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1 Genetics and the Human Genome Rae _ _ he questions are as oIct as humanity. Why do children resemble their parents? What is responsible for a person's blond hair, green eyes, stocky build? Why do certain diseases, in- clucling psychological diseases, run in families? Before the advent of molecular biology, geneticists approached such questions largely through the study of whole organisms. They brec! plants ant! animals with different traits and observer! how those traits appearec! in offspring. In sexually reproducing organisms, geneticists knew that these traits had to be inherited from something in egg and sperm cells, and towarc! the end of the nineteenth century their suspicions began to focus on the chromosomes long spindly objects seen through the mi- croscope in the nuclei of clividing cells. But for many years the exact nature of chromosomes remainec! a mystery. In the 1940s, 1950s, and 1960s, it became clear that the genetic information in each chromosome is carried in a long strand of cleoxy- ribonucleic acid, or DNA. The order of four simple molecules known as genetic bases along the strand specifies an organisms genes, its basic units of inheritance (see box, pages 6-7~. But only with the development of recombinant DNA in the 1970s could the nature of complex chromosomes be completely unravelecI. The new genetic tech- niques allow researchers to read an organisms genome, the full com- plement of its DNA, with unprecedented facility. For the first time, biologists have potentially unlimited access to the information that dictates the structure and function of all living things. 5

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Recombinant DNA and other new molecular techniques have `'pro- founcilly altered the practice of biology and medicine'" according to Leroy Hood, professor of biology at the California institute of Tech- nolo~v. Not only have they Ha the kindle nit ~til~li-~ that a'= L^;~ J ~ J - --~--~ _ _ _ ~ A ~ ^ ^ ~ A ~ %~ ~ ~ ~ ~ ~ ~ ~ ~ l A ~ ~ C41 ~ JO ~ 1 1 1 ~ 1_ ~ ~ 1 1 . 1 1 1 . . ~ ~ _ ups, DUE money nave greany increased the rate at which studies are being done. New findings in genetics and molecular biology are emerg- ing at an unprecedented] clip, and at least for the foreseeable future the rate of advance is only going to increase. ``T strongly believe that we will learn more about fundamental human biology in the next 20 years than we have in the last 2,000," says Hood. At the same time, new biological knowledge is raising ha host of perplexing ethical, social, and legal questions," Hoot! points out. Soon it will be possible to diagnose a person's susceptibility to many common diseases, including cancer and heart disease, generating thorny issues about how that information should be used. Specific genetic defects will become increasingly cletectable, forcing people to make unprece From Genes to Proteins DNA is the jewel in the crown of mo- lecular genetics. In structure, the DNA molecule resembles a set of railroad tracks twisted lengthwise into a spiral or helix. The rails consist of sugars and phosphate molecules. The ties connecting the rails consist of weakly linked pairs of four chem- icals known as genetic bases: adenine, guanine, thymine, and cytosine. Adenine (A) always pairs with thymine (T), while guanine (G) always pairs with cytosine (C). The double helix structure of DNA is el- egantly simple. Its complexity lies in its length. Within each human cell (except red blood cells, which expel their DNA, and sex cells, which contain only half the nor- mal complement of chromosomes) are about 6 billion base pairs of DNA. If DNA ac- tually were the size of railroad tracks, each cell would contain enough DNA to go around the world over 100 times. Genes are linear sequences of base pairs within DNA. When a gene is being ex- pressed, the DNA zips apart and a ribo 6 SHAPING THE FUTURE nucleic acid (RNA) copy is made of its genetic message (see figure). RNA is very similar to DNA, but it uses a different kind of sugar as a backbone and substitutes the chemical uracil (U) for thymine. Through a variety of metabolic steps, this RNA di- rects the construction of proteins, which consist of chains made up of 20 different amino acids. There is a strict correspon- dence between the sequence of bases on RNA and the order of amino acids in a protein. Therefore, by knowing the se- quence of bases in a gene, it is possible to derive the order of amino acids in a pro- tein, and vice versa (though there are some ambiguities when one is working in re- verse). If DNA is the blueprint for living things, proteins are the bricks and mortar from which living things are built. They give skin and bone its texture, they cause the con- traction of muscles, they carry small mol- ecules through the body, they combat foreign substances that enter the body, they generate and transmit nerve impulses, and they control the growth and differentiation

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dented decisions about how they should live their lives. Techniques are being developed to alter the genetic endowment of a subset of a person's cells to correct inherited diseases. These new capabilities will pose difficult questions for society, questions that reach to the core of what it means to be human. Genetics and Disease `'~et me begin with an assertion''' says Paul Berg, professor of bio- chemistry at Stanford University ant! director of Stanford's Center for Molecular and Genetic Medicine, ``that all human disease is genetic in origin, or, more accurately, that most diseases are the result of inter- actions between our genes and our environment." The cases for which this is most obvious are the diseases involving gross disturbances in the number or arrangement of a person's chromosomes. The classic of cells. Most important, in the form of en- zymes, they control nearly all of the chem- ical reactions that occur in living things, DNA _ ~Genetic Bases ~J / / / GENETICS AND THE HUMAN GENOME ~

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example is Down syndrome, in which an extra copy of one of the smallest human chromosomes causes mental retardation, congenital heart cle- fects, and increased susceptibility to infection. Other diseases of this type arise from a(lditional copies of other chromosomes, missing chro- mosomes, or chromosomes in which the genetic message has become garbled through insertions, (leletions, or other obvious rearrangements. Another category of hereditary diseases is macle up of those diseases associated with a defect in a single gene. These diseases are generally livide(1 into two groups, dominant and recessive, depending on their mode of inheritance. The DNA in human cells is clivided into 46 chromosomes organized into 23 pairs (Figure A-. Each pair consists of a copy of one of the 23 chromosomes in the father's sperm cell and a copy of one of the 23 chromosomes in the mothf~rtc ~ ~11 Ul;~h ~ 1 ~. ~1 =r ~ ~ ~ ~ ~ ~ ..~ ~ ~ 1 ~ 1 1 one exception ot the ~ and Y chromosomes in males, the two members of each pair are very similar. They contain virtually the same genes in virtually the same orcler. But the pairs of genes are not identical. Over 30 percent of the corresponding genes in a chromosome pair (1iffer in some way, reflecting the overall genetic variability of the human pop- ulation. A dominant genetic (lisease occurs when an individual ren~eivec ~ defective gene from either parent. in other words, the presence of a functioning gene on one member of a chromosome pair is not enough to overcome the effects of the defective gene on the other member. An example is familial hypercholesterolemia, in which an inability to clear cholesterol from the blood can cause children to suffer fatal heart attacks as early as 18 months. A person with a dominant genetic disease has a 50-50 chance of passing it on to a chilcl. Recessive genetic defects require that a person inherit defective copies of a gene from both parents. A person who has one defective and one functioning version of such a gene, known as a carrier, suffers little or no ill effects. But each child of two carriers has a one in four chance of inheriting two (defective genes and suffering from the (lisease. Examples of recessive diseases are sickle cell anemia, cystic fibrosis, Duchenne muscular dystrophy, and Tay-Sachs clisease. Over 3,000 single-gene, or monogenic, (diseases have been identified. Although individually rare, together they account for a great clear of human suffering. They affect more than ~ percent of liveborn infants anti cause almost TO percent of deaths among children. The role of genetics is not as clear-cut in m~1ti~enir. I thick ~ _ 1 _ . 1 . . ~ ~ I, ~= ~ 1~lvolvlng Ine interactions ot a number of genes with the environment. Examples of multigenic diseases include "hypertension, schizophrenia, manic (repression, juvenile diabetes' heart (liseases, rheumatoid ar 8 SHAPING THE FUTURE

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thritis, ant] a host of others " according to Berg. Biologists know that these diseases can be inherited, because they can cluster in families. But they clo not yet know the number or identity of most of the genes involves} in these diseases. Cancer should also be cllasse(1 as a genetic disease, Berg argues, even though it is not a hereditary disease. Cancer is causer] by defects in the genetic signals that regulate cell growth ant! reproduction, and human families clearly have genetic predispositions to certain kinds of A C D _ - _. 2 it:: ~ a 6 7 8 : ~ ~&-& ~_ IF 13 14 15 ~ ~5 I. B - _ 4 . - ~- s 9 10 11 12 ~- hi: 16 17 18 F '-' l-'- G i-`-i-l Of At, . 19 20 21 22 X / Y FIGURE 1-1 The DNA in human cells consists of 23 pairs of chromosomes. Twenty two of these pairs, which are numbered roughly in order of descending length, have very similar members, reflecting the equal genetic contributions of mother and father. In addition, each cell carries either two X chromosomes (in the case of a female) or an X and a Y chromosome I in the case of a male). Chromosome photographs such as the one shown here, known as karyotypes, are produced by arresting a cell during division (the chromosomes have doubled but not split apart), staining the chromosomes, and arranging photographs of the chromosomes by size. Karyotype courtesy of Dr. Patricia N. Howard-Peebles, Genetics & IVF Institute, Fairfax, Virginia. GENETICS AND THE HUMAN GENOME 9

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tumors. Also, genetic defects in the human immune system, in DNA repair systems, and in the ability to metabolize carcinogens are asso- ciatec] with a higher frequency of certain tumors. Even infectious diseases, according to Berg' can be seen as genetic diseases. But in this case' the genes of the infecting organism and their relationship with the host ant] with the environment determine the course of the disease. Tracking Disease to Its Source Molecular biology has already clemonstrated the amazing precision it can offer in analyzing genetic diseases. Research has shown, for instance, that sickle cell disease, which is characterized by anemia, impaired growth, and increaser] susceptibility to infection, is caused by a change in a single genetic base. This change lea(ls to the substi- tution of one amino acid for another in hemoglobin, the protein that carries oxygen in the blood, causing the molecule to misfunction when oxygen is scarce. Another example is phenylketonuria (PKU), which can cause severe mental retardation if uncliagnosed, it is caused by a efect in the gene co(ling for an enzyme that converts one amino acid into another. ``Every few months another gene is isolated and the struc- tural defect is identified," says Berg. "It's not too optimistic, ~ think, to predict that the genes and their (lefects for many of the monogenic diseases will be known within the next five years." The impressive accomplishments of the past emphasize how much remains to be learned, however. Of the over 3,000 monogenic (diseases now recognized, the responsible gene has been identified in only about 100 cases. The genes involved in multigenic diseases and disease susceptibilities remain largely unknown. The ideal situation would be to know the gene or genes involved in every human disease, their location on the chromosomes, the nature of the defect associated with the disease, and the way in which the defect contributes to the disease. This information is known for very few diseases, and attaining it for the majority of diseases will take many decades. But biologists are starting to systematically pursue some of the early stages of such a program. They are constructing maps of the human chromosomes that give the locations of known genes. By mapping these locations, researchers can clevelop genetic probes to (letermine whether a person has a normal or a defective gene, leading to great advances in the diagnosis and treatment of disease. Genetic maps can also reveal patterns in the way genes are organized and regulate(l, 10 SHAPING THE FUTURE

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leading to a greater understanding of how genes function in health and disease. The scientific community has also been considering a much more ambitious proposal: a plan to determine the exact sequence of the billions of genetic bases making up the human genome. This information would be an invaluable resource for biologists. It would allow them to identify all of the genes within a given region of a chromosome, including many currently unknown genes. Genetic differences among inclivicluals could be compared, revealing a great deal about the functioning of normal and defective genes. The mechanisms that control the expression of genes and the processes involved in development could be deci- pherecI. The structure and organization of genes in different species could be contrasted, leacling to insights into the evolutionary processes that resulted] in those species. The complete sequence would not answer every question in biology. it would not establish exactly how genes are controller! or how gene products function within a cell or organism. It would not completely ~rnl~in how n'?onle are different or how humans have evolved. But it is unlikely that these questions can be answered without a creep un- clerstanding of the human genome. AN ~ ~141 1~J ~ r~r ~ At, ~ __ Putting Genes on a Map Geneticists were mapping genes to chromosomes well before they knew how chromosomes are constructed. The first genetic map was made in 1913, for five traits carried on the X chromosome of the fruit fly Drosophila melar~ogaster. Today, maps of human chromosomes are being made using the same principles. The method used to make such maps involves analyzing how traits are passed down from generation to generation. in a parent's repro- ductive system, each chromosome pair separates during the formation of egg and sperm cells, reducing the original 46 chromosomes to 23. If this were all that occurred during reproduction, inheritance would be a straightforward matter, chromosomes would remain intact and be passed unchanged! between generations. But chromosomes do not re- main intact. Rather, before the formation of egg and sperm cells, each chromosome pair can exchange parts through a process known as cross ~ng-over. Imagine three different genes located on a pair of chromosomes (Figure T-2~. Each chromosome can have different versions of these genes, which interact according to the usual rules for dominant ant! . GENETICS AND THE HUMAN GENOME 11

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Without Crossing-Over ~ -~/~J'-~: Chromosome Each Chromosome \ Pairs Come Duplicates, Remaining \ Together in Linked at Centromere Cell \~_ a ~ Reproductive Divides l Cells Once _ b I Cells Divide Again to Yield Four Sperm Cells or Egg Cells With Crossing-Over ~ o ~ B\e bib ~/ ~J `1 Chromosome Pairs Come Together in Reproductive Cells Duplicated Chromosomes Exchange Pieces /~ Cell Divides Once 1~] 1 'A \ Cells Divide Again to Yield Four Sperm Cells or Egg Cells FIGURE 1-2 During the formation of egg and sperm cells, a process known as crossing-over can unlink versions of genes located on the same chromosome. In the above diagram, A and a are versions of a specific gene located on a chromosome pair, as are B and b and C and c. During crossing-over, A becomes unlinked from B and C in two of the four sperm or egg cells produced. If the resulting cells produce offspring, the genetic linkage will differ between parent and child. This information can be used to deduce the relative distance between the different genes. 12 SHAPING THE FUTURE

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recessive genes. For instance' gene B in Figure T-2 may be for brown eyes a dominant trait while gene b is for blue eyes. Tf crossing-over ant] other forms of genetic recombination never oc- curred, the versions of genes located on a given chromosome wouIc! always be inherited! together. In genetic terms, they would be perma- nently linked (top half of Figure Tub. But crossing-over can reshuffle the genes on a chromosome pair, resulting in new genetic combinations. The trick in genetic mapping is to observe how often this process separates the versions of genes on a chromosome. As shown in the ~ ~ ~ r r ~ ~ ~ bottom half of Figure T-2, genes that are close together tend to stay together, whereas distant genes are easier to separate. By noting the frequency of separation, researchers can calculate the distance between genes and assign them relative locations on a chromosome. Another way to map genes draws on the appearance of chromosomes under a microscope. If the chromosomes are stainer} during cell division with certain chemicals, alternating bands of light and `dark regions appear, with up to several (dozen bands on a single chromosome (Figure T-3~. These cytogenetic bancis distinguish the chromosomes and provide broad lanclmarks along their length. But they are still quite large' with each band containing an average of 100 genes. - .~ .~ - : - Neuroblastoma | ~ Gaucher Disease, Type 1 FIGURE 1-3 Several dozen disease-causing genes have been mapped to human chromosome 1. The gene responsible for Gaucher disease, type 1, a recessive disease characterized by an enlarged spleen, skin pigmentation, and bone lesions, has been mapped to a section of the q arm of the chromosome. One or more genes that control the spread of tumors in neuroblastoma, a cancer of nerve cells, have been localized to part of the chromosome's p arm. Karyotype of chromosome 1 courtesy of Dr. Patricia N. Howard-Peebles, Genetics & IVF Institute, Fairfax, Virginia. GENETICS AND THE HUMAN GENOME 13

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Some people with genetic disorders have recognizable abnormalities in their cytogenetic banding patterns. For instance, the gene for Duch- enne muscular (lystrophy was mapped to a specific region of the X chromosome by noting that some sufferers of the disease were missing that portion of the chromosome. TransIocations, in which part of one chromosome has broken away and become attacher] to another chro- mosome, and fragile sites that are susceptible to breakage have also pointer] to the locations of specific genes. If the base sequence of the gene being mapped is known, it can be located on a chromosome by making radioactive copies of part or all of the gene (by synthesizing or cloning the gene using radioactive constituents). These DNA probes can then be mixed with human chro- mosomes under chemical conditions that cause the DNA strands to temporarily separate. When a single-stran~le<1 probe encounters a matching single strand of DNA on a chromosome, the two combine or hybridize to produce double-stranclecl DNA. The radioactivity given off by the probe then functions as a marker to find the chromosome and the approximate location of the gene. Another method of locating genes on chromosomes involves an in- triguing technique known as somatic cell hybridization. When human cells and mouse tumor cells are grown together under the proper con- clitions, they tend to fuse and form hybrid cells. As these hybrid ceils grow and divide, they lose most of their human chromosomes. But often one or a few human chromosomes will become stably established in a particular cell. The result is a mouse tumor cell line containing specific human chromosomes. By looking for human proteins pro(luced by a given gene, that gene can be assigned to a chromosome carried in a cell line. Genes with known sequences can also be located using nNA hybridization. All of these techniques can establish the relative or approximate location of a gene on a chromosome. But to map a gene to an exact chromosomal location requires the techniques of recombinant DNA. ~_ . A Genetic Scalpel The ability of biologists to recombine DNA at will originated in the discovery of enzymes in bacteria that can cut DNA at specific sequences of four to ten genetic bases. These so-called restriction enzymes, which bacteria evolved to fight invasions of foreign DNA, were a marvelous gift to biologists. They allow researchers to slice DNA at specific lo- cations, so that [Large DNA molecules can be cut into smaller, more 14 SHAPING THE FUTURE

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manageable pieces. Then, using a class of enzymes known as ligases, biologists could splice together any two pieces of DNA formed with the same restriction enzymes. In this way, they couIcI create mosaics of DNA, known as chimeras, that have never before existed in nature. (In Greek mythology, the chimera is an animal with the head of a lion, the body of a goat, and a serpent for a tail.) Recombinant DNA has transformed the mapping of the human gen- ome. It has enabled researchers to cut complex genomes into pieces' each of which can then be cloned many times over. These pieces can be organized into a library, creating a complete collection of all the DNA in an organism. Indiviclual volumes in the library can be further analyzecl to map specific genes or pieces of DNA. The logical conclusion of this process is the next step beyond mapping: the sequencing of genetic bases in part or all of an organism's genome. One of the products of recombinant DNA technology has been a family of genetic markers much more precise and powerful than the banding patterns on chromosomes. Human beings are much more alike genetically than they are unalike. Only about one in every hundred base pairs of DNA are different between any two people. Many of these differences have no effect on the functioning of the genes, but others contribute to the crucial differences that make us unique: differences in physical appearance, aspects of personality susceptibility to disease. Without these genetic differences, people would all be as alike as identical twins. Using recombinant DNA, genetic differences among individuals can also serve as markers along human chromosomes. First DNA from a person's cells is cut into pieces using a specific restriction enzyme, and the resulting pieces are placed along the edge of a gel. Under ~ ~ , ~ ~ ~ ~ LIT 1 ~ 1 ~_ ~. ~ normal ColldltlOnS' L'1NIA has a sllgntly negative electric charge, so when an electrical potential is applied across the gel the DNAis attracted to the positive charge on the other side of the gel. The smaller pieces of DNA move faster through the gel than do the larger ones. When the DNA pieces are spread across the gel, this process, known as electro- phoresis, is stopped. The result is a virtually continuous series of bands, with each bane] corresponding to a DNA fragment of a different length. ~1 .] ~ ~ 1 ~ . ~cat - - - .r. ~ C~7 Researchers can then highlight specific bancts using radioactive DNA probes that combine with given DNA sequences. If two individuals have a difference, known as a polymorphism, in the region of DNA being tested, that difference can cause the restriction enzyme to produce DNA fragments of different lengths; For instance ~ - . . say a person has a difference in a sequence recognized by a restriction enzyme, causing the sequence to remain uncut (Figure T-4~. If so, the GENETICS AND THE HUMAN GENOME 15

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RFLPs and Disease Diagnosis The implications for genetics of RF~Ps are "extraordinary," accord- ing to Berg. Geneticists have aIreacly located hundreds of RF[Ps scat- tered throughout the human genome. At first, a RAP may be used as a flag to indicate the presence of a clisease-causing gene closely linked to the REAP. Later, RF~Ps may be used to track clown the actual location of that gene, so that the nature of its defect can be cletermined. The use of RF~Ps ant! other genetic probes to diagnose genetic iseases "will change medicine in a very profound way," according to Hood. For instance, it will eventually be possible to detect the genes responsible for virtually every monogenic ~lisor(ler. These genes could be detected after birth or prenatally, giving parents an option to ter- minate a pregnancy. Carriers of defective genes conic! also be identifiecI, allowing them to decide whether to have children and risk passing on the disease. The identification of carriers anc! prenatal testing conic] greatly reduce the toll that many monogenic diseases take on the human population, Berg points out. An example is Tay-Sachs disease, a recessive disease occurring most frequently among Jews of European crescent that causes retardation, paralysis, and early death. A test for Tay-Sachs disease that can identify carriers and affecter! fetuses has been available for a number of years. This test "has virtually eliminated Tay-Sachs disease from the Jewish population," Berg says. As scientists learn more about the role of genes in multigenic clis- eases, it will increasingly be possible to (letermine an inclividual's susceptibility to such diseases. In time, diagnostic tests shouIc} be available to determine a person's susceptibility to such common diseases as cancer, heart disease' ant! diabetes. Such tests coul(1 provide "a new opportunity for preventive medicine," says Hood. Now, genetic testing is confiner! largely to fetuses and expecting parents. But in the future it will be possible to diagnose the disease susceptibilities of anyone ant! enter that information into a medical record. That information could help a person to avoid certain habits, cliets, or environmental conditions that might leac! to the disease. New therapeutics might also be clevelopec! that could tower the chance of contracting a disease when a susceptibility exists. "We can start to think about changing some fundamental aspects of human health care," says Hood. As an interesting siclelight, RF~Ps and other DNA probes have also assumed a growing role in forensic medicine. The patterns in a person's DNA are just as distinctive as a fingerprint, and in some crimes DNA GENETICS AND THE HUMAN GENOME 17

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samples are easier to find than fingerprints. Already, genetic tests have been used to identify rape suspects, murcler suspects, and the parents of children applying for immigration. ``Very soon we will be in a position to translate genetic patterns into electronic databases and compare particular DNA fingerprints with any others that come up'" says Hood. ``This raises challenging social and ethical questions." The Price of Knowledge The social and ethical questions posed by genetic analysis extend far beyond DNA fingerprinting. As testing for diseases and disease susceptibilities increases, it will be possible for people to learn a great deal about themselves and their futures. if it were possible for a person to know what he or she was most likely to die of, would that person want to know? Today, genetic testing is often done as the prelude to some therapeutic intervention, from the abortion of a severely affected fetus to dietary or pharmacological interventions. But treatment may not be possible for some of the diseases that will be detectable in the future, at least until researchers apply new understandings of genetic information to the development of new therapies. A stark example of the dilemma posed by the gap between diagnosis and treatment is Huntington's disease, which threatens about 125,000 people in the United States. The disease is caused by a dominant gene inherited from either the mother or the father, therefore, a person with an affected parent has a SO-SO chance of also having the disease. The symptoms of the disease, beginning with loss of control over movement and proceeding to dementia and eventual death, generally do not appear until after the childbearing years. As with many monogenic diseases, there is no treatment for Huntington's disease. Through extensive analyses of RFI~Ps from families afflicted by Hun- tington's disease, geneticists have narrowed the location of the Hun- tington's gene to a million-base-pair region at the end of chromosome 4. By studying the RF~Ps of individual families, researchers can usually find markers that signal the presence of the defective gene with a high degree of accuracy. The question then becomes, Will a person at risk of Huntington's disease want to know whether he or she carries the gene for the disease? if a person is trying to decide whether to have a child and risk passing the gene on to the next generation, the information Is clearly useful. But if a person is past the childbearing years or does ~~~ ~ undergo a genetic test that has the not want to have children, why ~ v potential to predict future illness and early death? 18 SHAPING THE FUTURE

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. AS genetic testing becomes more powerful, similar dilemmas will emerge. The ability to test for the presence of a gene is an inevitable step in the process of unclerstanding a disease and developing ways to treat it. But some diseases will always remain untreatable, and for r 1 . ~ others the ability to diagnose a disease is imminent whereas the treat- ment for it is much less certain. Information from genetic testing will also raise a number of conten- tious social questions. Should employers or insurance companies have access to such information, even though in some cases it could lead to loss of a job or loss of insurance coverage? What kinds of traits or conditions should prospective parents be able to test for in deciding whether to have chilciren or abort a pregnancy? Guidelines will have to be established for the use of such information to preserve individual confidentiality and autonomy. Increaser] genetic testing will also require that people understand the information they are being given. An increased susceptibility to a disease does not mean that a person will inevitably get that disease. In fact, the chance of getting the (disease may be quite small. Yet such a diagnosis couIcl greatly increase a person's anxiety and affect important ~ . declslons. Even in cases in which a diagnosis is more certain, the potential for misinterpretation will be large. People who have a disease caused by a single gene can show enormous variations in the manifestations an severity of the (lisease. This variation is so great, says Berg, '`that some physicians who see these patients cannot accept that they're all causer] by the same genetic defect." In part, says Berg, these differences are caused by a person's broac! genetic background, which probably will not enter into the cliagnosis. They are also caused by the environmental influences a person experiences, which can be endlessly variable. Peo- ple will therefore have to understand the full range of outcomes that could accompany a specific (liagnosis. These are not new issues in human genetics. Similar situations have already been addressed by researchers, clinicians, patients, and pol- icymakers. But the issues will become more numerous and wiclespread as biologists learn more about the connection between a person's genetic heritage and disease. From Mapping to Sequencing From a purely scientific standpoint, biologists generally agree that a program to map the human genome through the construction of a large GENETICS AND THE HUMAN GENOME 19

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library of RF[Ps and other genetic markers is a worthwhile goal. But the new techniques of molecular biology allow more to be done. Within a few years it should be possible to begin systematically sequencing the 3 billion base pairs in the human genome. Tt would be the largest project ever undertaken in biology' with characteristics much different than those of normal biological research. There is much less consensus among biologists about how to undertake such a program. One thing is clear' however: it will be a formiciable task. Apart from the technical difficulties involved in sequencing' the project will pro- cluce a tremendous amount of information to store, analyze, and dis- seminate. There are about 3 billion bases that would neec! to be sequenced in the human genome (technically, only one member of each chromo- some pair needs to be sequenced, since the members of a pair are so similar) if every letter in this book represented one of these bases, it would take roughly 10,000 of these books to represent the entire se- quence. Furthermore, sequencing programs will leave to acquire se- quences from different individuals and from other species to make the best use of the human sequence. So far, only about 2 million base pairs of the human genome have been sequenced ant] storer! in a central data Building a Better Sequencer The standard laboratory method used to sequence DNA involves labeling fragments of the unknown DNA with a radioactive marker and passing the fragments through four different electrophoretic gels, one for each kind of base. Once the fragments have been separated on the gels, it is possible to read the base sequence from the order in which the fragments appear. The problem with this method is that it is tedious and expensive, requiring skilled scientists and the use of hazardous chem- icals and unstable radiosotopes. If se- quencing the entire human genome is to be practical, sequencing methods must be- come more efficient. One way to increase efficiency has been developed by Leroy Hood and his col- leagues at the California Institute of Tech- nology and Applied Systems, Inc. Instead 20 SHAPING THE FUTURE of labeling each DNA fragment with the same radioactive marker, the fragments are labeled with four distinct flourescent mark- ers, one for each kind of base. This makes it possible to run all of the fragments through a single electrophoretic gel. As fragments of different size move down the gel, a laser causes the passing bands of DNA to flu- oresce, and the emitted light is recorded by a computer (see figure). Since each flou- rescent marker produces a different color, the computer can read the DNA sequence from the passing DNA bands. This technique offers a number of ad- vantages over the conventional sequencing method. For one thing, it reduces the er- rors inherent in using four different gels. Also, because the sequencing is auto- mated, the process proceeds much more quickly. Commercial versions of the device can sequence about 6,000 bases per day, or approximately 1 million bases per year.

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base' less than 0.T percent of the total. Without a special effort to sequence the human genome' it will not be (lone for many years, if ever. There are several broad objections to a sequencing program that must be answered for the project to proceed' according to Hood. The first asks whether obtaining the full sequence would in fact be scientifically uninteresting ant] therefore a misallocation of resources. Hood agrees that much of the mapping and sequencing would be routine, repetitive work. Icleally' he says' much of it can be automated (see box' pages 20-211. It is the sequence itself that will generate lithe staggering anc] exciting kinds of scientific endeavors." Also, Hood questions whether the United States can afford] not to undertake a sequencing program. "I would argue that the impetus proviclec! by this program is going to have a major impact on whether the United States stays competitive in the inclustrial biotechnology area." A second objection is that such a project will draw funds from other areas of biology, and HoocI finds this to be "a much more serious objection.'' But the technologies and information developed through a sequencing program will find applications throughout biology, Hood Gel DNA Fragments Electrophorese Down the Gel Laser Beam ~/ Scanning Laser Excites Fluorescent Dye ( Filters Wheel Computer By using four different fluorescent markers Final Output to label the DNA fragments, the fragments can be run on a single gel and detected by a laser beam linked to a computer. GENETICS AND THE HUMAN GENOME 21

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points out. He also believes that the project will be "sufficiently com- pelling" to generate new sources of funding for itself. Several committees have studied sequencing proposals and have conclucled that funding in the neighborhood of $200 million per year would be appropriate for such a program. While this is a substantial amount of money' it is only about 3 percent of the total amount of funds spent by the federal government on biological research each year. A third objection is that the project will be "big science" anc] therefore at ocIds with the traditional approach to biological research, in which most work is done by small, indepenclent groups of scientists. But Hood contends that "it is not big science in the same sense that projects such as the superconducting supercollider and the space shuttle are big science." The costs of the instruments required are modest, and the instruments can be widely disseminated. Research proposals for aspects of mapping and sequencing will be peer reviewed, and this research will also be widely distributed. Furthermore, once the information is available, it will greatly increase the power and range of what small groups can do, and they will no longer need to spend great amounts of time on routine mapping and sequencing. The final objection is that the technology is not yet adequate to Lo the job, and this is the one objection that Hood finds convincing. As currently performed, sequencing is tedious, time-consuming, labor- intensive, and expensive, according to Hood. '`Technolo~v development ; ~Al ~11_ ~1~. 1 ~1 ~ all ~ ~-~4 ~ 1;~ Wlla'L WE Wholly ~llQUlU co~luenlrale on, ne says. "This is an area of major deficiency at this time." Sequencing the human genome will involve building up a library of overlapping pieces of the genome, which can be stored in a central location, easily reproduced, and sent to investigators to be sequenced and studied. This is now relatively easy to do with small pieces of DNA. They can be cut with restriction enzymes, inserted into pieces of bacterial or viral DNA known as vectors, and maintained in culture as clones. The problem is one of scale. Conventional vectors can hold a maximum of about 40,000 bases, meaning that with an average overlap of 10,000 bases, it would take over 100,000 different clones to store the entire human genome. Maintaining a culture library of this size would require an administrative structure much larger than any in existence today. Furthermore, some parts of the human genome are difficult to clone, and there are concerns about the stability of DNA in a culture library. Progress is being macie on many of these problems. Researchers are developing new vectors in yeast that may be able to carry 500,000 to ~ million DNA bases, over ten times the size of the fragments that can 22 SHAPING THE FUTURE

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be maintained with current systems. Instead of needing over 100,000 clones to encompass the entire human genome, it could be done with several thousand. Restriction enzymes have recently been discovered that cut DNA into very large pieces, and a new method of electrophoresis ~ 1 1 . r. ~ 1 using Pusey electric Feces can separate DNA fragments 200 times larger than the maximum possible with conventional electrophoresis. These are the kinds of technological developments that will be necessary to make sequencing the human genome economically and scientifically feasible. (The box on pages 24-25 discusses several other technologies that will be essential to sequencing efforts.) Hood advocates that en effort to sequence the human genome proceed In stages. During the first stage, new technologies would be developed to increase the efficiency of DNA sequencing five- to ten-fold. At the same ume, detailed mapping of the human genome could be under way, which would provide a framework for the sequencing effort. A phased approach would also allow systems to be developed to collect and disseminate DNA clones and to store and analyze the huge amounts of data that will be generated. Once this technological infrastructure is in place, Hood says, the complete sequencing of the genome could begin. . . Prospects for Human Gene Therapy The knowledge provided by mapping and sequencing the human genome may make it possible to achieve one of the most provocative of the new biotechnologies: human gene therapy. Interest in human gene therapy arises from a depressing fact: for the majority of monogenic diseases, no effective therapies are available. For some single-gene defects, dietary restrictions, the use of drugs or biologic agents, or transplantation of tissues or organs may alleviate part or all of the symptoms. But for many such genetic defects there is no alternative to "debilitating and progressive disease leading to suffering and early death," according to Berg. Any disease to be treated with human gene therapy must meet a number of "very formidable" criteria, Berg says. The gene responsible for the disease and the molecular nature of its defect must be known. The disease must involve cell types that are accessible and well-char- acterized. The disease cannot begin to exert its harmful effects until after birth, since a variety of technical and ethical constraints prohibit gene therapy before birth. Also, the defect must be limited to a single gene. `'We certainly enter the realm of wishful thinking when the therapy GENETICS AND THE HUMAN GENOME 23

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aims to moclify more than one gene or to rectify the defects resulting from chromosomal abnormalities," notes Berg. The gene therapy being considered for humans involves placing nor- mal genes into somatic, or bocly, cells, not into germ line, or sex, cells. In this way, `'the therapy stays with the treater} patient and is not transmitted to offspring," Berg says, Also any undesirable effects of the integration are not going to be propagated to f~,t~re ~en~r~tion~ " ~ . ~ . 1 . 1 1 1 Liven tne criteria tnat canct~ciate creases must meet, only a handful have received serious consideration. The most attention has focused on a rare disease caused by defects in the enzymes necessary for normal immune system development. ChilcIren with the disease, known as severe combiner! immunodeficiency disease, must live in totally sterile Protein Sequencers and Synthesizers Recombinant DNA technology is not the only driving force behind the rapid ad- vances now occurring in genetics. Another powerful influence has been the develop- ment of microchemical instruments that can analyze and synthesize genes and proteins with remarkable proficiency. One such instrument is known as a pro- tein sequenator. Essentially, this device is a chemical scissors that can clip off one amino acid at a time from the end of a protein and determine its identity. Since the development of the first sequenator in 1967, the amount of a protein needed for sequencing has steadily declined. Today, biologists are on the verge of being able to sequence the proteins purified by the most sensitive technique now available two- dimensional electrophoresis. This tech- nique separates complex protein mixtures in one dimension by size and in the other dimension by charge (see figure). Within a few years, it should be possible to sequence virtually every protein that appears in a two-dimensional gel. The result will be a dramatic increase in the number of pro- teins that can be analyzed. Once the sequence of amino acids in a protein is known, a synthetic gene can be made for the protein by converting the pro- tein's amino acid sequence into the corre- sponding base sequence. DNA synthesizers can now construct DNA fragments up to 200 bases long, and these fragments can be joined together to make longer sequences. A synthetic gene or part of a gene can be hybridized with chromosomal DNA to lo- cate the original gene for the protein. A synthetic gene or the cloned original gene can also be introduced into bacteria or other hosts to manufacture large quantities of the protein for research or therapeutic uses. Biologists are also using microchemical instrumentation to explore one of the most fundamental issues in biology: how the se- quence of amino acids in a protein deter- mines its structure and hence its function. Using DNA synthesizers, researchers are making synthetic genes that are slightly different from the original genes, resulting in proteins with different amino acids in particular locations. By observing how these modified proteins fold and operate, re- searchers hope to uncover the general prin- ciples that govern the structure and function of proteins. Researchers can also directly synthesize protein units over 100 amino acids long to examine which amino acids 24 SHAPING THE FUTURE

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``bubbles ''7 since their immune systems cannot protect them from com- mon viruses' bacteria' and fungi. Researchers have developed an experimental procedure in mice that could be applier! to humans if it proves effective and safe (Figure T-5~. First' bone marrow ceils are removed from the animals and mixed with an infectious agent known as a retrovirus. When retroviruses infect a cell, they insert a copy of their genetic information into the genome of the host. By genetically engineering the retroviruses to contain a normal version of the defective gene' scientists can insert the normal gene into the bone marrow cells. The ceils are then reimplanted into the mice where' pre- sumably, they will produce the missing gene product. Unfortunately' says Berg7 the results to date Shave been rather dis are important for particular functions. In time, such information may make it pos- sible to construct proteins with new and useful properties, inaugurating an era of protein engineering that is likely to have an even greater impact on science and medicine than genetic engineering has had. MOLECULAR CHARGE Two-dimensional electrophoresis can sepa- rate thousands of proteins extracted from a few hundred cells. Each dot in this separation indicates the presence of a single kind of pro- tein. Photograph courtesy of Leroy Hood. GENETICS AND THE HUMAN GENOME 25

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Retrovirus ^;W Hu man Cel I with Functional Gene (: DNA bye Nuclei DNA Equivalent of Retroviral ~ RNA with Ma for Genes Deleted DNA Segment Containi ng functional Gene Recombinant Retrovirus - Bone Marrow Cell with Defective Gene Bone Marrow Cell Containing Functional Gene FIGURE 1-5 Much of the research on human gene therapy has focused on re- troviruses, infectious agents that can insert their own genetic material into the DNA of cells they infect. The genome of a retrovirus consists of RNA, which is enzymatically copied into DNA when the virus invades the cell. In the scenario shown above, involving bone marrow cells with a defective gene, the major genes in a DNA copy of the retroviral RNA can be deleted and replaced with a functional version of the defective gene, along with the appropriate regulatory signals to ensure the expression of the gene. Once the recombinant molecule has been reconverted to RNA, the bioengineered retroviruses can be used to infect bone marrow cells withdrawn from an organism with the defective gene. The retroviruses insert the functional gene into a random location in the cells' DNA, and the transformed cells are reimplanted into the organism. 26 SHAPING THE FUTURE

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couraging and may prompt others to begin looking at other potential vectors." The genes can be inserted into mouse bone marrow cells and macle to produce the gene product, but only for a limited time. `'For reasons that are totally mystifying at the moment, the gene that has been introducec] by the virus is turned off over some varying perioc! of time. Although the animal has acquired the foreign DNA, it no longer expresses the gene of interest." Much effort has gone into trying to maintain the expression of the introduced gene, but without success. "My feeling is that this probably reflects our lack of knowledge about the development of the cell," says Berg. Human gene therapy has other potential problems that are only be- ginning to be addressed, notes Berg. One is that the retrovirus inserts its genetic message into the cell's DNA at random and uncontrollable locations. It is possible that the foreign DNA would integrate into the micIdIe of a gene essential for the survival of the cell, which wouIc] destroy it. Or the gene could] insert itself in such a way that it increases the activity of a gene regulating cellular growth, leading to tumors. `'The inability to control the site at which the vector DNA integrates is one of the major handicaps at the moment in this approach,'' Berg points out. As a result, he and his coworkers are examining ways to target the introduced DNA to specific locations. Perhaps vectors can be developed that would recognize sequences within the cell's genome and integrate its DNA in a predictable way around that sequence. Even more desirable would be some sort of agent that homes in on the defective gene ant] somehow repairs it in place. But knowing whether any of these approaches are feasible will require a much more sophis- ticatec! understanding of the workings of the human genome. GENETICS AND THE HUMAN GENOME 27