Chapter 5

Application of Human Genome

in Health and Medicine

The convincing rational for investing billions of Dollars to sequence the Human Genome was based on improving our ability to understand and treat diseases. It is time to make good on that investment.

Most diseases are caused by tiny mutation in our genes, like misprints, giving out the wrong instructions in the cell, resulting in production of a faulty protein. These defected instructions (mutations) can be inherited or could be caused by environmental factors such as radiation or pollution. Almost 5000 heritable disorders have been clinically characterised, up to 30% of hospital paediatric admission and 12% of adult hospital admission are associated with genetic problems. A single faulty gene could cause a disease, and currently there are a more than 4000 genetic diseases caused by a mutation in one single gene such as Sickle Cell Anaemia, Cystic Fibrosis, Muscular Dystrophy and Huntingdon’s disease. Up to 8% of hospital paediatric admission are due to single-gene disorder (Lee, 1993). On the other hand many of the more common diseases from Heart diseases and Cancer to Alzheimer and Parkinson’s are known to result from multifactorial factors among which is a genetic components.

In 1971 only 15 Human genes had been localised to specific chromosomes, most of them on easily identified sex chromosomes. By the mid 1990s, researchers had mapped the location of about 2000 genes, an impressive feat, but still only small percentage of the entire Human Genome (Grace, 1997). This exponential progress for gene hunting started since the advent of the DNA-based biology and first successful experiment in genetic engineering techniques, and the pace was further enhanced during 1990s with the Human Genome project initiative. To study the gene involved in a disease, first it is required to localise its position within a given chromosome, a process known as genetic map. Given the fact that the individual gene size within the whole length of Human Genome is similar in comparison to the size of an ant on “Mount Everset”. As Colin (the director of Human Genome Project) put it: “Locating a gene from scratch is like trying to find a burned-out light bulb in a house located somewhere between the East and the West coasts without knowing the state, much less the town or street the house is on (Elmer-Dewitt, 1994). There are many approaches to localise the gene of interest, either through metabolic study, chromosomal feature, or genetic markers. As there is different kind of maps, political maps, highway maps, topographic maps, so there are different kind of genomic maps depending on the degree of resolution. The genetic map is the lowest resolution map and generated by application of positional cloning which facilitated the hunting of hundreds of monogenic disease (genetic disease that is caused by defect in single gene). This method depends on studying the affected families and then worked out how the disease is inherited through generations. DNA samples from the affected families are taken, and then special sections scattered along the Genome called genetic markers are sequenced. In 1994 a comprehensive map was available that included more than 5800 markers including gene implicated in Cystic Fibrosis, Myotonic dystrophy, Hutington’s disease, Tay-Sach and several others. By seeing which marker sequence are found more frequent in family members affected by the disease, then the position of the responsible gene could be localised on a chromosome as well as its rough position on that chromosome,  a process known as genetic mapping (Fig.20). Then to work out the sequence of the whole gene to reveal its high resolution details in term of its base pairs sequence, a process known as physical mapping (Fig. 20). With the accumulated technologies of Human Genome project, now the scientists can easily work out the sequence by searching the computer database in the relevant region of the chromosome. In 1995, Ashworth and his team of the Lawrence Livermore National Laboratory completed 95% of the highest resolution physical map for Human chromosome 19, an estimated 54 million base pairs excluding the centromere. This achievement contributed directly to the characterisation of the genetic basis of the disease myotonic dystrophy and also to the description of the aberrant triplet repeats which is now known to be the major factor that contribute to the onset of at least 9 diseases, including Huntingdon’s disease. Likewise, Doggett (1995) had completed the physical map for chromosome 16 excluding the highly repetitive DNA in the centromere, which contributed immensely to the characterisation of other diseases, such as forms of breast and prostate cancers, fanconi’s anaemia and others. Both chromosomes 19 and 16 in addition to chromosome 5 have been sequenced completely by April 2000, whereas chromosome 21 has been completed on May 2000.

The benefits that will arise from having a complete sequence map of the Human Genome fall into the following categories :

5.1. Preventive Measure :

The sequencing of the Human Genome and ultimate knowledge of the all genes in the next decades to come will lead to a fundamental shift towards preventive medicine. It is predicted that in 2010 genetic tests would be routine practice, where blood samples are taken to extract the DNA materials. This material is then screened to determine the risk of developing various diseases. Paul Kelly, head of the British Company Gemini Genomics at Cambridge said “Screening will be more sophisticated than any thing we know today. We will not be able to say who is at risk, we will know whether their risk will be reduced by various interventions. For example, while one patient might be advised to eat a healthier diet to reduce their risk of heart disease, another’s gene might suggest that diet would make no difference and be prescribed exercise or a cholesterol-lowering drug instead”. (Marchant, 2000).

David Altshuler, a geneticist from the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, believes that the technique will simply extend familiar tests. “People are comfortable with the idea of measuring blood pressure or cholesterol and being told of any risk factors. It should be the same with genetic screening. For patients it is not a death sentence, it is information they can use”.

Once the risk is predicted early, the doctor can plan accordingly a preventive measure before problems develop. 

5.2. Develop New Diagnostic Test :

As a result of accumulation of large numbers of newly discovered genes as a consequence of Human Genome project’s efforts, scientists developed a new tool called “DNA chip” also known as “DNA micro array”. What silicon chips have done for computers, DNA chips may do for biological research.

DNA chips promise to carry the science of understanding Genome to a whole new level, and to bring tools for getting DNA-sequence information out of research labs into doctor’s and pharmacist’s day to day use, the better to tailor-fit medical treatments to an individual's particular genetic makeup.

Leroy Hood, a molecular biologist at the University of Washington in Seattle, said “DNA-chip technology will be the key in meeting one of the biggest scientific challenges of the coming century-the analysis of how all the genes in an organism work together as a very complex system.

"If we were to study each gene in isolation, we'd never know how the Genome functions as a whole. DNA chips are the prototype global technology for genetics, because they let us look at the behaviour of thousands of genes at once. System biology will be the challenge of the 21st century, and the only way to understand the biology of the systems will be to use tools such as these chips". (Daviss, 1998).

DNA chips will also help to tailor medicine for specific genetic character of any individual. The DNA chips will enable doctors to take a quick snapshot of a person’s genes to see which treatment is best for him and to avoid the side effects of other medicines (Daviss, 1998).

5.2.1. How the DNA Chips work ?

There are various DNA chips, but all depend on the same basic principle :

- Complementary DNA stands stick together

- Double-stranded DNA molecule can unzip into two complementary strands.

- Each of these can zip back together with its complementary sequence, either the old strand, or a new strand with the same sequence.

If we have a standard checkerboard, 7 squares on a side, 49 squares total. In each square, we tie down a different snippet of single-stranded DNA just three nucleotides long. We write down the sequence in each square. (We can make 49 different sequence variations from three nucleotides-ATG, CAT, GTG, TGC, ATA, and so on- so there’s enough room for all the possibilities.)

Now if we have an unknown sequence, also three nucleotides long and wanting to find out what this unknown sequence is, we set it loose on the array so that it moves from square to square. When the unknown sequence finds its complement, it sticks. The better the match, the more bonds there will be between the strands and the stronger the join. Next the chip is flushed with a chemical solution that breaks a part all but the best- matched double strand. The unknown sequence will be known by finding which square the unknown DNA stuck to. Because we know the sequence of the DNA we tied down to that square, we know that the unknown sequence is the complement. The percentage of error in this match is about 5%. Currently the typical chip has built-in redundancies; additional test sites with the same unique DNA fragment each offering a second opinion on the results of the others.

The real potential power of DNA chips lies in their flexibility, compact size, speed, and low cost. Scientists can put hundreds of thousands of distinct DNA sequences on a microscopic grid a few centimetres length. Then, using fluorescent molecular tags that light up when a complementary strand binds to a particular spot, a computer can read out which sequences on the chip find their complement in an unknown sample. The result is a complete catalogue of genetic ingredients.

DNA chips can gather an incredible data very quickly, and because they can be produced in mass-quantities, they will likely be very cheap in the future. That will allow easy collection of genetic information from many individuals, opening up all kinds of opportunities to help doctors diagnose and treat their patients.

5.2.2. Gene expression Analysis :

DNA chips allow the scientists to observe genes working together in what is called "expression analysis. Expression of gene means to synthesise a protein (refer to protein synthesis on Ch. 3.2). Cells first transcribe the gene's DNA sequence into a complementary mRNA copy. Then a ribosome translates the mRNA sequence into the string of amino acids, which makes up the protein. Cells constantly switch genes on or off in response to changing conditions. To understand a cell's behaviour in response to a stimulus- internally or externally the presence of a hormone, or a toxin, or some environmental signal-it would be handy and practical to have a minute-to-minute reading of which genes are turned on and in action. DNA chips are just about perfect for tracking this kind of minute-to-minute change in gene expression. For example, Patrick Brown and his colleagues at Stanford University wanted to find out the details of how yeast cells make spores. Other scientists had already determined the DNA sequence of every possible mRNA a yeast cell makes. So, Brown and his colleagues put the complements of each of these possible mRNA sequences onto a chip. Then, they ground up a bunch of resting yeast cells, which of course contained an mRNA corresponding to each gene that was active at the moment the cells hit the blender. Next, the researchers spread this mixture over the surface of the chip. Only the spots corresponding to genes that were actively churning out their mRNA lit up, because these were the only spots on the chip that had found their complementary sequence.

This first experiment gave Brown and his colleagues a baseline. Next, they stimulated the yeast to form spores (by taking away their food) and repeated their chip analysis six times over the next 12 hours. By looking at which genes turned on, and when, Brown and his colleagues got many new insights into how yeast cells genetically shift gears to make spores.

But the significance of Brown and company's work goes way beyond yeast physiology - it paved the way for using DNA chips to see how dozens of genes work in concert to change a cell's behaviour

(Jeremyhompage:http://www.washington.edu/).

Expression analysis has medical applications, too. For example, a team led by Eric Lander, director of the Whitehead Institute at the Massachusetts Institute of Technology, announced last October that they used expression analysis-made possible by a DNA chip-to develop a test to classify different types of leukemia. (To choose the best treatment, doctors need to know exactly what type of cancer a patient has.) These researchers looked at samples from about 50 patients already known to have one of two different kinds of leukaemia. Then, using the patterns of gene expression they found in the two groups, they correctly predicted which type of leukemia several patients had. In the near future, doctors may be able to use this test to decide which is the best treatment for a new leukaemia patient. Researchers also plan to develop similar tests to match treatments to patients for other kinds of cancer, too. (Patrick Brown’ home page: http://cmgm.stanford.edu/).

5.2.3. Human Differences

The DNA molecule is 99.9 percent identical in all Human population. The genetic basis of all of humanity's differences is only 0.1%, from our phenotype to the way some people get certain diseases to the fact that some patients respond to a certain drug while others don't. Scientists are now starting to use DNA chips to map out the one-letter variations in the 3 billion-nucleotide Human Genome. These pinpoint differences are called "single nucleotide polymorphisms," or SNPs. Identifying them will help the scientists to understand the genetic basis for Human variation.

But to map SNPs, a different kind of chip is needed. For expression analysis, a chip containing all possible genes is used. Whereas for SNP analysis, a chip with many possible variations of one gene is needed. Then a DNA sample from the person who wanted to be tested is taken, PCR technique is used to make multiple copies of the gene of interest, and put this "amplified" sample on the chip. The spot that lights up will correspond to the particular sequence variant the person has. Because the test is quick and not too expensive, you can do many of them. Then, you correlate different outcomes-response to a certain drug, for example, or the probability of getting heart disease-with the different genetic variations. (Affymetrix home page: www.affymetrix.com)

Francis Collins, director of the National Human Genome Research Institute in Bethesda, Maryland, is enthusiastic about SNP analysis. "There are only about 200,000 functionally important variants [SNPs] in the Human Genome that have reasonable frequencies", he says. "Nearly all of the genetic contributions to diabetes and heart disease and hypertension and all of the common illnesses are found in those 200,000 elements." Moreover, says Collins, once researchers know which SNPs correlate with higher risk for disease, people with these traits will be able to take extra steps to avoid getting sick. This might allow "medicine to move from its present mode, where we spend most of our resources treating people who are sick, to a preventive strategy, which is individualised", says Collins.

5.3. Develop Better Treatment or Personalised Medicine :

Decoding the 3 billion letter of the Human Genome will without any shadow of doubt revolutionise the treatment in 21st century as much as antibiotic did in mid of 20th century, and even more. Scientists will be able to predict inherited predisposition to various diseases and to be able to design tailor-made drugs. The information that the Genome provide will facilitate the development of personalised medicine, with each patient being treated according to his unique genetic make up. Michael Morgan, the chief executive of the Wellcome Trust’s Genome Campus in Cambridge, said that comparing a patient’s genomic differences with the Human Genome “gold standard” would enable doctors of the future to treat patients as real individuals. (Connor, 2000).

Genes are not only responsible for causing inherited diseases, but also deeply involved in most other diseases if they are not functioning properly. Cancer, Alzheimer’s, arthritis, are few examples developed as a result of specific changes in the activities of genes. Knowing where and when different genes are switched on and off in the Human body would lead the scientists to predict, prevent, design drugs and treat diseases. Technological advances associated with Human Genome project provided the scientists with the tools to discover rapidly which genes are expressed in any given tissue. Haseltine of Human Genome sciences (HGS) (1997) devised a strategy to identify genes of medical importance, which he described as quickest way in helping designing proper personalised medicine. He gave the example of atherosclerosis, in this common condition, a fatty substances called plaque accumulate inside arteries, notably those supplying the heart. Haseltinse’s strategy enabled him to generate a list of genes expressed in normal arteries, along with the measure of level of expression of each one. He can then compare the list with the one derived from the patients with atherosclerosis. The difference between the lists corresponds to the genes (and thus the proteins) involved in the disease. It also indicates how much the genes expression has been increased or decreased by the illness. Researcher can then make the Human proteins specified by those genes. Once a protein can be manufactured in a pure form, scientists can fairly easily fashion a test to detect it in a patient. A test to reveal overproduction of a protein found in plaque might expose early signs of atherosclerosis, when better options exist for treating it. In addition pharmacologists can use pure proteins to help them find new drugs. A chemical that inhibits production of a protein found in plaque might be considered as a drug to treat atherosclerosis. This approach, Haseltine called medical genomics. Thousands of genes were discovered by Haseltine company HGS, 300 have been identified that seemed to be highly likely to be medically important.

Drugs that single out genes expressed only in a few tissues may have fewer side effects than those that target genes expressed throughout the body. Studying changes in gene expression when a new drug is administered can help to explain how that drug works, and in clinical trails it can provide an early indication of whether the drug is effective, or has toxic side effects.

Increasingly, drug treatments will be customised to particular patients. Sometimes a disease can produce very similar symptoms in different patients, although the underlying genetic cause may be completely different. For example, two forms of leukaemia, known as AML and ALL, produce very similar symptoms, but the effective treatments for each are quite different. Using a kind of DNA chip scientists have found 50 genes that are expressed differently in the two forms. As a result, new cases of leukaemia can now be diagnosed accurately and treated appropriately (Bendall, 2001). Loius Staudt of the National Cancer Institute near Washington DC, hopes that knowing which gene are active in particular cell, will enable them to classify tumours more precisely. Staudt and his team used DNA chip to profile different samples of a cancer called diffuse large B-cells lymphoma, and found there are actually two distinct classes of disease, with different genes switched on in each. Cancer cells from the two groups look identical under the microscope, but one set of patients responded well to chemotherapy, while the other did not. Staudt hopes that in the future this technique will routinely guide cancer treatment (Marchant, 2000).

Whether their priorities are curing horrible illness or creating blockbuster products, the first organisations to capitalise on disease-specific gene susceptibilities, and the efficient delivery of chemical screening and clinical development will transform the world of medicine and, along the way, the pharmaceutical industries. As with the rest of scientific history, progress will be based on the changes instituted by few innovative individuals, not based on surveys of past performance based on stagnant paradigms. Commonly held group thinking has made the drug research and development process bigger and more expensive, but not as yet efficient. (Roses, 2001).

5.4. Correcting the Faulty Gene by Gene Therapy :

As early as 1967 and consequent years that lead to new development in the technology of genetic engineering, Marshall Nirenburg, who received Nobel prize for his critical contribution to deciphering the language of the genetic code predicted :

“My guess is that cells will be programmed with synthetic messages within 25 years… Man be able to programme his own cells long before he will be able to assess adequately the long term consequences of such alteration…”.  It has been a long and convoluted path from Nirenberg’s uncannily prescient remarks and the first approved gene therapy clinical trail, almost exactly 25 years later (Lee, 1993).

"Gene therapy" is the use of DNA to correct a genetic defect at the DNA level. Over the years, many scientists have written much about the great promise of this form of treatment, and the ethical issues surrounding it. Despite evidence of measurable success, gene therapy has in recent times attracted increasing scepticism because of its failure to deliver its promise 15 years of intensive research. A recent fatality attributed to an experimental gene therapy protocol has also raised serious concern. 18 year-old victim had a disorder that made his liver unable to break down ammonia, who took part in a trial at the University of Pennsylvania. 17 patients had no side effects, but Gelsinger liver began to fail, soon other organs also failed and his life support was switched off.

Clearly, the day of gene therapy is not here yet, and more focused research will be required to develop, above all, efficient and safer gene-delivery.

5.4.1. Basic Requirements for Gene Therapy

Potentially Gene therapy offers a new treatment paradigm for curing Human disease. Rather than altering the disease phenotype by using agents, which interact with gene products, or are themselves gene products, gene therapy can theoretically modify specific genes resulting in disease cure following a single administration. Initially gene therapy was envisioned for the treatment of genetic disorders, but is currently being studied in a wide range of diseases, including cancer, peripheral vascular disease, arthritis, neurodegenerative disorders and other acquired diseases.

Even though the range of gene therapy strategies is quite diverse, certain key elements are required for a successful gene therapy strategy. The most elementary of these is that the relevant gene must be identified and cloned. Upon completion of the Human Genome Project, gene availability will be unlimited, but until then the starting point for any gene therapy strategy remains gene identification and cloning for relevant genes related to the disease.

Once the gene has been identified and cloned, the next Consideration must be expression. Questions pertaining to the efficiency of gene transfer and gene expression remain at the forefront of gene therapy research. Currently many debates in the field of gene therapy revolves around the transfer of desired genes to appropriate cells, and then obtaining sufficient levels of expression for disease treatment.

Hopefully, future research on gene transfer and tissue-specific gene expression will resolve these issues in the majority of gene therapy protocols. Other important considerations for a gene therapy strategy include a sufficient understanding of the pathogenesis of the targeted disorder, potential side effects of the gene therapy treatment, and understanding of the target cells to receive the gene therapy.

The first approved Human gene therapy experiment was performed in the united state in 22 May 1989. It was used to treat cancer. The main objective of the protocol was two folds. First to demonstrate that the exogenous gene could be safely transferred into a patient and secondly, to demonstrate that the gene could be detected in cells taking back out of the patient. The primary results of this experiment were not encouraging, due to the following reasons: only 35-40% of the patients responded to this protocol. Large-scale tissue culture makes this procedure expensive and clinically difficult. In addition, those patients who do respond will often fail after 6-12 months. (Anderson, 1992).

In September 1990 Anderson and his team pioneered the first successful federally approved gene experiment on Ashanti DeSilva, a four year old girl who became the first patient to undergo such experiment. Ashanti was suffering from a kind of hereditary immunodeficiency syndrome called sever combined immunodeficiency (SCID). This disease resulted from inheritance of a defective gene from each parent. This gene normally synthesises an enzyme called adenosine deaminase (ADA), which is responsible for the proper functioning of the immune system. Without this enzyme the immune system rendered virtually useless, and the affected person has to be kept in an isolated sterile “bubble” environment, otherwise would be left vulnerable to a host of infections, a case usually referred to as “bubble-boy syndrome” (Anderson, 1992).

5.4.2. Methodology of Gene Therapy :

The general outline of Gene Therapy involved the following steps :

1- The healthy copy of the gene is isolated in the laboratory.

2- The gene vehicle, used in this instant is an attenuated virus called retrovirus which have an extraordinary capacity to enter a cell’s Genome and integrated within it’s machinery. Then with the help of recombinant DNA technology few genes removed from the virus to render them harmless to the recepient.

3- The corrected copy of the gene is inserted into the retrovirus, again through Recombinant DNA Technology.

4- The recombinant virus then infects a tissue culture of White blood cells of the immune system, which were removed from the patient. As a result the ADA gene was integrated in the white blood cells. The recombinant white blood cells, then given back to the girls through intravenous infusion. So successful was this experiment so that the corrected gene started to synthesis the ADA, which was lacking and the 4 years old girl was no longer constantly ill. These steps are illustrated in fig (21). This successful experiment opens the door wide for more gene therapy experiment to follow.

In other example, Gene Therapy turn spit into life-saving machine;

Researchers in California turned ordinary Salivary glands into Insulin Pump in rat.

Insulin gene inserted into salivary glands of the rat and make them produce insulin and secrete it in the saliva. They used naked DNA through small tube “catheter” into the saliva duct. The same trick was done with hGH.

5.4.3. Current Status of Gene Therapy

Currently there are at least 150 clinical gene therapy protocols worldwide. Since the approval process for these protocols is not as public outside the U.S., it is difficult to obtain an exact number of worldwide protocols. Of the publicised protocols, 125 are approved in the United States, 48 in Europe and at least 1 each in China and Japan. As of 31 December 1995, 1024 patients had been treated in either a gene transfer or gene therapy protocol. Much controversy exists regarding how many of these have benefited from their gene therapy, and no one has yet been cured (Jeremy-home page:http://www.washington.edu/).

Of the 125 approved protocols, 25 are marker protocols and 100 are therapy protocols. Marker protocols can be distinguished from therapy protocols in that a marker protocol transfers a gene into cells for the sake of identifying those cells.

Therapy protocols, on the other hand, involve the transfer of genes to cells, with the goal that expression of the transferred gene will treat the disease. The majority of the therapy protocols focus on treating acquired diseases such as cancer or HIV. Inherited disorders are the focus of 22 gene therapy protocols, which are aimed at treating 9 different genetic diseases. Three other gene therapies protocols round out the list of 125. These are aimed at treating peripheral vascular disease, rheumatoid arthritis, and arterial restenosis.

Cancer gene therapy protocols employ a wide variety of strategies and can be grouped as follows : in vitro insertion of a cytokine gene into tumor cells; in situ injection of an HLA gene; in situ insertion of a suicide gene into tumor cells; use of tumor suppressor genes or anti-on cogenes; and use of the multidrug resistant gene (Jeremy-home page : http://www.washington.edu/).

Two distinctions need to be made regarding gene therapy :

- Somatic cell therapy : where the target cell is a somatic cell and will die by the death of the person, and is less controversial.

- Germ-line therapy : where the target cell is the sex cell (sperms or eggs) and any genetic alterations will pass through generations. This is more controversial therapy and provokes wide ethical debates.

5.4.4. Current Gene Therapy Vectors :

The majority of these 125 approved protocols employ retroviral vectors (63%) to deliver the selected gene to the target cells. Other widely used Vectors include adenoviral vectors (16%), liposomes (13%) and adenoassociated vectors (2%). The remaining 6% employ a variety of vector systems, the majority of which include injection of naked plasma DNA.