant presence of different AD associated alleles increased several fold the risk of the disease (Licastro F., et al, Neurobiol Aging, 2006). Therefore, each allele has been assigned a differential score for the disease. A new methodology has been set up using a real time PCR method to perform the concomitant evaluation of several gene polymorphisms upon a small sample of DNA. To test this multi gene risk profile no blood sample is necessary. In fact, genomic DNA is purified by a small number of cells collected by brushing oral mucosa. Multi probes methodology by real time PCR technique has been set up to determine polymorphism in the following genes: apolipoprotein E (APOE), alpha-1- antichymotrypsin (ACT), interleukin-1 (IL-1beta), IL-10, vascular endothelial growth factor (VEGF) and hydroxy-methyl-glutaryl CoA reductase (HMGCR). A healthy subject can ask to be genotyped and each allele of the above multi gene profile will contribute to individual total score for the risk of developing the age associated cognitive decline and AD as assessed by a specifically developed statistical evaluation. According to our methodology it is possible to categorize healthy subjects in three classes with low, medium and high intrinsic risk of AD. Subjects with medium or high scores for the risk profile will be offered multi disciplinary medical, radiological, neurocognitive and laboratory further assessment along with a clinical follow up. This approach is useful to define individual risk profile which can lead to personalized early treatment with the aim to prevent or significantly delay the manifestation of age- related cognitive decline and clinical dementia.
P21
Mimovax: A novel approach for a safe vaccine for Alzheimer’s disease A. Álvarez, 1 R. Cacabelos, 2 M. Mandler, 2 F. Mattner
1
1 2
EuroEspes Biomedical Research Centre, Santa Marta de Babío, Bergondo, 15166-A Coruña, Spain; e-mail: neurofarma@euroespes.com, AFFiRiS GmbH; Campus Vienna Biocenter 2; Viehmarktgasse 2A, A-1030 Viena (Austria)
Alzheimer’s disease (AD) is characterized by the abnormal accumulation of Amyloid-ß peptides in the brain. In animal models it has been found that immunotherapeutic treatment targeting full length Aß reduces Aß burden and has a beneficial impact on the disease progression. However, the first phase II clinical vaccination trial using Aß42 as antigen had to be discontinued due to severe neuroinflammatory side effects including brain infiltration by autoreactive T-cells. Therefore, a safe and effective AD vaccine has to avoid the formation of autoreactive T cells and should exclusively induce an Aß specific antibody-based immune response. The aim of the Mimovax project is to develop a novel and effective vaccine avoiding the development of immunological complications. The technology of mimotopes (AFFITOPES) has been developed to create antigens, lacking native Aß peptide sequences. With this vaccination approach the antibodies generated are exclusively reacting with the pathological Aß molecules but not with parental structures like APP. Since these mimotopes do not contain potential T-cell epitopes the induction of autoreactive T-cells is avoided. Several mimotopes have been used to vaccinate transgenic mice overexpressing human APP (hAPP). Vaccination of hAPP transgenic mice with mimotopes leads to a significant reduction of amyloid plaque load and associated pathological alterations, improves cognitive functions, and does not induce detectable Aß specific T-cells in treated animals. Thus the AFFITOPE technology could provide a novel treatment strategy with improved safety for use in AD patients. The next step is to evaluate the safety and tolerability of mimotopes developed during the Mimovax project in a Phase I clinical trial with AD patients. Supported by the Sixth Framework Program of the EC (Contract No. LSHB-CT-2006-037702)
P22
Presenilin-related biochemical and brain hemodynamic parameters in patients with Alzheimer’s disease Carmen Fraile, Rocío Martínez, Natalia Cacabelos-Pérez, Lola Corzo, Lucía Fernández-Novoa, Ramón Cacabelos-Pérez, Ramón Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain The presenilin-1 (PS1) gene (14q24.3) has been associated with Alzheimer’s disease (AD). More than 100 PS1 mutations have found in AD cases worldwide. Polymorphic variants in the PS1 gene have also been associated with increased risk for AD. We have studied the distribution of PS1 polymorphic variants in 100 AD patients (54F, 46M, age range: 51-93 years) as well as biochemical and brain hemodynamic parameters associated with different PS1 variants. The distribution of PS1 genotypes among AD was 31.68% PS1-1/1, 48.96% PS1-1/2, and 11.52% PS1-2/2. Patients harbouring the PS1-1/1 genotype were the youngest of the AD population and those carrying the PS1-2/2 genotype were the oldest of the cluster. December 2008
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PS1-2/2 cases also showed the most deteriorated cognitive conditions. No differences among different PS1 genotypes have been found in the serum levels of ApoE, total cholesterol, LDL-cholesterol, VLDL-cholesterol, HDL-cholesterol, triglycerides and Ace; however, serum Nitric Oxide (NO) levels were found significantly higher in patients with the PS1-1/2 genotype as compared with PS1-1/2 cases (p<0.05). Blood histamine levels and serum beta-amyloid levels were higher in PS1-2/2 than in PS1-1/1 and PS1-1/2. We have also studied PS1-related brain hemodynamic parameters. Standard hemodynamic parameters assessed by non-invasive transcranial Doppler ultrasonography included the following: (a) mean velocity (Mv), (b) systolic velocity (Sv), (c) diastolic velocity (Dv), (d) the pulsatility index of Gossling (PI = (Sv-Dv)/Mv), and (e) the resistance index of Pourcelot (RI = (Sv-Dv)/Sv). These parameters have been measured in the left middle cerebral artery (LMCA) of the patients. PS1-2/2 carriers exhibited higher values in Mv, Sv, and Dv than both PS1-1/1 and PS1-2/2 carriers. These data together seem to indicate that PS1 variants differ in vascular hemodynamic function. It is likely that NO- and histamine-related mechanisms may influence cerebrovascular disorders in PS1-associated AD cases. (Supported by the International Agency for Brain Research and Aging, IABRA)
P23
CYP2D6-related biochemical and brain hemodynamic parameters in patients with Alzheimer’s disease Carmen Fraile, Ruth Llovo, Rocío Martínez, Natalia Cacabelos-Pérez, Lola Corzo, Lucía Fernández-Novoa, Ramón Cacabelos-Pérez, Ramón Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain CYP2D6 variants, expressing phenotypes of extensive (EM), intermediate (IM), poor (PM), or ultra-rapid metabolizers (UM), influence biochemical parameters, liver function, and vascular hemodynamic parameters which might affect drug efficacy and safety. We have studied 155 AD cases (81F, 74M, age range: 42-90 years). Blood glucose levels are found elevated in EMs (*1/*1 vs *4/*10, p<0.05) and in some IMs (*4/*10 vs *1xN/*4, p<0.05), whereas other IMs (*1/*5 vs *4/*4, p<0.05) tend to show lower levels of glucose compared with PMs (*4/*4) or UMs (*1xN/*4). The highest levels of total-cholesterol are detected in the EMs with the CYP2D6*1/*10 genotype (vs *1/*1, *1/*4 and *1xN/*1, p<0.05). The same pattern has been observed with regard to LDL-cholesterol levels, which are significantly higher in the EM-*1/*10. In general, both total cholesterol levels and LDL-cholesterol levels are higher in EMs (with a significant difference between *1/*1 and *1/*10), intermediate levels are seen in IMs, and much lower levels in PMs and UMs; and the opposite occurs with HDL-cholesterol levels, which on average appear much lower in EMs than in IMs, PMs, and UMs, with the highest levels detected in *1/*3 and *1xN/*4. The levels of triglycerides are very variable among different CYP2D6 polymorphisms, with the highest levels present in IMs (*4/*10 vs *4/*5 and *1xN/*1, p<0.02). In general terms, the best cerebrovascular hemodynamic pattern is observed in EMs and PMs, with higher brain blood flow velocities and lower resistance and pulsatility indices, but differential phenotypic profiles are detectable among CYP2D6 genotypes. More striking are the results of both the pulsatility index (PI=(Sv-Dv)/Mv) and resistance index (RI=(Sv-Dv)/Sv), which are worse in IMs and PMs than in EMs and UMs. We have also studied the association of GOT, GPT, and GGT activity with the most prevalent CYP2D6 genotypes in AD. UMs and PMs tend to show the highest GOT activity and IMs the lowest. Significant differences appear among different IM-related genotypes. The *10/*10 genotype exhibited the lowest GOT activity with marked differences as compared to UMs (p<0.05 vs *1xN/*1; p<0.05 vs *1xN/*4)(42). GPT activity was significantly higher in PMs (*4/*4) than in EMs (*1/*10, p<0.05) or IMs (*1/*4, *1/*5, p<0.05). The lowest GPT activity was found in EMs and IMs. Striking differences have been found in GGT activity between PMs (*4/*4), which showed the highest levels, and EMs (*1/*1, p<0.05; *1/*10, p<0.05), IMs (*1/*5, p<0.05), or UMs (*1xN/*1, p<0.01)). These data together seem to indicate that CYP2D6-related biochemical parameters as well as brain hemodynamic parameters and transaminase activity may influence the drug metabolizing capacity of specific phenotypes. (Supported by the International Agency for Brain Research and Aging, IABRA)
P24
CYP genes and drug metabolism: major substrates for antidepressants, neuroleptics and benzodiazepines Rocío Martínez, Natalia Cacabelos-Pérez, Ramón Cacabelos-Pérez, Ramón Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain Retail prescription drugs filled at pharmacies in the USA (annual per capita by age) in 2007 were 3.9 (0-18 yrs), 11.2 (19-64 yrs), and 28.6 (>65 yrs). About 25% of the 100 most prescribed drugs in the USA and the European Union are psychotropic drugs, including antidepressants, neuroleptics and benzodiazepines. Most of these drugs are major substrates for enzymes of the Cytochrome P450 (CYP genes) superfamily. More than 80% of psychotropic drugs are metabolized by enzymes known to be genetically variable, including: (a) 78
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esterases: butyrylcholinesterase, paraoxonase/arylesterase; (b) transferases: N-acetyltransferase, sulfotransferase, thiol methyltransferase, thiopurine methyltransferase, catechol-O-methyltransferase, glutathione-S-transferases, UDP-glucuronosyltransferases, glucosyltransferase, histamine methyltransferase; (c) Reductases: NADPH:quinine oxidoreductase, glucose-6-phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehydehydrogenase, monoamine oxidase B, catalase, superoxide dismutase, trimethylamine N-oxidase, dihydropyrimidine dehydrogenase; and (e) cytochrome P450 enzymes, such as CYP1A1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A5 and many others. Polymorphic variants in these genes can induce alterations in drug metabolism modifying the efficacy and safety of the prescribed drugs. Drug metabolism includes phase I reactions (i.e., oxidation, reduction, hydrolysis) and phase II conjugation reactions (i.e., acetylation, glucuronidation, sulfation, methylation). The principal enzymes with polymorphic variants involved in phase I reactions are the following: CYP3A4/5/7, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1B1, CYP1A1/2, epoxide hydrolase, esterases, NQO1 (NADPH-quinone oxidoreductase), DPD (dihydropyrimidine dehydrogenase), ADH (alcohol dehydrogenase), and ALDH (aldehyde dehydrogenase). Major enzymes involved in phase II reactions include the following: UGTs (uridine 5’-triphosphate glucuronosyl transferases), TPMT (thiopurine methyltransferase), COMT (catechol-O-methyltransferase), HMT (histamine methyl-transferase), STs (sulfotransferases), GST-A (glutathion S-transferase A), GST-P, GST-T, GST-M, NAT2 (N-acetyl transferase), NAT1, and others. We have identified that 18% of neuroleptics are major substrates for CYP1A2 enzymes, 40% for CYP2D6, and 23% for CYP3A4; 24% of antidepressants are major substrates for CYP1A2 enzymes, 5% for CYP2B6, 38% for CYP2C19, 85% for CYP2D6, and 38% for CYP3A4; 7% of benzodiazepines are major substrates for CYP2C19 enzymes, 20% for CYP2D6, and 95% for CYP3A4. About 10-20% of Caucasians are carriers of defective CYP2D6 polymorphic variants that alter the metabolism of many psychotropic agents. When psychotropic drugs are administered by trial-and-error to patients with neuropsychiatric disorders who did not undergo pharmacogenetic assessment, it is likely that about 40-60% of this class of drugs are wrongly prescribed. Other 100 genes participate in the efficacy and safety of psychotropic agents. (Supported by the International Agency for Brain Research and Aging, IABRA)
P25
Genetic polymorphisms of Cytochromes P450: CYP2D6, CYP2C19, CYP2C9 and CYP3A5 in the Spanish population 1
R. Llovo, 1 I.Tudoli, 1 L. Fernández-Novoa, 1 S.Seoane, and 2 R. Cacabelos Department of Clinical Genetics and Genomics, EBIOTEC. 15165 Polígono I. de Bergondo, Bergondo, A Coruña, Spain; 2 Institute for CNS Disorders, EuroEspes Biomedical Research Center, Bergondo, La Coruña, Spain 1
Introduction: Cytochrome P450 (CYP) enzymes are involved in the biotransformation of large number of xenobiotic compounds. The major polymorphic CYP enzymes, CYP2D6, CYP2C19, CYP2C9 and CYP3A5 metabolize about 80% of commonly prescribed drugs. The activity of these enzymes is characterized by a broad inter-individual and inter-ethnic variability. The mutations in CYPs genes can encode enzyme products with abolished (poor metabolizer, PM), reduced (intermediate metabolizer, IM), normal (extensive metabolizer, EM) or increased (ultrarapid metabolizer, UM) or rapid metabolizer (RM in CYP3A5) enzyme activity. Objective: To determine the prevalence of the most common allelic variants of the Cytochrome P450 enzymes CYP2D6 (*1, *3, *4, *5, *6 and *1xN), CYP2C19 (*1, *2, *5), CYP2C9 (*1, *2, *3) and CYP3A5 (*1, *3) and to assess genotype and phenotype frequencies in the Spanish population. Materials and Methods: Genotyping was performed by a combination of long-PCRs, and allele-specific Real-time PCRs in a total of 1672, 1628, 1563 and 498 Spanish individuals for CYP2D6, CYP2C19, CYP2C9 and CYP3A5 studies, respectively. Results: The frequencies distribution of CYPs polymorphic alleles, genotypes and phenotypes corresponded to the frequencies of other European populations. Genotyping for polymorphic CYP2D6 reveals that 4.7% of total subjects are poor metabolizers and 6.4% are ultrarapid metabolizers. The most common inactive allele in this population is CYP2D6*4 (frequency 0.16). In the CYP2C19 study only the CYP2C19*2 mutant allele (frequency 0.14) was detected. The phenotype frequencies of CYP2C19 were 1.3% of poor metabolizers and 26% of intermediate metabolizers. The phenotype frequencies of CYP2C9 were 5.3% of poor metabolizers and 33.1% of intermediate metabolizers. The phenotype frequencies of CYP3A5 were 1.2% of rapid metabolizers and 15.5% of intermediate metabolizers. The Hardy-Weinberg equilibrium was confirmed for all genotype frequencies. Conclusions: The CYP2D6, CYP2C19, CYP2C9 and CYP3A5 genotype frequencies found in the Spanish population were similar to those found in other European studies. The data suggest that the treatment of Spanish patients can be improved via the clinical use of CYP2D6, CYP2C19, CYP2C9 and CYP3A5 genotyping and the pharmacogenetic assessment of drug metabolic pathways. December 2008
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P26
Pharmacogenetic patterns of CYP2C9 and VKORC1 in the Spanish population 1
R. Llovo, 1 L. Fernández-Novoa, 1 S.Seoane, 1 I. Tudoli and 2 R. Cacabelos 1 Department of Clinical Genetics and Genomics, EBIOTEC. 15165 Polígono I. de Bergondo, Bergondo, A Coruña, Spain; 2 Institute for CNS Disorders, EuroEspes Biomedical Research Center, Bergondo, La Coruña, Spain
Introduction: Some anticoagulant drugs like Acenocoumarol and Warfarin (coumarin anticoagulants) have a narrow therapeutic window. Small variations in dosing may result in hemorrhagic or thrombotic complications. There are some clinical factors that affect the maintenance of a stable dose such as age, gender, body surface, diet, and liver function. The incorporation in the recent years of genetic factors like polymorphisms of CYP2C9 and VKORC1 genes has improved drug dosage. The CYP2C9 isoenzyme is responsible for the formation of inactive metabolites of this kind of anticoagulants. The CYP2C9 gene is polymorphic, with a series of alleles that encode enzymes with null or minimum catalytic activity. CYP2C9 alleles affect the half life of coumarinic anticoagulants. VKORC1 is the target of this type of anticoagulants. It is well established that common polymorphisms in regulatory regions of the VKORC1 gene correlate strongly with the therapeutic response to coumarin treatments. To reduce the risk, clinicians can estimate the initial therapeutic dose by genotyping these genes and using dosing algorithms. With these tools it is possible to identify patients who need a moderate or a marked dose adjustment. Moreover, genotypic or phenotypic studies of these genes do not exist in the Spanish population. Objective: The objective of this investigation is to determine the prevalence of the most common allelic variants of the genes involved in the safety and effectiveness of anticoagulant therapy, such as CYP2C9 (*1, *2, *3) and VKORC1 (G-1639A), and to predict genotype and phenotype frequencies in the Spanish population. Materials and Methods: Genotyping was performed using real-time PCRs in 265 DNA samples obtained from Spanish individuals. The different phenotypes were calculated analyzing the relative contribution of the genotype of each gene. This classification was realized inferring a probable “initial dose adjustment” (IDA) of the treatment based on the contribution of age, CYP2C9 and VKORC1 genotype and body size, using a dosing algorithm (Sconce et al, 2005). The three different phenotypes are designated as patients with low-dose requirements, moderate-dose requirements and patients with normal-dose requirements. Results: The frequency distribution for CYP2C9 and VKORC1 polymorphic alleles and genotypes corresponded to the frequencies of other European populations. Genotyping for CYP2C9 revealed that 5% of total subjects were poor metabolizers and 33% were intermediate metabolizers. The most common inactive allele in this population was CYP2C9*2 (frequency 0.16). The genotype frequencies of VKORC1 were 56.67% of G-1639 allele and 43.33% of A-1639 allele. The distribution of phenotypes in the Spanish population is about 47.55% for normal-dose patients, 39.25% for moderate-dose patients and 11.7% for low-dose patients. Conclusion: Both allelic and genotypic frequencies of the CYP2C9 and VKORC1 gene are similar to other European populations. These data suggest that there is a proportion of the Spanish population that could improve the safety and effectiveness of anticoagulant therapy by means of pharmacogenetic tools.
P27
Important role of novel vascular risk factors to decrease vascular risk in patients with dementia Lola Corzo, Susana Rodríguez, Ramón Cacabelos Euroespes Biomedical Research Center. Santa Marta de Babío s/n. 15166 Bergondo, La Coruña, Spain Increasing evidence indicates that vascular disease contributes to cognitive impairment and dementia. Novel vascular risk factors (NVRF) and their markers represent a new strategy to discriminate vascular risk in populations in which traditional risk factors are not present. These factors are related to proinflammatory state, prothrombotic state and endothelial dysfunction. To clarify the role of NVRF in dementia is important because most of them are modifiable, in contrast to other risk factors of dementia. We analysed Apolipoprotein A1, Apolipoprotein B, Homocysteine (HCY), Lipoprotein (a) [Lp(a)], Fibrinogen and ultrasensitive reactive C protein in the general population older than 50 years (n=55), in non-demented subjects (n=26) and in patients with mild cognitive impairment (MCI) and dementia (n=25). All of them showed low-moderate vascular risk according to SCORETM scale for low risk European countries. In the general population, we found high levels of NVRF, increasing vascular risk respect to SCORETM vascular risk stratification scale. In MCI/ dementia, data showed a higher frequency of high levels of HCY (36.0% vs. 15.4%) and Lp(a)(40.0% vs. 15.4%) compared with non80
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dementia group. The other NVRFs presented similar frequencies in the two groups. Elevated serum HCY (>12 µmol/L) is associated with endohelial-related mechanisms, contributing to atherosclerosis. Lp(a) is an independent vascular risk factor and concentrations higher than 30 mg/dL increase two-fold the risk of vascular events promoving thrombosis and interfering with fibrinolysis. All these NVRFs were associated with increased risk to develop dementia. The HCY and Lp(a) associated-higher vascular risk observed in dementia could be modified by HCY and LP(a)-lowering treatments or by changing daily habits. According to our data, we suggest that NVRF analysis, especially HCY and Lp(a) analysis, can improve the prediction of vascular risk status in dementia and their knowledge might be very useful to decrease vascular risks in these patients.
P28
IL-6 and IL-6R gene polymorphisms and risk of stroke and neurodegeneration 1
Juan C Carril, 1 Indara Tudoli, 2 Ramón Cacabelos Department of Genetics, EBIOTEC, A Coruña, Spain; 2 Institute for CNS Disorders and Genomic Medicine, EuroEspes Biomedical Research Center, A Coruña, Spain 1
Circulating markers of inflammation are associated with risk of atherosclerosis, myocardial infarction, stroke, and the progression of autoimmune disease, although the reasons for these associations remain unclear. It is now widely recognized that atherosclerosis is a specific example of a chronic inflammatory response mainly to dyslipidemia and other risk factors. The foam cells and activated endothelium may also produce proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumour necrosis factor alpha (TNFA), which promote the further development of the inflammatory response. We have genotyped two single nucleotide polymorphisms located in the interleukin 6 (IL-6) promoter region (-573 C>G), that affect gene transcription; and, the 1510 A>C transversion inside the interleukin 6 soluble receptor (IL-6R) sequence. The study population comprised 74 patients with history of cerebral vascular disease, 65 neurodegenerative patients, and 37 control subjects without history of ischemic or hemorrhagic stroke, or other cerebral diseases, aged over 50 years. Significant associations with both the -573 C>G and 1510 A>C SNPs and cerebral vascular disease were observed, but not for the IL-6R polymorphism and neurodegenerative patients. This association was attributable to an increased prevalence of the IL6*-573G allele (P<0.05) and the IL6R*1510A allele (P<0.05) in cerebral vascular patients versus healthy controls.
P29
Logistic regression as a useful tool to explain genetic risk of stroke 1
Juan C Carril, 1 Ruth Llovo, 1 Lucía Fernández-Novoa, 2 Ramón Cacabelos Department of Genetics, EBIOTEC, A Coruña, Spain; 2 Institute for CNS Disorders and Genomic Medicine, EuroEspes Biomedical Research Center, A Coruña, Spain 1
Vascular diseases are the most common cause of mortality worldwide, accounting for approximately 30% of all deaths. About 25% of the population aged above 65 years has evidence of silent brain infarcts. 25% of men and nearly 20% of women aged 45 years can expect to have a stroke if they live to their 85th year. Prevalence is higher in Asian, Hispanic and Afro American populations. In Spain, the incidence is about 156 new cases per 100.000 inhabitants each year. We use the Likelihood Ratio (LR) to estimate the posterior probability of disease that is influenced by genetic and environmental factors. The LR reflects the probability that a patient with the disease has an observed test result, compared with the probability that a patient without the disease has the same result. The study population comprised 263 patients with history of cerebral vascular disorders (126 men and 137 women) and 43 control subjects (26 men and 17 women) without history of ischemic or hemorrhagic stroke, or other cerebral diseases, aged over 50 years. Six genetic polymorphisms well-established as genetic variants of vascular risk are genotyped and included in the analysis as genetic factors (APOE*4, A2M*D, ACE*I, AGT*174M, NOS3REP*a, NOS3POL*T) as well as three systemic risk factors (HTA, BMI, and LDL-Cholesterol). Assuming a dominant model for the disease transmission, presence or absence of one risk allele means risk phenotype, and a conservative prevalence value of stroke in Spain, or a priori probability of 8,5%, we can observe the additive effect of genetic risk variants in the total genetic risk or a posteriori probability. The model of logistic regression shows the individual gene contribution to the overall genetic risk. In this way we can explain the higher or lower genetic predisposition of any genotypic combination in function of the presence of more or less risk variants and we can establish a classification of genotypic combinations of stroke risk in three different status of individual genetic predisposition: low genetic risk (L), moderate genetic risk (M) and high genetic risk (H). Genetic counsellors can use this tool to explain the level of genetic predisposition to disease (non-modifiable) for patients acting over the systemic factors (modifiable) to decrease the cumulative risk to develop the disease, i.e., prescribing exercise and diet to prevent obesity, hypertension and hypercholesterolemia, and, in this way minimize the impact of unfavourable genetic susceptibility. December 2008
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P30
Vascular genetic risk factors in Alzheimer’s disease 1
L. Fernandez-Novoa, 1 S. Seoane, 1 R. Llovo, 1 I. Tudoli and 2 R. Cacabelos 1
Genetics Unit, EBIOTEC, Bergondo, A Coruña, Spain. 2 EuroEspes Medicine, Santa Marta de Babio, Bergondo, A Coruña, Spain
There is growing evidence that Alzheimer’s disease (AD) and cerebrovascular disease commonly coexist, and it is also known that vascular risk factors may increase the risk of AD. In this study, we analyzed four polymorphisms in four genes related to vascular function (APOE, ACE insdel, AGT M235T and eNOS3 E298D) in AD patients. A total of 198 AD patients were included in the study, 110 classified as possible AD and 88 as probable AD. A diagnosis of probable AD was made when the dementia could not be explained by any other disorder; a diagnosis of possible AD was made when the most likely cause of dementia was AD, but there were other disorders that could contribute to the dementia such as cerebrovascular disease or Parkinson’s disease (Luchsinger J et al). A Genetic Predisposition Score (Yiannakouris N et al) was developed for the four polymorphisms studied, each polymorphism contributed to 1 unit if the subject was homozygous for the risk allele, 0.5 units when the subject was heterozygous and 0 units if the subject was homozygous for the low-risk allele. According to this, AD patients could have score values between 0 (very low genetic risk) and 4 (very high genetic risk). The final objective of this study was to search for genetic risk factors associated with possible AD rather than probable AD. Our results showed significant differences in GPS between probable AD and possible AD, with a score above or equal to 2.5 we found an over representation of probable AD (chi-square = 4.17; p=0.04). The odds ratio for having probable AD with a GPS equal or above 2.5 was 0.493 (95% confidence interval: 0.248-0.978). According to these results, it seems that vascular genetic risk factors are more related to probable AD rather than possible AD.
P31
Effect of ACE genotype on brain bioelectrical activity I. Tellado, R. Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, EuroEspes Chair of Biotechnology and Genomics, Camilo Jose Cela University, Coruña, Spain. info@euroespes.com The ACE gene (17q23) encodes the angiotensin I-converting enzyme, which is a major component of the renin-angiotensin system. This system participates in the control of electrolytic balance and blood pressure and it may be associated with cerebrovascular and neurodegenerative pathologies, and with hypertension and arteriosclerosis, as well. This gene displays a polymorphism I/D expressed by insertion (I) or deletion (D) of Alu sequence of 287 base pairs in intron 16. There are 3 possible genotypes in the general population: D/D, I/D, and I/I. The allele D is associated with risk of heart attack, hypertension, diabetes mellitus, stroke, and cerebrovascular diseases. The ACE-I allele is associated with increased risk of developing late onset Alzheimer’s disease, and associations between the ACE I/D and Alzheimer’s disease have been reported by different studies in different populations. We have investigated the influence of ACE on brain bioelectrical activity using quantitative EEG. We applied qEEG in 1697 subjects: 605 ACE DD, 258 ACE II and 834 ACE ID. Brain mapping recordings were performed in all subjects in resting conditions with eyes closed by 19 scalp electrodes fixed in a elastic cap, located according to the International 10-20 system (Fp1, Fp2, F7, F3, Fz, F4, F8, T7, C3, Cz, C4, T8, P7, P3, Pz, P4, P8, O1 and O2). The electrode impedance was below 10 KΩ. All electrodes were referenced to linked ears [(A1+A2)/2]. The EEG signal was analog-filtered with a band pass of 0.5-25 Hz. EEG was visually inspected and free-artifact EEG epochs of 1020 ms were selected for analysis performed with Fast Fourier Transform (FFT). The following frequency bands were studied: delta (0.5-3.5 Hz), theta (4-7.5 Hz), alpha (8- 12.5 Hz) and beta (13-25 Hz). The averaged power spectrum (µV/ Hz) for each electrode was used as an analysis parameter. Statistic analysis shows no significant differences in brain bioelectrical activity among different ACE genotypes. This study suggests that the ACE genotype has no influence on brain bioelectrical activity.
P32
Polygenic risk factors related to neurodegeneration in Alzheimer’s disease: calculation of a genetic predisposition score (GPS) 1
L. Fernandez-Novoa, 1 S. Seoane, 1 R. Llovo, 1 I. Tudoli, 2 L. Corzo and 2 R. Cacabelos 1
Genetics Unit, EBIOTEC, Bergondo, A Coruna, Spain. 2 EuroEspes Medicine, Santa Marta de Babio, Bergondo, A Coruna, Spain
There is a great interest in the study of polymorphic markers at DNA level for the study of complex, multifactorial human diseases. The final goal of this strategy is to identify and characterize the effects of mutations influencing phenotypic variation and interindividual susceptibility to complex disorders. Using a genetic predisposition score (GPS) approach, based on the method reported by Yiannakouris N et al, we analyzed four polymor82
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phisms associated with AD predisposition. A total of 552 individuals were included in the study, 222 control subjects and 330 AD cases (176 classified as probable AD and 154 as possible AD). The GPS was developed for a total of four polymorphisms (APOE, ACE insdel, A2M V1000I and PSEN1 rs165932), each polymorphism contributed to 1 unit if the subject was homozygous for the risk allele, 0.5 units when the subject was heterozygous and 0 units if the subject was homozygous for the low-risk allele. According to this, individuals could have score values between 0 (very low genetic risk) and 4 (very high genetic risk). Our results showed significant differences in GPS between cases and controls, with a score above or equal to 2 we found an over representation of cases (chi-square = 14.89; p=0.000). The odds ratio for having probable AD with a GPS equal to or above 2 was 2.207 (95% confidence interval: 1.47-3.31). According to these results, calculating a GPS integrated by four polymorphims may help to identify people at risk of developing AD.
P33
Chronic activation of PBMCS in Alzheimer´s disease patients Lombardi VRM, Etcheverría I, Fernández-Novoa L, Seoane S, Cacabelos R EBIOTEC, Biotechnology Division, La Coruña, Spain Introduction: Reactive microglia appear in almost every type of central nervous system (CNS) disorder, including infection, trauma, stroke, degeneration, and demyelination. These inflammatory cells are the major source of CNS-derived cytokines, help to regulate wound healing in neuronal tissues, and serve as an important link between systemic immune responses and the CNS. The activation of brain-resident macrophages, the microglia, is thought to be a central event in the pathogenesis of Alzheimer´s disease (AD), but the primarily activating agent(s) is unknown so far. In order to better understand the disease-specific mechanism of microglial activation, we examined patients’ peripheral blood for both a related macrophage:lymphocyte activation and an accelerated apoptosis. Material and Methods: Blood samples of 10 ml were drawn from 60 patients clinically diagnosed for probable AD, 20 control individuals and 20 patients with vascular dementia (VD). The mononuclear cells were separated by standard centrifugation and studied for their phagocytosing capacity in a cell adhesion assay, for CD26, CD69, CD72 expressions and for their apoptotic activity by flow cytometry. Results: In the majority of AD cases (66%), both accumulation of apoptotic cells and correlated macrophage and lymphocyte activation were found dependent on the clinical severity. In the mild and moderate stages of dementia, the fraction of macrophages was increased and only a weak accumulation of apoptotic cells was seen. In the severe stages, the number of macrophages declined and the removal of circulating apoptotic cells was impaired. Short-term cultures of unstimulated PBMCs made these processes more evident. Conclusions: This study provided information on the expression of activation markers in peripheral immune cells. In the late-onset sporadic form of AD the peripheral blood macrophage system is chronically activated in response to an excess of apoptotic cells. Brain macrophages, critically involved in the immune:inflammatory regulation within the CNS, are suggested to undergo the same mode of activation and thus could trigger the chronic inflammatory condition found in the brain of AD patients.
P34
Microencapsulated VEGF-expressing cells as a potential treatment for Alzheimer´s disease 1 1
Aitziber Portero, 1 Gorka Orive, 1 Rosa María Hernández, 2 Carlos Spuch, 2 Desiré Antequera, 2 Eva Carro, Jose Luis Pedraz 1 2
Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria-Gasteiz, Spain Group of Neurodegenerative Diseases, Laboratory of Neuroscience, Research Center, Hospital “12 de Octubre”, Madrid, Spain. 1gorka.orive@ehu.es
Alzheimer´s disease (AD) is characterized by elevated levels of amyloid beta peptide in the brain that are associated with neuronal toxicity and vascular toxicity. There is increasing evidence that this disorder may be related to cerebrovascular dysfunction. Also, reduced microvascular density has been described in AD. Since vascular endothelial growth factor (VEGF) is known to be involved in brain angiogenesis, neuroprotection and cerebromicrovascular exchange of substrates and nutrients, replacement therapy with VEGF may have therapeutic effects in the treatment of AD. Cell microencapsulation technology is a promising approach for the controlled, localized and long term in vivo delivery of therapeutic peptides to the host. This technology allows cells to be surrounded by a semi-permeable membrane which protects them from mechanical stress and isolates them from the host’s immune response. We have evaluated the use of cell microencapsulation as a tool for the continuous release of vascular endothelial growth factor (VEGF). Initially, the angiogenic activity of VEGF-secreting BHK cells immobilized within 400-μm-sized microcapsules, releasing approximately 4 ng. of VEGF/24h/100 microcapsules, was evaluated in vitro. The subsequent intracerebral implantation of the microcapsules in December 2008
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both C57BL/6 and transgenic APP/PS1 mice showed a significant increase in the density of brain vasculature, and this effect was kept at least for 3 months after the implantation. Additionally, neuronal loss in the cortex was significantly reduced, as it was the expression of caspase-3, whose activity is related to increased cell apoptosis. Results obtained in the behavior test suggest that reducing the neuronal loss in the cerebral cortex may improve cognitive functions such as spatial memory. These results indicate that continuous VEGF delivery in the cortex could be a useful therapy in AD. References: (1) Yang, SP et al., Neurobiol. Aging., 25: 283-290, 2004. (2) Storkebaum, E et al., BioEssays, 26: 943-954, 2004. (3) Galvan, V et al., Mini. Rev. Med. Chem., 6: 667-669, 2006. (4) Zlokovic, BV et al., Trends. Neurosci., 28:202-208, 2005. (5) Humpel, C et al., Curr. Neurovasc. Res., 2: 341-347, 2005. (6) Orive, G et al., Nat. Med., 9: 104-107, 2003. (7) Lim, F et al., Science, 210: 908-909, 1980. (8) Folin, M et al., Int. J. Mol. Med. 17: 431-435, 2006. (9) Ayala-Grosso, C et al., Neuroscience, 141: 863-874, 2006 (10) Schwab, C et al., Exp. Neurol., 188: 52-64 2004. (11) Harguindey, S et al, Curr Alzheimer Res., 4(1): 53-65, 2007 (12) Harguindey, S et al., Neuropsych Dis & Treatment, 2008 (download link: - 3688 OLE-NDT-OA2008-Harguindey.pdf). Total number of words: 466
P35
Association of novel vascular risk factors and cognitive function in dementia Lola Corzo, Susana Rodríguez, Lucía Fernández-Novoa*, Marta Laredo, Verónica Couceiro, Ramón Cacabelos Euroespes Biomedical Research Center. Santa Marta de Babío s/n. 15166 Bergondo. La Coruña, Spain. * EBIOTEC, Bergondo Industrial State, c/ Parroquia de Guísamo, Parcela A-6, Nave F. 15165 Bergondo, La Coruña, Spain. Vascular dysfunction is associated with the pathogenesis of dementia; however, some studies have shown unclear evidence for an association between vascular risk factors and poorer cognitive performance. During the past years, in addition to the cholesterol metabolism-associated factors, several vascular risk markers related to processes of inflammation, coagulation dysfunction or platelet aggregation have emerged as independent risk parameters involved in atherosclerosis. In order to evaluate the possible association between cognitive status and emergent vascular risk markers (EVRM), we analysed ultrasensitive reactive C protein (RCP-us), fibrinogen (F), lipoprotein (a) (Lp(a)) and homocysteine (HCY) concentrations in serum/plasma from patients with dementia and non-demented control subjects. MMSE, ADAS-cog and GDS scales were used to evaluate cognitive function. Results showed that folate levels in healthy subjects were negatively associated with HCY and that RCP-us positively correlated with F in the two groups. These correlations were independent of APOE and MTHFR677C polymorphisms. Data did not show an association between EVRM and cognitive performance. To find changes in EVRM between mild cognitive impairment (MCI) and dementia, we classified patients as controls (MMSE= 28,77±1,2; GDS= 1,8±0,47), MCI (MMSE= 24,57±3,3; GDS= 3,0±0.68) and dementia (MMSE= 17,43±6,1; GDS= 4;0±0.73). Higher concentrations of HCY were found in dementia when we analysed them versus controls (P< 0.02) and MCI (P< 0.03). Lower RCP-us levels were observed in dementia versus controls (P< 0.02) and MCI (P< 0.02). No significant differences were observed in the other EVRM. According to genetic vascular risk markers, we found significantly higher levels of Lp(a) in APOE4 carriers (P= 0.031) and lower levels of RCP-us in CC carriers versus CT carriers of the MTHFR 677C polymorphism. Based on these findings, we conclude that inflammation, atherotrombotic and HCY-related vascular mechanisms seem to be associated with dementia, and these mechanisms are interrelated with each other. Only RCP-us and Lp(a) concentrations in serum, among all EVRM, are affected by genetic risk markers. No association exists between EVRM and cognitive impairment according to our data.
P36
Impact of APOE genotype on brain bioelectrical activity I. Tellado, R. Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain The APOE gene (19q13.2) encodes the apolipoprotein E that plays a role in lipid homeostasis. APOE has been identified as a major risk factor for dementia. Furthermore, it is involved in neurite outgrowth and differentiation, neural repair, and immunoregulation. Functional genomic studies have revealed the association of APOE with the phenotypic expression of biological traits (e.g. brain atrophy, cognitive decline rate, b-amyloid deposition, lipid metabolism dysfunction, immunologic dysregulation or therapeutic outcome). This gene presents 3 major alleles (e2, e3, and e4) associated with risk (APOE-4 allele) or protection (APOE-2 allele) for Alzheimer’s disease and other CNS disorders. We have investigated the influence of APOE on brain bioelectrical activity using quantitative EEG. We applied qEEG in 1914 subjects: 148 ApoE 23, 22 ApoE 24, 1308 ApoE 33, 392 ApoE 34 and 43 ApoE 44. Brain mapping recordings were performed in all subjects in resting conditions with eyes closed by 19 scalp electrodes fixed in a elastic cap, located according to the International 10-20 system (Fp1, Fp2, F7, F3, Fz, F4, F8, T7, C3, Cz, C4, T8, P7, P3, Pz, P4, P8, O1 and O2). The electrode impedance was below 10 KΩ. All electrodes were referenced to linked ears [(A1+A2)/2]. The EEG signal was analog-filtered with a band pass of 0.5-25 Hz. EEG was visually inspected and free-artifact EEG epochs of 1020 ms were selected for analysis performed with Fast Fourier Transform (FFT). The following frequency bands were studied: delta (0.5-3.5 Hz), theta (4-7.5 Hz), alpha (812.5 Hz) and beta (13-25 Hz). The average power spectrum (µV/ Hz) for each electrode was used as an analysis parameter. Statistic analysis shows 84
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significant differences in brain bioelectrical activity among different APOE genotypes: Theta 33vs34 in Central, Parietal and Occipital electrodes; Alpha 23vs24 in Temporal, Parietal and Occipital electrodes; Alpha 23vs33, Alpha 23vs34, Alpha 23vs44, Alpha 33vs44 and Alpha 34vs44 in all regions; Beta 23vs33 in Frontal, Parietal and Occipital electrodes; Beta 33vs44 in Parietal and Occipital electrodes; Beta 23vs34, Beta 23vs44, Beta 24vs44, Beta 33vs44 and Beta 34vs44 in all regions. Our results indicate that genotypes 24, 34 and 44 show a diminished fast activity as compared with other APOE genotypes. This study suggests that the APOE genotype influences the phenotypic expression of brain activity and indicates that the presence of the APOE-4 allele has a deleterious effect on brain function.
P37
Influence of APOE genotype on brain function as assessed by Optical Topography I. Tellado, C. Fraile, R. Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, EuroEspes Chair of Biotechnology and Genomics, Camilo Jose Cela University, Coruña, Spain. info@euroespes.com The APOE gene (19q13.2) encodes the apolipoprotein E that plays a role in lipid homeostasis. APOE has been identified as a major risk factor for dementia. Furthermore, it is involved in neurite outgrowth and differentiation, neural repair, and immunoregulation. This gene has 3 major alleles (ε2, ε3, and ε4) associated with risk (APOE-4 allele) or protection (APOE-2 allele) for Alzheimer’s disease and other CNS disorders. Functional genomic studies have revealed the association of APOE-related alleles with the phenotypic expression of biological traits (e.g. brain atrophy, cognitive decline rate, β-amyloid deposition, lipid metabolism dysfunction, immunologic dysregulation or therapeutic outcome). ( Cacabelos, R.; Aging Health, 2005, 1(2):303-348). We have investigated the influence of APOE on brain hemodynamics using Optical Topography (OT), which measures the hemodynamic response following brain activation and offers the spatial and temporal variability in the concentration and oxygenation of hemoglobin on the brain cortex. Brain optical topography patterns have been assessed in response to photic stimulation in 159 patients: 18 APOE 2/3 (11.25 %), age: 51.61±20.36 years (range: 18-79 years); 4 APOE 2/4 (2.5 %), age: 66±17.81 years (range: 44-83 years); 104 APOE 3/3 (65%), age: 54.95±19.69 years (range: 7-89 years); 25 APOE 3/4 (15.625%), age: 61.35±16.2 years (range: 25-83 years); and 8 APOE 4/4 (5%), age:63.4±10.39 years (range: 41-74 years). Statistic analysis shows significant differences in brain hemodynamics among different APOE genotypes: BDHb-2/3 vs BDHb-4/4 p<0.009; SDHb-2/3 vs SDHb 4/4 p<0.02; BTHb-2/3 vs BTHb 4/4 p<0.03; STHb-2/3 vs STHb-4/4 p<0.03; BOHb-2/3 vs BOHb-3/3 p<0.05; SOHb-2/3 vs SOHb-4/4 p<0.03; SDHb-3/3 vs SDHb-4/4 p<0.03; BTHb-3/3 vs STHb-3/3 p<0.03; STHb-3/3 vs STHb-4/4 p<0.001; BOHb-3/3 vs SOHb-33 p<0.05; SOHb-3/3 vs SOHb-4/4 p<0.007; BDHb-3/4 vs BDHb-4/4 p< 0.02; STHb-3/4 vs STHb-4/4 p<0.02; SOHb-3/4 vs SOHb-4/4 p<0.01. According to our results, the presence of the APOE-4 allele has a deleterious effect on brain hemodynamics altering brain oxygenation which may contribute to poor cerebrovascular perfusion and chronic brain damage.
P38
In vitro cellular model for Alzheimer’s disease with simultaneous overexpression of amyloid precursor protein, tau and amyloid-beta peptide Carmen Vale, 1 Juan A. Rubiolo, Eva Alonso, 2 Helga Rivas, 2 Lydia Giménez-Llort, 3 Mercedes R. Vieytes and 1 Luis M. Botana
1
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Spain. 2 Instituto de Neurociencia, Universidad Autónoma de Barcelona, Spain. 3Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Spain
1
Alzheimer’s disease (AD), is the most common cause of dementia and afflicts more than 15 million individuals worldwide. During the past two decades, the elucidation of susceptibility and causative genes for Alzheimer’s disease as well as proteins involved in the pathogenic process has greatly facilitated the development of genetically-altered mouse models. These models have played a major role in defining critical disease-related mechanisms and for evaluating novel therapeutic approaches, with many treatments currently in clinical trials owing their origins to studies initially performed in mice. Most AD cases occur sporadically, although inheritance of certain susceptibility genes enhance the risk. A small minority of AD cases (<1%) is inheritable and referred to as familial AD, and caused by mutations in genes encoding for either the amyloid precursor protein (APP), presenilin-1 (PS1), or presenilin-2 (PS2). Definitive diagnosis of AD occurs during post-mortem examination upon detection of two hallmark pathologies: amyloid plaques, which consist of the amyloid-β peptide (Aβ) and intraneuronal aggregates composed of the microtubule associated protein Tau. In the year 2003, researchers from the University of California, Irvine, December 2008
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developed the triple-transgenic mouse model (3×Tg-AD) for Alzheimer’s disease, which harbors the PS1M146V, APPSwe, and tauP301L human transgenes, and this mouse model recapitulates the most salient features of human AD. Here, we have characterized the presence of the main pathologic hallmarks of AD (overexpression of tau and Ab in an in vitro neuronal model obtained from 3xTg-AD mice) and compared the results with the expression of these proteins in the same cultures from non 3xTg-AD mice. As evidenced by inmunocytochemistry combined with confocal microscopy, primary cultures of cortical neurons obtained from 3xTg-AD mice showed overexpression of APP, Ab, total tau and three different phosphorylated isoforms of tau. Western blot analysis of the levels of tau and beta-amyloid in cultures from 3xTg-AD and non 3xTg-AD mice corroborated the results obtained in inmunocytochemistry experiments. In addition primary neuronal cultures from 3xTg-AD mice showed increased levels of extracellular b-amyloid and decreased levels of the synaptic marker synaptophysin. Therefore, primary neuronal cultures from 3xTgAD mice provide the first in vitro model with simultaneous overexpression of APP, tau and Ab and could provide a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed in vitro.
P39
Neuropsychiatric symptoms and APOE-4 in cognitively impaired patients M. Laredo, V. Couceiro, A. Álvarez, C. Sampedro, R. Cacabelos. Coruña, Spain EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain Frequency (NPIF), severity (NPIS) and total (NPIT) neuropsychiatric symptom (NPS) scores, and their correlations with cognitive and functional performance were investigated in APOE-genotyped vascular dementia (VaD), Alzheimer´s disease (AD) and mild cognitive impairment (MCI) patients. Patients with AD (N=160), VaD (N=46) and MCI (N=45) were assessed with the Neuropsychiatric Inventory (NPI), the Alzheimer´s disease Assessment Scale (ADAScog) and the disability in AD scale (DAD). The ApoE genotype was determined in all patients. Results of the present study indicate that: 1) in comparison with MCI, apathy and agitation were more frequent in AD and VaD patients, whereas aberrant motor behaviour was more prevalent only in AD cases; 2) irritability and sleep disorders appeared with a higher frequency in AD patients without the ApoE4 allele than in ApoE4 carriers; 3) NPIF, NPIS and NPIT scores were higher in AD and VaD than in MCI, and showed a significant increase in non-ApoE4 AD patients in contrast with ApoE4 carriers; 4) NPIF and NPIS scores correlated negatively with functional performance, but not with cognitive measures in both AD and VaD; 5) apathy, agitation, delusions and aberrant motor activity were the best correlates for disability, whereas delusions and agitation correlated negatively with cognitive performance. According to the present results it seems that NPS can contribute to differentiate senile dementia (VAD and AD) from MCI and have a stronger impact on functional than on cognitive impairment; and that the APOE genotype might influence the frequency of NPS in AD, other than those differentiating AD from MCI.
P40
Immuno-trophic regulation in Alzheimer’s disease: tnf-alpha/igf-i interactions A. Álvarez, C. Sampedro, L. Corzo, R. Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain Tumor necrosis factor-alpha (TNF-alpha) and insulin-like growth factor I (IGF-I) are involved in the pathogenesis of Alzheimer’s disease (AD). TNF-alpha concentrations are increased and IGF-I levels are reduced in the brains and CSF of AD patients. Experimental studies demonstrated that TNF-alpha antagonizes neuronal survival and brain amyloid clearance induced by IGF-I, whereas this peptide reverses neuronal apoptosis produced by TNF-alpha. According to these scientific data we postulated a hyperactivity of the peripheral TNF-alpha system and a negative interaction with IGF-I in patients with AD. Serum levels of TNF-a, soluble TNF receptors (sTNF-R I and II), total, dissociable and free IGF-I, and IGF binding proteins (IGFBP-1, IGFBP-3) were determined in patients with AD, vascular dementia (VaD) or mild cognitive impairment (MCI), and in controls. The effect of the treatment with a neurotrophic compound (Cere) on the expression of these factors was also investigated in AD patients. Serum TNF-alpha levels were significantly increased (p<0.01) and correlated negatively with dissociable IGF-I values in AD patients (Álvarez et al, Neurobiol Aging, 2007); while total, but not dissociable and free IGF-I levels appeared reduced (p<0.05) in comparison with controls. Concentrations of sTNF-RI and IGFBP-3, both with pro-apoptotic activity, were higher (p<0.01 and p<0.05) in AD patients than in controls. Cere treatment reduced circulating TNF-a and sTNF-RI, and increased free IGF-I levels as compared with placebo and on a dose-dependent manner in patients with mild-moderate AD. These results showing a negative interaction between serum TNF-alpha and IGF-I suggest that the opposite influence of inflammatory and growth factors on AD pathology has a peripheral representation, which can be modified with neurotrophic drugs such as Cere. 86
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P41
Nutraceutical properties of E-SAR-94010 (LipoEsar®): APOE-Related Nutrigenomic Profile Rocío Martínez, Ramón Alejo, Valter Lombardi, Lucía Fernández-Novoa, Lola Corzo, Margarita Alcaraz, Laura Nebril, Ángela Casas, Carmen Fraile, Ana Vallejo, Ramón Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain E-SAR-94010 (LipoEsar®) is a nutraceutical product extracted from Sardina pilchardus. In previous studies we have demonstrated the biological properties of this lipoprotein obtained by non-denaturing biotechnological processes from a biomarine source. E-SAR-94010 is a powerful lipid lowering compound, regulating lipid metabolism at the physiological level. This nutraceutical product is also able to reduce the size of atheroma plaques on the aortic wall of patients with atherosclerosis. In the present study we have investigated the influence of different APOE genotypes on the hypolipemic effects of E-SAR-94010 as well as on plasma transaminase activity in patients with chronic dyslipidemia. We have included 420 subjects (age: 55.24±17.71 yrs; range: 9-96 yrs; females=213; age: 57.04±17.68 yrs; range: 15-96 yrs; males=207; age: 53.38±17.58 yrs; range: 9-91 yrs), who received 500-1000 mg/day of ESAR-94010 in capsules of 250 mg each for one month. E-SAR-94010 significantly reduced the levels of total-cholesterol, LDL-cholesterol and triglycerides, with a mild enhancing effect on HDL-cholesterol levels. The effects of E-SAR-94010 on total-cholesterol and LDL-cholesterol were significantly relevant in patients harbouring the APOE-3/3 (66.38%)(p<0.00001) and APOE-3/4 genotypes (21.55%)(p<0.05), but not in carriers of the APOE-4/4 genotype (2.01%). In contrast, only APOE-4/4 carriers showed a significant increase in HDL-cholesterol levels. E-SAR-94010 significantly reduced triglyceride levels in all patients (APOE-3/3>APOE-3/4>APOE-4/4). Plasma transaminase activities (GOT, GPT, GGT) were also reduced in the total population of dyslipidemic patients; however, the best responders were those patients carrying the APOE-3/3 and APOE-4/4 genotypes, who showed a significant reduction in GOT activity. Reduced values of GPT activity were observed in APOE-3/4 and APOE-4/4 carriers; and GGT activity was only significantly reduced in homozygous APOE-3/3 patients. These data clearly confirm the lipid lowering effect of E-SAR-94010 in a large sample of patients with chronic dyslipidemia, as well as its effect on transaminase activity, probably reflecting an effect on liver steatosis, and also demonstrate that these effects are differentially influenced by APOE variants. (Supported by the EuroEspes Foundation and the International Agency for Brain Research and Aging, IABRA)
P42
APOE-related phenotypic profile in Alzheimer’s disease and vascular dementia Rocío Martínez, Carmen Fraile, Natalia Cacabelos-Pérez, Ramón Cacabelos-Pérez, Lucía Fernández-Novoa, Lola Corzo, Valter Lombardi, Iván Tellado, Rogelio Pérez, Marta Laredo, Verónica Couceiro, Antón Álvarez, Julio Des, Ramón Cacabelos EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, 15165-Bergondo, Coruña, Spain The inheritance of the APOE-4 allele is a major risk factor for dementia, either degenerative (Alzheimer’s disease, AD) or dementia with vascular component (DVC). We have studied the influence of the APOE genotype on the biological phenotype of patients with AD and DVC. The phenotypic profiles in randomly selected patients with AD (N=465; 296 females and 169 males; age: 70.90±10.12 years) or DVC (N=475; 260 females and 214 males; age: 69.59±10.42 years) included the following parametric variables: (i) age, (ii) gender, (iii) weight, (iv) height, (v) family history of dementia, (vi) ECG, (vii) Chest X-ray, (viii) CT-Scan, (ix) blood pressure, (x) blood biochemistry, (xi) thyroid function, (xii) metabolic factors, (xiii) hematology, (xiv) brain atrophy measures, (xv) brain hemodynamic parameters, and (xvi) immune markers. Age was similar in both groups. Females were more prevalent in AD (63.65% vs 36.35%) than in DVC (54.85% vs 45.15%). DVC patients were taller (p<0.05) and showed a higher body weight (71.48±13.48 Kg) than AD patients (67.47±11.88 Kg; p<0.001). The family history of dementia was similar in AD (76.80%) and in DVC (70.47%). An abnormal ECG was present in 40% of AD cases and in 54% of DVC patients. An atherosclerotic aortic arch was found in 79% of AD and in 80% of DVC in chest X-ray films. Blind CT-scan analysis by an independent neuroradiologist revealed that 42% of AD cases show an apparently normal CT-scan (2% normal in DVC); 41% AD cases exhibit microvascular pathology (56% DVC), and 17% of the AD cases show macrovascular pathology (42% DVC). Both systolic (p<0.001) and diastolic blood pressure (p<0.001) as well as the heart rate (p<0.05) were significantly higher in DVC than in AD. Blood glucose, cholesterol (CHO), LDL-CHO, urea, creatinine, proteins, albumin, phosphorous, bilirubin, CPK, sodium, potassium, chloride, TSH, T4, folate, vitamin B12, iron, and ferritine levels were similar in both groups, whereas HDL-CHO levels (p<0.03) were higher in AD; and triglyceride (p<0.02), uric acid (p<0.001), and calcium levels (p<0.02), as well as GOT (p<0.007), GPT (p<0.001), GGT (p<0.001), alkaline phosphatase (p<0.03), and lactate dehydrogenase (LDH) activities (p<0.03) were significantly higher in DVC than in AD. The values of the most relevant hematological parameters, including RBC (p<0.03), hematocrit (p<0.03), hemoglobin (p<0.03), leukocytes (p<0.007), lymphocytes (p<0.04), monocytes (p<0.03), and granulocytes (p<0.03) were found higher in DVC as December 2008
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compared with AD. Brain atrophy measures were similar in AD and DVC except left temporal lobe atrophy (p<0.05) and the inter-hippocampal distance (p<0.05) which were both significantly larger in AD. Surprisingly, most brain hemodynamic parameters were identical between AD and DVC except systolic blood flow velocity (p<0.04) and diastolic blood flow velocity (p<0.05) in the territory of the left anterior cerebral artery of AD patients, showing a diminished blood flow velocity in the anterior lobe of the dominant hemisphere. In contrast, all immune markers studied by flow cytometry analysis (CD3, CD4, CD7, CD8, HLA-DR, CD25, CD28, CD56, CD3+/HLA-DR+, CD4/CD8) showed similar values in AD and DVC. According to the results obtained with these biological parameters, the phenotypic profile of patients with DVC differs from that of AD patients in the following: (a) anthropometric values (weight, height), (b) cardiovascular function (ECG, heart rate), (c) blood pressure, (d) lipid metabolism (HDL-CHO, TGs), (e) uric acid metabolism, (f) peripheral calcium homeostasis, (g) liver function (GOT, GPT, GGT), (h) alkaline phosphatase, (i) lactate dehydrogenase, (j) red and white blood cells, (k) regional brain atrophy (left temporal region, inter-hippocampal distance), and (l) left anterior blood flow velocity. Some gender differences are also predicted, since the number of females with AD is almost twice higher than males, whereas in patients with DVC the number of females is only 10% higher than males. The distribution of the APOE genotypes in AD/DVC was the following: APOE-2/2: <0.1%/<0.1%; APOE-2/3: 4%/7%; APOE-2/4: 1%/2%; APOE-3/3: 55%/62%; APOE-3/4: 35%/26%; and APOE-4/4: 5%/3% (Table 3). The proportion of females/males (F/M) per genotype in AD(DVC) was 81.25%/18.75% (43.75%/56.25%) in APOE-2/3, 50%/50% (77.77%/22.23%) in APOE-2/4, 61.37%/38.63% (52.18%/47.82%) in APOE-3/3, 61.48%/38.52% (61.82%/38.18%) in APOE-3/4, and 75%/25% (66.67%/33.33%) in APOE-4/4 (Table 3). Gender: The distribution of females and males in the APOE-3/4 and APOE-4/4 genotypes of both AD and DVC was identical, and in the APOE3/3 genotype the number of females doubled that of men in AD and was similar in DVC (Table 3). Age: According to the age of onset, the youngest population was the APOE-4/4 in AD, but not in DVC, with a significant difference between AD44 and AD23(p<0.01), AD33 (p<0.02), and AD34 (p<0.004), and also between AD44 and DVC44 (p<0.05), indicating that the presence of the APOE-4 allele in homozygotes is a major risk factor for AD, probably anticipating the age of onset; however, this effect is not observed in DVC. Weight and Height: In general, DVC patients tend to be heavier than AD patients, except DVC patients with the APOE-4/4 genotype (DVC44) who are lighter than AD44. In both groups homozygous and heterozygous APOE-4 carriers showed a lower body weight, suggesting that potential changes in lipid metabolism associated with APOE gene products or metabolic effects may influence overall body mass. Besides these global observations, significant differences in body weight have been found in AD23 vs AD34 (p<0.05), AD33 vs AD34 (p<0.04), AD33 vs DVC33 (p<0.006), AD34 vs DVC34 (p<0.02), DVC23 vs DVC24 (p<0.05), DVC23 vs DVC33 (p<0.04), DVC23 vs DVC34 (p<0.01), DVC23 vs DVC44 (p<0.003), DVC33 vs DVC44 (p<0.01), and DVC34 vs DVC44 (p<0.05). Height was similar in AD and DVC, except in patients with the APOE-3/3 genotype where DVC33 patients were taller than AD33 (p<0.05). In general, APOE-3 carriers tend to be taller than APOE-4 carriers. In this regard, no major differences of height have been found among APOE genotype-related AD patients; in contrast, significant differences were identified in DVC23 vs DVC (p<0.05), DVC23 vs DVC34 (p<0.01), DVC23 vs DVC44 (p<0.05), and DVC33 vs DVC34 (p<0.03), suggesting that APOE might indirectly influence growth and body mass through different metabolic pathways and that other powerful regulators of growth and body weight are more influential in DVC than in AD. ECG : The ECG record was found similarly abnormal in all APOE genotypes in both groups and it does not appear that the presence of the APOE-4 allele exert a deleterious effect on cardiac function, except in APOE-2/4 carriers where abnormal ECG accumulates. Aortic atherosclerosis: More than 80% of the patients with AD or DVC show atherosclerotic signs in the aortic arch as revealed by visual inspection of chest X-ray films. This observation is slightly more frequent in DVC than in AD with no major APOE genotype-related differences, although APOE-4(+) carriers tend to show more aortic atherosclerosis signs than APOE-4(-) carriers. Cerebrovascular lesions : Cerebrovascular lesions in AD and DVC are not APOE-related. No cerebrovascular lesions are seen in the CT-Scan of 23% AD23, 60% AD24, 55% AD33, 35% AD34, and 47% AD44, and only in 7% DVC23, 4% DVC33, and 3% DVC34. Microvascular lesions are observed in 53% AD23, 20% AD24, 29% AD33, 49% AD34, and 47% AD44; and macrovascular lesions are apparent in 24% AD23, 20% AD24, 16% AD33, 16% AD34, and 6% AD44. In contrast, microvascular and macrovascular lesions are permanently present in DVC irrespective of the APOE genotype. Blood pressure: Systolic blood pressure (SBP) is significantly higher in DVC than in AD irrespective of APOE. SBP tends to be higher in AD34 and AD44; however, in patients with DVC, SBP is higher in DVC33 than in DVC34 and DVC44, indicating that other genetic factors that accumulate in DVC are responsible for hypertension. Significant differences in SBP values have been found between several APOE-related genotypes and between AD and DVC APOE-related genotypes, including AD33 vs DVC33 (p<0.001), AD34 vs DVC34 (p<0.05), DVC23 vs DVC24 (p<0.05), DVC-23 vs DVC33 (p<0.02), DVC24 vs DVC33 (p<0.004), DVC24 vs DVC34 (p<0.01), and DVC24 vs DVC44 (p<0.01). High diastolic blood pressure (DBP) values accumulate in DVC33, DVC34, and DVC44, with minimum variation in AD. Significant differences in DBP are present between AD33 and DVC33 (p<0.001), AD34 and DVC34 (p<0.03), AD44 and DVC44 (p<0.04), DVC23 and DVC33 (p<0.003), 88
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DVC23 and DVC34 (p<0.05), DVC23 and DVC44 (p<0.05), DVC24 and DVC33 (p<0.01), DVC24 and DVC34 (p<0.04), and DVC24 and DVC44 (p<0.05). Heart Rate: The pulse rate seems to be slower in APOE-4(+) than in APOE-4(-) in DVC and it is faster in DVC33 than in AD33. APOE-4(+) in AD and DVC shows the slowest pulse rate, suggesting that APOE might indirectly influence heart function. Significant differences in pulse rate have been observed between AD23 and AD44 (p<0.05), AD33 and DVC33 (p<0.02), AD23 and AD33 (p<0.01), and AD23 and AD34 (p<0.03), with no significant differences among APOE-related DVC patients. Blood biochemical parameters: Glucose levels are higher in DVC than in AD with APOE-related differential variation. In AD the lowest glucose levels are observed in APOE-4(+), whereas in DVC all APOE-genotypes tend to show indifferent high glucose levels, this indicating that APOE might slightly influence glucose metabolism in AD and that prodiabetogenic factors accumulate in DVC but not in AD. Significant differences in glucose levels have been found between AD44 and DVC44 (p<0.05), and between DVC33 and DVC34 (p<0.05)(Table 3). Total cholesterol (CHO) levels are very similar between AD and DVC, and tend to be higher in APOE-3/4 and APOE-4/4, demonstrating a direct influence of APOE-4 on CHO metabolism in both AD and DVC. The lowest CHO levels are detected in APOE-2/3 and APOE-2/4 carriers. Significant differences in CHO levels have been observed in the following groups: AD23 vs AD33 (p<0.007), AD23 vs AD34 (p<0.001), AD23 vs AD44 (p<0.005), AD33 vs AD34 (p<0.005), DVC23 vs DVC33 (p<0.05), DVC23 vs DVC34 (p<0.02), DVC23 vs DVC44 (p<0.01), DVC24 vs DVC44 (p<0.03), DVC33 vs DVC44 (p<0.05), and DVC34 vs DVC44 (p<0.05). No significant differences have been found in HDL-CHO in either AD or DVC APOE-related genotypes; however, LDL-CHO levels followed a similar pattern to that of total CHO with the highest levels expressed in APOE-3/4 and APOE-4/4 carriers in both AD and DVC. Significant differences in LDL-CHO levels have been identified in AD23 vs AD33 (p<0.002), AD23 vs AD34 (p<0.001), AD23 vs AD34 (p<0.001), AD23 vs AD44 (p<0.003), AD24 vs AD33 (p<0.05), AD24 vs AD34 (p<0.03), AD24 vs AD44 (p<0.05), and AD33 vs AD34 (p<0.004), with no major differences between AD and DVC, and no differences among APOE-related DVC patients, indicating that in DVC additional risk factors are regulating LDLCHO and/or that APOE particularly influences LDL-CHO in AD. The behavior of triglyceride (TG) levels in AD and DVC is completely different to that of CHO, with the lowest TG levels present in APOE-4/4 and the highest levels in APOE-3/4 and APOE-3/3 carriers, and also with a tendency to higher TG levels in DVC than in AD. Significant differences in TG levels have been found in AD33 vs AD44 (p<0.05), AD33 vs DVC33 (p<0.04), AD34 vs AD44 (p<0.05), AD34 vs DVC34 (p<0.04), DVC33 vs DVC44 (p<0.05), and DVC34 vs DVC44 (p<0.03). The levels of urea are similar in DV and DVC, but the lowest levels of urea are seen in APOE-4/4 carriers, especially in AD (AD33 vs AD44, p<0.04). Creatinine levels are also lower in APOE-4/4 than in any other APOE genotype in both AD and DVC. Significant differences in creatinine levels are found in AD23 vs AD44 (p<0.05), AD33 vs AD44 (p<0.05), DVC23 vs DVC33 (p<0.05), DVC23 vs DVC34 (p<0.02), and DVC23 vs DVC44 (p<0.01). Likewise, the levels of uric acid are also lower in APOE-4/4 carriers than in other genotypes, and higher in DVC than in AD, with significant differences in AD23 vs AD44 (p<0.05), AD33 vs AD44 (p<0.01), AD33 vs DVC33 (p<0.02), AD34 vs AD44 (p<0.05), and AD44 vs DVC44 (p<0.05). The blood protein levels also show some APOE-related differences in AD without changes in DVC and no differences between AD and DVC. The lowest levels of serum proteins are seen in APOE-4/4 carriers. Significant differences in protein levels have been found in AD23 vs AD24 (p<0.05), AD23 vs AD33 (p<0.02), AD23 vs AD34 (p<0.05), AD24 vs AD44 (p<0.05), AD33 vs AD44 (p<0.01), and AD34 vs AD44 (p<0.03). Albumin levels do not show any APOE-related variation in either AD or DVC. The levels of calcium also show differences between AD23 and AD44 (p<0.01), and between AD33 and AD44 (p<0.03) because of low calcium levels in AD44. In contrast, the levels of calcium are significantly higher in DVC34 as compared with AD34 (p<0.01). The levels of phosphorus do not vary in AD, and show significant differences in DVC24 vs DVC33 (p<0.04), and DVC33 vs DVC34 (p<0.05). Liver function is markedly deteriorated in DVC with important APOE-related differences in both AD and DVC. The lowest levels of transaminase activity are observed in APOE-4/4 carriers. GOT activity shows significant differences in AD33 vs AD34 (p<0.05), AD33 vs AD44 (p<0.01), and AD44 vs DVC44 (p<0.05). GPT activity is also significantly different in AD33 vs AD34 (p<0.001), AD33 vs AD44 (p<0.002), AD33 vs DVC33 (p<0.05), AD34 vs DVC34 (p<0.01), and DVC33 vs DVC44 (p<0.05). GGT activity shows differences between AD23 and AD24 (p<0.02), AD23 and AD44 (p<0.001), AD23 and DVC23 (p<0.05), AD24 and AD33 (p<0.03), AD24 and AD34 (p<0.02), AD33 and AD44 (p<0.001), AD33 and DVC33 (p<0.004), AD34 and AD44 (p<0.001), AD44 and DVC44 (p<0.01), DVC23 and DVC24 (p<0.05), DVC23 and DVC33 (p<0.02), DVC23 and DVC34 (p<0.004), DVC23 and DVC44 (p<0.003), and DVC33 and DVC44 (p<0.05). Alkaline phosphatase activity is also lower in APOE-4/4 carriers than in any other APOE genotype, with similar values in AD and DVC, except in APOE-3/4, and significant differences in AD24 vs AD44 (p<0.05), AD23 vs AD44 (p<0.01), AD34 vs AD44 (p<0.05), AD34 vs DVC34 (p<0.02), DVC33 vs DVC44 (p<0.05), and DVC34 vs DVC44 (p<0.05). Bilirubin levels tend to be higher in APOE-3(+) carriers, with significant differences in AD33 vs AD34 (p<0.05), DVC23 vs DVC33 (p<0.001), DVC23 vs DVC34 (p<0.01), and DVC33 vs DVC34 (p<0.01). Creatine phosphokinase (CPK) activity is lower in APOE-4(+) in AD, but not in DVC, without major differences among genotypes, as well as lactate dehydrogenase (LDH) activity which only shows significant differences between AD33 and DVC33 (p<0.01), but not significant variation among genotypes. Sodium levels are higher in AD44 with significant differences in AD23 vs AD44 (p<0.05), AD33 vs AD44 (p<0.05), and AD34 vs AD44 (p<0.05), and no changes in APOE-related DVC. Potassium levels show differences in AD24 vs AD33 (p<0.05), AD24 vs AD34 (p<0.04), December 2008
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and AD24 vs DVC24 (p<0.02) due to an elevated concentration of potassium in AD24; and chloride levels do not show any APOE-related variation in either AD or DVC. Thyroid function: Approximately 4% of AD patients and 5% of DVC patients exhibit thyroxin (T4) levels below 0.60 ng/dL which are compatible with clinical hypothyroidism. The proportion of APOE-related AD/DVC cases with T4 levels below this threshold is the following: AD23 0%, DVC23 6%, AD24 0%, DVC24 0%, AD33 6%, DVC33 4%, AD34 3%, DVC34 7%, AD44 6%, and DVC44 17%. Cases of hyperthyroidism (T4>1.5 ng/dL) accounted for 13% in AD and 13% in DVC, with the following APOE-related distribution: AD23 8%, DVC23 6%, AD24 26%, DVC24 0%, AD33 15%, DVC33 13%, AD34 12%, DVC34 20%, AD44 6%, and DVC44 8%. It is striking to observe the high rate of hypothyroidism (4-5%) and hyperthyroidism (13%) in patients with AD and DVC. In absolute values, the levels of TSH show significant differences in DVC24 vs DVC33 (p<0.01), DVC33 vs DVC34 (p<0.04), and DVC24 vs DVC44 (p<0.05). T4 levels were different in AD24 vs DVC24 (p<0.03), DVC24 vs DVC33 (p<0.05), and DVC24 vs DVC34 (p<0.02), with a clear hormonal disequilibrium in DVC and minor variation in AD. Folate and vitamin B12 levels: Cases of folate deficiency (folic acid levels<3.0 ng/ml) accounted for 5% in AD and 6% in DVC with the following distribution: AD23 8%, DVC23 6%, AD24 25%, DVC24 0%, AD33 5%, DVC33 7%, AD34 6%, DVC34 6%, AD44 22%, and DVC44 8%. No significant differences in folic acid levels have been found among APOE genotypes in either AD or DVC. Vitamin B12 deficiency (vitamin B12 levels<150 pg/ml) was found in 4% of the cases with AD and in 3% of the cases with DVC, according to the following distribution pattern: AD23 8%, DVC23 3%, AD24 0%, DVC24 0%, AD33 3%, DVC33 2%, AD34 8%, DVC33 5%, AD44 8%, and DVC44 0%. The lowest vitamin B12 levels have been found in DVC44, and significant differences were identified in AD24 vs DVC24 (p<0.04), DVC24 vs DVC33 (p<0.04), DVC24 vs DVC34 (p<0.03), and DVC24 vs DVC44 (p<0.02), basically due to the presence of high levels of vitamin B12 in DVC24. Hematologic parameters: Red blood cell (RBC) counting did not show any difference among APOE genotypes nor between AD and DVC. In contrast, hematocrit values tend to be lower in APOE-4(+), with significant differences in AD23 vs AD44 (p<0.04), AD33 vs AD44 (p<0.05), DVC23 vs DVC24 (p<0.05), and DVC33 vs DVC34 (p<0.03). In a similar manner, hemoglobin levels were found lower in APOE-4(+) in both AD and DVC, with significant differences in AD33 vs AD44 (p<0.05), DVC23 vs DVC24 (p<0.05), DVC23 vs DVC34 (p<0.05), and DVC33 vs DVC34 (p<0.05). Minor changes have been found in other RBC-related parameters, such as MCV, MCH, MCHC, and RDW. Significant changes were identified in white blood cell (WBC) counting with differences in AD23 vs AD33 (p<0.01), AD23 vs AD34 (p<0.01), AD23 vs AD44 (p<0.01), AD23 vs DVC23 (p<0.03), AD24 vs DVC24 (p<0.04), AD33 vs DVC33 (p<0.04), and AD24 vs DVC34 (p<0.02). The lowest WBC levels are seen in AD44. The number of lymphocytes is similar in all APOE genotypes with only significant differences between AD34 and DVC34 (p<0.02). The number of monocytes differs in AD23 vs AD33 (p<0.01), AD23 vs AD34 (p<0.01), AD23 vs AD44 (p<0.01), AD23 vs DVC23 (p<0.02), and AD33 vs DVC33 (p<0.03), indicating significant differences between AD and DVC in monocyte numbers. Similarly, the number of granulocytes tends to be smaller in APOE-4(+), especially in AD, with significant differences in AD23 vs AD33 (p<0.03), AD23 vs AD34 (p<0.01), AD23 vs AD44 (p<0.01), AD23 vs DVC23 (p<0.03), and AD34 vs DVC34 (p<0.05). The number of platelets and their MPV do not differ among APOE genotypes nor between AD and DVC; however, the GSV is particularly high in DVC with only significant differences in AD23 vs AD24 (p<0.04), and AD23 vs AD33 (p<0.05). Brain hemodynamic parameters: Standard hemodynamic parameters assessed by non-invasive transcranial Doppler ultrasonography include the following: (a) mean velocity (Mv), (b) systolic velocity (Sv), (c) diastolic velocity (Dv), (d) the pulsatility index of Gossling (PI = (Sv-Dv)/ Mv), and (e) the resistance index of Pourcelot (RI = (Sv-Dv)/Sv). These parameters have been measured in the left (LMCA) and right middle cerebral arteries (RMCA), in the left (LACA) and right anterior cerebral arteries (RACA), and in the vertebrobasilar system (VBS) of patients with AD and DVC. The RMCA Mv did not show major differences between AD and DVC, with only significant differences in AD23 vs AD44 (p<0.04), and in AD24 and DVC24 (p<0.05). The Sv in RMCA tended to be lower in DVC than in AD, with significant differences in AD23 vs AD24 (p<0.02), AD23 vs AD34 (p<0.03), AD23 vs AD44 (p<0.02), AD23 vs DVC23 (p<0.05), and AD24 vs DVC24 (p<0.05). The Dv in RMCA was also found diminished in DVC with significant differences in AD23 vs AD44 (p<0.05), AD24 vs AD34 (p<0.05), AD24 vs DVC24 (p<0.05), and AD44 vs DVC44 (p<0.05), indicating that brain blood flow is poorer in DVC than in AD with certain APOE-related vascular component. The PI in RMCA was different in AD23 vs AD34 (p<0.05), AD23 vs DVC23 (p<0.01), DVC23 vs DVC33 (p<0.05), DVC23 vs DVC34 (p<0.05), and DVC24 vs DVC34 (p<0.05); and the RI in RMCA differed in AD23 vs DVC23 (p<0.05), and DVC23 vs DVC33 (p<0.04), confirming an increased resistance to flow in DVC. In the RACA only Dv showed differences between AD44 and DVC44 (p<0.05) with also a diminished Dv in DVC. In the territory of the RACA no significant changes have been found in either PI or RI. The most relevant findings in brain blood flow were detected in the LMCA with a significantly poorer performance in DVC, the lowest Mv in DVC44, and significant differences in AD23 vs AD44 (p<0.02), AD33 vs AD44 (p<0.01), AD34 vs AD44 (p<0.01), and AD44 vs DVC44 (p<0.02). The Sv in LCMA showed a very similar pattern to that of Mv in the same arterial territory, with significant differences in AD23 vs AD44 (p<0.007), AD23 vs DVC23 (p<0.05), AD33 vs AD44 (p<0.006), AD34 vs AD44 (p<0.002), and AD44 vs DVC44 (p<0.01). The Dv also behaved in a similar manner in LMCA, with significant differences in AD23 vs AD44 (p<0.04), AD23 vs AD44 (p<0.05), and AD34 vs AD44 (p<0.05). LMCA PIs were significantly different in AD33 vs AD34 (P<0.05), and DVC23 vs DVC33 (p<0.05); and the LMCA RIs only differed in AD33 vs AD34 (p<0.03), with no variation in DVC. In the LACA territory, AD patients showed worse hemodynamic parameters than DVC, with only significant differences in AD34 vs AD44 Mv (p<0.05), AD33 vs DVC33 Sv (p<0.03), and AD34 vs AD44 Dv 90
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Poster Presentation Abstracts
(p<0.05). PI and RI in LACA were increased in AD34 with respect to AD33 (p<0.04), and no major differences between AD and DVC. In the VBS, most hemodynamic parameters were worse in DVC than in AD, with significant differences in AD23 vs AD34 Mv (p<0.05), AD23 vs AD34 Sv (p<0.02), AD23 vs AD44 Sv (p<0.05), AD33 vs AD34 Sv (p<0.05), and no differences in either PI or RI. Immunological markers : The percentage of total lymphocytes (CD3) is significantly reduced in DVC44 as compared with AD44 (p<0.01), and also shows differences in DVC23 vs DVC44 (p<0.05), DVC24 vs DVC44 (p<0.03), DVC33 vs DVC44 (p<0.008), and DVC34 vs DVC44 (p<0.004), indicating a clear depletion of active lymphocytes in DVC44 but not in AD. CD4 lymphocytes were also different in AD34 vs DVC34 (p<0.05), and DVC23 vs DVC34 (p<0.01). CD7 lymphocytes (+NK) showed significant differences in AD34 vs AD23 (p<0.04), AD33 vs AD34 (p<0.04), AD33 vs DVC34 (p<0.04), and DVC33 vs DVC34 (p<0.05) mainly due to reduced numbers in APOE-3/4 carriers in both AD and DVC, probably indicating a functional defect in natural killer activity. CD8 lymphocytes (suppressor/cytotoxic) showed specific poor values in DVC44 with significant differences between AD44 vs DVC44 (p<0.05), DVC23 vs DVC24 (p<0.05), DVC23 vs DVC44 (p<0.05), DVC24 vs DVC33 (p<0.05), DVC24 vs DVC34 (p<0.02), DVC24 vs DVC44 (p<0.009), and DVC33 vs DVC44 (p<0.04), indicating a quantitative disbalance in DVC44. No APOE-related differences in MCH class II antigens (HLA-DR) have been found in AD or DVC neither between AD and DVC. IL-2 receptors (CD25) were found reduced in APOE-4(+), and especially diminished in DVC34, with significant differences in AD34 vs DVC34 (p<0.007), and DVC33 vs DVC34 (p<0.007). CD28, including 60-80% activated CD3+, did not show clear differences among genotypes nor between AD and DVC. Natural killer cells (CD56) tended to show reduced values in APOE-3(+) and in AD44, with significant differences in AD24 vs AD33 (p<0.05), and AD24 vs AD34 (p<0.05). Activated T lymphocytes (CD3+/HLA-DR+) were found to be diminished in APOE-4/4 carriers in both AD and DVC, with significant differences in AD23 vs AD24 (p<0.05), AD24 vs AD33 (p<0.01), AD24 vs AD34 (p<0.01), and AD24 vs AD44 (p<0.005). Finally, the CD4/ CD8 ratio was especially low in AD44, and DVC24, with significant differences in AD44 vs DVC44 (p<0.04), DVC23 vs DVC44 (p<0.05), DVC24 vs DVC33 (p<0.05), DVC24 vs DVC34 (p<0.04), DVC24 vs DVC44 (p<0.01), and DVC33 vs DVC44 (p<0.05). All these data together appear to indicate (a) that APOE-related products may influence the immune function in AD; (b) that cellular immunity-mediated processes may be altered in AD; and (c) that distinct pathogenic mechanisms differentially alter lymphocyte-regulated immune function in AD and DVC. In general terms, APOE-4/4 carriers show a biological disadvantage as compared with other APOE genotypes. References: Cacabelos, R., Fernández-Novoa, L., Corzo, L., Pichel, V., Lombardi, V., Kubota, Y. Genomics and phenotypic profiles in dementia: Implications for pharmacological treatment. Meth. Find. Exp. Clin. Pharmacol., 26(6): 421-444 (2004). Cacabelos, R., Fernández-Novoa, L., Corzo, L., Amado, L., Pichel, V., Lombardi, V., Kubota, Y. Phenotypic profiles and functional genomics in Alzheimer’s disease and in dementia with a vascular component. Neurol. Res., 26: 459-480 (2004). Cacabelos, R. Genomic characterization of Alzheimer’s disease and genotype-related phenotypic analysis of biological markers in dementia. Pharmacogenomics 5(8):1049-1105 (2004). (Supported by the EuroEspes Foundation and the International Agency for Brain Research and Aging, IABRA).
P43
Genetic polymorphisms of several genes associated with key metabolic pathways in obesity 1
R. Llovo, 1 L. Fernández-Novoa, 1 I.Tudoli, 2 A.I.Vallejo and 3 R.Cacabelos 1 Department of Clinical Genetics and Genomics, EBIOTEC. Bergondo Industrial Estate. 15165 Bergondo, A Coruña, Spain. 2 Nutrition and Nutrigenomics Unit, EuroEspes Biomedical Research Center, Sta. Marta de Babio, s/n. 15166 Bergondo, A Coruña, Spain. 3 Institute for CNS Disorders, EuroEspes Biomedical Research Center, Sta. Marta de Babio, s/n. 15166 Bergondo, A Coruña, Spain.
Introduction: Nutrition has the most important life-long environmental impact on human health. While nutrigenetics addresses how an individual’s genetic makeup predisposes dietary susceptibility, nutrigenomics poses the question of how nutrition influences the expression of the genome. It is known that there are hundreds of genes associated to obesity pathways. Some of them are LEP and LEPR related to the Leptin hormone and its receptor; NPY codifying the Neuropeptide Y that stimulates ingestion, Insulin secretion and Lipoprotein lipase activity; PPARG: the protein encoded by this gene is PPAR-gamma and is a regulator of adipocyte differentiation. Additionally, PPAR-gamma has been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer. On the other hand polymorphisms in the peroxisome proliferator-activated receptor (PPARG) and ß-adrenergic receptor (ADRB3) genes have been linked to increased body mass index (BMI), obesity, and more recently dietary nutrients and preferences. In addition, common variation in ADRB3 interacts with PPARG to modulate adult body weight. Regarding UCP2, a common polymorphism in the promoter of this gene is associated with decreased risk of obesity in middle-aged humans. To sum up, it is necessary to analyze the most common polymorphisms of those genes associated with lipid metabolism, such as APOE; APOB; APOC; CEPT and LPL. December 2008
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Objective: To determine the prevalence of the most common allelic variants of some genes associated with the main metabolic alterations associated with overweightness and obesity. Materials and Methods : Genotyping was performed by a combination of long-PCRs, and allele-specific Real-time PCRs in a total of 40 adult individuals with BMI > 29. Results: Gene
Polymorphism Studied
Genotype
Frequency of GENOTYPE (%)
LEP
A19G
A/A
14.64
A/G
39.02
G/G
46.34
Gln/Gln
29.27
Gln/Arg
29.27
Arg/Arg
41.46
Leu/Leu
95
Leu/Pro
5
Pro/Pro
0
Trp/Trp
93.18
Trp/Arg
6.82
Arg/Arg
0
G/G
0
G/A
68.18
A/A
31.82
Pro/Pro
88.64
Pro/Ala
11.36
Ala/Ala
0
3/2
10.64
3/3
10.64
3/4
12.76
C/C
40.42
C/T
44.68
T/T
14.89
C/C
40.42
C/T
44.68
T/T
14.89
B1/B1
46.81
B1/B2
42.55
B2/B2
10.64
Ser/Ser
76.59
Ser/Stop
21.28
Stop/Stop
2.13
LEPR
NPY
Leu7Pro
ADRB3
UCP2
PPARG
APOE
APOC3
APOB
CEPT
LPL
92
Gln223Arg
Trp64Arg
G-866A
Pro12Ala
C112T/C158T
C3175G
C2488T
G+279A
C1421G
December 2008 - international edition
Genotyping for polymorphic NPY and ADRB3 reveals that more than 93 % of the subjects show no polymorphisms in these cases. The highest frequency is present in the UCP2 gene (100% of the cases), followed by CEPT, LEPR, APOB and LEP with 89.36 %; 70.73%; 59.57% and 53.66% respectively for each allele studied. The genes related to lipid metabolism (APOE; APOC3; APOB; CEPT and LPL) show important frequencies of polymorphisms. Conclusions: From the results it could be inferred that it is very useful to know the genetic profile in cases of obesity, because the frequency of several polymorphisms is very high. Some of the polymorphisms studied could show antagonistic effects on the same metabolic pathway, so a careful study in order to clarify as much as possible the best healthy guidelines in each case is required.
Poster Presentation Abstracts
P44
Inflammatory activation of circulating macrophages from patients with type 2 diabetes can be reduced by a PPARγ - Agonist Pfützner E. 1 Weise A. 2, Prause S. 3, Eidens M. 3, Musholt P. 2, Weber M.M. 1; Forst T. 2, Pfützner A. 2, 3 Clinic of the Johannes Gutenberg University, Mainz; Germany 2 Institute for Clinical Research and Development, ikfe GmbH, Mainz; Germany 3. PharmGenomics GmbH, Mainz; Germany
1
Background: Monocytes/Macrophages of patients with type 2 diabetes have been shown to be in a proinflammatory state as demonstrated by excess expression and secretion of cytokines and other inflammatory markers. This study investigates the influence of treatment with pioglitazone (vs. metformin/sulfonylurea) on mRNA expression of inflammatory markers in monocytes, in 63 patients with well controlled orally treated type 2 diabetes (62 male, 11 female, age(Mean±SD): 66±7 yrs., disease duration: 6.6±9.6 yrs., HbA1c: 6.7±0.6 %). Methods: The patients were randomized to receive either 30 mg of pioglitazone or placebo in addition to their current therapy in a doubleblinded study. Blood samples for isolation of peripheral monocytes were taken at baseline and after 4 weeks. After isolation of mRNA and transcription, the cDNA for IκBß, IκBα, MMP9, TNFα, MIF, NFK-B (Rel-A and p105), IL-6R, and PPARγ was quantified in relation to a housekeeping marker (ß-actin) using real-time PCR (LightCycler, Roche Diagnostics). Results; Starting from comparable baseline levels, mRNA expression of proinflammatory markers in peripheral monocytes was downregulated by pioglitazone while further up-regulation was seen in the comparator arm as shown in the Table 1. As expected from the mode of action of pioglitazone, an increased expression of PPARγ was observed in the verum arm only. Discussion: Pioglitazone down-regulated the activated state of peripheral moncytes/macrophages as demonstrated by mRNA expression of inflammatory markers independent from glycemic control. Our results may contribute to the overall understanding on the cardiovascular risk reduction observed in type 2 diabetic patients treated with pioglitazone. Conclusion: Quantification of the above determined marker panel by means of new microarray methods may be a possible way for routine assessment of chronic systemic vascular inflammation.
Table 1: % Change in mRNA expression of inflammatory mRNA markers Treatment
IκBß
IκBα
MMP9
TNFα
MIF
P105
RelA
IL-6R
PPARγ
Pioglitazone
-16 %
-11%
-33%
-12%
-11%
-19%
-20%
-11%
+128%
Placebo
-11 %
+8%
+75%
+13%
+2%
+5%
-2%
+11%
-37%
p
n.s.
n.s.
p<0.05
p<0.05
n.s.
p<0.05
n.s.
p<0.05
p<0.001
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Index of Authors
A
Aguilar Aldrete, ME............................................. 74 Alcaraz, Margarita................................... 69, 70, 85 Alejo, Ramón...................................................... 85 Alonso, Eva........................................................ 83 Alonso Cerezo, C............................................... 71 Álvarez, Antón........................................ 75, 84, 85 Arlucea, Jon....................................................... 66 Arranz, Jose Luis................................................ 66
B
Ballesteros, J...................................................... 73 Basinski, T.......................................................... 73 Blanco, Lorena............................................. 66, 67 Bolla, E............................................................... 72 Bortolaso, P....................................................... 72 Botana, Luis M................................................... 83
C
December 2008 - international edition
F
Fernández Millares, V.......................................... 71 Fernández-Novoa, L............. 69, 70, 71, 75, 76, 77 ...................................... 78, 79, 80, 81, 82, 85, 89 Fernández-Sánchez, M...................................... 65 Ferrari, M............................................................ 72 Fraile, Carmen...................... 70, 71, 75, 76, 83, 85 Galimberti, Damiano..................................... 59, 74 Garcia - Orad, A................................................. 73 Garcia de Sola, R............................................... 71 Garoi, Flavio....................................................... 74 Gil, Javier............................................................ 67 Giménez-Llort, Lydia........................................... 83 Gracia Marin,Maria............................................. 65 Gutiérrez Rubio, SA............................................ 74
D
E
Etcheverría, I........................................... 68, 69, 81
Cacabelos, Ramón............... 66, 68, 69, 70, 71, 74 ........................................... 75, 76, 77, 78, 79, 80 ........................................... 81, 82, 83, 84, 85, 89 Cacabelos-Pérez, Natalia....................... 75, 76, 85 Cacabelos-Pérez, Ramón....................... 75, 76, 85 Candenas, Luz................................................... 67 Candenas, ML.................................................... 65 Canuet, Leonides............................................... 72 Carril, Juan C..................................................... 79 Casas, Angela.................................................... 85 Casis, Luis.......................................................... 66 Catalán, José..................................................... 66 Ceccon, F........................................................... 72 Chacón, R.......................................................... 68 Chiappelli, Martina.............................................. 74 Corzo, Lola........................... 70, 71, 75, 76, 78, 80 .............................................................. 82, 84, 85 Cosentino, M...................................................... 72 Couceiro, Veronica................................. 82, 84, 85
Des, Julio........................................................... 85 Díaz de Otazu, Ramón........................................ 66
94
H
Harguindey, Salvador.......................................... 66 Hayashi, Noriyuki................................................ 72 Hernando Requejo, V......................................... 71 Herrera Peco, I................................................... 71
I
Irazusta, Jon................................................. 65, 67
J
Jutel, M.............................................................. 73
K
Kamino, Kojin..................................................... 72 Kiejna, A............................................................. 73 Kudo, Takashi..................................................... 72
L
Laredo, Marta......................................... 82, 84, 85 Larrinaga, Gorka........................................... 64, 67
G
Index of Authors
Lecchini, S.......................................................... 72
Leszek, J............................................................ 73
Ravina, CG......................................................... 65 Reshkin, Stephan J............................................ 66 Rivas, Helga....................................................... 83 Rodríguez, Susana....................................... 78, 82 Rodríguez-Vega, MC.......................................... 74 Rosales-Gómez, RC........................................... 74 Rubiolo, Juan A.................................................. 83
Licastro, Federico............................................... 74 Llovo, Ruth........................... 76, 77, 78, 79, 80, 89 Lombardi, VRM................................ 68, 69, 81, 85 López, Jose I................................................ 66, 67 López-López, E.................................................. 67 Lucchini, Vittorio................................................. 74 Luigi Ioro,Eugenio......................................... 63, 65
M
Mandler, M......................................................... 75
R
S
Sampedro, Carolina............................................ 84 Seoane, S.................................. 69, 77, 78, 80, 81 Subirán, N.......................................................... 65
Marino, F............................................................ 72 Martínez, E......................................................... 68 Martínez, Rocio...................................... 75, 76, 85 Martin-Guerrero, I............................................... 73 Mattner, F........................................................... 75 Moran Moguel, MC............................................. 74 Mori, Koji............................................................ 72
T
Tagami,Shinji...................................................... 72 Takeda, Masatoshi.............................................. 72 Tanaka, Toshihisa............................................... 72 Tellado, I........................................... 80, 82, 83, 85 Tudoli, Indara.............................. 77, 78, 79, 80, 89
Morihara, Takashi............................................... 72
N
U
Ugalde, Aitziber.................................................. 66
Navajas, A.......................................................... 73 Nebril, Laura . .................................................... 85 Noshi, Eriko........................................................ 72
C
Okochi, Masayasu.............................................. 72
V
Vale, Carmen...................................................... 83 Vallejo, Ana................................................... 85, 89 Varona, Adolfo.............................................. 66, 67 Vender, S............................................................ 72 Vieytes, Mercedes R........................................... 83
Orive, Gorka................................................. 66, 81
P
Y
Yokokoji, Mikiko.................................................. 72
Parra Bernal, MC................................................ 74 Pastor Gómez, J................................................ 71 Pérez, Itxaro................................................. 66, 67 Pérez, Rogelio.................................................... 85
Z
Zubero, Jaime.................................................... 67
Pinto, FM............................................................ 65 Pinto, Francisco.................................................. 67 Piñán, MA........................................................... 73
December 2008
95
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