Indi an Journal of Experimental Biology Vol. 39, October 2001, pp. 955-96 1

Review Article

Human genome project : Pharmacogenomics and drug development

N K Ganguly, Rahmat Bano & S D Seth* Indian Council of Medical Research, New Delhi

Fax:9 I -11-685779 I ; E-mail : icmrhqds @sansad.nic. in

Now that all 30,000 or so genes that make up the human genome have been deciphered, pharmaceutical industries are emerging to capitalize the custom based drug treatment. Understanding human genetic variation promises to have a great impact on our ability to uncover the cause of individual variation in response to therapeutics. The study of association between genetics and drug response is called pharniacogenomics. The potential implication of genomics and pharmacogenomics in clinical research and clinical medicine is that disease could be treated according to the interindividual differences in drug disposition and effects, thereby enhanc ing the drug discovery and providing a stronger scientific basis of each patient' s genetic consti tution. Sequence information derived from the genomes of many individuals is leading to the rapid discovery of si ngle nucleotide polymorphisms or SNPs. Detection of these human polymorphi sms will fuel the discipline of pharmacogenomics by developi ng more personalized drug therapies. A greater understanding of the way in which individual s with a particular genotype respond to a drug allows manufacturers to identify population subgroups that will benefit most from a particular drug. The increasing emphasis on pharmacogenomics is likely to rai se ethical and legal questions regarding, among other things, the design of research studies, the construction of clinical trials and the pricing of drugs.

Human genome The human genome is the twisted strand of

biological text that carries all the instructions for making and growing a human being. Enors in that text cause or contribute to the vast majority of human diseases. Genomics is the study of the genome as a whole: the total sequence of DNA in the cell and how this provides the information for the cell to function and reproduce itself in an organism or individual. Watson and Crick deduced that DNA within the nucleus of the cell is a double helix framework, and positioned along the rungs of the two DNA ladders are four chemical bases : adenine(A), cytosine(C), guanine(G), and thymine(T) . The four letters in this simple alphabet specify all the biological properties of a human being. In normal DNA, an A on one strand always pairs with aT on the other, and G always pairs with C. If we know the sequence of one strand, we know the sequence of the other. Genome has gene as well as 'junk' DNA. Genome is a pair of 23 chromosomes, and the average chromosome has around 100 million bases 1• It is assumed that there are 30,000 genes atTanged on the chromosomes . Genome controls the day-to-day function of the body's 60 trillion cells and guides an embryo's growth into a li ving, breathing and thinking human . The average gene, which is a packet of information that carries out

*Correspondent author

a particular instruction, varies from a few thousand to few hundred thousand bases. Unity is in racism is out - all human beings share an incredible 99.99 percent of all genetic material2

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Human genome project The Human Genome Project was initiated on

October 1,1990, and successfully completed, and announced a draft sequence of the human genome on 26 June, 2000 simultaneously by the publicly funded Human Genome Project (HGP) and the privately funded Celera Genome Corporation. The HGP is a publicly funded consortium that includes four large sequencing centers in the US, as well as the Sanger Center near Cambridge, England, and laboratories in Japan, France, Germany and China. Working together for more than a decade over 1,100 scientists have crafted the map of the three billion DNA base pairs, or uni ts, that make the human genome. In April , 2000 a brash young company called Celera Genomics, Rockville, Md., beat the public consortium to the punch, announcing its own rough draft of the human genome2

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The sequencing of the human genome is a brilliant techno-managerial exercise, which has succeeded in bringing together molecular biology. genetics and engineering, especially automation technology and bioinformatics3

. The HGP used a hierarchical mapping and sequencing approach, involving generation of a series of overlapping clones that cover

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the entire genome and shotgun sequencing of each clone. The genome sequence was reconstructed by assembling the fragments on the basis of sequence overlap and mapping and chromosomal position information on the clones. Celera took a shorter route: shredding the encyclopedia all at once. Celera Genomics used a whole-genome shotgun sequencing approach , without generating a series of overlapping clones, but also incorporated HGP information where avail able. All of the results of this analysis are available on web site maintained by the University of California at Santa Cruz (http://genome.ucsc.edu) and National Center for Biotechnology Information (NCBI; http:// www. ncbi .nlm.nih .gov)4

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The goals of the HGP were to first construct a comprehewive genetic map of the human genome. Also, libraries of overlapping clones spanning the entire genome were to be constructed . The final goal was the sequencing itself. This was to be complemented by developments in bioinformatics so as to organize the tremendous amount of data generated in a meaningful manner3

. Beginning with blood and sperms, the team separated out the 23 pairs of chromosomes that hold human genes. Scientists then clipped bits of DNA from every chromosome, identified the sequence of DNA bases in each bit, and finally matched each snippet up to the DNA on either side of it in the chromosome. And they went on gradually crafting the sequences for individual gene segments , complete genes, whole chromosome and, eventually, the entire genome. Wilson compares thi s approach to taking out one page of an encyclopedia at a time, ripping it up and putting it together agai n. Other applications of HGP include pharmacogenomics and patient counseling about individual heal th ri sks. Concerns include how to integrate genetic technology into clinical practice and how to prevent genetic-based discrimination4

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Equally striki ng is how little of the genome actually codes fo r prote ins and how those exons are distributed . Celera calculates that just 1.1% of the genome codes fo r proteins; the public fig ure is 1.5%. Human genome contains in tervening sequence, so metimes extending thousands of bases, between exons. Not only does this make for big genes, but it complicates the task of gene identification . Moreover, genes themselves can be separated by vast "deserts" of noncoding DNA, the so-called junk DNA. Celera scientists est imate that between 40% and 48% of the genome consists of repeat sequences: DNA in which a particular pattern of bases occurs over and over,

sometimes for long stretches of a chromosome. One of the more common repeats, called Alu's, cover 288 megabases in the Celera human genome-nearly 10% of the total. And the public consortium's analysis shows that older Alu's tend to concentrate in gene­rich areas, suggesting that those Alu's located near genes may serve some useful purpose and thus were retained by the genome. "It's like looking into our genome and finding a fossil record, what came and went," says Collins5

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Among the most common DNA fossils are transposones - pieces of DNA that appear to have no purpose except to make copies of themselves and often jump from place to place along the chromosomes. They typically contain just a few genes - those needed to promote the transposone 's proliferation. Both drafts confirm that transposones may also be a source of new genes. Celera found 97 coding regions that appear to have been copied and moved by RNA-based transposones called retrotransposones. Once in a new place, these condensed genes often decay through time for lack of any clear function, but some may take on new roles . And transposone genes themselves become part of the genome. Until recently, 19 of these tranposone­derived genes were known. The public consortium just found 28 more. " It almost looks like we are not in control of our own genome," notes Phil Green, a bioinformatics expert at the University of Washington, Seattle5

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Drug discovery aud development There are important differences between drug

discovery and drug development. Drug discovery is an expensive process invol vi ng research into the mechani sms of disease, the selection of biological targets, and the identification of compounds that modulate the di sease. Drug development is focused on es tabl ishi ng the efficacy and safety of a single compound through phased clinical trials to ach ieve marketi ng approval. Drug development is constrained by the high cost of clinical investi gation and the fact that each day required to achieve marketing approval can reduce the economic value of a product by many mill ions of dollars6

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The drug discovery and developmen t process can be divided into several phases. In the discovery phase lead compounds are identified by the use of high throughput screening against chemical libraries, rational design based on knowledge of the three­dimensional structure of the target, modification of known chemical structures or the production of

GANGULY et al.: HUMAN GENOME PROJECT 957

therapeutic proteins . These are then screened through a series of increasingly complex assays, eventually leading to a demonstration of efficacy in an animal model representing the relevant disorder. The concerns that exercise the minds of scientists involved in this process often center around the choice of target and the drug candidate specificity. Ideally one would select the molecular target based on its intimate involvement in the disorder to be targeted and its limited involvement in the other biological process that might, if disturbed, lead to unwanted side-effects. In addition, eveh when the appropriate target is chosen and lead compounds identified, concerns will remain as to whether other gene products will be inhibited or activated by these compounds7

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Pre-clinical safety and toxicity testing begins immediately following the drug discovery process, although some of these activities often take place concurrently with the final stages of drug discovery. Safety and toxicity testing comprises acute and chronic toxicity tests, mutagenicity assays, reproductive toxicity and drug disposition studies. They are normally carried out in rats and one to two additional mammalian species, although the scientists involved in these safety assessments are acutely aware that these species do not necessarily respond to drugs in the same manner as humans. Compounds that pass the safety hurdles and are suitable for commercial production enter clinical trials. However, even after these efforts, only about I 0-30% of compounds reach the marketplace. Furthermore, differences in the pathobiology of a disorder, the underlying cause, might lead to very similar clinical manifestations. This would be of significant concern for those di sorders for which no clear biochemical marker can be used to measure the pathological process such as behavioral disorders7

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One of the most recent revolutions in biology has begun to generate both the knowledge base and tools to address many of the current concerns of pharmaceutical researchers. This revolution comprises the ability to explore and characterize the structure and activity of whole genome in a high throughput or massively parallel manner and as such has been called genomics. However, in addition to the scientific benefits it brings, genomics also brings with it concerns from both the ethical and legal point of view as individuals are defined, to a great extent. by their genomes. From the commercial perspective one of the current major concerns is the rapid patenting of human genes to protect their use as therapeutics and

more importantly as therapeutic targets. Therefore, in their estimation only about 0.5-1% of the genome has been targeted by therapeutics. Obviously not all genes will encode viable therapeutic targets but it is likely that a significant number of genes will, and they remam undiscovered or, as yet undiscovered. Genomics actiVlttes such as high throughput sequencing are already reducing this number (as available targets) through new gene discovery and patenting. It is also clear that, in the not too distant future, every human gene will be identified and characterized, at least by its sequence. In order to remain compettttve the modern pharmaceutical company must recognize the fact that it must not only continually increase the efficiency of its drug discovery efforts but, in addition, must ensure its freedom to operate by creating its own intellectual property rights for the new therapeutic targets in its pipeline. The tools of genomics will contribute significantly to both of these activities 7.

HGP and drug development Presently the human genome sequencing project is

reported to be about 6% completed and is projected to be fully complete in about 2003. One of the most valuable contributions that the human genome project will make to biomedical research is the ability to study the natural genetic variations in human. The scale of effort required to fully categorise the genome comprising 23 pairs of chromosomes made up of 3 x 109 base pairs of DNA is immense. In addition to generating a complete sequence of the human genome, the HGP has other objectives: (i) to further develop sequencing technology towards higher throughput and reduced cost, (ii) to develop functional genomics technologies through establishing full length eDNA resources, developing the tools for the study of nonprotein coding regions of genes, advancing gene expression analysis, improving methods for genome wide mutagenesis, developing the technology for global protein analysis, (iii) to advance comparative genomics through sequencing the genome of other organisms such as C. elegans. Drosophila and the mouse, (iv) to address the ethical , legal and social implications of the data. specially the data on human genome variations, (v) to train scientists in the specialties created by this and related efforts8

. For pharmaceutical industry it will mean that every potential pharmaceutical target will be known (at least by neucleic acid and protein sequence) and mapped. All homologous (paralogous) will immediately be known for a new target gene so a high

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degree of drug specificity can be designed in the very early stages of development program and mechanism based adverse effects will be avoidable, or the very best explainable9

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In the postgenomic era, dramatic increase in the amount of genomic information will have a tremendous impact on biomedical research and on the way that medicine is practiced. When all the human genes are truly known, scientists will have produced a Periodic Table of Life, containing the complete list and structure of all genes and providing us with a collection of high- precision tools with which to study the details of human development and disease 10

. As most of the genome is sequenced, changes in genes and proteins in disease will be better identified for the rational designing of drugs. The surprising number of genetic polymorphism identified in the human genome indicates the genetic basis of the tremendous amount of variations in the species. The identification of the genetic basis of variations in response to drugs, both in terms of efficacy and toxicity, holds out hope for individualized therapy. Why a person is more likely to react adversely than another to a particular drug, is not well understood.

Proteomics, offers an alternative, and complimentary approach to genomic-based technologies for the identification and validation of protein targets and for the description of changes in protein expression under the influence of disease or drug treatment. Much interest has been expressed by the pharmaceutical industry in proteomics, in anticipation of the value of this technology to both discovery and development of new drugs 11

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Proteomics involves the identification and quantitation of gene expression at the protein level. Additionally, proteomics may help to identify protein interaction partners and members of multiprotein complexes. Furthermore, this technique may assist in following time-dependent changes in protein expression levels resulting from selective excitation of a biological pathway, and thereby delineating from selective excitation of a biological pathway, and thereby delineating a cellular protein network, a methodology that has been referred to as functional proteomics. Recently, however, considerable progress has been made in improvi ng detection of low copy proteins through enhancement of gel-loading techniques and enrichment strategies such as affi nity -based purification two-dimensional gel separation. Finally, enhanced protein stai ning/detection methods are now becoming available, and mass spectrometry

is pushing the bounds, are now becoming available, and mass spectrometry is pushing the bounds of detection to even more sensitive limits. Notwithstanding the technical difficulties that remain, sufficient evidence exists, even at this early stage of technology to warrant that proteomics will provide crucial information for the discovery and development of novel therapeutic targets 12

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Post-genome project era has given the name "Functional Genomics", which will begin early in millennium and will encompass the many efforts needed to elucidate gene function. Indeed, the phenotyping of genetically manipulated animals will be critical in the determination of biological function of a particular gene. But, in reality, the discipline of functional genomics has its foundation in the physiological and pharmacological sciences. This is gratifying to the "traditional" pharmacologist, whose expertise will be drawn on even more in the future to unravel the mysteries of genetics. Although the evaluation of genetically manipulated animals will require a thorough understanding of physiology and pharmacology, the experimental approach will involve many new technologies. These methods will include in vivo imaging (i.e. magnetic resonance imaging, micro-positron emiSSions tomography, ultrafast computed tomography, infrared spectroscopy), mass spectrometry, and microarray hybridization, all of which should enhance the speed and accuracy at which functional genomics is achieved 12

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The major interest of the pharmaceutical industry in "gene therapy" will undoubtedly be centered around in vivo treatment protocols, although more invasive ex vivo methods (whereby cells are removed from the patient, transfected with the gene of interest, and then placed back into the patient) may be acceptable for certain serious diseases (e.g. cancer). Currently, genetic information can be transferred into cells by a number of protocols, including the use of DNA plasmids, DNA liposomes, or a variety of viruses. The most effective transforming agents are viral vector, such as adenovirus, adenoassociated viruses, and retroviruses. Although retroviruses require cell division to incorporate the new information into the genome, adenovirus and adenoassociated viruses will transfer their information into nonreplicating cells 12

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HGP and pharmacogenomics

The new technologies created 111 the Human Genome Project have changed the face of genetics

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(like gene identification), creating genomics and pharmacogenomics, the blending of high technology and pharmacogenetics. Whereas, pharmacogenetics takes advantage of high throughput DNA sequencing, gene mapping, and bioinformatics, the result is a quantum leap in the ability to discover genes, which are associated with physical attribute, disease susceptibility, or the response to drugs 13

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What are the pharmacogenomic tests likely to be developed recently? There has been much discussion about "genetic bar codes" and "genetic profiles", suggesting that it will be possible to include all gene sequence information relevant to an individual within a single test. At this point, we have neither the knowledge nor the technology to develop such a test. For the foreseeable future, tests based on pharmacogenomics will be directed towards single response. The tests will currently focus on three key attributes: therapeutic need, clinical utility, and ease of use 13

. Therapeutic need is a combination of the number of patients likely to be tested for a particular drug response, the consequence of that response, and the alternate means of obtaining an equivalent answer. For instance, a drug used by many people but which is frequently ineffective, and has a high incidence of therapeutic failure, would have a high medical need for a test to predict efficacy in individual patients 13

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Although a massive quality of sequence information is accumulating, the functions of thousands of genes remain undetermined. Functional genomics refers to the methods for assessment of gene function by making use of the information provided by structural genomics. Pharmacogenomics, an offshoot of genomics, refers to the application of genomic technologies in drug discovery and development. The vision of pharmacogenomics is to study genetic variances that affect drug action. This will lead to the development of new diagnostic procedures and therapeutic products that will enable drugs to be prescribed selectively to patients for whom they will be effective and safe. Pharmacogenomics is the application of genomic technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. It applies the large-scale systematic approaches of genomics to speed the discovery of drug response markers , whether they act at the level of the drug target, drug metabolism, or di sease pathways. The potential implication of genomics and pharmacogenomics in clinical research and clinical medicine is that the

disease could be treated according to genetic specific individual markers, selecting medications and dosages that are optimized for individual patients 14

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Two recent developments are responsible for the increased interest in pharmacogenomics. The first is the recent recognition that systematic discovery of genetic variance can provide important, achievable opportunities for developing new therapeutic and diagnostic products from genomics. The second is the emergence of appropriate methods for discovery and analysis of genetic variation in human populations that may be employed within the time limits and constraints of drug development 14

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Genetics and pharmacogenetics Genetic variation within a population is the base

upon which evolution operates. It is clear from even the most cursory observations that a great deal of genetic variation exists within human populations and one would expect that variations will be found within the genes that are involved in both disease processes and those involved in drug responses. Indeed, it has been accepted that polymorphism within the genes involved in drug metabolism can play a critical role in the variable responses to drugs within patient populations 15

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. The application of genetics to pharmaceutical discovery and development can be understood into two broad categories. Firstly, the application of genetics to the discovery of diseased genes and describe some of the tools available for these analyses. The second category IS

pharmacogenetics, that addresses the application of human genetics in understanding drug response.

Over the last few years genetic markers have become available in ever increasing numbers. These reagents are used for linkage studies, the aim of which is to determine how often two loci are separated by meiotic recombination. If two loci are on different chromosomes they will segregate independently at meiosis. However, if they are present on the same chromosome they have a greater chance of segregating together, this chance being inversely promotional to their distance apart on the chromosome (assuming recombination is randomly distributed along chromosomes). In order to identify disease loci in humans, one needs to make use of genetic markers. These are the Mendelian characters having sufficient polymorphism such that randomly selected individuals are heterozygous. The genetic markers presently in use are DNA polymorphisms which can be typed by the same techniques and can be mapped directly onto their chromosomal location

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through physical mapping. DNA polymorphism, such as microsatellites, are mostly comprised (CA)n repeats, commonly observed in the 15-30 repeat range and are found throughout the genome. The advantage of these markers is that they are highly polymorphic and therefore highly informative 17

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Single Nucleotide Polymorphisms (SNPs), common variations among the DNA of individuals, are being uncovered and assembled to large SNP databases that promise to enable the dissection of the genetic basis of disease and drug response (i.e. pharmacogenomics). The health care industry is clamoring for access to SNP databases for use in research in the hope of revolutionizing the drug development process. The next phase of HGP will focus on, among other things, creating a genome-wide map of I 00,000 of the most common type of genetic variation i.e.SNPs. Similarly, a consortium of 10 pharmaceutical companies and the Wellcome Trust are working toward identifying 300,000 SNPs within the next two years 18

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SNP as an alternative set of markers have been developed, based to a large extent on the data coming out of the human genome projec t. The single nucleotide polymorphisms or SNPs represent single base variations in the genomic DNA sequence at defined positions that are found at a frequency of over I% in the human population 19

. SNPs represent the most common type of human genetic variation. It is anticipated that SNPs will aid in the identification of disease genes by family linkage studies, linkage disequilibrium studies in isolated populations20 and even association studies of patients and control healthy subjects21

• As SNPs can only have two alleles, they are less informati ve than microsatellite markers, however, they are more abundant and lend themselves to automation(SNPs can be analysed through direct hybridization). In a recent study SNPs were used to design genotyping microarray chips to demonstrate the feasi bility for high throughput genotyping 19

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Studies of SNPs and disease have become more effic ient when a few more problems are solved. First, although 82% of SNP variants are found at a frequency of more than 10% in the global human population, the 'microdistribution' of SNPs in individual populations is not known. Second not all SNPs are created equal, and it will be essential to know as much as possible about their effects from computational analysis before studying their involvement in disease. For example, each SNP can be classified by whether it alters the sequence of the

protein encoded by the altered gene. Changes that alter protein sequences can be classified by their effects on protein structure. The non-coding SNPs can be classified according to whether they are found in gene-regulating segments of the genome - many complex diseases may arise from quantitative, rather than qualitative, differences in gene products. Third, technology of patients and controls, is not yet fully developed, although there are some creative ideas around22

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Pharmacogenomics and drug development Pharmacogenomics is a distinct di scipline within

genomics. It is concerned with genetic effects on drugs themselves and with the genetic variances that contribute to the variable effects of drugs in different individuals. Pharmacogenomics aims to satisfy this clinical need by focusing on genetic contributions to drug action as opposed to the genetic causes of disease. Focusing on those genes and variances that are most likely to have significant pharmacological effects rather than on randomly selected genetic markers can further reduce complexity. lnformatic tools and experimental models of drug action can be used to identify genes that are most likely to affect the action of a drug. Molecular methods can also be used to identify all common variances within a gene and characterize those variances that alter the structure and function of the expressed product or its level of expression 14

• The application of genomic technologies has expanded the opportunity for identifying genetic effects on drug action. Studies have begun to identify common variances in genes that are the targets for drug action as well as in genes that control the activation , distribution, or elimination of many drugs. These discoveries are expected to lead to the development of diagnostic test drugs in individual patients. It is likely that such tests will be generally applicable in medical practice to determine which drugs, in the armamentarium available to the physician, are most likely to be effective and safe for an individual patient. Drugs that will be potentially toxic will be avoided, effective therapies will be prescribed sooner, and diseases will be more effectively and economically managed 14

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The potential benefits of pharmacogenomics on drug development are profound. Achieving these benefits requires a clear focus on technologies that can be applied within the paradigm of conventional development. With the economics of drug development already constrained to the point that many approved drugs never recover their

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development costs, it is unlikely that pharmacogenomic strategies that require any significant increase in the scope or cost of development be adopted by the industry. The challenge then for pharmacogenomics is to invent and implement the novel technologies that can meet drug development' s needs 14

• The leading strategies fo r pharmacogenomics involve selecting candidate genes on pathways for drug action, activation, and elimination in humans (or other species) and identifying variances in the gene sequences. These variances can then be studied both on a biochemical level, to assess their functional significance in drug action, and on a population level, to establish statistical association which observe phenotypic variance m drug action. In a managed-care environment, pharmacogenomic strategies can provide a competitive advantage and increase the market potential of certain drugs. The preferred strategy involves identifying variances that define the population of patients in whom a drug will be safe and effective, and marketing the drug together with a diagnostic product that enables the drug to be prescribed selectively to these patients. The use of such products would provide r·0st savings to the health care provider and payers by increasing the effectiveness of the initial prescri bed therapy, reducing the number of doctor visits , eliminating the cost of prescribing ineffective pharmaceuticals, and eliminating avoidable toxicity 14

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Pharmacogenomics also offers strategies for more efficacious and economical use of conventional pharmaceutical products without the risks and cost of this iterative process as well as opportunities for developing new products that take advantage of the normal variability of human populations to provide safer, more effective therapies. Products based on genetic variances that contribute to drug efficacy and safety are likely to be the first, and may be the most important, clinical application of genomic science 14

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Our understanding of the structure and function of the human genome has, within the last ten years, increased beyond the most optimistic of predictions. It is likely that, over the next ten years, our insights into genome function in development, health and disease will develop in many obvious and unexpected ways. Presently our increased knowledge and the application of genomics tools are poised to optimize drug discovery and development activities as well as add value to the resulting drugs. Ethical issues are now a major concern for scientists. Like an ID a

person's gene sequence record can be made available on tap. In the near future, this means an insurance company, for example may charge a higher premium from a person who shows a susceptibility for developing hypertension. Or at the work place, Darwinian selection may be replaced by managerial selection as employees are hired or fired, on the basis of their potential as seen from their genetic profile.

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