Personalized Medicine Europe: Health, Genes

Personalized Med. Vol. 2 No. 2 (2005)

Abstracts presented during: The Yoran Institute for Human Genome Research, Tel-Aviv University / European Science Foundation Workshop

Personalized Medicine Europe: Health, Genes & Society June 19–21, 2005 Tel-Aviv University, Tel-Aviv, Israel

Scientific Organizers: Gregory Livshits Department of Human Anatomy & Anthropology David Gurwitz Department of Human Genetics & Molecular Medicine Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel Sponsors: Adams Super Center for Brain Studies, Tel-Aviv University, Israel Roche Diagnostics, Switzerland Dyn Diagnostics, Israel Teva, Israel

ISSN 1741-0541

www.futuremedicine.com

Contents Personalized Medicine Europe: Health, Genes & Society

Workshop Abstracts Arranged in alphabetical order

Workshop Introduction 143 Personalized medicine: desirable, affordable,

attainable? D Gurwitz & G Livshits

Abstracts 145 A comprehensive study on the molecular genetic

basis of hereditary hearing loss KB Avraham, D Amiel, O Atar et al. 145 Preventive programs in Israel: an ethnic-based

personalized medicine G Bach 146 Sequenom MassArray technology and its uses in

152 Personalized medicine: ethical and social

consequences FW Frueh 153 Individualization of isoniazid doses based on NAT2

genotype: design features of a randomized clinical trial U Fuhr 154 Pharmacogenetics of glatiramer acetate therapy for

multiple sclerosis reveals drug-response markers I Grossman, N Avidan, C Singer 154 Understanding the pathogenesis of human

Israel

hereditary deafness by expression profiling of inner ears from mutant mice

E Ben-Asher, D Amann, T Olender, T Koch, D Lancet

R Hertzano, M Montcouquiol, S Rashi-Elkeles et al.

146 Pharmacogenetics of fluvastatin in familial

155 Novel Palestinian mutations in deafness-related

hypercholesterolemic patients

genes

D Bercovich, V Meiner, Y Friedlander

M Kanaan, T Walsh, AA Rayan et al.

147 Cross-cultural genetic counseling R Carmi 148 Automated mass spectrometry in personalized

medicine CR Cantor 148 The role of the APOE genotype in

immunomodulation J Chapman, G Dallal, DM Michaelson 149 Possible differences in the immune response to the

common environmental disease factor Onchocerca volvulus in two ethnic communities living in the Ecuadorian rainforest GF De Stefano 149 The LIM domain transcription factor LHX3 is a

putative target of POU4F3 in the inner ear AA Dror, R Hertzano, M Montcouquiol 150 Promoting Arab and Israeli cooperation: peace

building through health initiatives Z El Nasser, H Skinner, Z Abdeen 150 Genetics analysis of human infertility M Fellous 151 Pharmacogenomic research in drug development

– the ethical concern, bridging the gap between public perceptions and industries realities J Friedman

2005 © Future Medicine Ltd ISSN 1741-0541

156 Gene–environment interactions on bone mass: The

Framingham Study D Karasik, RR McLean, DP Kiel et al. 157 The CYP2C9 polymorphism: from enzyme kinetics

towards genotype-adjusted drug therapy J Kirchheiner 158 The meaning of free and informed consent in

biomedical research personalizing medicine V Kucinskas, G Andrulionis, D Steponaviit 158 Identification of A-to-I RNA-editing sites in the

human transcriptome EY Levanon, E Eisenberg, Y Kinar et al. 160 Evaluating attitudes towards genetic screening

programs among orthodox Jewish students S Lieberman, A Frumkin, M Sagi 161 Personalized medicine: health, genes, and society K Lindpaintner 162 CYP2D6 multiplication in Spanish healthy

volunteers and schizophrenic patients A LLerena, P Dorado, MC Caceres, E Penas-Lledo, A De la Rubia 163 Polymorphism in signal transduction as a major

contributor to osteoarthritis genetic risk J Loughlin

CONTENTS CONT.

164 Personalized medicine: new perspectives, new

177 A human rights perspective on personalized

dilemmas?

medicine& justice

JE Lunshof

C Shalev

164 Genetic testing for common diseases in clinical

177 ErbB4 shows a highly significant association with

practice

schizophrenia in Ashkenazi Jews

LI Minaicheva, OA Makeeva, VA Stepanov, LP Nazarenko, MG Spiridonova, VP Puzyrev

G Silberberg & R Navon

165 Integration of pharmacogenetics into the

therapeutic drug monitoring clinical service of large hospitals VG Manolopoulos 166 Sniffing SNPs – the genetic basis of human olfactory

178 Polymorphism in cancer patients' DNA repair

capacity as a factor in determining the dosimetry of radiation and chemotherapy H Slor, S Batko, W Mueller, H Schroeder, I Zaveliyuk, I Ron 179 Neurogenetics of acetylcholinesterase: from stress

variability

reactions to Parkinsonism

I Menashe, Y Gilad, O Man, R Aloni, D Lancet

H Soreq, L BenMoyal-Segal, Y Ben Shaul et al.

167 Biobanking and personalized medicine A Metspalu 168 Mitochondrial genetics, longevity, adaptation and

disease D Mishmar, J Feder, A Bachrat 168 The prospects and bioethical dimensions of

expanding the meaning of pharmacogenomics to encompass individualized pharmacotherapy C Møldrup 169 ESF EMRC contribution to building a public–private

platform for clinical research in Europe C Moquin-Pattey 170 Public health genetics in Germany: Pandora’s perils

or Panakeia’s promise? NW Paul 172 Personalized medicine USA: pros, cons, but no way

back I Peter 173 Personalized medicine in times of “Global Genes”:

making sense of the “hype” B Prainsack 174 In silico structural analysis of cytochrome P450-

dependent monooxygenase APA Rani & A Aysha Mahmoodha 175 Abundant A-to-I-editing sites in the human

transcriptome: relevance to disease G Rechavi

179 Cancer pharmacogenomics: the field that studies

the role of an individual’s genetics in the response to drugs L Soussan-Gutman 180 The use of twins in genetic research: implications

for personalized medicine TD Spector 180 Is there a link between personalized medicine and

community genetics? LP ten Kate 181 Analysis of genetic variation in complex endocrine

diseases AG Uitterlinden, J van Meurs, F Rivadeneira, HAP Pols 182 Individual sensitivity to warfarin could be predicted

from genetic profiles of the components and effectors of vitamin K-dependent ã-carboxylation system M Vecsler, R Loebstein, S Almog et al. 183 The ethics of clinical prediction MA Weingarten 184 Pharmacogenetics of citalopram in pediatric

anxiety and depression A Weizman, S Kronenberg, A Apter, A Frisch 184 Genetic screening for Gaucher disease in Israel:

genetic screening program for a low penetrant, treatable disease S Zuckerman, E Levy-Lahad, A Lahad, M Sagi

175 Bioethical limits of prenatal genetic testing M Revel 176 Genetic breast cancer – a top secret information? A Rosen & R Ben-Yosef

142

Personalized Med. (2005) 2(2)

WORKSHOP I NTRODUCTION

Personalized medicine: desirable, affordable, attainable? David Gurwitz1† & Gregory Livshits2,3 †Author

for correspondence of Human Genetics and Molecular Medicine, Tel-Aviv University, Faculty of Medicine, Tel-Aviv 69978, Israel Tel.: +972 3640 7611; Fax: +972 3640 7611; E-mail: gurwitz@ post.tau.ac.il 2Yoran Institute for Human Genome Research 3Department of Anatomy & Anthropology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel 1Department

The Human Genome Project, the most celebrated human consortium effort in biomedicine and one of the greatest scientific achievements of modern time, has yet to deliver the anticipated improvements in healthcare [1]. The blueprint of a human being, the complete 3.2 billion nucleotides of the human DNA sequence, has been available since April 2002. Up to the present day, however, we still understand very little about the biology of common complex disorders, such as diabetes, hypertension, osteoarthritis, osteoporosis, Alzheimer’s disease, schizophrenia, and depression, despite the fact that the genetic determination of each of these conditions is reliably established. Besides the individual burden of suffering and reduced quality of life, these diseases have an increasing impact on the healthcare system. Pharmacotherapy for such chronic diseases, which are primarily age-dependent and whose prevalence across Europe keeps mounting due to demographic factors, typically burdens the national healthcare systems with costs of thousands of Euros per patient annually in direct and indirect drug and hospitalization costs, and treatments continue for many years. However, such treatments typically allow only limited relief from symptoms for large segments of patients [2–4]. In addition to suffering from suboptimal efficacy, drugs might sometimes do more harm than benefit. Large studies performed in the UK and Germany, published during 2004 [5,6], have both found that approximately 6% of new hospital admissions to internal medicine wards were related to adverse drug reactions (ADR). About 4% of total UK hospital bed capacity reflects ADR-related hospitalizations. The associated annual costs for the UK healthcare system alone were estimated at €706 million [5]. The disillusionment about the direct benefits from the Human Genome Project and their capacity to allow rapid improvements in the quality of clinical care is, in a large part, a consequence of our realization that human genome diversity is much larger than originally envisaged. For example, we now know that each of us is likely to differ from our fellow humans at over 10 million nucleotides on average – roughly

10.1517/17410541.2.2.143 © 2005 Future Medicine Ltd ISSN 1741-0541

each nucleotide every 300 letters in our common genetic blueprint. In other words, at most, humans share 99.7% of their genetic code; the other 0.3% makes us genetically unique, and this uniqueness, in part, determines how each individual responds to pharmacotherapy [7]. Both drug safety and drug efficacy could be improved by personalized medicine, which would allow pharmacotherapy to be selected for individual patients according to their genetic profiles [7,8]. Pharmacogenetics (PGx), the marriage of pharmacology and clinical genetics, forms the cornerstone of personalized medicine. It is expected, in the long run, to significantly improve the practice of medicine by allowing individualized diagnosis and pharmacotherapy, as well as preventive medicine, adjusted to the profile of the individual patient. Such personalized diagnosis and treatment would be based, to a large extent, on each individual’s set of relevant polymorphic gene alleles and their epigenetic interactions. Thus, PGx is claimed to hold the key for better medicine, where safer and more effective drugs would become a reality for many patients. This would replace the current ‘trial and error’ pharmacotherapy approach for patient treatment. While PGx is far from being a new discipline, and will celebrate 50 years in 2007 [8], its implementation is lagging behind original expectations [7]. It seems that the number of PGx applications currently available at the clinic is limited, and the few available diagnostics are costly, raising questions about costeffectiveness [9]. Several bottlenecks have been identified in the road to implementation of personalized medicine. Primarily, there is a lack of sufficient data about correlations between individual patient genotypes and drug safety and efficacy phenotypes. Another problem arises from the conservative nature of medical practice, and the lack of incorporation of PGx into the core teaching curricula of medical schools [10]. Meanwhile, public concerns are being voiced, particularly in Europe, about the concept of personalized medicine being in possible conflict with the moral demand for equity. We are all entitled to the best Personalized Medicine (2005) 2(2), 143–144

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healthcare, but if certain diagnostics would indicate that some patients cannot be treated at all, and others can only be treated at exceptionally high cost, how could we maintain an equal healthcare for all? During the European Science Foundation/Tel-Aviv University Workshop, ‘Personalized Medicine Europe: Health, Genes, & Society’, we hope to examine this apparent conflict and discuss ways for resolving it. The workshop will also consider, among other issues, routes that will be most useful for removing bottlenecks on the road towards the implementation of personalized medicine in the clinic. We hope that the workshop will form a stage for discussing the Bibliography 1.

2.

3.

4.

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Collins FS, Green ED, Guttmacher AE, Guyer MS: US National Human Genome Research Institute. A vision for the future of genomics research. Nature 422, 835–847 (2003). Roses AD: Pharmacogenetics and the practice of medicine Nature 405, 857–865 (2000). Kalow W: Human pharmacogenomics: the development of a science. Hum. Genomics 1, 375–380 (2004). Evans WE, Relling MV: Moving towards individualized medicine with pharmacogenomics. Nature 429, 464–468 (2004).

5.

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8.

impact of pharmacogenetics on clinical practice, while taking into account regulatory, policy and bioethics aspects. Personalized medicine has so much to offer for improving both individual and community health: we must strive for the most sound and cost-effective ways to make it happen, while maintaining a fair balance between the interests of all parties involved [11,12]. If we deem personalized medicine to be desirable for society it should be affordable and, with time, an attainable good for all who need it. Even so, we believe it can be accomplished, for the benefit of society, without compromising the European values of human equality: liberte, egualite, fraternite.

Pirmohamed M, James S, Meakin S et al.: Adverse drug reactions as cause of admission to hospital: prospective analysis of 18 820 patients. Br. Med. J. 329, 15–19 (2004). Dormann H, Neubert A, Criegee-Rieck M et al.: Readmissions and adverse drug reactions in internal medicine: the economic impact. J. Intern. Med. 255, 653–663 (2004). Meyer UA: Pharmacogenetics – five decades of therapeutic lessons from genetic diversity. Nat Rev Genet. 5, 669–676 (2004) Frueh FW, Gurwitz D: From pharmacogenetics to personalized medicine: a vital need for educating health professionals and the community. Pharmacogenomics 5, 571–579 (2004).

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Veenstra DL: Bringing genomics to the bedside: a cost-effective pharmacogenomic test? Pharmacogenetics 14, 333–334 (2004). Gurwitz D, Weizman A, Rehavi M: Education: Teaching pharmacogenomics to prepare future physicians and researchers for personalized medicine. Trends Pharmacol. Sci. 24, 122–125 (2003). Melzer D, Detmer D, Zimmern R: Pharmacogenetics and public policy: expert views in Europe and North America. Pharmacogenomics 4, 689–691 (2003). Smart A, Martin P, Parker M: Tailored medicine: whom will it fit? The ethics of patient and disease stratification. Bioethics 18, 322–342 (2004).

Personalized Medicine (2005) 2(2)

WORKSHOP ABSTRACTS

A comprehensive study on the molecular genetic basis of hereditary hearing loss Karen B Avraham, Dror Amiel, Orna Atar, Zippora Brownstein, Orit Dagan. Adi Dgani, Orit Hermesh, Ronna Hertzano, Joel P Jacobson, Tali Landau, Eran Ophir, Natalia Urshanski, Hashem Shahin, Ella Shalit, Alina Starolovsky, Tama Sobe Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel [email protected]

The first and crucial step in sensory processing, the transduction of stimuli, such as odor, light and sound, into a cellular response, are all regulated by genetic pathways. The past years have provided a significant increase in our understanding of some of these pathways, due in large part to the genes found to be associated with inherited hearing loss (HL). This has been no easy task, as human HL is extremely heterogeneous. The intricate structure and multiple cell types of the inner ear requires a range of proteins with different functions, including maintenance of structural integrity, neuronal innervation, and mechanoelectrical transduction. There is great variability in the clinical features of human HL, and mutations in the same genes can contribute to syndromic, non-syndromic, prelingual and postlingual HL. In the past 10 years, mutations in 42 genes have been found, and these have provided clues about auditory transduction, ion homeostasis and inner ear development. We have taken a comprehensive approach to understanding the molecular and functional basis of HL. Our laboratory has identified several human deafness genes, including POU transcription factors and myosins, and are studying the expression and function of the proteins they encode. We are continuing to search for new genes in Israeli cohorts, both for non-syndromic HL and otosclerosis. We have performed a microarray expression profile of embryonic mouse inner ears and have identified downstream targets of the Pou4f3-deafness gene. A yeast two-hybrid cDNA library has been constructed from RNA derived from sensory epithelia of neonatal mice in order to identify protein–protein interactions that are unique in the ear. We are cloning genes responsible for deafness in mice, which serve as models for human HL. We are studying several aspects of connexin involvement in deafness by following the prevalence and genotype–phenotype correlations of connexin deafness in the Israeli population, examining whether there is a connection between connexin mutations and cochlear efficacy with use of a cochlar implant, and performing experimental assays to validate the transmembrane domain structure of a cryo-electron microscopy-based model of gap junction proteins by examination of disease-causing mutations. Our laboratory is also addressing the impact of genetics and molecular diagnosis on the hard-of-hearing and deaf communities. Together, we expect these experimental approaches to provide us with better knowledge of the intricate mechanisms of the inner ear. Deciphering the function of the proteins the deafness genes encode will help us to understand the complexities of auditory function and may lead to therapeutic advances for HL. Bibliography 1.

Gottfried I, Landau M, Glaser F et al.: A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein. Hum. Mol. Genet.11, 1311–1316 (2002).

Preventive programs in Israel: an ethnic-based personalized medicine Gideon Bach Department of Human Genetics, Hadassah University Hospital, Jerusalem 91120, Israel E-mail: [email protected]

Disease-causing mutations can be found only in rare, isolated, patients while in other cases there is a recurrent appearance of a specific mutation. The latter phenomenon is a result of a variety phenomenon, mostly this stems from either mutation ‘hot spots’ or, alternatively, founder mutations in isolated populations or communities in which periods of bottlenecks on the one hand and massive expansions on the other occurred during its history, resulting in a genetic drift. Thus, a typical occurrence of a few inherited disorders, mostly recessive, in high frequency is found in these instances. The unique social constitution in Israel of a variety of ethnic groups, Jews and non-Jews, presents an important medical challenge in this respect, since many of the recessive disorders are either fatal or seriously devastating conditions. Medicine at present is short-handed in the treatment of most of these conditions, and this is particularity true for those affecting the central nervous system. Thus, prevention programs are essentially the most meaningful tools in the reduction of disease occurrence. These programs can be related as ethnic


145

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community-based personalized medicine, since they utilize the existence of a relatively small panel of mutations to detect most of the mutation carriers. A variety of prevention programs has been implicated in Israel, including population screening for the detection of carriers of frequent disorders in each ethnic group as well as newborn screening for the detection of affected patients. These programs are targeted for early patient treatment or for preventive measurements such as prenatal diagnosis or marriage decisions. The programs contributed to a significant reduction of morbidity in Israel.

Sequenom MassArray technology and its uses in Israel Edna Ben-Asher, Daniela Amann, Tsviya Olender, Tamar Koch, Doron Lancet Department of Molecular Genetics, The Weizmann Institute of Science, Israel E-mail: [email protected]

The Sequenom MassARRAY® technology is designed to rapidly distinguish genotypes with a high level of precision and sensitivity. Using Matrix Assisted Laser Desorption/Ionization – Time-of-Flight (MALDI-TOF) mass spectrometry, the MassARRAY system measures target DNA associated with SNPs and other forms of genetic variation directly. Sequenom (San Diego, California, USA) has developed the MassARRAY system to overcome some limitations of traditional approaches to SNP analysis. By combining MassEXTEND primer extension chemistries with high-density SpectroCHIP arrays, the MassARRAY system offers high-throughput SNP analysis. Use of this instrument allows for unambiguous identification of the variant – only one oligonucleotide can fit to the molecular mass determined. This technology is being used at the Weizmann Institute for association studies aiming to identify the genes involved in polygenic traits or diseases on one hand and drug response on the other hand. In these association studies hundreds of SNPs representing tens of candidate genes are being genotyped in search of diseaseassociated genes and their corresponding alleles. Among the various studies are some major projects, in collaboration with leading physicians and researches, that include the search for genes associated with schizophrenia, smoking addiction, diabetes, colon cancer and even tomato fruit quantitative trait locus.

Pharmacogenetics of fluvastatin in familial hypercholesterolemic patients Dani Bercovich1, Vardiela Meiner2, Yechiel Friedlander3, Asi. Houminer3, Amnon Hoffman4, Lilach Kleinberg4, Chen Shochat1, Eran Leitersdorf5, Sigal Korem1 1HMG & Pharm, MIGAL-Galilee Bio-Technology Ctr, 3Dept

of Social

Medicine, Hebrew University; 4School

Israel; 2Dept of HG, Hadassah University Hospital; University Hospital, Jerusalem, Israel.

of Pharmacy; 5Dept of Medicine, Hadassah

Familial hypercholesterolemia (FH) characterized by tendon xanthomas, corneal arcus, and premature atherosclerosis, is commonly treated with HMG-CoA reductase inhibitors. Yet, therapeutic response is frequently also variable among patients with identical low-density lipoprotein cholesterol (LDL-C) receptor mutations. This heterogeneity may partially result from differences in pharmacodynamic (PD) and pharmacokinetic (PK) characteristics, which may be genetically determined. The research reported here focuses on genetic polymorphisms in the cholesteryl-ester transfer protein (CETP) and the Pglycoprotein drug transporter (MDR1), representing PD and PK determinants respectively, and their association with variable response to fluvastatin in 77 molecularly characterized FH patients. Lipid levels were determined in a compliance-monitored clinical study at baseline and following 16 weeks of treatment with escalating doses of fluvastatin. CETP and MDR1 SNP genotyping was performed using denaturing high performance liquid chromatography and sequence analysis. Linear regression was used to examine the associations between common SNPs and haplotypes (based on tagSNPs that capture the majority of common genetic variation) and lipids response. Treatment with fluvastatin resulted in mean LDL-C reduction of 21.48%; mean triglyceride (TG) reduction of 8.33%; and a mean high-density lipoprotein cholesterol (HDL-C) increase of 13.42%. Five tagging SNPs in both genes were used to reconstruct 5 and 6 haplotypes accounting for 71.4 and 90.2% of the observed haplotypes in the CETP and MDR1 genes, respectively. An increase in LDL-C response was associated with CETP-H13 (mean LDL-C reduction 29.38% p = 0.026) and with MDR1-h4 (mean LDL-C reduction 26.56% p = 0.025). Similarly, CETP-H5 was shown to be significantly associated with decreased TG and HDL-C response, while MDR1-h10 was associated with a decrease in TG response. A multivariate regression model indicated an independent additive effect of CETP-H5 and MDR1h10 on the level of TG response. SNP-haplotypes in CETP and MDR1 have significant independent effects on lipid changes following a fluvastatin treatment in FH patients. 146


WORKSHOP ABSTRACTS

Further studies on genetic determinants correlated with adverse effects to statin treatment of FH patients are underway. The results of the reported study can lead to the identification of genetic determinants relevant to drug response and is applicable to improved diagnosis and optimization of drug therapy.

Cross-cultural genetic counseling Rivka Carmi Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel E-mail: [email protected]

The unprecedented advances of genetic technology and the rapid accumulation of genetic data have created a great need for genetic counseling. This communicative process differs from other healthcare encounters since it typically incorporates the entire family and sometimes even a larger circle of the community and often deals with issues that are emotionally charged and very much influenced by education, culture and religious beliefs. Furthermore, as genetic information becomes more complex, this process involves the communication of complicated risk estimates and multiple options that keep changing based on newly acquired scientific data. One of the most important barriers to the provision of genetic counseling and genetic services at large is ethno-cultural distinctiveness. Patients have their own cultural interpretations of health and disease, societal-based preferences for health behavior and cultural and religious considerations formulating their decision-making processes. Western-style genetic counseling often lacks deep awareness of non-Western cultural values and responses of distinct ethnic communities and thus may results in decreased utilization of genetic services and low compliance with communicated recommendations. Examples of genetic counseling programs implemented over the years in various ethnic communities have illustrated the limitation of the prevailing Western-style genetic counseling model and served to emphasize the notion that any counseling or intervention program should be tailored to the given population, based on the identification of its specific cultural and societal barriers. The sickle cell anemia screening program in African–Americans, which was not sensitive to some basic cultural issues and was not accompanied by a proper educational campaign, resulted in stigmatization and further discrimination of the target population. Other programs such as the thalassemia screening in Southeast Asia and in the Mediterranean basin, and the Dor Yesharim program in the orthodox Jewish community were preceded by ethical, psychological and cultural investigations and thus proved very successful in lowering the prevalence of genetic diseases. Some of these programs challenge basic tenets of Western-style genetic counseling such as non-directive counseling, individual consent and right of an individual for access to his/her own genetic information. However, addressing specific ethno-cultural considerations made those programs the most appropriate and thereby the most successful for specific communities wishing to benefit from new genetic technologies within the context of their unique societal fabric. Our own experience with providing genetic counseling services to the Negev Bedouin-Arabs, a highly traditional, consanguineous community, illustrates the effectiveness of adopting specific guidelines when working with populations with diverse backgrounds. Among them are: respect and acknowledgment of beliefs and practices concerning the presented problem, attention and respect to expressed suspicion or distrust, sensitivity to the possibility of stigmatization, and provision of guidance to enable the formulation of plan of action that will be culturally acceptable. The basic approach of our program is to engage the community in the design of what seems to be the most culturally appropriate genetic programs, educate the public on genetic issues as well as train semiprofessionals from within the community to provide the services. The results are encouraging though a comprehensive evaluation warrants a longer follow-up as changes in health behavior and in disease prevalence are long-term goals. Finally, most contemporary discussions address cross-cultural issues from a perspective of presumably defined ethno cultural groups. However, the reality is much more complex since even within defined communities there are differences among individual members related, among others, to education, economy and societal status within the culture group. Each of those factors creates complex dynamics of self identity, beliefs and affiliations that transcend those discussed under the unitary ethno-cultural categories. These issues should be recognized and properly addressed when considering any type of genetic-related intervention. Bibliography 1. 2. 3. 4. 5.

Dixson B, Dang JO, Cleveland PRM: An educational program to overcome language and cultural barriers to genetic services. J. Genet. Counsel 1, 267–274 (1992) King P: The past as a prologue: race, class and gene discrimination. In: Gene mapping: using law and ethics as guides. Annas G, Elias S (Eds), Oxford University Press, pp 94–111 (1992). Wang V, Marsh F: Ethical principles and cultural integrity in healthcare delivery: Asian ethno cultural perspectives in genetic services. J. Genet. Counsel 1, 81–92 (1992). Weil J: Multicultural education and genetic counseling. Clin. Genet. 59, 143–149 (2001). Weitzman D, Shoham-Vardi I, Elbedour K, Belamker I, Siton Y, Carmi R: Factors affecting the use of prenatal testing for fetal anomalies in a traditional society. Comm. Genet. 3, 61–70 (2000).


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Automated mass spectrometry in personalized medicine Charles R Cantor SEQUENOM, Inc., San Diego, CA 92121, USA

Personalized medicine is a laudable goal but it is realistic only if, simultaneously, it allows a reduction in the overall cost of healthcare. There may be several ways to achieve this: by profiling individual disease risk susceptibility, the frequency of testing for disease onset can be tuned to individual needs; by replacing invasive tests for disease risk or progression with non-invasive tests, considerable cost savings can be realized; by using inexpensive biomarkers as indicators of disease progression, more expensive testing of people who are actually well may be avoidable. In addition, a great deal of individual responsiveness to particular therapies such as pharmaceuticals may be predictable before these medications or procedures are implemented. SEQUENOM approaches individualized medicine in two ways. Its automated mass spectrometry platform for the analysis of nucleic acids provides a uniquely sensitive and quantitative way to detect both genetic risk markers and disease onset and progression markers. The system is totally flexible and allows many tests to be multiplexed and run simultaneously on a single sample. During 2005 the first diagnostics tests using the SEQUENOM platform will be launched commercially. These are likely to include such tests as non-invasive fetal sex determination, by detecting Y-chromosomal markers in the maternal circulation, and highly multiplexed tests of common genetic variations that predispose to cystic fibrosis. Because automated mass spectrometry coupled to PCR provides sensitivity down to single molecules, it promises improvements in such tests as viral load, infectious disease agent typing, and markers in blood, urine, or the mouth that reflect disease progression. While RNA profiles can be studied in this way, our experience thus far suggests that corresponding methylated DNA markers may be more powerful and more easily utilized. SEQUENOM’s second contribution to the prospects of individualized medicine comes as a result of the large program of human genetic studies that has been executed over the past few years. More than 11 whole-genome single nucleotide polymorphism scans have been carried out to measure disease association in the general population. Included are studies of breast cancer, lung cancer, prostate cancer, Type II diabetes and schizophrenia. In every study, multiple disease predisposing genes have been discovered that can be replicated in multiple independent populations and thus present true positive associations and not false-positive Type I errors. A number of these gene discoveries will be described. What is intriguing is that many of them, although discovered purely as disease risk markers, also appear to have predictive or monitoring value in disease progression. Some genes are found to play a role in multiple disease indications such as breast cancer and prostate cancer. In diagnostic testing a major source of error is sample mix up. Because of the high multiplexing power of SEQUENOM’s analytical platform, it may become cost effective to carry out a personal identity test on every diagnostic sample under examination for a particular purpose. If this aspect of personalized medicine can be realized, it promises to improve the overall standard and reduce the cost of medical care in a simple but direct way.

The role of the APOE genotype in immunomodulation Joab Chapman1,2, Galia Dallal2, Daniel M Michaelson3 1Department

of Neurology, Sheba Medical Center, and 2Department of Physiology and Pharmacology, Sackler Faculty of Medicine, 3Department of Neurobiochemistry, Wise Faculty of Life Sciences, Tel Aviv University, Israel

The effect of APOE genotype on neurodegenerative disease is well-established by epidemiological data. Both primary degenerative disorders, such as Alzheimer’s disease, and neuroimmunological disorders, such as multiple sclerosis, are influenced by the specific genotype of common APOE ε3 or ε4 alleles. The mechanisms in which this genotype influences neurological disease is complex and probably involves lipid pathways and regeneration in the CNS, as well as immunological and inflammatory factors in the periphery and in the CNS. In the present study we established a lymphocyte-based assay to examine the interaction of APOE with immune activation. Normal human lymphocytes were isolated by a standard commercial gradient kit and stimulated by concanavalin A (ConA) or lipopolysaccharide (LPS). Experiments were performed in the presence of media conditioned by astrocytes cell lines producing APOE ε3 or ε4. The effects of treatments on the activity of the lymphocytes were assayed by both proliferation (BrdU) and tumor necrosis factor (TNF)-α secretion (ELISA). Stimulation of lymphocytes with either ConA or LPS produced significant tenfold increases in TNF-α secretion. This was reduced by up to 50% by exposure to APOE-enriched media. The effect was similar in APOE ε3 and ε4 in these preliminary experiments. This experimental system may provide insights into the mechanism by which APOE influences immune function both in the periphery and in the CNS.

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Possible differences in the immune response to the common environmental disease factor Onchocerca volvulus in two ethnic communities living in the Ecuadorian rainforest GF De Stefano Department of Biology, University of Rome Tor Vergata, Rome, Italy E-mail: [email protected]

Host factors are known to be important in determining the pathological response to a number of infectious diseases. The degree to which genetic factors determine host response appears uncertain for a number of infectious diseases, and it remains unknown for onchocerciasis in spite of such factors being shown to be important in other tropical diseases. Geographical differences in disease presentation have been demonstrated in Africa, but within a single ethnic group. Such differences might be accounted for by different parasite (Onchocerca volvulus) strains, different Simulium species or environmental factors. Differences have also been described between clinical disease in Africa and that which is encountered in Latin America involving groups of different ethnic origin. However, it is still difficult to distinguish ethnically related genetic factors from geographical differences already listed. The opportunity to study two distinct ethnic groups living side-by-side and exposed to similar transmission variables have been afforded in the Esmeraldas province, Northwestern Ecuador. Here, blacks (of African descent) and Chachi (Cayapa) Amerindians inhabit a rainforest focus of onchocerciasis. Previous epidemiological studies of the disease and its clinical presentation suggested that the Ecuadorian focus is relatively new and expanding geographically. This changing epidemiological picture and continuing evolution of the disease fostered the study of previously unobserved ethnically related immune genetic differences. The possible relationship between such observed differences and the results of an ongoing study, which take into account nuclear families of the two ethnic groups and putative immune individuals, will be discussed on the basis of preliminary results on the DQA1*0103 and DQB1*0603 allele molecular variability.

The LIM domain transcription factor LHX3 is a putative target of POU4F3 in the inner ear Amiel A Dror1, Ronna Hertzano1,2,5, Mireille Montcouquiol2, Sharon Rashi-Elkeles3, Rani Elkon3, Gidi Rechavi4, Thomas B Friedman5, Matthew W Kelley2, Karen B Avraham1 1Dept

of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, on Developmental Neuroscience, NIDCD-NIH, Rockville, MD, USA, 3The David and Inez Myers Laboratory for Genetic Research, Dept of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 4Dept of Pediatric Hemato-Oncology and Institute of Hematology, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 5Section on Human Genetics, NIDCD-NIH, Rockville, MD, USA

2Section

A mutation of POU4F3, a class IV POU domain transcription factor, underlies human autosomal-dominant non-syndromic progressive hearing loss DFNA15. We used Affymetrix oligonucleotide microarrays to generate expression profiles of inner ears of Pou4f3ddl/ddl mutant and wild-type mice in order to identify downstream targets of this inner ear hair cell-specific transcription factor. We identified and validated the gene encoding the LIM domain transcription factor, Lhx3 (also known as lin11, Islet-1, mec-3), as an in vivo target gene regulated by Pou4f3. Lhx3 is known to be involved in motor neuron and pituitary cell specification. Mice null for Lhx3 are embryonic lethal due to pituitary dysgenesis, and humans with mutations in Lhx3 have combined pituitary hormone deficiency. Semi-quantitative RT-PCR revealed that a deficiency of Pou4f3 leads to a statistically significant reduction in Lhx3 expression levels in the inner ear. Furthermore, Lhx3 protein is specifically expressed in the sensory hair cells of the auditory and vestibular systems. Our results demonstrate that a transcription profiling experimental strategy, starting with inner ears of embryonic mice, can reveal gene expression changes that are specific for hair cells.


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Promoting Arab and Israeli cooperation: peace building through health initiatives Harvey Skinner1, Ziad Abdeen2, Hani Abdeen2, Phil Aber1, Mohammad Al-Masri12, Joseph Attias4, Karen B Avraham5, ProfRivka Carmi6, ProfCatherine Chalin1,7, Ziad El Nasser3, Manaf Hijazi8, Rema Othman Jebara2, Moien Kanaan9, ProfHillel Pratt10, Firas Raad11, Yehudah Roth5, A Paul Williams1 and Arnold Noyek1,7 1University of Toronto, Toronto, ON, Canada, 2Al Quds University, Abu Dis, West Bank, 3Jordan University of Science and Technology, Irbid, Jordan, 4University of Haifa, Haifa, Israel, 5Tel Aviv University, Tel Aviv, Israel, 6Ben Gurion University of the Negev, Beer Sheva, Israel, 7Mount Sinai Hospital, Toronto, ON, Canada, 8Royal Medical Services of Jordan, Amman, Jordan, 9Bethlehem University, Bethlehem, West Bank, 10Technion–Israel Institute of Technology, Haifa, Israel, 11Harvard University, Boston, MA, USA, l2Amman University, Amman, Jordan E-mail: [email protected]

This article describes a positive experience in building Arab and Israeli cooperation through health initiatives. Over the past 10 years Israeli, Jordanian, and Palestinian health professionals have worked together through the Canada International Scientific Exchange Programme (CISEPO). In the initial project, nearly 17,000 Arab and Israeli newborn babies were tested for early detection of hearing loss, an important health issue for the region. The network has grown to address additional needs, including mother–child health, nutrition, infectious diseases, and youth health. Our guiding model emphasizes two goals: project-specific outcomes in health improvement, and broader effects on crossborder cooperation. Lessons learned from this experience and the model provide direction for ways that health professionals can contribute to building peace.

Genetics analysis of human infertility M Fellous INSERM U709, Hôpital Cochin, Pavillon Baudelocque, 123, bd de Port Royal, Paris, France E-mail: [email protected]

Introduction Infertility affects about 15% of couples. In most cases, the genetic causes are still unknown. Female factor infertility accounts for about half of infertile couples. Results and discussion Female infertility

Premature ovarian failure (POF) is a rare disease affecting 1–3% of women < 40 years. The clinical features are a primary or secondary hypergonadotropic amenorrhea, leading to infertility that can usually be solved only by ovum donation. Although the detection of genetic anomalies and mutations in some familial cases of POF points to a genetic origin of the syndrome, the precise etiology of the disease is still unknown in > 90% of patients. The Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES), a rare disease associated with POF, is induced by mutations in FOXL2, the earliest and recently discovered marker of ovarian development. Through characterization of the transcription factor FOXL2, we have obtained results providing new insights into the pathogenic mechanisms associated with the most frequent mutation in this gene. In addition, the wholegenome screening of genetic linkage in rare familial forms of POF has uncovered a large region of interest, where we expect to identify, with a positional cloning approach, new genes associated with POF. Male infertility

Male factor infertility accounts for about half of the cases of couple infertility. In over 60% of cases the origin of reduced testicular function is unknown but may have an unidentified genetic anomaly. Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb and AZFc) that are recurrently deleted in infertile males. Several genes have been identified within this region and have been proposed as candidates for infertility. Many of these genes encode proteins involved in post-transcriptional gene expression and, therefore, could participate in the sperm maturation process. About 10–15% of azoospermic and about 5–10% of severely oligozoospermic men have Yq microdeletions. The deletions are associated with a wide range of histological pictures ranging from Sertoli cell only syndrome (SCOS) to spermatogenic arrest and severe hypospermatogenesis. Assisted reproduction techniques such as in vitro fertilization (IVF) and

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intracytoplasmic sperm injection (ICSI) alone, or in association with testicular sperm retrieval, represent an efficient therapy for these patients. However, the potential of these techniques to transmit genetic defects causing male infertility raises the need for systematic genetic screening and genetic counseling of these patients. Bibliography 1. 2. 3. 4. 5. 6.

Calogero AE, Garofalo MR, Barone N et al.: Spontaneous transmission from a father to his son of a Y chromosome microdeletion involving the deleted in azoospermia. (DAZ) gene. J. Endocrinol. Invest. 25, 631–663 (2002). Choi JM, Chung P, Veeck L et al.: AZF microdeletions of the Y chromosome and in vitro fertilization outcome. Fertil. Steril. 81, 337–341 (2004). Kent-First MG, Kol S, Muallem A et al.: The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol. Hum. Reprod. 2, 943–950 (1996). Krausz C, Forti G, McElreavey K: The Y chromosome and male fertility and infertility. Int. J. Androl. 26, 70–75 (2003). Patsalis PC, Sismani C, Quintana-Mursi L et al. Effects of transmission of Y chromosome AZFc deletions. Lancet 360, 1222–1224 (2002). Simpson JL, Rajkovic A. Ovarian differentiation and gonadal failure. Am. J. Med. Genet. 89(4), 186–200 (1999).

Pharmacogenomic research in drug development – the ethical concern, bridging the gap between public perceptions and industries realities Julie Friedman, MPH, BScN Pharmacogenomic Clinical Liaison, Group Leader, Clinical Discovery, Bristol–Myers Squibb, USA [email protected]

The pharmaceutical industry continually evaluates advances in science to improve the selection of new chemical entities while striving to identify compounds which can survive the arduous development process and eventually make it to market as novel efficacious drugs. It is therefore not surprising that following the complete sequencing of the human genome, scientists have increasingly focused on identifying novel gene sequences, studied the impact of DNA sequence variation on gene function and expression, and are trying to understand the impact genetic variations have on drug safety and efficacy. What has perhaps been surprising are the levels of concern appearing in public advocacy groups, institutional ethics committees and Ministries of Health around genetic based research as compared to more ‘traditional’ medical research models. Indeed, many groups have placed a significantly greater ‘risk’ to individual privacy on genetic data than on traditional clinical data. While well intentioned, these concerns are likely to be misguided. With the exception of rare Mendelian single trait diseases, genotyping and mRNA expression profiling typically provide much lower informational value for predicting disease risk and susceptibility than well-accepted ‘routine’ clinical measures such as blood pressure, glucose levels or lipid levels. Ironically, despite this disparity in predictive value, very few groups are asking that routine clinical information be anonymized or held by a third party. In pharmaceutical development, all clinical data are handled extremely carefully as it would be highly damaging for any company to develop a reputation for not abiding by all necessary laws and regulations governing investigatory drug trials. Despite this fact, some current and proposed restrictions on genetic research appear to stem from misconceptions as to the types of information being generated, the ultimate value of these data, and the intended use. It is fair to evaluate why such scrutiny is currently being applied to genomics data, and in the abstract, one concludes that it is simply fear spawned by lack of balanced information regarding this emerging science. Public perception is that in the near future everyone will carry their genetic sequence on a ‘credit card’ and that with this information physicians will identify disease susceptibility and prescribe appropriate medicines. While the promise of personalized treatment has a high public profile and is a worthy goal for the future it is not the primary focus of genetic research in the drug development process. On average it takes 10–15 years to develop a new marketable drug and the failure rate from new clinical entity to marketable compound remains around 90%. The high failure rate and growing cost of drug discovery is simply not a tenable model for the future and, new discovery approaches and innovations are required. Tools to improve drug candidate selection and improve safety profiles are essential and there are a multitude of genomic research strategies in use today. Some of the most common approaches are aimed at: • • • • •

increasing knowledge of the mechanism of action of a new drug identifying how a drug interacts with its target defining the safety profile of a novel compound and off target effects understanding how genetic polymorphisms affect the drugs interaction with its intended target mRNA expression profiling can identify groups (for example tumor types) that are similar to one another in the way that they respond to chemotherapy


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• using a variety of techniques when screening multiple drug candidates to make more informed decisions on those with the best profile, the most likely to show efficacy while avoiding major toxicity issues • increase the understanding of disease pathology in order to identify novel 'drugable' targets Genomic research is in its infancy but already provides significant information which facilitates the drug development process. To become more comfortable with genomic data sets and the benefits to patients, academic investigators, organizational oversight committees and the general public must be provided with balanced information regarding process, expected outcomes, benefits and overall risk of genomic research. When well informed, individuals will hopefully allay existing concerns and promote the further exploration of this science and allow genetic research to fill its potential for improved patient care and drug development.

Personalized medicine: ethical and social consequences Felix W Frueh, PhD Associate Director for Genomics, Office of Clinical Pharmacology and Biopharmaceutics, FDA/CDER/OCPB, USA E-mail: [email protected]

The human genome has been sequenced, the SNP consortium has published 1.8 million SNPs, and the HapMap program is well underway. Trying to identify which genetic variations are most important to better understand human disease is one critical aspect of these efforts, another one is to explore this genetic variability to comprehend differences in drug response among individuals. Whether or not this effort will lead to ‘Personalized Medicine’ in the near future can be debated; the conception that drug development will deliver products tailored to an individual’s genome is likely a vision that is many years or decades away. Nevertheless, along the road of personalizing medicine, we will use the human genome as a map and, consequently, encounter issues that are reaching beyond the science associated with the discovery of new, important genetic variations. Genetic markers most often display different frequencies in different populations. If such markers are used to identify individuals for which therapy is likely to be successful (efficacy markers), or, alternatively, might pose a health risk (safety markers), we have generated a situation in which a specific drug therapy can benefit one population group more than another group. This creates an ethical dilemma, in particular if no alternative treatment is available. Besides the use of genetic markers, the publicity around the use of skin color to identify a specific population is of controversy as well; BiDil, a drug used for the treatment of advanced heart failure, has been shown to have superior efficacy in the African-American population (as shown by a significant decrease in one-year mortality rates), whereas the Caucasian population sees no benefit. Discussions around racial discrimination sparked. Do we need to identify the molecular mechanism of action before we can justify bringing this product to market or can self-identified race/ethnicity be used as a ‘biomarker’? The controversy illustrates the tight connection between markerdriven drug therapy and ethical and social implications. However, it would be wrong to delay the marketing of drugs that are effective in certain populations, but not others, only because we do not yet have an understanding of the molecular mechanism(s) of response or action. Recent discoveries (i.e., on ‘Whole Genome Patterns of Common DNA Variation in Three Human Populations’) illustrate that we are getting a better understanding of underlying molecular mechanisms (genetic variability) by which differences in drug therapy can be explained, but it will take many years more to translate these findings into clinical practice. To achieve this goal, we must educate the public and raise awareness of what can, but also cannot, be achieved by using genetic analysis to guide drug therapy. In February 2005, the US Congress passed a law against genetic discrimination: an important step towards this objective. The US FDA released the final ‘Guidance for Industry: Pharmacogenomic Data Submissions’, encouraging industry to use new tools provided by pharmacogenomics to develop better, more targeted drug therapies. We are entering the ‘genome era’ – and within this exciting era rests the responsibility to address issues surrounding the science in order for the science to reach and impact public health. The views expressed in this article are the ones of the author and may not represent the view or policies of the US Food and Drug Administration.

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Individualization of isoniazid doses based on NAT2 genotype: design features of a randomized clinical trial Uwe Fuhr Department of Pharmacology, Clinical Pharmacology Unit, University of Cologne, Gleueler Strasse 24, 50931 Koeln, Germany E-mail: [email protected]

Isoniazid is a pivotal agent in the treatment of tuberculosis. The daily standard oral dose in adults is 300 mg. Hepatotoxicity is observed in approximately 10% of all patients and occasionally results in severe hepatitis. On the other hand, in approximately 15% of patients, no therapeutic success is achieved by standard treatment after 8 weeks, with final treatment failure in approximately 5% of patients. Isoniazid is acetylated by the genetically polymorphic enzyme arylamine N-acetyl-transferase Type 2 (NAT2). Patients phenotyped as ‘rapid acetylators’ carry one (‘intermediate acetylator’) or two high-activity alleles (NAT2*4, NAT2*12), whereas ‘slow acetylators’ have two low-activity variants. Individual isoniazid concentrations following administration of the standard dose depend on NAT2 activity [1]. Slow acetylators have a clearly higher risk to develop hepatotoxicity [2,3], and NAT2 genotype also affects therapeutic activity [4]. To assess the potential therapeutic benefit of a dose adjustment of isoniazid according to NAT2 genotype, we designed a prospective, randomized, double-blind, controlled clinical study. The study design differs from that of other Phase III trials: • the patients in the intervention arm will have the different treatment changes: the control treatment will be compared to a dose reduction in slow acetylators but to a dose increase in rapid acetylators, whereas no adjustment is made in intermediate acetylators. Accordingly, genotype specific hypotheses are needed. • since there is wide variation in the frequency of NAT2 genotypes between different populations, recruitment of patients for the individual groups has to be focused to different geographical areas. • it is difficult to derive a quantitative estimate for the expected benefit from a dose adjustment as the basis for sample size calculations, because factors beyond isoniazid plasma concentrations may play a role in differences of isoniazid effects between genotypes. We make the following assumptions: • frequency of 0.45 for high activity alleles in the mixed study population in which incidence of adverse events and treatment failure has been reported • 10.0% hepatotoxicity overall with risk multiplier 1/3 between slow–intermediate and intermediate–rapid genotypes and between adjusted/not adjusted treatment within acetylator groups • 15.0% early treatment failure overall with corresponding risk multiplier 2. For slow acetylators, we calculated a sample size of n = 2 × 121 to demonstrate superiority of the dose adjustment regarding hepatotoxicity. Likewise, for rapid acetylators with regard to treatment failure a sample size of n = 2 × 158 would be needed. Other details of the planned study will be discussed The proposed study is considered as a ‘proof of concept’ study to assess the therapeutic impact of the intervention ‘dose adjustment according to a genetic polymorphism of a drug metabolizing enzyme’ in a disease with global importance. Bibliography 1. 2. 3. 4.

Kinzig-Schippers M, Tomalik-Scharte D, Jetter A et al.: Should we use NAT2 genotyping to personalize isoniazid doses? Antimicrob. Agents Chemother., (In Press) (2005). Huang YS et al.: Polymorphism of the N-acetyltransferase 2 gene as a susceptibility risk factor for antituberculosis drug-induced hepatitis. Hepatology 35, 883-889 (2002). Ohno M, Yamaguchi I, Yamamoto I et al.: Slow N-acetyltransferase 2 genotype affects the incidence of isoniazid and rifampicin-induced hepatotoxicity. Int. J. Tuberc. Lung. Dis. 4, 256–261 (2000). Donald PR et al. The influence of human N-acetyltransferase genotype on the early bactericidal activity of isoniazid. Clin. Infect. Dis. 39, 1425–1430 (2004).


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Pharmacogenetics of glatiramer acetate therapy for multiple sclerosis reveals drug-response markers Iris Grossman1,2, Nili Avidan2, Clara Singer2, Dan Goldstaub3, Liat Hayardeny3, David Ladkani3, Eli Eyal3, Shaul Kadosh3, Edna BenAsher2, Muriel Chemla2, Tamar Paperna1, Jacques S. Beckmann2, Doron Lancet2, Ariel Miller1 1Division

of Neuroimmunology and Multiple Sclerosis Center, Rappaport Faculty of Medicine and Research Institute, Technion and Carmel medical center, Haifa, 2Department of Molecular Genetics, Crown Human Genome Center, Weizmann Institute of Science, Rehovot, 3TEVA Pharmaceutical Industries Ltd, Kiryat Nordau, Netanya E-mail: [email protected]

Evidence is accumulating to indicate that response patterns to drugs are at least partly under genetic control. For a disease such as multiple sclerosis the evaluation of patients’ response to drug treatment may take up to 2 years. During this time a significant deterioration in health may amass. For this reason, a diagnostic tool that will allow the allocation of drugs and their doses, based on objective genetic screening, may have a considerable impact on patient care and health management. The field of pharmacogenetics is aimed at seeking such solutions by testing the potential associations between genetic markers, such as SNPs or their haplotypes, and drug response. In an exploratory study with this future goal in mind, we have genotyped 63 SNPs within 27 candidate genes likely to be involved in the presumed mode-of-action of the multiple sclerosis drug glatiramer acetate and its clinical response features. DNA samples were obtained from two different clinical trials, each of which was analyzed separately, since they involved different populations and primary clinical end points. A number of candidate genes were found to be significantly associated with different response definitions. An overlap of associations between the two independent cohorts tested corroborates the robustness of the results. The fact that significant association is found despite the rather limited number of patients and SNPs studied, may indicate that the glatiramer acetate drug response is under the genetic control of a limited number of genes, each potentially having a rather pronounced effect. Taken together, our results indicate that the goal of personalized treatment for multiple sclerosis, and possibly other autoimmune diseases, may be within reach.

Understanding the pathogenesis of human hereditary deafness by expression profiling of inner ears from mutant mice Ronna Hertzano1,2,7, Mireille Montcouquiol2, Sharon Rashi-Elkeles3, Rani Elkon3, Amiel Dror1, Raif Yücel4, Wayne N. Frankel5, Gidi Rechavi6, Tarik Möröy4, Thomas B. Friedman7, Matthew W. Kelley2, Karen B. Avraham1 1Dept.

of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 2Section on Developmental Neuroscience, NIDCD-NIH, Rockville, MD, USA, 3The David and Inez Myers Laboratory for Genetic Research, Dept of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 4Institut für Zellbiologie, Universitätsklinikum Essen, Essen, Germany, 5The Jackson Laboratory, Bar Harbor, ME, USA, 6Dept. of Pediatric Hemato-Oncology and Institute of Hematology, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 7Section on Human Genetics, NIDCD-NIH, Rockville, MD, USA E-mail: [email protected]

Inherited hearing loss is a common sensory defect, affecting approximately 1 in 2000 newborns and a significant portion of the elderly population. The ear is the mammalian auditory and vestibular sensory organ. The outer and middle ears comprise the sound conductive system. The inner ear is a fluid-filled organ consisting of bony and membranous labyrinths that contain the auditory (cochlea) and vestibular sensory end-organs (utricle, saccule and ampluae of the semicircular canals) connected to cranial nerve (CN) VIII. The sensory organs contain sensory epithelia that are comprised of a mosaic of sensory and supporting cells that are organized in a highly ordered fashion. The sensory cells, named hair cells, are cells with actin-rich apical projections named stereocilia, which function as mechanosensors and transducers of sound and movement in the auditory and vestibular systems, respectively. Pou4f3 (Brn3.1, Brn3c) is a class IV POU domain transcription factor that has a central function in the development and survival of all hair cells in the human and mouse inner ear sensory epithelia. Our laboratory has previously reported that a mutation of POU4F3 underlies human autosomal dominant non-syndromic progressive hearing loss DFNA15. In the absence of Pou4f3, hair cells begin to form, but undergo apoptosis beginning at E17, resulting in a complete depletion of hair cells from all inner ear sensory epithelia by early postnatal stages. We sought to identify and validate the downstream in vivo target genes of Pou4f3. We used Affymetrix oligonucleotide microarrays to generate expression profiles of inner ears of Pou4f3ddl/ddl mutant and wild-type mice, and successfully identified growth 154


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factor independence 1 (Gfi1), Lhx3 and brain derived neurotrophic factor (Bdnf ) as the first downstream targets of an inner ear hair cell-specific transcription factor. We further validated these results using a combination of semiquantitative RT-PCR, in situ hybridization and comparative immunohistochemistry and scanning electron microscopy. Our results demonstrate that a deficiency of Pou4f3 leads to a statistically significant reduction in Gfi1 expression levels that most probably lead to the outer hair cell degeneration observed in Pou4f3 mutants. We further show that STAT3, a signaling molecule known to function downstream of Gfi1, is localized to the OHC of the mouse inner ear, suggesting that Pou4f3 might act through Gfi1 and ultimately STAT3 to promote OHC survival. Our results demonstrate that a transcription profiling experimental strategy, starting with inner ears of embryonic mice, can reveal gene expression changes that are specific for hair cells and provide the first insights into a hair cell-specific signaling cascade that is involved in promoting hair-cell survival. Most important, our data provide the first insights into the mechanism of POU4F3-related deafness in vivo and may aid in the future in the development of therapeutics for progressive hearing loss.

Novel Palestinian mutations in deafness-related genes M Kanaan1, T Walsh2, AA Rayan1, H Shahin1,3, K Hirschberg3, M Tekin4, KB Avraham3 & M-C King2 1Bethlehem

University, Bethlehem, Palestinian Authority, 2University of Washington, Seattle WA 98195 USA, 3Tel Aviv University, Tel Aviv 69978 Israel, 4Ankara University, Ankara 06650, Turkey E-mail: [email protected]

We are searching for genes for hearing loss in the Palestinian population. Of the 15 multiply affected, consanguineous kindreds characterized thus far, deafness in most kindreds maps to genomic regions likely to harbor novel hearing-related genes. In contrast, hearing-loss phenotypes in five kindreds were linked to locales of known deafness genes: TMPRSS3, pendrin (SLC26A4), and otoancorin. Sequencing revealed a novel allele of each gene in each kindred. We characterized the mutations and determined allele frequencies among 136 other, unrelated Palestinian children with prelingual hearing loss and among 200 Palestinian adults with normal hearing. TMPRSS3 In family W, all 15 individuals with prelingual, bilateral, severe-to-profound hearing loss were homozygous for a frameshift mutation in exon 10 of TMPRSS3: 988delA (357stop). The mutation presumably abrogates TMPRSS3 serine protease activity. The allele frequency of TMPRSS3.988delA was 2% among other Palestinian probands with prelingual hearing loss and 0.5% among Palestinian controls. The TMPRSS3 Palestinian β satellite mutation, which defined DFNB10, was not present in any deaf families or controls in our series. Pendrin Severe-to-profound hearing loss and enlarged vestibular aquaduct (EVA) in families Y and BF are due to two novel mutations in pendrin (SLC26A4), a missense 716 Threonine>Alanine (V239D) and a splice mutation (1001[+1] Glycine>Threonine) respectively. Thyroid function was normal in affected individuals in both families. We used green fluorescent protein (GFP) chimeras of wild-type pendrin and of the mutant protein 716T>A to study their intracellular trafficking in living cells. The mutant protein is retained in the endoplasmic reticulum, whereas the wild-type protein targets to the plasma membrane. In family BR, we have shown that 1001(+1)Glycine>Threonine abrogates normal splicing of SLC26A4 exon 8 and unmasks a cryptic splice site in intron 8, leading to insertion of 43 bp into the message and premature truncation of the pendrin protein. Both pendrin mutations did not occur in other Palestinian probands with hearing loss nor among Palestinian controls. Otoancorin In family BR, all six individuals with prelingual, bilateral, moderate-to-severe hearing loss were homozygous for a missense mutation in otoancorin: 1067Alanine>Threonine (D356V). Otoancorin D356V is a nonconservative change at a highly conserved site. The predicted consequence of D356V is disruption of the transmembrane domain structure. The allele frequency of otoancorin D356V was zero among other Palestinian probands with hearing loss and 0.025% in the Palestinian hearing population. The otoancorin Palestinian mutation ivs12(+2)Threonine>Cysteine, which defined DFNB22, was not present in any probands or hearing controls in our series. Inherited hearing loss in the Palestinian population is highly heterogeneous both in the number of genes involved and in the number of alleles at each gene. With the exception of mutations in GJB2, alleles responsible for hearing loss in this population are individually very rare. Families with hearing loss who are wild type for GJB2 are likely to harbor novel alleles, either of known hearwww.futuremedicine.com

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ing-related genes (as in these three families) or of previously unknown genes. Studies of multiple Palestinian kindreds are in progress to identify and characterize the previously unknown genes. Genetic heterogeneity of deafness in the Palestinian population may offer a model for complex traits generally. That is, other complex traits may also be due to many different mutations, with only one or a few mutations present in any one family but many mutations in the population as a whole. The high rate of consanguinity in the Palestinian population reveals such alleles that lead to recessive traits. The lesson of heterogeneity however, may apply to dominant as well as to recessive traits, and therefore to populations regardless of their demographic structure. Bibliography Walsh T, Walsh V, Lee MK, Avraham KB, King M-C, Kanaan M: A new locus for non-syndromic hearing loss on 11q14.3-q21 identified in a consanguineous Palestinian kindred. Abstract, Mol. Biol. Hearing and Deafness, (Oct 2004). Kanaan M, Walsh T, Abu Rayan A et al:. Novel Palestinian mutations in deafness-related genes. Abstract, Mol. Biol. Hearing and Deafness (2004). Shahin H, Walsh T, King M-C, Avraham KB, Kanaan M: DFNB28, a novel locus for prelingual nonsyndromic recessive hearing loss maps to 22q13 in consanguineous Palestinian families. Abstract, Mol. Biol. Hearing and Deafness (2004).

1. 2. 3.

Gene–environment interactions on bone mass: The Framingham Study David Karasik1, Robert R McLean1, Douglas P Kiel1, Serge L Ferrari2, Amanda M Shearman3, L Adrienne Cupples4 Rehabilitation Center for Aged and Harvard Medical School, Boston, MA, USA, 2Geneva University Hospital, Switzerland, 3Massachusetts Institute of Technology, Cambridge, MA, USA, 4Boston University School of Public Health, Boston, MA, USA [email protected]

1Hebrew

Background Common diseases are often a result of the complex interplay between genetic and environmental factors. Genetic variations do not necessarily cause complex disease but rather influence a person’s susceptibility to detrimental environmental factors. A good example of this concept is a genetic and environmental network that contributes to osteoporosis. Osteoporosis is a major public health problem. Bone mineral density (BMD) measured with dual X-ray absorptiometry is considered to be among the best predictors of the risk for osteoporotic fracture. In our recent whole-genome linkage analysis we have shown that quantitative trait loci for BMD differ among sub samples stratified on the known biological contributors to bone mass, such as sex and age [1]. We then examined possible interactions between polymorphisms in several candidate genes and environmental factors on BMD in a framework of our group’s ongoing search for genes responsible for osteoporosis. Methods Femoral and lumbar spine BMD were assessed in unrelated women (n = 817) and men (n = 737, mean age ±standard deviation, 60 ± 9, range 32–86 yrs) from the Framingham Offspring Study. Genotyping was performed in several genes, including interleukin (IL)-6, methylenetetrahydrofolate reductase (MTHFR), estrogen receptor α (ESR1), and low-density lipoproteinrelated protein (LRP)5. We collected information on age, height, body mass index (BMI), physical activity, smoking status, dietary intakes of calcium and folate, and menopausal status and oestrogen replacement therapy in women. These traits were used as covariates or effect modifiers in our analyses. Results IL-6

In women, many interactions were significant between IL-6 promoter genotype -174 and calcium intake, estrogen status, and years since menopause. Thus, BMD was higher in CC compared with GG women with calcium intake below 940 mg/d and in women who were ≥15 years after menopause. In estrogen-deficient women with poor calcium intake, BMD differences between genotypes CC and GG were 10.2% at femoral neck (p = 0.012) and 12.0% at trochanter (p = 0.012). In contrast, no difference was observed in women who were either estrogen-replete, reached menopause less than 15 years ago, or whose calcium intake was above 940 mg/d. No interactions were found in men [2]. ESR1

Interactions were significant (p = 0.003 to 0.03) between ESR1 genotypes XbaI and PvuII and current smoking on femoral BMD in men only. Thus, among male smokers the XX homozygotes had the lowest BMD, while among male nonsmokers, this genotype was associated with the highest BMD. 156


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MTHFR

A statistically significant interaction also was found between the common C677T mutation in the MTHFR gene and plasma folate concentration (p ≤ 0.05) for femoral BMD phenotypes. Thus, in the folate < 4 ng/ml group, BMD was lower in TT homozygotes, while among individuals with folate (4ng/ml), TT individuals had significantly higher BMD [3]. LRP5

There was also significant interaction between two polymorphisms (SNPs) in this gene and physical activity with spine BMD in men (p for interaction 0.02–0.05), but not in women. Conclusions Although there often were no significant main effects of genotypes of the above genes on BMD, we observed multiple significant interactions between genotypes and environmental factors contributing to BMD. Gender specificity, as we noted, conforms to known differences in genetics between men and women. Taken together, the above findings of gene polymorphisms interacting with lifestyle and nutritional factors highlight the importance of always considering interactions between genes and environmental factors. Understanding the complex interplay between risk factors involved in the development of complex disease will ultimately direct strategies for risk reduction. Bibliography 1. 2. 3.

Karasik D, Cupples LA, Hannan MT et al.: Age, gender, and body mass effects on quantitative trait loci for bone mineral density: the Framingham study. Bone 33(3), 308–316 (2003). Ferrari SL, Karasik D, Liu J et al.: Interactions of interleukin-6 promoter polymorphisms with dietary and lifestyle factors and their association with bone mass in men and women from the framingham osteoporosis study. J. Bone Miner. Res. 9(4), 552–559 (2004). McLean RR, Karasik D, Selhub J et al: Association of a common polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene with bone phenotypes depends on plasma folate status. J. Bone Miner. Res. 19(3), 410–418 (2004).

The CYP2C9 polymorphism: from enzyme kinetics towards genotype-adjusted drug therapy Julia Kirchheiner Department of Pharmacology, University of Cologne, Gleuelerstr. 24, 50931 Köln, Germany E-mail: [email protected]

CYP2C9 is the major human enzyme of the cytochrome P450 2C subfamily and metabolizes about 10% of all therapeutically relevant drugs. Two inherited single nucleotide polymorphisms termed CYP2C9*2 (arginine144cysteine) and *3 (isoleucine359leucine) are known to affect catalytic function. About 35% of the Caucasian population carry at least one mutant *2 or *3 allele; whereas in Africans and Asians, these alleles are very rare. CYP2C9 metabolizes several oral antidiabetics, oral anticoagulants, nonsteroidal antiinflammatory drugs (NSAIDs) and other drugs such as phenytoin, losartan, fluvastatin and torsemide. CYP2C9*3 affects the substrate binding site of the enzyme and shows a marked decrease in CYP2C9 activity. Individuals carrying the homozygous genotype *3/*3 were shown to have between a five- and tenfold reduced activity depending on substrate whereas activity of the enzyme coded by the CYP2C9*2 allele was only moderately reduced compared with the wild-type allele CYP2C9*1. The clinical pharmacokinetic implications of these polymorphisms are more variable depending on the enzyme’s contribution to total clearance. Studies in patients or healthy volunteers revealed differences of up to tenfold in pharmacokinetic parameters such as oral clearance or elimination halflives. These differences in drug metabolism can lead to substantial differences in drug response. For oral antidiabetics, decreased oral clearances can lead to hypoglycemia, which is a severe adverse drug effect in diabetic patients. In oral anticoagulant therapy, CYP2C9 polymorphisms have already been shown to cause differences in anticoagulant efficacy measured by the international normalized ratio (INR), which lead to differences in the frequency of bleeding complications. NSAIDs have a high risk for gastrointestinal bleeding complications and ulcerations, and differences in pharmacokinetic parameters of these drugs caused by CYP2C9 polymorphisms might result in a higher risk for these adverse effects in individuals carrying genetic polymorphisms. Data appear established enough for routine consideration of CYP2C9 genotypes in therapy with drugs such as acenocoumarol, phenytoin, warfarin and others. Nevertheless, before routine genotyping for these CYP2C9 alleles can be incorporated into the clinical setting, the benefits of genotype-based therapeutic recommendations have to be tested by a randomized, controlled clinical trial comparing therapy with pharmacogenetic diagnostics, with therapy as usual in a randomized controlled fashion. The typical primary outcome parameters in such studies should be efficacy parameters and parameters such as longterm outcome, adverse drug effects and direct cost-related parameters such as duration of hospital stay or disability.


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The meaning of free and informed consent in biomedical research personalizing medicine Vaidutis Kucinskas1, Gytis Andrulionis2, Danguol Steponaviit1 1Vilnius

University, Vilnius, Lithuania; 2Mykolas Romeris University, Vilnius, Lithuania E-mail: [email protected]

Development of personalized medicine and its efficient practical application is impossible without a wide-scale biomedical research involving human subjects as well as clinical trials in the creation of new drugs and treatment remedies, which must precede introduction for clinical application. In general, any experimentation on human subjects must only be carried out with the informed consent of the person being tested. This is an obligation enshrined in international regulations starting with the Nuremberg Code (1947), which was followed by the Geneva Convention (1949) and additional protocols, including the United Nations Organization International Covenant on Civil and Political Rights (1966). A number of states, including the Republic of Lithuania, entrenched the above-stated international provisions in their national acts of law. The requirement for informed consent conforming to the interests of a human subject is particularly aimed to protect the rights of persons who do not have the capacity to give their free and informed consent to an intervention because of an infant or senile age, a mental disability, a disease or similar reasons. On the other hand, specific physical and mental conditions of such persons need specific personal approach in treatment to obtain appropriate healthcare based on personalized medicine. This implies the necessity of biomedical investigation of this particularly vulnerable group of humans. The international community seeks to overcome the limitations imposed by the strict requirement for informed consent by developing new regulations such as the Helsinki Declaration (1964, Articles 24 and 26), Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine (1997, Articles 6 and 17). Such provisions on an international level establish the consensus between patients, societies, pharmaceutical groups, governments, science and other interest groups. Nevertheless, analogous regulations at a national level are not yet adopted by a number of states and still prohibit biomedical research involving certain individuals of their population. As a consequence, such populations are excluded from the programs for the investigation of the multifactorial basis of a number of complex diseases, thus reducing the possibilities of developing an efficient personalized means of treatment and prevention of a severe physical and/or mental disability caused by these diseases. On the other hand, a number of modern means of treatment are oriented to a definite genotype. As the prevalence rates of some genotypes differ across populations, a medication shown to be efficient on the basis of clinical trials in one population might be less efficient when applied in another population with a different genetic history. In summary, inflexible regulations at a national level in some states still entrenching strict requirement for informed consent to an intervention finally lead to the reduction of the right to appropriate healthcare and retardation of development of personalized medicine, thus limiting those human rights that were initially aspired to be protected.

Identification of A-to-I RNA-editing sites in the human transcriptome EY Levanon1,2, E Eisenberg3, Y Kinar2, M Hallegger4, R Shemesh2, K Djinovic-Carugo4, S Nemzer2, R Sorek2, MF Jantsch4, G Rechavi1 1 Department of Pediatric Hemato-Oncology, Safra Children’s Hospital, Sheba Medical Center and Sackler School of Medicine, Tel Aviv University

Tel Aviv, Israel; 2 Compugen Ltd, Tel Aviv, Israel; 3 School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University Tel Aviv, Israel; 4Max F. Perutz Laboratories, Department of Chromosome Biology, University of Vienna, Rennweg 14, A-1030 Vienna, Austria E-mail: [email protected]

RNA editing is a yet unappreciated mechanism that dramatically increases the complexity of the human transcriptome. The essence of this phenomenon is the enzymatic substitution of single nucleotides either in coding or noncoding sequences of RNA in the course of the transcription and splicing processes. Site-selective adenosine to inosine (A-to-I) RNA editing is carried out by members of the double-strand A-to-I RNA-editing ADAR family predominantly acting on precursor messenger RNAs. Altered editing patterns were found in various neurological conditions, epileptic mice, suicide victims suffering chronic depression, amyotropic lateral sclerosis and in malignant gliomas. 158


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Genetic recoding by editing was observed up until recently for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using millions of available, expressed sequences, we mapped 12,723 A-to-I editing sites in 1637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude [1]. Most of these sites occur in noncoding regions of the RNA, typically in Alu repeats. Next, we used comparative genomics and expressed sequence analysis to identify and experimentally verify four evolutionary conserved, non-Alu, human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. The editing pattern observed suggests that some of the affected proteins might have altered physiologic properties, raising the possibility that they can be related to the phenotypes of ADAR1 knockout mice [2]. In addition [3] we found that the abundant A-to-I editing in noncoding regions of the human genome is at least an order of magnitude higher than that of mouse, rat, chicken or fly. The extraordinary frequency of RNA editing in humans is explained by the dominance of the primate-specific Alu element in the human transcriptome, which increases the number of double-stranded RNA substrates. See Figure 1. Figure 1. Editing in the F11 receptor (JAM1) gene.

Top: some of the publicly available expressed sequences covering this gene, together with the corresponding genomic sequence. The evidence for editing is highlighted. Bottom: Results of sequencing experiments. Matching DNA and cDNA RNA sequences for a number of tissues. Editing is characterized by a trace of guanosine in the cDNA RNA sequence, where the DNA sequence exhibits only adenosine signals (highlighted). Note the variety of tissues showing editing, and the variance in the relative intensity of the edited guanosine signal.


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Bibliography 1. 2. 3.

Levanon EY, Eisenberg E, Yelin R et al.: Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nature Biotechnol. 22, 1001 (2004). Levanon EY, Hallegger M, Kinar Y et al.: Evolutionarily conserved human targets of adenosine to inosine RNA editing. Nucleic Acid Res. 33, 1169 (2005). Eisenberg E, Nemzer S, Kinar Y, Sorek R, Rechavi G and Levanon EY: Is abundant A-to-I RNA editing primate-specific? Trends Genet. 21, 77 (2005).

Evaluating attitudes towards genetic screening programs among orthodox Jewish students S Lieberman, A Frumkin, M Sagi Department of Human Genetics, Hadassah University Hospital E-mail: [email protected]

Background Carrier screening for frequent genetic diseases is offered in Israel by The Health in Genetic Centers. The standard screening is a two-step procedure: one of the couple is tested first and gets the test results, if found to be a carrier of one of the diseases the spouse is tested. If both of the spouses are carriers, they are offered an option of prenatal diagnosis and termination of pregnancy if the fetus is affected. The ultra-orthodox community (Haredim) uses a unique screening program, which is operated by Dor-Yesharim organization. These tests are couple based, meaning that only a carrier couple will know their genetic status, the others will only know if the match is safe. Among the orthodox population (non-Haredim) the estimated compliance rate to any genetic screening is low. Therefore, we developed an educational programme for religious high-school students and evaluated its effect on the attitudes towards genetic screening, carrier status and abortions within. Likewise, we examined their preference between the different screening programs (standard approach vis-à-vis Dor-Yesharim). Methods The participants were 769 students from eight orthodox high-schools and four post high-school educational institutions, and girls serving in the national service. The research compared two groups: 269 students who did not take part in the educational program, who filled questionnaires following a brief explanation, and 500 students who attended the educational program, consisting of a lecture that included information about genetic diseases, carrier status and the attitudes of Judaism towards abortions. Following the lecture they also filled in questionnaires. Results The educational programme was found to have a significant effect in all examined parameters: 81% of the program participants were interested in genetic test, compared with 53% of nonparticipants. In addition, 70% of the programme participants would date with a carrier, as compared with 45% of nonparticipants, and 87% of the programme participants chose the standard approach of testing (as opposed to ‘Dor Yesharim’) compared with 66% of the nonparticipants. Likewise, a significant difference was found in the attitudes towards abortions between these two groups: program participants tended more to accept rabbinic consultation, rather than absolutely oppose abortion. Regarding the timing of the genetic tests, 68% of the students think that tests should be offered to high-school students, 28% preferred being offered the tests while dating, and only 4% think the tests should be offered after marriage during pregnancy. Discussion An educational programme was found to have a short-term effect on interest in genetic screening, and to change students’ attitudes. In particular, it was found to reduce the stigmatization of carriers and to change attitudes towards abortions. The students would be interested in getting their test results, therefore most of them did not prefer the anonymous approach (‘Dor-Yesharim’). We suggest increasing the motivation for screening in the orthodox population by implementing education programs in high-schools. Owing to the relatively young age of marriage in this population and according to their preferences, actual screening should be offered during the short dating period. On the other hand, because pregnancy termination may not be absolutely rejected by this community, a couple who missed premarital screening should still be offered it after marriage.

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Bibliography 1. 2. 3. 4. 5.

Broide E, Zeigler M, Eckstain J, Bach G: Screening for carriers of Tay-Sachs disease in ultraorthodox ashkenazi jewish community in Iarael. Am. J. Med. Genet. 47, 213–215 (1993). Clow CL, Scriver CR: Knowledge about and attitudes toward genetic screening among high-school students: the Tay-Sachs experience. Pediatrics 59(1), 86–90 (1997). Gasson AA, Sheffield E, Bankier A et al.: Evaluation of Tay-Sachs disease screening program. Clin. Genet. 63, 386–392 (2003). Mitchell JJ, Capua A, Clow C, Scriver CR: Twenty-year outcome analysis of genetic screening programs of Tay-Sachs and ß-Thalassemia disease carriers in high schools. Am. J. Med. Genet. 59, 793–798 (1996). Sagi M: Ethical aspects of genetic screening in Israel. Sci. Context 11, 419–429 (1998).

Personalized medicine: health, genes, and society Klaus Lindpaintner, MD, MPH Roche Distinguished Scientist, VP Research; Head, Roche Genetics & Roche Center for Medical Genomics, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland

The tools of molecular cell biology research are providing us with an increasingly sophisticated and differentiated basic understanding of the molecular pathology of disease. This should, in due time, translate into more effective medicines, which presumably will need to be applied in an equally more differentiated fashion to newly defined subsets of conventional clinical diagnoses; in effect creating a further branching of the tree of differential diagnosis. The molecular in vitro diagnostics that will define these new branches will, in some cases, no longer only provide merely associative data, but will disclose causative or contributory mechanisms of disease in a manner heretofore largely restricted to infectious diseases. This will result in a rising importance of molecular diagnostics as a key tool to diagnose pathological entities. Furthermore, an enhanced and more causative understanding of disease will allow the targeting of novel, contributory mechanisms in our search for new medicines. The application of these medicines to clinical practice may thus often depend on first establishing the molecular diagnosis for which the drug is applicable. The development of the Her-2-neu antibody, trastuzumab (Herceptin®), in conjunction with a ‘companion diagnostic’ may serve as a paradigmatic example of this approach. Likewise, harnessing variation in absorption, distribution, metabolism and excretion (ADME)-relevant genetic variation by means of developing clinically applicable test systems, as well as the use of pharmacodynamic biomarkers to accelerate and enhance the accuracy of decision making along the drug research and development pipeline is expected to add value. Whilst we look forward to additional, incremental progress being made along these lines, it is prudent not to overstate the rate at which this will happen. We are still ignorant of the biological function of the vast majority of all genes, let alone of the clinical impact of any one of the millions of DNA variants found so far. In addition, we must be careful not to overestimate the overall role that genetic predispositions and susceptibilities play in the multifactorial concert of common complex disease etiology and treatment, where lifestyle and environment, by and large, remain, not only more important, but are in principle also much more easily modulated to reduce risk. A more comprehensive and integrated, real-time estimation of disease risk, and/or a more accurate determination of the severity of subclinical pathology (i.e., the status of progression from a state of health to a state of clinically manifest disease) are expected to rely increasingly on more complex algorithmic modeling. Such models will have to take into account the combined effect of both inherited and acquired risk factors, such as genetic data, environmental, life-style and demographic parameters, as well as, importantly, the measurement of dynamically regulated novel biomarkers which are expected to provide important insights as present-day surrogates of future disease burden. Given the complexity of common disease etiology, we may still have to grapple with less than desirable performance of these models and tests, such as positive and negative predictive values that are far from perfect. To generate a more realistic expectation than is commonly portrayed, a renewed effort at public education about genetics, long neglected by the scientific community, as well as about the actual promise of progress based on the personalized medicine approach is urgently needed, to avoid unwarranted hopes as well as fears, and to combat genetic exceptionalism, the sentiment that genetics represents a fundamentally different and new aspect of biology and medicine. Success in providing realistic perspectives for genetics, genomics and associated disciplines, including robust healthcare economic models of incremental cost–benefit ratios, will be essential to realize their important potential for the future of human health.


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CYP2D6 multiplication in Spanish healthy volunteers and schizophrenic patients A LLerena1, P Dorado2, MC Caceres1, E Penas-Lledo1, A De la Rubia 1Department

of Pharmacology and Psychiatry, Faculty of Medicine, University of Extremadura, Merida Psychiatric Hospital, Spain. 2Department of Medical Sciences, University of Beira Interior, Covilhã, Portugal. E-mail: [email protected]

Introduction The cytochrome P450 enzyme, CYP2D6, is a polymorphic enzyme that contributes significantly to the pharmacokinetics of several psychotropic drugs. The CYP2D6 gene is highly polymorphic with alleles causing absent (PMs), decreased (IMs), normal (EMs), and increased activity (URs) due to the presence of two or more copies of a functional CYP2D6 allele existing in tandem on the same chromosome. Previously we reported the high frequency of phenotypically PMs among psychiatric patients. We have shown the relevance of CYP2D6 for the metabolism of haloperidol, thioridazine and risperidone during treatment in psychiatric patients. We also described the relationship of CYP2D6 activity and personality traits. A high frequency of CYP2D6 gene multiplication in the Mediterranean area has been reported. Aims The present study was aimed at analyzing the frequency of CYP2D6 allele frequencies among schizophrenic patients and healthy volunteers as a control group (n = 135). The study was also aimed at analyzing the frequency of CYP2D6 multiplication alleles in a Spanish Population. Material and methods Schizophrenic patients (n = 89) and healthy volunteers (n = 135) were of the same ethnic group and from the same geographic area. The CYP2D6 genotype was analyzed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) techniques for the CYP2D6 *3, *4, *5, *6, *10 and duplicated alleles. Results and discussion The frequencies of functional alleles (*1 or *2) were 2.2 and 3.3% for healthy volunteers and patient,s respectively. A total of 3.3% (95% confidence interval 0.7–9.9%) schizophrenic patients were classified genotypically as PMs; this frequency was lower than the control group, 8.9% (95% confidence interval 7.3–11.9%) (Table 1). This finding might be due to the fact that the patients were selected from a clinical setting under regular pharmacologic treatment. We also found a high frequency of CYP2D6 allele multiplication, although the percentage of individual UMs in this Spanish population was lower than 7–10% previously reported. These preliminary results support the interethnic variability in CYP2D6 alleles multiplication, and suggest that there is no difference in the frequency of CYP2D6 genotypes in schizophrenic patients. The functional implication of CYP2D6 multiplication among EMs and IMs remains to be clarified. Supported by a Junta de Extremadura, Consejería de Sanidad y Consumo Grant (SC.URM.04.11). Table 1. CYP2D6 genotypes in Spanish schizophrenic patients and healthy volunteers. CYP2D6 genotype (no. active genes)

CYP2D6 genotypes

Schizophrenic patients (n = 89)

Healthy volunteers (n = 135)

UR (>2)

wt/wtx2

4 (4.5%)

6 (4.4%)

EM (2)

wt/wt; wtx2/*4; wt/*10; wtx2/*10

60 (67.4%)

75 (55.5%)

IM (1)

wt/*4; wt/*4x2; wt/*6;wt/*5; *5/*10,

22 (24.7%)

42 (31.1%)

PM (0)

*4/*4; *4/*6; *5/*6; *6/*6

3 (3.3%)

12 (8.9%)

EM: Normal activity; IM: Decreased activity; PM: Absent activity; UR: Increased activity; wt: Wild type.

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Polymorphism in signal transduction as a major contributor to osteoarthritis genetic risk John Loughlin PhD University of Oxford, Institute of Musculoskeletal Sciences, Botnar Research Centre, UK E-mail: [email protected]

Osteoarthritis (OA) is the most common form of the arthritides, and a leading cause of musculoskeletal disability in developed countries. It is characterized by the degeneration of articular cartilage and has long been shown to be a different entity from the aging process alone. Clinically, it manifests itself with pain, stiffness and often leads to disability and sometimes the need for joint replacement. Primary OA is a form of OA that is characterized by being of late-onset and has no obvious cause, unlike the secondary form that often has an earlier onset and has definite identifiable causes, such as injury or developmental abnormalities. Cross-sectional and longitudinal studies have demonstrated familial clustering of primary OA, implying a genetic component to the disease. However, such clustering could also be the result of shared environmental factors within a family. Twin studies have since been performed that demonstrate a clear heritability to OA at a number of skeletal sites, including hands, hips, knees and the spine [1]. Other epidemiological studies have also been performed, investigating the nature of OA transmittance from parents to offspring and the prevalence of disease between relative pairs, particularly siblings. These studies have confirmed a major genetic component to OA, which is transmitted in a non-Mendelian, complex manner. It has gradually become apparent that the nature of the genetic risk is likely to vary somewhat between different skeletal sites and may also vary between the sexes, although this latter observation is based on a small number of studies and needs further investigation to confirm its veracity. With a genetic component, established the next step was a hunt for the risk alleles. Investigators initially focused on genes encoding the major structural components of the cartilage extracellular matrix, such as aggrecan and Type II collagen. These studies did not provide the expected breakthroughs and prompted a rethink on the nature of OA susceptibility: instead of the cartilage matrix being poorly constructed, could the susceptibility be acting in those pathways and processes that develop the cartilage and then help maintain the tissue throughout life? Subsequent genome-wide linkage and association studies have revealed genes whose proteins do regulate cartilage development and homeostasis. Three particularly compelling results are: • An association to the secreted frizzled-related protein 3 (SFRP3) gene FRZB on chromosome 2q32.1 in a UK population • An association to the asporin gene ASPN on chromosome 9q22.31 in a Japanese population • An association to the calmodulin 1 gene CALM1 on chromosome 14q32.11, also in a Japanese population. SFRP3, ASPN and CALM1 all regulate chondrogenesis and help to maintain the cartilage matrix. However, they do this through distinct pathways: SFRP3 acts as an antagonist of extracellular Wnt ligands; ASPN influences TGF-f-mediated signaling; CALM1 influences intracellular calcium signaling. These recent genetic findings suggest that OA genetic risk is acting on chondrocyte differentiation, proliferation and the general homeostatic balance of the articular cartilage ECM rather than through an inadequately constructed cartilage ECM. This is an important observation since signalling pathways are modifiable. The new genetics has therefore identified targets for new drug development, as well as loci, which can now be genotyped to identify at-risk individuals for more focused clinical trials. This complex disease, whose etiological understanding many considered intractable, is now beginning to reveal some of its secrets. This is an exciting time and the future looks promising for scientists and for patients. Bibliography 1. 2. 3. 4.

Spector TD, MacGregor AJ: Risk factors for osteoarthritis: genetics. Osteoarthritis Cartilage 12 (Suppl. A), S39–S44 (2004). Loughlin J et al.: Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females. Proc. Natl Acad. Sci. USA 101, 9757-9762 (2004). Kizawa H et al.: An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis. Nature Genet. 37, 138–144 (2005). Mototani H et al.: A functional single nucleotide polymorphism in the core promoter region of CALM1 is associated with hip osteoarthritis in Japanese. Hum. Mol. Genet. (In press) (2005).


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Personalized medicine: new perspectives, new dilemmas? Jeantine E Lunshof VU University Medical Center, Department of Clinical Genetics & Human Genetics, Section Community Genetics, Amsterdam, The Netherlands E-mail: [email protected]

Personalized medicine is a multilayered concept. The final goal of improving individual health by the optimization of pharmacotherapy, can only be reached by way of stratification of disease phenotypes and drug-reaction genotypes. Current biomedical ethics has a strong focus on individual rights and interests. The new perspective of personalized medicine, as applied pharmacogenomics based on stratification, requires that group interests are adequately addressed. This raises new and special dilemmas. Do we need new concepts in biomedical ethics to keep pace with scientific development in pharmacogenomics? Personalized medicine: a disputed notion The notion of personalized medicine as such has been disputed. This criticism has a point insofar as the goals of medicine traditionally are directed towards the health and wellbeing of individuals. Personalized medicine in the form of individual-adjusted, pharmacogenetics-guided pharmacotherapy, however, can only be attained by means of group-oriented pharmacogenomics-guided drug development. Shifting perspectives: new dilemmas The essential feature of personalized medicine is stratification. The only situation where the individual is addressed is the clinical patient–physician encounter. At all other levels, the focus is on groups. Group interests in fundamental, clinical and translational research present us with new dilemmas that are fundamentally different from the classical cases in biomedical ethics. In data collection, biobanking, and in study design, individual and group interests must be reconciled. Adjusting ethics? Ethical concepts specifically addressing group- and community-related normative issues are currently being developed. Emphasis is on reciprocity, mutuality, solidarity, citizenry and universality. A principle of genetic equity has been proposed for the protection of human dignity. With regard to the regulation of both research and clinical implementation the question is to what extent genetic exceptionalism – developed for the protection of the individual – can still be justified. Adjusting ethical approaches in pace with the scientific developments in pharmacogenomics poses an enormous cross-cultural challenge. Cost effectiveness, the good life and community values Cost effectiveness will always remain a decisive criterion for the broad implementation of personalized medicine in clinical practice. The costs can be calculated, but what about effectiveness? Defining ‘effectiveness’ presupposes a basic consensus on the constituents of health, good medicine and the good life. But, will we ever reach agreement about the ranking of the values and preferences that are at stake? In these matters, not only the views of individuals, but also, and even more, of communities differ, posing huge dilemmas for research as a global endeavor. Safeguarding the way to rational pharmacogenomics based individualized drug therapy in an equitable, ethically justifiable manner should define the role of ethics in the development of personalized medicine.

Genetic testing for common diseases in clinical practice LI Minaicheva, OA Makeeva, VA Stepanov, LP Nazarenko, MG Spiridonova, VP Puzyrev

Research Institute of Medical Genetics, Siberian Branch of Russian Academy of Medical Science, Tomsk, Russian Federation E-mail: [email protected]

Human Genome Project completion provided new insight into the understanding of the molecular basis of human diseases. Much has been done to improve molecular diagnostics for monogenic disorders,with several hundreds of genetic tests introduced to medical practice. It is obvious that genetic factors play an important role in predisposing to common diseases. A growing number of genetic variants have been implicated in the development of pathological features of complex conditions. Disease-related polymorphisms should be taken into account together with the other known risk factors (such as 164


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blood pressure, cholesterol levels, smoking or diabetes for cardiovascular disease) to define at-risk individuals. Risk stratification based on genotyping could significantly impact on healthcare. In this respect, the introduction of new genomic tools to clinical practice is of the greatest importance. Relying on previous experience of more than 20-years study in the field of common diseases, several genetic panels were developed at the Institute of Medical Genetics (Tomsk, Russia). Diagnostic panels for cardiovascular disease, diabetes complications, bronchial asthma severity, venous thrombosis and pregnancy complications include key subsets of genetic markers that are clinically informative. During a patient’s previous medical consultation, a medical genetic specialist determines which diagnostic test should be performed and makes a decision about the most appropriate set of genetic markers. These can be made on the basis of family history, should take into account the patient’s preferences or include genetic variants with known pharmacogenomic effect. To assess disease risk, a method based on the calculation of a number of unfavorable alleles was proposed. According to such an evaluation, an individual can be at high or low risk for a particular disorder. Clinical validity of diagnostic testing depends on the occurrence of prophylactic measures and preventive drug treatment. In many cases genetic testing not only predicts further disease development, but also makes it possible to perform effective preventive measures, which in terms of cost-effectiveness are comparable with drug treatment of the later disease stages. For example, body mass monitoring, ceasing smoking, blood pressure control, physical activity, and low-salt diet in the case of cardiovascular disease. However, diagnostic testing should not be undertaken where no effective treatment strategies yet exist.

Integration of pharmacogenetics into the therapeutic drug monitoring clinical service of large hospitals Vangelis G Manolopoulos1,2 1Laboratory

of Pharmacology, Medical School, Democritus University of Thrace, and 2Laboratory of Clinical Pharmacology Academic General Hospital of Alexandroupolis, Alexandroupolis, Greece E-mail: [email protected]

Aim To explore the clinical utility of pharmacogenetics/ pharmacogenomics, see how it correlates with therapeutic drug monitoring (TDM), and define the necessary parameters to provide optimum pharmacogenetic testing in specific clinical settings. Since the inception of the term pharmacogenetics in 1959, evidence has constantly been accumulating on the genetic variability in drug response among individuals. Several drug-metabolizing enzymes, drug transporters and drug targets exhibit genetic polymorphisms that can affect the response of certain individuals to specific drugs. A few of these polymorphic enzymes have been validated for their clinical utility (e.g., the drug metabolizing enzymes cytochrome (CY)P2D6, CYP2C9, CYP2C19 and thiopurine methyltransferase). This evidence, however, has yet to receive wide acceptance by clinicians and has only been minimally translated into an applied clinical tool. Pharmacogenetic testing is currently performed for only a few drugs (e.g., mercaptopurine, azathioprine, thioguanine, trastuzumab and tacrine), in a limited number of teaching hospitals and specialist academic centers, despite the fact that there is ample evidence supporting its use for several other drugs (e.g. warfarin, codeine and phenytoin) or classes of drugs (e.g. antidepressants and antipsychotics) [1,2]. TDM is a tool that can guide the clinician to provide effective and safe drug therapy in the individual patient. Although clinical drug measurement services have been provided for more than 30 years, routine TDM was established during the 1980s when the importance of drug concentration–effect relationships became widely recognized and simple immunoassay-based commercial drug assays were introduced. TDM is indicated when: • an expected therapeutic effect of a drug has not been observed • drug toxicity related to high plasma drug concentration is suspected • the manifestations of disease are life threatening and the trial and error approach to modification of dosing regimen would better be avoided • it needs to be established whether optimum therapeutic drug concentrations have been achieved for drugs characterized by a response that is difficult to detect Both pharmacogenetics and TDM share the similar goal of improving pharmacotherapy through better explanation of individual variability in drug response. In this paper the idea already suggested by others [1,3–5], that pharmacogenetic biomarkers offer a unique opportunity to complement and expand the scope of conventional TDM in several clinical areas,


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most notably psychopharmacology, is supported. The advantages and difficulties in combining pharmacogenetics with TDM are discussed. More specifically, attempts are made to: • • • • •

define the necessary parameters to provide optimum pharmacogenetic testing in specific clinical settings define the potential links in the roles of pharmacogenetics and TDM in clinical settings discuss possible guidelines for clinical laboratories introducing pharmacogenetic testing services discuss issues related to optimization of clinical assays needed in pharmacogenetic testing address the role of third-party payers and regulators of the pharmacogenetic laboratory


Ensom MHH, Chang TKH, Patel P: Pharmacogenetics: the therapeutic drug monitoring of the future? Clin. Pharmacokinet. 40, 783–802 (2001). Kirchheimer J, Brosen K, Dahl ML et al.: CYP2D6 and CYP2C19 genotype-based dose recommendations for anti-depressants: a first step towards subpopulation-specific dosages. Acta Psychiatr. Scand. 104, 173–192 (2001). Dahl ML, Sjoqvist F: Pharmacogenetic methods as a complement to therapeutic monitoring of antidepressants and neuroleptics. Ther. Drug Monit. 22, 114–117 (2000). Albers LJ, Ozdemir V: Pharmacogenomic-guided rational therapeutic drug monitoring: conceptual framework and application platforms for atypical antipsychotics. Cur. Medicinal Chem. 11, 297–312 (2004). Eap CB, Sirot EJ, Baumann P: Therapeutic monitoring of antidepressants in the era of pharmacogenetics studies. Ther. Drug Monit. 26, 152–155 (2004).

Sniffing SNPs – the genetic basis of human olfactory variability Idan Menashe1, Yoav Gilad1,3, Orna Man2, Ronny Aloni1, Doron Lancet1 1Dept 3Dept

of Molecular Genetics, the Weizmann Institute, Rehovot 76100, Israel of Genetics, Yale University School of Medicine, New Haven, CT, USA

It has long been known that humans are afflicted with highly prevalent specific deficits in their smelling faculties [1]. Practically, every individual has a diminished sensitivity towards one or more odorants. Such deficits are known as specific anosmia and have often been rationalized in terms of genetic polymorphisms in individual olfactory receptor (OR) proteins [2]. Experimental evidence for this hypothesis requires a demonstration that the genome of each individual contains a different assortment of ORs. The olfactory receptor gene superfamily has undergone a tremendous functional diminution in recent primate evolution. Consequently, humans have only approximately 400 intact ORs, compared with more than 1000 for the mouse [3]. It is conjectured that due to this recent evolutionary process, a significant fraction of the human ORs might segregate between intact and pseudogene alleles. To study this hypothesis, 59 OR pseudogenes with a single coding disruption, likely to show functional segregation, were examined, as well as 18 database-derived nonsense single nucleotide polymorphisms (SNPs) in intact OR genes. These were genotyped in DNA samples of 189 human individuals from different ethnic origins, acquired from the National Laboratory for the Genetics of Israeli Populations [101] and Coriell Cell Repositories, NJ, USA [102] using high throughput SNP scoring (Sequenom). A total of 29 functionally segregating loci were revealed [4]. These SNPs reflected a high degree of interindividual diversity in which almost every human in the sample was carrying a different combination of disrupted ORs. This analysis revealed one of the most pronounced cases of functional diversity in the human genome, suggesting that no two people perceive the environment of odorants in the same way. Still, the identified nonsense segregating pseudogenes (SPGs) are likely only to be a tip of a functional variability iceberg, which may consist of other deleterious polymorphisms. To expose more types of OR SPGs, a probabilistic algorithm that predicts the consequences of amino-acid substitutions with respect to protein functional viability was developed. The algorithm can distinguish between active and inactive OR genes/alleles, with 67% sensitivity and 95% specificity. This algorithm was then used to analyze 759 nonsynonymous SNPs in human intact OR genes from the HORDE database [103] and revealed another 30 missense SPGs, thus bringing the total number of such human OR loci to 59. These genomic polymorphisms may underlie at least some of the widespread phenotypic variation in human odorant sensitivity thresholds. Ongoing genotype– phenotype association studies are carried out to substantiate the detailed relationships between individual OR gene disruptions and defined cases of odorant-specific olfactory threshold variability. Such association would shed new light on the interaction between ORs and their ligands and hence would provide new opportunities to employ novel biotechnology approaches in the food and fragrance industry.

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Bibliography 1. 2. 3. 4.

Amoore JE, Steinle S: A graphic history of specific anosmia. Chemical Senses 3, 331–351 (1991). Amoore JE: Specific anosmia: a clue to the olfactory code. Nature 214, 1095–1098 (1967). Gilad Y, Wiebe V, Przeworski M, Lancet D, Paabo S: Loss of olfactory recepror genes coincide with aquisition of full trichromatic vision in primates. PLOS 2, 0120–0125 (2004). Menashe I, Man O, Lancet D, Gilad Y: Different noses for different people. Nature Genet. 34(2), 143–144 (2003).

Websites 101. http://nlgip.tau.ac.il 102. http://locus.umdnj.edu/ 103. http://bip.weizmann.ac.il/HORDE/

Biobanking and personalized medicine Andres Metspalu Department of Biotechnology, IMCB, Estonian Biocenter, MDC of the Joint Laboratories of University of Tartu Clinicum and Estonian Genome Project Foundation, Tartu, Estonia E-mail: [email protected]

If the definition of personalized medicine is used to describe the idea of modifying therapy of common diseases according to the individual patient’s genetic variability, then there are not many examples to speak about. We could think of pharmacogenetic testing using Roche AmpliChip CYP450 and gene-expression profiling using microarrays. The latter is in the development and discovery phase and although huge datasets are emerging, there is still a lot of noise in the system [1]. Pharmacogenetic testing could perhaps be the first truly useful medical application of human genetic variability in predicting drug metabolism and efficacy [2]. It could not only save patients but also the industry by keeping drugs from withdrawal, if genetic testing could identify a small percentage of patients who are likely to develop an adverse drug reaction. However, as we all know, genetics is important, but is not the whole story. In order to apply personalized medicine we have to be more personal than just the individual SNP map [3]. We have to know the patient’s other variables as well, such as environmental factors, history of exposure, health status, lifestyle, diet and other classical epidemiologic data. And we have to keep strongly in mind the confounding factors, bias and reproducibility, when designing and performing these studies. This brings objectivity to the large, prospective population-based cohorts – biobanks – as one of the last missing resources after the HapMap and genotyping technology together with the bioinformatics in order to move toward personalized medicine. This fusion of different disciplines brings together high-tech genetics and large-scale epidemiology and will be the discovery field for the next few decades. Ideally, we should all be in the biobank, and by implementing the ‘e-health’ concept widely enough this will eventually happen. The UK Biobank [101] has started a pilot study after careful preparation and the main project should start in 2006. The Estonian Genome Project [102] has 1% of the adult population (10,300 gene donors) in the database so far and the first research projects have started [4]. There are more biobank projects in different stages with different scope, design and goals. The international consortium Public Population Project in Genomics (P3G) [103] has been organized in order to promote collaboration between the different biobanks. Why cohorts? To avoid bias and to be cost effective. This design allows the collection of information prior to development of disease and many different intermediate or end phenotypes can be studied. Why prospective? Samples collected before the event can be studied for early markers for the disease. This allows the study of conditions hard to investigate retrospectively. The future technologies and hypothesis are applicable. Value is increasing with the time. Why large scale? Better statistics, adequate power. Allows the nested case–control studies and provides appropriate information on a variable range of health conditions. Why population-based? This information has direct relevance to health in the general population, direct data on incidence and prevalence of intermediate phenotypes, risk factors and disease. Less severe phenotypes can be included in the study. Biobanks and personalized medicine form a part of the future and therefore it is difficult to fund them today. Large databases, biobanks or not, containing medical, personal or other information related to the individual demand strict legal rules and a contemporary ethical environment [5]. Everyone, including the mass media, has something to say here and therefore the biobanks are under close inspection from society. The question is: how personal the personalized medicine can be, and when will we be ready for it?


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Michiels S, Koscielny S, Hill C: Prediction of cancer outcome with microarrays: a multiple random validation strategy. Lancet 365, 488–492 (2005). Roses AD: Pharmacogenetics and drug development: the path to safer and more effective drugs. Nature Rev. Genet. 5, 645–656 (2004). Hinds DA, Stuve LL, Nilsen GB et al.: Whole-genome patterns of common DNA variation in three human populations. Science 307, 1072–1079 (2005). Metspalu A: The Estonian Genome Project. Drug Dev. Res. 62, 97–101(2004). Knoppers BM, Chadwick R: Human genetic research: emerging trends in ethics. Nature Rev. Genet. 6, 75–79 (2005).

Websites 101. www.ukbiobank.ac.uk 102. www.geenivaramu.ee 103. www.p3gconsortium.org

Mitochondrial genetics, longevity, adaptation and disease Dan Mishmar, Jeanette Feder, Anna Bachrat Department of Life Sciences, National Institute of Biotechnology (NIBN), Ben-Gurion University, Beer-Sheva, Israel 84105 E-mail: [email protected]

Mitochondria were incorporated into ancient eukaryotes to utilize oxygen in energy production more than a billion years ago, resulting in far more efficient cellular energy (ATP) production. As a by-product of cellular energy production, the mitochondrion also produces reactive oxygen species (ROS), the cumulative damage of which is implicated in aging. Previously it has been shown that mitochondrial DNA (mtDNA)-linked sets of common variants (haplogroups) are associated with age- related disorders. In contrast, it was also shown that common mtDNA variants are associated with prolonged aging. We have shown that such common variants associate with the adaptation of humans to different climates [1–3]. Taken together, these observations led us to the hypothesis that common mitochondrial variants differ in their oxidative phosphorylation (OXPHOS) performance, probably in the efficiency of ATP production and in the load of ROS production. This variation in cellular metabolism may underlie the population-based difference in the kinetics of gradual deterioration in metabolic activity over time. As the first step towards testing this hypothesis we have undertaken a population genetic approach to study the possible association of common mtDNA variants with Type II diabetes and diabetic complications as a model for age-related disorders. In addition, we compare OXPHOS performance in cell lines carrying different common mtDNA variants. Bibliography 1. 2. 3.

Mishmar D, Ruiz-Pesini E, Golik P et al.: Natural selection shaped regional mtDNA variation in humans. Proc. Natl Acad. Sci. USA 100, 171–176 (2003). Ruiz-Pesini E, Mishmar D, Brandon M, Procaccio V, Wallace DC: Effects of purifying and adaptive selection on regional variation in human mtDNA. Science 303, 223–226 (2004). Wallace DC, Ruiz-Pesini E, Mishmar D: mtDNA variation, climatic adaptation, degenerative diseases, and longevity. Cold Spring Harb. Symp. Quant. Biol. 68, 479–486 (2003).

The prospects and bioethical dimensions of expanding the meaning of pharmacogenomics to encompass individualized pharmacotherapy Claus Møldrup, PhD, MSc (pharm.), Associate Professor The Danish University of Pharmaceutical Sciences, Department of Social Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Phone: +45 35 30 6452 E-mail: [email protected] Web: http://www.dfuni.dk/index.php/Moeldrup_Claus/755/0/

Although we have had great expectations for pharmacogenomics, very few of them have been met to date. The reasons are many and complex, spanning a range of scientific, political and economic problems. One crucial and perhaps the most important realization, however, is that the complex character of most diseases and the attendant complexity of pharmacotherapeutic

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management and treatment often makes it difficult to achieve an optimal therapeutic outcome using a classic pharmacogenomics approach, because individual and situational factors play an equally important role [1]. In fact, seen from the perspective of the pharmaceutical sciences, it makes more sense to expand the meaning of pharmacogenomics to encompass individual pharmacotherapy, including all treatment phases from diagnosis to therapeutic monitoring and support. Thus, the following aspects of pharmacotherapy would be individualized: • • • • •

indications that emerge through dialog between doctor and patient choice and adaptation of the form of administration that best suits the patient dosages that are constantly adapted by doctor and patient on the basis of daily experience therapeutic monitoring that generates evidence for treatment and can supplement data from clinical examinations support that strengthens the patient’s daily treatment through compliance systems, for example [2]

As an integral part of individualized pharmacotherapy, pharmacogenomics would have a chance to go public, rather than being restricted to university hospitals as it is today. In the process, it is important to consider the bioethical issues involved. It has been documented that the bioethical issues regarding pharmacogenomics are comparable to those concerning other genetic developments in general [3]. However, two main issues are unique to pharmacogenomics: society, industry, patient groups and individuals view the prospects offered by pharmacogenomics very differently, and there is a lack of research on the post-marketing implications [3]. It has been argued that the postmarketing implications of pharmacogenomics will crystallize the bioethical implications of genetics in general for a wider public, and that these implications will be stretched to their limits when commonly used pharmaceuticals also become public genetic information markers due to their therapeutic specificity [4]. Thus it is imperative to place extensive focus on the ethical, social and legal implications of pharmacogenomics, including pre-marketing as well as post-marketing issues. A multidisciplinary approach that openly includes individual and group opinions in the research and development process is also essential [5]. Otherwise, we run a substantial risk that the positive therapeutic prospects for pharmacogenomics and thus individualized pharmacotherapy will not survive due to lack of acceptance, understanding and fear on the part of the general public. Bibliography 1. 2. 3. 4. 5.

The European Society of Human Genetics (ESHG) TIfPTSI: Polymorphic sequence variants in medicine: Technical, social, legal and ethical issues with pharmacogenetics as an example. Eur. J. Hum. Genet. (2004) (In Press). Møldrup C: Livsstilsmedicin og farmakogenetik – individualiseret lægemiddelterapi. Ugeskrift for Læger, Accepted for Publication (2005). Møldrup C: Ethical, social and legal implications of pharmacogenomics - a critical review. Community Genetics 4, 204–214 (2001). Møldrup C: When pharmacogenomics goes public. New Genetics in Society 21(1), 29–37 (2002). Møldrup C: Medical Technology Assessment of the ethical, social, and legal implications of pharmacogenomics – a proposal for an Internet Citizen jury. Int. J.Technol. Assess. Health Care 18(3), 728–732 (2002).

ESF EMRC contribution to building a public–private platform for clinical research in Europe Dr Carole Moquin-Pattey, PharmD PhD European Science Foundation (ESF) – Head of the European Medical Research Councils (EMRC) E-mail: [email protected]

The process of research and development for innovative medicines in human health is facing new challenges. According to numerous reports, after decades of growth, pharmaceutical industry, the major actor of translational research in the biomedical field, is under enormous pressure. Pharma is on the brink of a scientific and technological revolution that will ultimately transform both the nature and the production of medicines. A better understanding of the molecular sciences and massive advances in computing power would eventually enable the industry to develop targeted treatment solutions, or healthcare packages for patients with specific disease subtypes. These targeted treatment solutions will use biological methods of discovery and development, targeted at particular patient subpopulations, and aimed at measurably modifying the diseases for which they will be prescribed. They will also include biomarkers, devices, preventative medicines and a network of services for diagnosing, treating, monitoring and supporting patients, which will improve persistence and compliance. Last year, the World Health Organization (WHO) reported on priority medicines for Europe, highlighting the still unmet needs for new and effective medicines according to 17 priorities: • future public health threats: infections due to antibacterial resistance; pandemic influenza; • diseases for which better formulations are required: cardiovascular disease (secondary prevention); diabetes; postpartum www.futuremedicine.com

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hemorrhage; pediatric HIV/AIDS; depression in the elderly and adolescents; • diseases for which biomarkers are absent: Alzheimer’s disease; osteoarthritis; • diseases for which basic and applied research is required: cancer; acute stroke; • neglected diseases or areas: tuberculosis; malaria and other tropical infectious diseases such as trypanosomiasis, leishmaniasis and Buruli ulcer, HIV vaccine; • diseases for which prevention is particularly effective: chronic obstructive pulmonary disease including smoking cessation; alcohol use disorders: alcoholic liver diseases and alcohol dependency. The report suggests that Europe can and should play a global leadership role in public health, as reflected by its history of social services provision and social safety nets for all citizens. However, Europe has lost its attractiveness as a prime location for Research & Development, and a radical rethink is necessary to regain it. This challenge is tackled by the European Commission which asked the industry to identify key barriers to innovation that hamper translation of research results into therapeutic benefits as well as other stakeholders such as SMEs, patient organizations, academia, clinical researchers, regulatory agencies, funding bodies and ethical experts. The European Commission intends to play a catalytic role in the development of an industry-driven European Technology Platform (ETP) for Innovative Medicines; the implementation of the research agenda now remains to be explored The European Medical Research Councils (EMRC), one of the five Standing Committees of European Science Foundation (ESF), is looking forward to contributing to this initiative as part of its Strategy Plan 2006–2010. EMRC, which is the association of 38 funding agencies in the medical field in 30 countries in Europe, is taking steps to develop and implement a public–private platform for clinical research in Europe. The platform will consist of: • setting up a Central Clearing House to make the inventory of the current initiatives, stakeholders, and infrastructures which promote clinical research in Europe, • providing the academic researchers with an independent and high-level expertise to evaluate their projects feasibility and implement their developmental plan. This will be achieved thanks to an Expert Committee composed of scientists, representatives of the regulatory authorities, pharmaceutical and biotechnological companies as well as patient associations, • working in coordination with a Network of Clinical Research Centers and Coordination Units or Organizations. By increasing the visibility, support and coordination of the European initiatives in translational research, this public–private platform for clinical research in Europe should prevent duplication of efforts and help shorten the time frame necessary for development of innovative medicines.

Public health genetics in Germany: Pandora’s perils or Panakeia’s promise? Norbert W Paul Institute for History, Philosophy, and Ethics of Medicine, Johannes Gutenberg-University Medical School, Am Pulverturm 13, D-55131 Mainz, Germany E-mail: [email protected]

Background In 2030, a third of citizens in European countries will be 65 years and older. Age-associated ailments, particularly those related to cell or tissue degeneration and having a high potential for creating chronic illness, will be a major challenge for healthcare. Genome-based approaches to predictive and preventive medicine can help to better identify individuals at risk and to individualize strategies of disease control and prevention. This requires profound changes in our healthcare systems and the resolving of a number of serious social and ethical issues under uncertainty. Health benefits of public health genetics (PHG) will only be measurable in long-term epidemiological cohort studies. In this situation a clear understanding of the reach of more individualized PHG and of related ethical issues are essential for informed decision making about the future of health [1]. Individualization and geneticization In Germany the horrors of biological discrimination during the Nazi regime raise the question whether PHG might promote genetic discrimination and an unjust allocation of healthcare resources. Geneticization has become a metaphor for increasingly judging health, disease and quality of life based on genetic knowledge [2]. Geneticization, however, is neither

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intrinsic to nor a necessary effect of PHG. The premier goal of PHG is using genetic and genomic knowledge to better target populations at risk with strategies of behavioral and environmental prevention. Sufficiently dense sets of genetic markers (such as SNPs) are feasible for assessing individual physiological variants, susceptibility, and health risks of individuals. Without integrating genetic and genomic knowledge with environmental and behavioral factors, however, prevention would miss its main target. Deterministic views of genetic risk leading to genetic discrimination are scientifically obsolete and not at all suited for PHG [3]. It has become clear that health risks and gene–environment interaction will hardly become controllable on the genetic level. For this conceptual reason, individualized prediction and prevention cannot and will not strive for “genotypic” prevention, where genotypic prevention would be the interruption of transmission of genetic variation related to risk or disease from generation to generation [4]. Individualized prevention and the social achievability of health How can more individualized, “phenotypic” strategies of prevention improve the social achievability of health? As evidenced by early studies in pharmacogenomics and the genetic epidemiology of obesity and cardiovascular disease, genome-based individualization supports the more intelligent allocation of scarce resources [5]. We are currently observing a profound shift of health markets from economies of scale to economies of scope. This is heavily impacting on the affordability of health. Patient’s preferences and criteria of health care professional are demanding a re-allocation of health care resources. The demand will increasingly conflict with communitarian criteria such as distributive or enabling justice. Hence, introducing PHG will not only require social accountability regardless of particular interests but also a transformation of current interest-based decision making into an evidence-based model (Figure 1 and 2). Only if central issues of health can be related to findings in genetic epidemiology and only if they can be successfully addressed by the means of environmental and behavioral prevention suited to target genetic susceptibility, PHG will contribute to improving the social achievability of health and thus become medically useful, socially desirable, ethically justifiable, and affordable. Until now, nobody can reasonably answer if we are opening Pandora’s Box, setting free perils of geneticization and social discrimination, or if we are holding the panacea for our plagued Western health care systems in our hands.

Figure 1. Internet-based model of decision making based on competing assessments.

POLICY MAKING RELATED TO PUBLIC HEALTH GENETICS

PROFESSIONAL POLITICAL

ETHICAL

COMMERCIAL

LEGAL SOCIAL

CONSUMER

COMPETING ASSESSMENTS OF UTILITY

PUBLIC AND PROFESSIONAL COMPROMISE DETERMINES TECHNOLOGY USE

UTILIZATION AND REIMBURSEMENT DRIVES INNOVATION AND STANDARD OF CARE © Norbert W. Paul, 2003


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Figure 2. Pragmatic and normative assessment of clinical goals in a reflexive mode.

GENOTYPE RELATED EPIDEMOLOGICAL AND PRECLINICAL STUDIES IN PUBLIC HEALTH GENETICS

EMPIRICAL ASSESSMENT

NORMATIVE ASSESSMENT

Pragmatic Issues

ELSI* Issues GOALS OF PUBLIC HEALTH

EPIDEMIOLOGICAL AND PUBLIC HEALTH STUDIES (Empirical Data on Pragmatic & ELSI Issues)

PUBLIC AND PROFESSIONAL CONSENSUS DEFINES STANDARD OF CARE

STANDARD OF CARE DRIVES UTILIZATION AND REIMBURSEMENT © Norbert W. Paul, 2003

* ELSI = Ethical, Legal, and Social Issues


Khoury MJ, Burke W, Thomson EJ, eds: Genetics and Public Health in the 21st Century. Using Genetic Information to Improve Health and Prevent Disease. Oxford University Press, New York, NY (2000). Lippman A: Prenatal genetic testing and screening: constructing needs and reinforcing inequities. Am. J. Law Med. 17, 15–50 (1991). Paul NW: Auswirkungen der Molekularen Medizin auf Gesundheit und Gesellschaft. Bonn, Friedrich Ebert Stiftung (2003). Juengst ET. 'Prevention' and the goals of genetic medicine. Hum. Gene Ther. 6, 1595–1605 (1995). Paul NW, Roses AD: Pharmacogenetics and pharmacogenomics: recent developments, their clinical relevance and some ethical, social, and legal implications. J. Mol. Med. 81, 135–140 (2003).

Personalized medicine USA: pros, cons, but no way back Inga Peter Institute for Clinical Research and Health Policy Studies, Tufts-New England Medical Center, 750 Washington St., Boston, MA 02111, USA E-mail: [email protected]

Physicians, in view of their Hippocratic oath, are obligated to do no harm. Can this obligation be fulfilled when the information available to physicians is limited? In the USA alone, adverse drug reactions are thought to kill about 100,000 hospitalized patients and cause more than two million serious side effects annually. It is believed that many of these reactions are due to genetic variants and thus can be avoided by testing people before drug prescription. Therefore, the promise of pharmacogenomics lies in its potential ability to identify sources of inter-individual variability in drug response (both efficacy and toxicity) to treat the people who stand to benefit, weeding out those who could develop adverse reactions.

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Over the past several years, both the Food and Drug Administration (FDA), a public health organization responsible for the evaluation, approval and surveillance of foods and drugs in USA, and the pharmaceutical industry have recognized the importance of pharmacogenomics in drug development. Voluntary submissions of related research information are highly encouraged and guided by the FDA, which pledges not to use it for regulatory decisions. Instead, genomic submissions from industry will continue to serve as useful tools for both researchers and regulators to gain experience in reviewing and analyzing pharmacogenomics data. Despite efforts to make use of genetic information in medical decision-making, there are remarkably few examples of genetic technology that have been validated to the point where they can be used in a clinically licensed and regulated manner. So far, trastuzumab, an engineered monoclonal antibody used as a treatment for metastatic breast cancer in tumors positive for the human epidermal growth factor receptor (HER2) protein, is the only FDA-approved drug which is prescribed based upon a patient’s HER2 status. Another example is BiDil®, a nitric oxide-enhancing medicine, first rejected by FDA for use in the general population because of its ineffectiveness in the treatment of heart failure. However, BiDil appeared to be effective in African–Americans due to the drug’s effect on nitric-oxide deficiency, more common in black patients. BiDil is expected to be approved by the FDA, whereas new biomarkers will have to be determined to explore the biological differences between Americans of different descent given the high diversity and genetic heterogeneity of this population. Some advocates have been lobbing the FDA for mandatory genetic tests before prescribing numerous drugs, however, the medical establishment remains deeply suspicious, citing various problems, including patient privacy, risk frequency, dosing levels, physician education, and cost. While overall costs are expected to initially rise during the transition to genetically guided drug therapy, in the long run, the potential benefits will reduce this burden by shortening the time of clinical trials due to a higher response rate, bringing drugs to market faster, and in many cases, vastly increasing research and development productivity. Additional spending will include extra training of clinicians in molecular biology and genetics to interpret genetic tests for prescription of individually suited drugs, and allocation of funding by medical health providers to cover these expenses. There is also an enormous danger of the misuse of genetic data. In order to protect individuals, the Genetic Information Nondiscrimination Act issued by the US Senate bars employers from using people’s genetic information or family histories in hiring, firing or assigning workers. According to this act, insurance companies may not use genetic records to deny medical coverage or set premiums. In summary, the future holds incorporation of pharmacogenomics into clinical practice to benefit public health, however more research, funding and regulation policies must be directed at protecting patient privacy, nondiscrimination, while expending affordability for these individually-tailored treatments.

Personalized medicine in times of “Global Genes”: making sense of the “hype” Barbara Prainsack (Dr Phil) Department of Political Science, University of Vienna, Austria E-mail: [email protected]

The disparity between the number of articles presenting original research in the field of personalized medicine (pharmacogenomics), and articles dealing with the potential societal and ethical implications of this newly emerging discipline [1], is puzzling. The considerably larger quantity of publications on personalized medicine in the ethical, legal, and social implications field and in public media than in life science suggests the use of the word “hype” in this context. What attracts so much attention to the topic of personalized medicine outside of life sciences? What intrigues the larger public? And what is it that the concept, practices and tools of personalized medicine “really do”? These are the key questions which this paper is trying to answer. Common attempts to explain the “pharmacognomics `hype´” range from the objective to reduce drug development costs [2] to the claim that personalized medicine is “hot” because it is part of a larger paradigm shift from industrial mass production to targeted fabrication [3]. While these explanations are certainly helpful in understanding the phenomenon, I argue that they are not sufficient. Personalized medicine as a concept, and some of its practices and tools (such as genetic databanks), receive so much public attention because of the problems that they are capable of solving symbolically. Apart from the promise of better responses to individual needs in healthcare, they suggest answers to some pressing problems in healthcare and the larger social field. For example, irrespective of all criticism with regard to justice and privacy concerns, personalized medicine embodies a “morally sound” version of globalization instead of what is seen by many as the profit-driven accumulation of economic power. It avoids the antagonistic power struggle between different levels of governance (such as supranational governance emerging at the cost of the the national level) by being distributed to all of them: personalized medicine and its auxiliary apparatuses, such as genetic databanks, are www.futuremedicine.com

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regional, national and global at the same time. Genomes are national resources (Iceland) and the “heritage of humanity” (UNESCO Universal Declaration on the Human Genome and Human Rights 1997) simultaneously, thereby enabling and entailing - despite being situated in delimited local contexts - transnational data sharing and accessibility. Furthermore, although personalized medicine entails a strong involvement of for-profit enterprises, it is still seen by many as morally sound because it is “for health”. The quest for growth and efficiency, which is often criticized as a manifestation of ruthless capitalism in other contexts, in case of personalized medicine is embedded in the overall objective of making “drugs and medical services... cheaper, safer, and more ethical” [4]. A second tension that personalized medicine seems to be able to bridge is the contradiction between individual interests and common good. The “common good” is partly determined by a core message of the genomic era: the conviction that “we all share the same basic human genome” [5]. Because we are basically all the same, support for medical research will eventually enable everybody to profit. Despite all that we share, however, there are also individual genetic variations. Unity/sameness and diversity co-exist: while the interests of individuals might have particular manifestations which do not coincide with what others want, there is no longer any room for a conceptual dissonance between individual and common benefit. In this sense, the “pharmacogenomics `hype´” is not simply an overstated enthusiasm based on unrealistic expectations. The articulation and discussion of hopes, expectations, and fears does important work as it draws both the boundaries around what constitutes the field and predetermines public health strategies in the future. Bibliography 1. 2. 3. 4. 5.

Hedgecoe A: Terminology and the construction of scientific disciplines: the case of pharmacogenomics. Science, Technology and Human Values 28(4), 513–537 (2003). Regalado A: Inventing the pharmacogenomics business. Am. J. Health-Systems Pharmacy 56, 40–50 (1999). Felcht U-H: The future shape of the process industries. Chemical Engineering & Technology 4, 345–355 (2002). European Commission: Communication from the Commission to the Council, The European Parliament, the Economic and Social Committee and the Committee of the Regions: Life Science and Biotechnology – A Strategy for Europe. COM final. Brussels, January 23 (2002). Human Genetics Commission: Inside Information: Balancing Interests in the Use of Personal Genetic Data, May (2002).

In silico structural analysis of cytochrome P450-dependent monooxygenase APA Rani1 and A Aysha Mahmoodha 1Lecturer

in Zoology, Lady Doak College, Madurai, Tamil Nadu, India. E-mail: [email protected]

Monooxygenases are classes of enzymes that play a major role in detoxification of xenobiotics, including drugs and pesticides (as well as in the anabolism and catabolism of endogenous compounds including hormones and pheromones). Cytochrome P450 (P450) monooxygenases metabolize a large number of substrates because of the presence of numerous P450s in each species (~90 P450s in Drosophila melanogaster [1] and approximately 111 in Anopheles gambiae [2] and the broad substrate specificity of some P450s [3]. Structural characterization of this enzyme would help in delineating the role played by this enzyme in the detoxification process. Drug toxicity and sensitivity differs from person to person and so an insight into this molecule would help in designing new drugs, which are specific to individuals. This would also help in manipulating the monooxygenase and favor the binding and better detoxification of the substrate. This study was carried out by retrieving the protein sequence (P78329) from the National Center for Biotechnology Information database and subjecting it to various proteomics tools pertaining to protein characterization, topology, secondary and tertiary structure and 3D structure analysis. Using SWISS MODELLING the structure of the molecule is derived. Based on the various studies it is found to be a protein made of 520 amino acids with a molecular weight of 59814.79. It is localized to the extracellular region including the cell wall (77.8%) and the vacuole (22.2%) and is a secretory molecule carrying a cleavable signal peptide of about 32 amino acids. The protein seems to be a transmembrane protein with the amino acids 1–14 spanning outside the membrane, 15–37 within the transmembrane and 38–520 lying inside the membrane. Regarding the possible transmembrane helices observed, it is found to contain four inside to outside helices and three outside to inside helices, with the actual transmembrane helice region running from aminoacids 8–27 [4]. All the cysteines and methionines are in reduced form. When we consider the domains, it is found to contain P450 domain (52–515). It might play an oxidoreductase role and help in degradation. The redox potential and the structural motifs together play a major role in the function of monooxygenase. Hence this study would help in understanding the mechanism of detoxification. Monooxygenase could be applied as efficient biomarker in testing the effect of drugs, pesticides for example. A population molecular genetic study would give a deep insight into the genetic susceptibility of organisms to drugs and pesticides.

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Bibliography 1. 2. 3. 4.

Tijet N, Helvig C, Feyereisen R: The cytochrome P450 gene superfamily in Drosophila melanogaster: Annotation, intron-exon organization and phylogeny. Gene 262, 189–198 (2001). Ranson H, Claudianos C, Ortelli F et al.: Evolution of supergene families associated with insecticide resistance. Science 298, 179–181 (2002). Rendic S, Di Carlo FJ: Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab. Rev. 29, 413–580 (1997). Tsunády G.E and Simon I: Bioinformatics 17, 849–850 (2001).

Abundant A-to-I-editing sites in the human transcriptome: relevance to disease G Rechavi MD, PhD Head, Sheba Cancer Research Center, Israel

RNA editing by members of the adenosine deaminases acting on RNA (ADAR) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Until recently, only a limited number of human ADAR substrates were known, including the glutamate and serotonin receptors. Several diseases were associated with altered editing including depression-associated suicide and amyotrophic lateral sclerosis. We conducted a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability. Widespread editing could be relevant to various disease states.

Bioethical limits of prenatal genetic testing Michel Revel, Chair National Bioethics Council of Israel Weizmann Institute of Science, Rehovot, 7610 Israel

Different categories of genetic tests in the unborn child need different ethical evaluation. Genetic alterations causing severe diseases, often lethal in childhood, raise little ethical concern whereas prenatal diagnosis for late-appearing diseases – that will affect the individual after 40 or 60 years – poses much more difficult ethical questions. Genes that cause a predisposition to a disease, increasing its risk but not determining absolutely the disease to occur – as is the case for many cancer genes – raise additional queries. Finally for multigenic and multifactorial diseases, including mental disorders such as schizophrenia, and the most common cardiovascular diseases, it is not clear what the predictive value of the genetic test is, since it appears difficult to predict all the interactions between the genetic condition, the environment, nutrition and other modalities of life. The definition of what is a serious genetic disorder in the unborn child is often subjective and depends on individual decisions of the prospective parents. This is particularly true for malformations and handicaps: who can decide objectively what is acceptable and what is not? There may be two extreme views of genetic testing. On the one hand, Associations of Handicapped advocate “the right to be different”. In this view, the existence of a prenatal genetic test should not be an incentive for interrupting pregnancy, and society has the duty to make the necessary investments to allow active participation of the handicapped in professional life. At the other extreme, some would say: every birth with a genetic defect is an error of prenatal diagnostics as evidenced by lawsuits for ‘Wrongful Life’ against the geneticist or even against the parents. The claim in these lawsuits sounds like ‘it would be better not to have been born’, even though the judicial process is essentially about financial compensation for the suffering child. Many handicapped would rather say: “Thank God that my parents did not take the genetic test and make a decision to abort me because, despite my severely impaired condition, I know today all what I would have missed would I not have been born”. Real life lies somewhere between these two extreme viewpoints. Genetic testing is not about seeking an ideal genetic make-up or an ideal baby. Some fear that the multiplication of genetic tests may distance the mother from her fetus. Too often today, mothers avoid an affective relation to the fetus before being


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certain that he/she has no genetic defect: a sort of ‘tentative pregnancy’. The unborn child is not a consumer good, which is accepted only if defect-free. One should understand that prenatal genetic diagnosis, at least with present technologies, cannot ensure a perfect genome. Even if in the future, DNA chip technology will allow genome-wide testing, the question will remain of whether the goal of genetics is to eliminate all ‘bad’ genes. The notion of ‘good’ and ‘bad’ genes has an uncertain scientific basis. Many pathogenic gene variants have been selected for, rather than being just accidental. Eradicating these mutations from the human species may be dangerous in some environments and at some times in history. With all the undeniably beneficial progress in human medical genetics, the public needs to know that there is no ideal human genome. As put by the Quebec Genetic Group: “It is important to conceive man in his complexity and originality, to recognize that all humans carry abnormal recessive genes and susceptibility genes (5–30 in each of us). No one can be qualified as genetically sane or genetically deficient”.

Genetic breast cancer – a top secret information? A Rosen1 & R Ben-Yosef2 1Department

of Surgery “A”, Edith Wolfson Medical Center, Holon and Oncologic department, Holon 58100, Israel Tel.: 972 3 502-8604; Fax: 972 3 503-6408, 2Soraski Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel †E-mail: [email protected]

The first indication of a possible genetic linkage to breast cancer emerged in 1994 when the BRCA1 and 2 genes had been isolated. This finding was followed by a press conference on September 28,1995 where the Ashkenazi Jews became the main target of this mutation. It is well known that 5–10% of all breast cancers are inherited and a significant proportion of these are related to the BRCA1 and BRCA2 genes. Carriers of this mutation are estimated to have an 85% lifetime risk of breast cancer and a 40–50% risk of ovarian cancer [1]. As a result of this incoming information we face, as surgeons and oncologists, a daily dilemma of how to address this subject: which women should be encouraged to undergo the genetic testing and what are going to be the direct consequences of a possible positive result? From the medical point of view, these patients, BRCA1,2 carriers, should have a specific follow-up program and offered preventive therapeutic modalities [2–5]. Current recommendations regarding surveillance include a monthly breast self–examination beginning by ages 18–21(instead of 35–40), clinical breast examination semiannually, and annual mammography beginning at 25 years (instead of 40 years) [4]. Recently, a multicenter study from Rotterdam emphasized that magnetic resonance imaging (MRI) – a modality which is not in use for breast screening, appears to be more sensitive than mammography (79.5 vs 33.3%) in detecting tumors in women with an inherited susceptibility to breast cancer [5]. As for treatment modalities, the optimal local therapy for BRCA1-positive patients remains controversial. It is not clear whether the accepted breast-conserving surgery for sporadic cancer is justified in genetic cases or should these patients undergo unilateral or bilateral mastectomy, as local recurrence and second primaries in the same breast and controlateral breast tumors are significantly more common in this group [2,4]. There is also a debate concerning prophylaxis: prophylactic bilateral mastectomy is an option that can reduce risk but not eliminate it in BRCA1 and 2 carriers. Prophylactic oophorectomy can reduce ovarian cancer in BRCA1 and 2 patients [3]. Women who are eligible should be offered, though very carefully, to receive chemo preventive drugs which may significantly reduce breast cancer risk (38–50%) in a selected group of patients [3]. But, rather than focusing only on the medical risks and benefits, in regards to the fact that many of our patients belong to the Ashkenazi community, we have to consider as well, the psychological and societal problems of any individual and family receiving the information. A patient receiving a positive genetic result may face anxiety, decreased self image, alteration in family relations, fear and confusion. This also may create a stigma and severely interfere with normal social life such as marriage, child bearing, employment, insurance, and more complicated problems concerning organ donation and in vitro fertilization [1]. As we address these patients, most of whom are in severe anxiety – willing on one hand to know whether they are carriers or not, but on the other are reluctant to face the social consequences and demand to exclude any of this information from their chard, it is today recommended that all genetic testing should be done within a Cancer Genetics Clinic after personal genetic consultation under very specific criteria [3].

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Rothenberg KH: Breast Cancer the Genetic "Quick Fix" and the Jewish Community. Health Matrix 7, 97–124 (1997). Robson M, Svhn T, McCormick B et al.: Appropriateness of Breast-Conserving treatment of breast carcinoma in women with germ line mutation in BRCA1 or BRCA2. Cancer 103, 44-51 (2005). Sauven P: Guidelines for the management of women at increased familial risk of breast cancer. Eur. J. Cancer 40, 653–665 (2004). Rhei E,.Nixon AJ, Iglehart JD: Surgical Management of high risk patients. Breast Disease 12, 3–12 (2001). Kriege M,.Brekelmans CTM, Boetes C et al.: Efficacy of MRI and mammography for breast cancer screening in women with a familial or genetic predisposition. N. Engl. J. Med. 351, 427–437 (2004).

A human rights perspective on personalized medicine & justice Dr Carmel Shalev Adjunct Professor, Tel Aviv University Faculty of Law, Israel

Growing health gaps between rich and poor countries at the global level, as well as within societies, pose challenging questions of justice for the development of new medical technologies. While the prospect of personalized medicine holds promise for individuals, it also raises ethical issues of equitable access and the fair distribution of benefits. Some of these issues are recognizable from other areas of genetics, medicine and drug development, but have added meaning in the specific context of individual genotyping. This paper addresses these concerns from a human rights perspective. The Universal Declaration of Human Rights, 1948, recognizes a universal right to health, as well as a right to enjoy the benefits of scientific progress. The Universal Declaration on the Human Genome and Human Rights, 1997, provides that benefits from advances in genetics and medicine should be made available to all, and that applications of research shall seek to improve the health, not only of individuals but also of humankind as a whole. The International Declaration on Human Genetic Data, 2003 makes further provision inter alia for non-discrimination and non-stigmatization, and for sharing of benefits. In particular, there is a concern that personalized medicine and pharmacogenetics will classify individuals into new subgroups and create new forms of difference, which will interact with existing forms of prejudice and result in stratification into genetically defined groups, with the risk of racial and ethnic profiling.

ErbB4 shows a highly significant association with schizophrenia in Ashkenazi Jews Gilad Silberberg & Ruth Navon Department of Human Genetics and Molecular Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

E-mail: [email protected] NRG1 has been found to be associated with schizophrenia in several diverse populations [1–5]. Consistently, mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia [1]. These observations raised the hypothesis that impaired NRG1–ErbB4 signaling may contribute to schizophrenia susceptibility. Nineteen SNPs encompassing the ErbB4 gene were selected from the HapMap database [101] defining six linkage disequilibrium (LD) blocks and genotyped in genomic DNA from 60 Ashkenazi schizophrenic patients and 130 matched controls. Case-control comparisons were based on SNP genotypes and allele frequencies, as well as on haplotype frequency estimations. A highly significant difference between patient and control groups was observed in three SNPs from one LD block (rs707284, rs839523 and rs7598440) both in allele frequencies (p-value = 0.0086, 0.0031, 0.0043 respectively) and in genotype frequencies (p-value = 0.000087, 0.000013, 0.00024 respectively). Haplotypes constructed from these SNPs showed a powerful association as well (p-value for risk haplotype = 0.0005). Results obtained on ErbB4 in Ashkenazi Jews strongly indicate for the first time, that this gene plays a role in susceptibility to schizophrenia. Bibliography 1. 2.

Stefansson H, Sigurdsson E, Steinthorsdottir V et al.: Neuregulin 1 and susceptibility to schizophrenia. Am. J. Hum. Genet. 71, 877–892 (2002). Stefansson H, Sarginson J, Kong A et al.: Association of neuregulin 1 with schizophrenia confirmed in a Scottish population. Am. J. Hum. Genet. 72, 83–87 (2003).


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3. 4. 5.

Williams NM, Preece A, Spurlock G et al.: Support for genetic variation in neuregulin 1 and susceptibility to schizophrenia. Mol. Psychiatry 8, 485–487 (2003). Li T, Stefansson H, Gudfinnsson E et al.: Identification of a novel neuregulin 1 at-risk haplotype in Han schizophrenia Chinese patients, but no association with the Icelandic/Scottish risk haplotype. Mol. Psychiatry 9, 698–704 (2004). Petryshen TL, Middleton FA, Kirby A et al.: Support for involvement of neuregulin 1 in schizophrenia pathophysiology. Mol. Psychiatry 10, 366–374 (2005).

Website 101. http://www.hapmap.org/index.html.en

The Hapmap database website.

Polymorphism in cancer patients' DNA repair capacity as a factor in determining the dosimetry of radiation and chemotherapy Hanoch Slor1, Sima Batko1, Werner Mueller2, Hans Schroeder2, Irena Zaveliyuk3, Ilan Ron3 1Department

of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, of Molecular Biology, University of Mainz School of Medicine, Mainz, Germany, 3Tel Aviv Sourasky Medical Center, Division of Oncolgy, Sackler School of Medicine, Tel Aviv University, Israel 2Institute

Radiation therapy and most chemotherapeutic agents target the DNA of the treated tumors. Various types of DNA damage occur, including single- and double-strand breaks and the formation of various types of DNA adducts. DNA damage can be repaired in most human cells by various mechanisms such as nucleotide excision repair (NER), base excision repair (BER), mismatch DNA repair (MMR) and homologous and non-homologous recombination repair. Altogether, nearly 150 human genes are implicated in the various aspects of DNA repair. It is expected that variations in DNA repair capacity may exist among the normal population. The current radiation and chemotherapy dosimetry for cancer treatment takes into account only the physical parameters of the patient (body weight or body surface area) and no consideration is given to DNA repair capacity. Our hypothesis is that anticancer treatments depend on the cellular DNA repair capacity. Cells that have reduced DNA repair capacity are expected to be more sensitive to the treatment and may show extensive side effects. Cells that have elevated levels of DNA repair capacity may quickly repair the damage to DNA induced by the radiation or chemotherapy, and cancer cells may escape the killing effects of the therapy. We have analyzed DNA repair capacity in nearly 100 lymphocyte cultures obtained from consented normal cancer-free donors. Large variations in DNA repair capacity were observed, indicating that DNA repair is indeed a "factor" that should be considered when anti-cancer radiation or chemotherapy are administered. A major study is underway at the radiation therapy unit, Mainz university medical center, Germany. Lymphocytes drawn from consented cancer patients before the initiation of radiotherapy are tested for DNA repair capacity. Patients are treated under the currently accepted protocols, and their response (remission, recurrence, survival, and side effects) are registered for several years and correlated with their DNA repair capacity. Transformed cancer cells may express DNA repair capacity that is different from the unaffected normal cells. We suggest that tumor biopsy can be tested for DNA repair capacity using the different modes of repair (e.g. NER or BER). In those cases in which the tumor cells show reduced DNA repair capacity in one of the tests, this tumor may be treated best by a drug (or radiation) that induces specifically the type of DNA damage repaired by that particular mode of DNA repair. Several ovarian tumors and the surrounding normal tissues were analyzed recently for their NER. Two of five tumors showed reduced NER in comparison to the normal cells of the patients. It is possible that such tumors may respond best to cisplatinum (or carboplatinum), a drug that forms mono and di-adducts with guanine in the treated DNA. Cisplatinum-induced DNA damage is repaired by the NER system. This research was supported by the German Krebshilfe and by the Israel Cancer Association.

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Neurogenetics of acetylcholinesterase: from stress reactions to Parkinsonism Hermona Soreq1, Liat BenMoyal-Segal1, Yoram Ben Shaul1, Ella Sklan1, Shani Ben Arie1, Eran Meshorer 1 and Hagai Bergman2 1Department

of Biological Chemistry, Safra Campus-Givat Ram, 2Department of Physiology, Hadassah Medical School, The Faculty of Medicine, Ein Kerem Campus, Jerusalem, The Hebrew University of Jerusalem E-mail: [email protected]

Mammalian stress responses and neurodegenerative diseases involve numerous neurotransmission pathways. Because of the modulatory role of acetylcholine, we are studying the potential contributions of acetylcholinesterase (AChE) to these processes. The ACHE gene is subject to stress-induced modifications in promoter usage and alternative splicing [1]. Human genotype-phenotype analyses further point to polymorphisms in cholinesterase genes as causally associated with anxiety and stress-associated disease risk. Specifically, polymorphism(s) in the adjacent PON1 and ACHE genes interactively affect the expression levels of both these genes and reflect the variations in human anxiety parameters [2]. Polymorphisms which debilitate PON activity constitutively and cause impaired AChE over-production under anticholinesterase exposure were jointly over-represented in Parkinsonian patients from agriculturally exposed areas in Israel. Additionally, serum AChE and PON activities were significantly lower in patients compared to healthy individuals and in carriers of the risk associated polymorphisms as compared with other Parkinsonian patients [3]. In transgenic mice, failure to adapt the alternative splicing configuration of AChE mRNA splice variants to a changing environment impairs brain diffusion and blood–brain barrier functioning [4]. Moreover, while exposure to the neurotoxin MPTP notably destroys dopaminergic neurons in mice, transgenic mice representing robust splicing shift of AChE mRNA showed neuroprotection. Microarray analyses of neuronal gene expression, using both discrete and continuous bioinformatics approaches [5], point at an organismal capacity to confront damaging insults by re-adjusting the spliceosomal configuration as a neuroprotection strategy. Our findings demonstrate the relevance of this concept for ACHE gene expression and suggest that the capacity for neuroprotective alternative splicing may be exhausted once a threshold of multiple insults has been surpassed. Bibliography 1. 2. 3. 4. 5.

Meshorer E, Toiber D, Zurel D et al.: Combinatorial complexity of 5' alternative acetylcholinesterase transcripts and protein products. J. Biol. Chem. 279(28), 29740–29751 (2004). Sklan EH, Lowenthal A, Korner M et al.: Acetylcholinesterase/paraoxon-nase genotype and expression predict anxiety scores in Health, Risk Factors, Exercise Training, and Genetics study. Proc. Natl Acad. Sci. USA. 101(15), 5512–5517 (2004). Benmoyal-Segal L, Vander T, Shifman S et al.: Acetylcholinesterase/paraoxonase interactions increase the risk of insecticide-induced Parkinson's disease. FASEB J. 19(3), 452–454 (2005). Meshorer E, Biton I, Ben-Shaul Y et al.: Chronic cholinergic imbalances promote brain diffusion and transport abnormalities. FASEB J. (In Press) (2005). Ben-Shaul Y, Bergman H, Soreq H: Identifying Subtle Interrelated Changes in Functional Gene Categories using Continuous Measures of Gene Expression. Bioinformatics (In Press) (2005).

Cancer pharmacogenomics: the field that studies the role of an individual’s genetics in the response to drugs Lior Soussan-Gutman Oncotest-TEVA, Israel E-mail: [email protected], [email protected]

In an era where cDNA arrays and proteomic technology are becoming increasingly available and gene expression profiles in the tumor are used to identify molecular signatures of prognosis and prediction of response to conventional chemotherapy, it is becoming increasingly clear that the response to drugs is largely determined by a patient’s genetic make-up. Genetic analysis of tumors is the key to improving the treatment of cancer patients. To be able to better understand and, thus, treat cancer, insight into the complex characteristics of tumors is essential. Deeper knowledge of tumor structure and behavior is of clinical significance, as it can be used to predict the tumor’s ability to metastasize or its response to anticancer drugs. Breast cancer can serve as a paradigm for tailored medicine. This presentation will focus on the application of molecular oncology and pharmacogenomics in the treatment decision process of this disease. Current practice in breast cancer involves the use of a histological profile of the tumor, and drug choices that are based on population prognostic and predictive factors. The emerging practice involves the molecular profile of the tumor, with treatment and drug choice based on individual prognostic and predictive factors that are derived from the unique molecular profile of the tumor and the unique genetic make-up of the patient.


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The use of twins in genetic research: implications for personalized medicine Tim D Spector Twin Research & Genetic Epidemiology Unit, St Thomas’ Hospital, London SE1 7EH www.twin-research.ac.uk

Twins are a unique natural genetic experiment that allow us to answer many fundamental questions about ourselves. They have been used since the 1920s in formal experiments to test the relative importance of nature versus nurture in a wide variety of human diseases, traits and behaviors. In the traditional or classic twin study a group of identical twin pairs sharing all of their genes are compared with a group of non-identical or fraternal twins sharing only half of their genes. If there is greater similarity for the trait or disease within the identical twins than the non-identical twins then the trait has a genetic component. There are formal tests of this which provide estimates of the additive genetic, common environmental, and unique environmental components, the latter of which includes error variance. Twin research has accelerated in the last few years with most European countries having large twin registers and productive research groups (www.genomeutwin.org). Our group have studied a wide variety of traits over the years and as an example have shown the following disease related traits to be heritable ; osteoarthritis, back pain, pain thresholds, wrist fracture and bone density, body fat and shape, migraine, raynauds, soft tissue injuries, myopia, cataract, clotting factors, hypertension, asthma, moles and freckles, sun sensitivity, C-reactive protein (CRP) levels, and leptin levels. In addition, behavioral traits such as depression and anxiety, smoking history, religious beliefs, divorce, infidelity and sexual satisfaction were also heritable. Twins are also used to answer other questions in genetics – such as epigenetic and gene expression phenomena within identical twin pairs, or by using non-identical twins as sib-pairs for linkage studies, or for examining pleiotropy between traits. They have also been used to test for genetic responses to drugs. These studies have shown us that nearly everything we can measure accurately has a genetic component. We are therefore not all created equal. The implications for personalized medicine are that every individual has their own genetically predisposed starting points or baselines which are not the same as the ‘average’. In the future we will start to realize and exploit this fact in medicine to our own advantage. Bibliography 1. 2. 3.

Martin N, Boomsma D, Machin G: A twin-pronged attack on complex traits. Nature Genet. 17(4), 387–392 (1997). MacGregor AJ, Snieder H, Schork NJ, Spector TD: Twins. Novel uses to study complex traits and genetic diseases. Trends Genet. 16(3), 131–134 (2000). Spector TD. Your Genes Unzipped. Robson Books, London, UK (2003).

Is there a link between personalized medicine and community genetics? Leo P ten Kate VU university center, PO Box 7057, NL-1007 MB, Amsterdam, The Netherlands

At first glance these two concepts seem to have little in common. This is already apparent from their naming. The term ‘personalized medicine’ stresses individuality, while the name ‘community genetics’ stresses collective interests. At second thought no man is an island, entire in itself; every man is a piece of the continent, a part of the main [1]. So the interest of the individual and the interest of the community may parallel each other. In this talk I want to focus on potential disparities between the interest of one individual and the others in the community and on disparate interests between communities. Before doing so I want to draw your attention to the work of Seige, who described a cyclic pattern in the history of most, if not all, medicines, consisting of a first phase which is characterized by hope, promises and enthusiasm, a second phase with skepticism and growing criticism, and a third phase of disappointment, resignation and hope for a new promise. Will pharmacogenetics escape this cycle? The words personalized medicine already show that the first phase has begun. It is not a scientific label but a promotional one. The aim of pharmacogenetics – distinguishing between people who will benefit from a certain medication and those who will not benefit from it or even will be harmed – is a laudable goal. However, we are concerned with its side-effects here. One possible side effect is the finding of variant genotypes that predict not only the ability of an individual to handle a certain drug in one way or the other, but also a susceptibility for a disease which is unrelated to the condition the drug is meant

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for. In this case, not only the person themself but also family members are at risk of developing this disease. If such an association is known it should be discussed with the patient before testing. In many cases it will be possible to give a higher dose of drug to those who are high metabolizers and a lower dose to those who are at risk for an adverse reaction. But will this always be the case? What if some people cannot be treated at all with existing drugs? Will they become the new orphans? Will industry be interested in developing drugs for this small market too? Is there a parallel with screening in the workplace where the enterpriser is more interested in selecting against susceptible persons than in cleaning the workplace? And how will insurers react? Will they still insure people who cannot be treated with existing drugs? And what will happen to people who belong to the wrong community? Will the poor and people of undeveloped countries be able to afford the pharmacogenetic tests and the ever increasing prices of new tailor made drugs? And then, we know that there are impressive differences in allele frequencies not only between ‘racial’ and ethnic groups, but also between geographic communities if there is reproductive isolation. Some people will belong to communities in which there is a high chance of adverse reaction or no benefit at all, while others belong to communities that are more fortunate. Will this cause stigmatization or discrimination? And what if belonging to a community or ethnic group is used as a proxy for pharmacogenetic label? Finally, communities may start negotiating with industry and health care in order to obtain pharmacogenetic testing and appropriate drugs. This may lead to competition, which will not always result in a just distribution of health care resources. Bibliography 1. 2.

Knoppers BM: Of biotechnology and man. Community Genet. 7, 176–181 (2004). Seige M: Klinische Erfahrungen mit Neuronal. Dtsch Med Wochenschr 38, 1828 (1912).

Analysis of genetic variation in complex endocrine diseases André G Uitterlinden1,2,3, Joyce van Meurs1, Fernando Rivadeneira1,2, Huibert AP Pols1,3 Departments of 1Internal Medicine, 2Clinical Chemistry and 3Epidemiology & Biostatistics Erasmus MC, Rotterdam, The Netherlands E-mail: [email protected]

Endocrine factors change during aging and play an important role in the pathogenesis of many common diseases in the elderly, such as diabetes, obesity, osteoporosis and cardiovascular disease. These all have strong genetic influences and therefore intense efforts are ongoing to identify the underlying genetic factors. One approach in this respect has involved genetic linkage analysis in large collections of related individuals by “genome searches”, but this approach is being abandoned because of very limited success so far in spite of large global investments in terms of time and money. A more promising approach to identify genetic determinants of disease involves direct association-analysis of “candidate” gene polymorphisms in populations. This is because the “full-length” human genome sequence is available, large-scale information on polymorphisms has been generated (dbSNP), and information on the linkage disequilibrium (LD) block/haplotype structure is being completed (HapMap). Large (public) databases now contain information on DNA polymorphisms, the most frequent form of which are SNPs), including functional variations in coding and regulatory regions and haplotype tagging SNPs. Subsequently, epidemiological analyses evaluate the contribution of DNA polymorphisms to phenotypic endpoints of interest in large population studies. We study several biological pathways, and these involve estrogen [2,4,5], vitamin D [7], homocysteine [3], and bone matrix molecules [1] and bone regulatory factors [6]. Our laboratory is focusing on key proteins in these pathways including the vitamin D receptor (VDR) gene and the estrogen receptor α (ESR1) gene. Variations of these genes are analyzed in large collections of DNA samples, including those from the Rotterdam Study, a large prospective cohort study of chronic diseases in the elderly, which involves 7,983 men and women. In addition, we are coordinating a large multicenter EU-sponsored collaboration (GENOMOS; QLK6-CT-2002-02629) involving over 25,000 subjects to identify genetic risk factors for osteoporosis by prospective meta-analyses of candidate gene polymorphisms [5]. In past years, we have reported several associations (see bibliography) but these do not establish “cause and effect”, so additional evidence is needed to definitely implicate genetic variations in etiology of disease. We are therefore pursuing several lines of further research: • find all (relevant) sequence variations in a candidate gene and determine haplotypes (e.g.,VDR) • collect genetic association data of different study-populations and run prospective meta-analyses to find replication and measure true effect size (e.g., ESR1 in EU GENOMOS consortium). • perform functional studies, e.g., in cellular test systems, to determine molecular mechanisms (e.g., VDR) www.futuremedicine.com

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Finally, we are building risk models to predict disease risk based on multiple genetic risk factors, also in combination with existing risk factors. Eventually, this combined information will lead to genetic “markers” predicting risk-of-disease and response-totreatment with medication, and as such this will allow tailored and individual-specific preventive and therapeutic medicine to be developed. Bibliography 1. 2. 3. 4. 5. 6. 7.

Uitterlinden AG, Burger H, Huang Q et al.: Relation of alleles of the collagen type Ialpha1 gene to bone density and the risk of osteoporotic fractures in postmenopausal women. N. Engl. J. Med. 338, 1016–1021 (1998). van Meurs JBJ, Schuit SC, Weel AE et al.: Association of 5' estrogen receptor alpha gene polymorphisms with bone mineral density, vertebral bone area and fracture risk. Hum. Mol. Genet. 12(14), 1745–1754 (2003). van Meurs JBJ, Dhonukshe-Rutten RA, Pluijm SM et al.: Homocysteine levels and the risk of osteoporotic fracture. N. Engl. J. Med. 350(20), 2033–2041 (2004). Schuit SCE, Oei HH, Witteman JC et al.: Estrogen receptor alpha gene polymorphisms and risk of myocardial infarction. JAMA 291(24), 2969–2977 (2004). Ioannidis JP, Ralston SH, Bennett ST et al.: Differential genetic effects of ESR1 gene polymorphisms on osteoporosis outcomes. JAMA 292(17), 2105–2114 (2004). Uitterlinden AG, Arp PP, Paeper BW et al.: Polymorphisms in the sclerosteosis/van Buchem disease gene (SOST) region are associated with bone-mineral density in elderly whites. Am. J. Hum. Genet. 75(6), 1032–1045 (2004). Uitterlinden AG, et al. In: Vitamin D, 2nd Edition. Feldman, Pike, Glorieux (Eds). Ac Press, 1121–1157 (2005).

Individual sensitivity to warfarin could be predicted from genetic profiles of the components and effectors of vitamin K-dependent γ-carboxylation system Manuela Vecsler 1,3, Ronen Loebstein 2, Shlomo Almog 2,3, Daniel Kurnik 2, Boleslav Goldman 1,3, Hillel Halkin 2,3, Eva Gak 1,3 1Danek

Gertner Instititute of Human Genetics and, 2Institute of Clinical Pharmacology, Sheba Medical Center, Tel Hashomer; 3Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Email: [email protected]

In view of expanding indications for long-term oral anticoagulation and disapproval of the first oral direct thrombin inhibitor (ximelagatran) by the Food & Drug Administration, warfarin is expected to remain the main oral anticoagulant for the next decade. In order to better individualize warfarin therapy and to avoid the detrimental consequences associated with warfarin overand/or under-anticoagulation, it is essential to elucidate molecular mechanisms underlying large inter-individual variability in warfarin dose response. We hypothesized that genetic variations in the components and effectors of vitamin K-dependent γ-carboxylation system could contribute to the inter-individual variability in vitamin K cycle activity and, thereby to the variability in warfarin dose response. We recruited 100 warfarin-treated patients with stable international normalized ratio (INR) that were characterized with demographic and clinical parameters, as well as biochemical indices of vitamin K cycle activity. In this set, we investigated gene variations whose role in vitamin K cycle has been established, such as Q325R polymorphism in γ-carboxylase (GGCX) and mild mutations in the major warfarin metabolizing enzyme CYP2C9*2 and *3. In addition, we included polymorphisms in genes that were recently discovered in the context of vitamin K cycle, such as G73A polymorphism in the regulator of the cycle, calumenin (CALU) and C1767G polymorphism in vitamin K epoxide reductase component 1 (VKORC1), as well as two other polymorphisms in putative cycle effectors, T612C and A691G in microsomal epoxide reductase (EPHX1) and T-631G and T-567G in glutathione S-transferase (GSTA1). We studied the individual and compound effects of these polymorphisms on various indices of vitamin K cycle activity and warfarin dose response. The most significant factors in which genetic variations had the most profound effect on warfarin dose response were CYP2C9, VKORC1 and CALU. While polymorphisms in CYP2C9 and VKORC1 were associated with lower warfarin doses (both with p < 0.0001) even in the heterozygous individuals, the polymorphism in CALU was related to higher warfarin doses only in the homozygotes (p = 0.045). Patient age, body weight and dietary vitamin K intake had an additional minor contribution to warfarin dose response, altogether explaining 70% of the inter-individual variability (multiple regression r = 0.70). Biological effect of these genes was also evident by various indices of vitamin K cycle activity, most significantly by KO/W (oxidized vitamin K per plasma warfarin concentration) which expresses the rate of vitamin K cycle under the inhibition of warfarin (p = 0.002, p = 0.003 and p = 0.013, respectively), and was moreover significantly associated with the presence of GGCX polymorphism (p=0.013). Compound genotype analysis revealed that patients with wild-type CYP2C9 and VKORC1

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and mutant CALU genotypes required the highest warfarin daily doses (8.3±2.1mg/day) as compared to the patients with CYP2C9 and VKORC1 mutant and CALU wild-type genotypes (2.9±0.3mg/day). Our findings suggest that genetic profiles of VKORC1, calumenin together with CYP2C9 could predict with sufficient degree of certainty individual requirement of warfarin dose. In addition, understanding of the molecular basis and underlying function of enzymes involved in vitamin K metabolism may lead the better prediction of individual sensitivity to warfarin.

The ethics of clinical prediction Michael A Weingarten Sackler Faculty of Medicine, Tel-Aviv University, Israel E-mail: [email protected]

This paper will challenge the notion that genomics involves special ethical problems that are any different from other forms of clinical prognostication. The major issues that will be considered are complexity, and the rights of future generations. Clinicians are challenged with phenotypes, not genotypes. Knowledge of the genome may predict, following well-established probability patterns, the genotype of future generations. The phenotype of future generations will always remain less certain. The future clinical course of the present generation – the predicted expression of an existing genome – is also uncertain. These uncertainties are functions of the immense complexity of the environment and of the interaction between the individual and the environment. Complexity implies the existence of unsuspected, and therefore unpredictable, patterns of behavior. The best we can do is to amass empirical observational data and use it to calculate the statistical likelihood of future events – much as in the long-term weather forecast. In this respect genetics is not particularly different from other areas of clinical medicine, such as cardiovascular risk profiling, or ante-natal care. We can provide the patient with a group-risk assessment but not with an accurate personal clinical prediction. The more the patient resembles the group the closer these estimates will be, but in the end the only truly relevant group has an n of 1, that particular patient. The ethics of prognostication involves two main but interdependent considerations – informing the patient, and the uncertainty of the information. People are entitled to know the clinical information relevant to their own welfare. How far does group-based information fit this requirement? People should only be given groupbased information together with the essential analytic tools they need to interpret it and apply it to themselves. This is not usually part of routine clinical talking. Statistical data is routinely expressed in terms of the uncertainty inherent in the observational data, such as confidence intervals. People who are trying to gain an assessment of their future clinical course need to appreciate that reality might not reflect what they expect from the data presented to them. This concept of the uncertainty of data is generally alien to a non-scientific person, and extremely difficult to convey. So the communication process by which the information is transmitted from expert to layman is not conducted on a symmetrical basis, and cannot easily be described as fully honest. A second ethical point to be considered is the extent to which we may be said to have any duties at all towards future generations. Or, put another way, on what basis do as-yet-unborn people have any rights at all? On the basis of the Golden Rule – do not unto others what you would not have done unto yourself – it seems reasonable at first sight to protect the ecosystem in our lifetime so that it is available in good condition for future generations. On the other hand it is difficult to understand how a person not yet born can impose any duties on me. In the same way, the question arises as to the extent to which I have a duty of concern for the genetic makeup of future generations. I may reasonably wish my own children to be spared of the genetic disadvantages potentially conferred by some of my own genes, but that might be adequately explained as self-interest to save myself from the burden of bringing up a disabled child. Any imposition of my own value judgments on the engineering of the genotype of the next generation is eugenics, the dangers of which have been amply demonstrated in twentieth century Europe. It seems that I have no specific duties towards the next generation, nor even do I have the right to meddle in their fate. Bibliography 1. 2. 3.

Heyd D: Genethics. Berkeley, University of California Press, USA (1992). Buchanan A, Brock D, Daniels N, Wikler D: From Chance to Choice: Genetics and Justice. Cambridge University Press, UK (2002). Kimmelman J: Recent developments in gene transfer: risks and ethics. Br. Med. J. 330, 79–82 (2005).


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Pharmacogenetics of citalopram in pediatric anxiety and depression Abraham Weizman, Sefi Kronenberg, Alan Apter, Amos Frisch Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel AvivUniversity, Tel Aviv, Israel

We evaluated, in an open-label study, the relationship between polymorphisms in genes coding serotonergic components and the clinical response to citalopram in children and adolescents with major depression or anxiety disorders. We found an association between the serotonin transporter allele and genotype polymorphism and the initial onset of the antidepressive activity, as well as the efficacy of citalopram. The genotype also predicted the sensitivity of the children to the side effects of citalopram. It seems that serotonin transporter genotype can be used as a genetic marker for the efficacy of selective serotonin reuptake inhibitors (SSRIs) in anxious and depressed pediatric population. Further large-scale double-blind randomized clinical trials are needed to substantiate our observation.

Genetic screening for Gaucher disease in Israel: genetic screening program for a low penetrant, treatable disease Shachar Zuckerman1,2, Ephrat Levy-Lahad1,2, Amnon Lahad3, Michal Sagi2 1Medical

Genetics Unit, Shaare Zedek Medical Center, 2Hebrew University - Hadassah Medical School, 3Dept. of Family Medicine, Hebrew University E-mail: [email protected]

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder. The most common type, type 1, is prevalent among Ashkenazi Jews (1:17 carrier frequency). It is characterized by splenomegaly, hepatomegaly, anemia, bone fragility, and lack of neurological involvement. Presentation in type 1 is highly variable, and 60–80% of homozygotes remain virtually asymptomatic. Enzyme replacement therapy has proven to be effective in reducing most of the symptoms of type 1. Type 2 and type 3, are the rare and severe forms of GD. The purpose of carrier screening is to detect couples at risk for an affected child. Among the prerequisites for carrier screening, as formulated by the World Health Organization (WHO), are severe disease and high heterozygote frequency. Therefore, carrier screening that may lead to prenatal diagnosis and pregnancy termination for GD is controversial. In Israel, in spite of the recommendation of both the 'GD committee' in the Ministry of Health and the Organization of Medical Geneticists to exclude GD from carrier screening programs, it has been offered since 1995 in most genetic institutes. We conducted a nationwide study of the outcome of this screening so far, in order to draw conclusions for the future. Objectives • • • •

to collect the data on GD carrier screening in Israel. to document steps taken by couples at risk and assess their decision making process. to evaluate the genetic consultation and participants’ satisfaction. to re-evaluate the carrier screening program for GD in Israel and consider its consequences.

Methods Data was collected from all genetic institutes, and all couples identified at risk were contacted, 78% of them were interviewed. Between 1995 and 2003, about 35,000 individuals were tested for GD and 83 carrier couples were identified (in three one spouse was homozygote). We interviewed 65 couples at risk for a child with GD type 1, 82% for a mild and 18% for a moderate disease. 78% of the couples performed prenatal diagnosis, and among 68 pregnancies 13 fetuses were homozygotes for a mild mutation (N370S), two of them were aborted (15%). Three fetuses were compound heterozygotes and two (66%) were aborted. The main reason for performing GD prenatal diagnosis was reducing uncertainty. The main predictors for positive attitude towards pregnancy termination were perception of GD as severe disease and perception of high risk for having a homozygote child.

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Most subjects (76%) did not regret testing for GD and 68% think the screening should be continued. Nevertheless, only half of the subjects would have prenatal diagnosis for GD in future pregnancies and only 25% would terminate a pregnancy if the fetus was affected by GD. Only 55% of the subjects said that the genetic counseling session was informative and clear. We show that in spite of expert recommendations, genetic screening for GD is widely used in Israel. Although most couples utilized prenatal diagnosis, only a few terminated the pregnancy of a fetus with mild mutations. The information received was insufficient to make an informed decision. Although for most couples no substantial harm was detected and they were satisfied with the procedure, two out of four couples who chose pregnancy termination regret their decision. We conclude that the benefits of this screening are very limited and future harm will be minimized only if such programs include extensive genetic counseling and consultation with professionals familiar with the diseases. This study presents the attitudes of the population most influenced by the screening program, and contributes to the understanding of controversial genetic screening for a mild disease with low penetrance. Carrier screening for GD raises questions about the moral justification of screening for non-life-threatening, treatable diseases. Bibliography 1. 2. 3. 4. 5.

Beutler E, Grabowski GA: Gaucher disease. In: The Metabolic and Molecular Basis of Inherited Disease, 7th Edn. Scriver CR, Beaudet AL, Sly WS, Valle D (Eds.), McGraw-Hill, New York, USA, 2641–2670 (1995). Horowitz M, Pasmanik-Chor M, Borochowitz Z et al.: Prevalence of Glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population. Hum. Mut. 12, 240–244 (1998). Beutler E: Enzyme replacement therapy for Gaucher’s disease. Clin. Haematol. 10, 751–763 (1997). Zlotogora J, Leventhal A: Screening for genetic disorders among Jews: How should the Tay-Sachs screening program be continued? IMAJ 2, 665-667 (2000). Zimran A, Zaizov R, Zlotogora J: Large scale screening for Gaucher’s disease in Israel – a position paper by the National Gaucher Committee of the Ministry of Health. Harefuah 133, 107–108m (1997) (Article in Hebrew).


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