Genes And The Human Genome Project

Anthony Perszyk, M.D.
Anthony Perszyk, M.D. is a Medical Geneticist at Nemours Children's Clinic in Jacksonville.

The half way point in the 15 year Human Genome project was passed in April 1998. The first half has shown good progress, in that about 30,000 genes have now been identified. This is less than half of the 60 to 80,000 genes predicted to compose the human genome. The first half has also yielded physical mapping of markers or sign-posts throughout the genome. This will allow more rapid progress during the second half. The first half has also seen great strides in methodologies, biological informatics, organization and collection of data, and major technical enhancements that should allow completion to be on time and perhaps under budget unless surprises are encountered along the way. So where do we stand?

The following covers some of the new discoveries, names of techniques, and treatments proposed. The basic terminology will not be reviewed here.

THE HUMAN GENOME PROJECT

What can be said about the Human Genome Project?

Rather then begin this article extolling the possibilities of what genetics might do, or what the genetic revolution in medicine will bring, or go on and on about the future of genes and DNA, let's take inventory of what has happened since the last genetics issue was published. Here are some basic practical questions one might ask about the Human Genome Project.

What has been spent?

There are 18 nations involved in the consortium of scientists and institutions of the Human Genome Project (HGP). Since October 1990, formal sequencing under the HGP began. Prior to this date about 5000 genes had been found and sequenced. Additional studies to confirm these and to begin mapping the remainder of human, mouse, several bacteria, and nematodes were undertaken. Included in the list of nations are Canada, Russia, Sweden, Denmark, Netherlands, France, Germany, Italy, Japan, Australia, Brazil, Mexico, United Kingdom, and the United States of America. The Department of Energy (DOE) and the National Institutes of Health (NIH) have together spent $300 million dollars through the end of 1998. This is estimated to be 20% of the total cost. On a per gene basis this works out to be 50 cents per base. The average cost per gene would be $2000 dollars.

What institutes are involved in the sequencing of the human genome?

As of November 1998 over 200,000,000 bases out of an estimated 3,000,000,000 bases have been sequenced. These institutions and universities have been the major contributors. The Sanger Centre, Whitehead Institute for Biomedical Research / MIT Center for Genome Research, Lawrence Livermore National Laboratory Human Genome Center, Stanford Human Genome Center, The Institute for Genomic Research, Washington University Genome Sequencing Center, German Consortium (Institute of Molecular Biotechnology, Max Planck Institute for Molecular biology, and GBF), University of Tokyo, Keio University, University of Washington in Seattle, Los Alamos National Laboratory, University of Oklahoma, Baylor College of Medicine Human Genome Sequencing Center. Each of these centers have websites that give a general overview of their activities, who's involved and their plans.

What organism have been fully sequenced?

All of these genomes (the entire genetic information of an organism) have been fully sequenced: Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilus, Synechocystis sp., Archaeoglobus fulgidus, Pyrobaculum aerophilum, Haemophilus influenzae, Methanobacterium thermoautotrophicum, Helicobacter pylori, Methanococcus jannaschii, Aquifex aolicus, Borrelia burgdorferi, Treponema pallidum, Mycoplasma pneumoniae, and Mycoplasma genitalium. Which is your favorite pathogen?

What organisms are nearing completion?

Several organism that are nearing completion are: Arabidopsis thaliana, Caenorhabditis briggsae, Caenor-habditis elegans, Drosophila melanogaster (<50%), Mycobacterium tuberculosis, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. The genomes being chosen include disease pathogens and lab animals to allow potential treatment of certain infectious diseases, as well as to gain behavior and clinical correlations and insights.

What disease genes have been identified and sequenced?

About 7000 disease genes in humans have been characterized as of November 1998. Some of these are included in Table 1. The known sequence will help further our understanding of these diseases. The diseases named here do not mean that commercial labs are available for routine testing. For many diseases the sequencing of the gene and its correlation with a human disorder is the first step. However, much work is needed to understand what the gene does and how mutations effect the disease state. For many conditions this is a monumental task that still lies ahead. The sequenced genes are being compiled and are available without copyright restriction. When complete, the GenBank database will be free to use and the human genome will not be owned by any one person or institution.

Table 1. Identifiable Disease Genes
Adrenoleukodystrophy Apolipoprotein E Ataxia telangiectasia Burkitt lymphoma Cystic fibrosis DiGeorge syndrome Diastrophic dysplasia Duchenne muscular dystrophy Ellis-van Creveld syndrome Gaucher disease Gyrate atrophy of the choroid and retina Huntington disease Juvenile onset diabetes Long QT syndrome Marfan syndrome Menkes syndrome	Multiple endocrine neoplasia Myotonic dystrophy Neurofibromatosis, type 2 Phenylketonuria Polycystic kidney disease Retinoblastoma, type 1 Severe combined immunodeficiency Spinocerebellar atrophy Testis-determining factor Tuberous sclerosis Von Hippel-Lindau syndrome Waardenberg syndrome Werner syndrome Wilson disease X-linked mental retardation, Fragile X syndrome Zellweger syndrome

What new lab techniques have been developed?

Sequence tagged sites (STS) have been found throughout the genome. A STS is composed of two short synthetic sequences. These derive from a known regional sequence. The STS can act as a PCR primer for that site. That is to say that the length of DNA can be checked to see if it is intact by looking for this sign-post. STS's are now located on all chromosomes at approximately every 1000 bases. These markers will act as sign posts for a refined level of mapping.

"DNA chips" are solid little blocks to aide in analysis of sequences. This has greatly advanced the speed and automation of sequencing. A "chip" is an array of short DNA pieces with known, overlapping sequences. A long sample DNA (unknown sequence) is allowed to bind to the chip. The specific binding pattern can be read by a computer, and the sequence determined. The development of DNA chips has allowed for sequencing at 100 times the rate of previous systems. These are additional types of sequencing by hybridization (SBH):

Micro-capillary electrophoresis techniques help speed the process and reduce costs. Less materials are consumed. Less sample is needed. Less heat is generated and the assay is `cleaner'. This technology will have application long after the genome project.
Radiation Hybrid Mapping is used in conjunction with STS sign-posts and PCR assay. Radiation hybrid (RH) mapping uses a panel of somatic cell hybrids. Each cell line has a random set of human DNA fragments formed after irradiation of genomic DNA in a hamster cell. Calculations identify the continuous linkage along each arm of a chromosome. The markers are assembled into the map as a single linkage group.
Electronic PCR (e-PCR) is the computational procedure of findings STS segments that identify the location of the gene. This provides a simple means to verify sequence positions. Likewise, mapped sites may be found in the already accumulating human genomic sequence data. This type allows the non-lab researcher to run a Polymerase chain Chain Reaction (PCR) without getting his/her hands wet.

THE GENETIC DISCOVERIES AND THEIR IMPACT ON MEDICINE

While each of the genes found and reported raise new hopes for new therapies and new understanding of the disease mechanisms — what is going on now?

Genes For The Treatment Of Genetic Conditions

The success of gaucher's disease gives many great hope of finding treatments, if not a cure, for previously untreatable diseases. Gaucher's disease is a metabolic condition with splenomegaly and lytic bone lesions. It may present with avascular necrosis of the hip, or anemia with displacement of the bone marrow. The blood level of acid phosphatase is often elevated at presentation. The enzyme that is deficient is glucocerebrosidase. This disease is one of the more common metabolic disorders in humans. Several different forms are known. Bone marrow transplantation had been shown to be beneficial. But since the early 1990's enzyme replacement therapy has been the main stay of treatment. The disease is `curable' with repeated infusion of a modified glucocerebrosidase enzyme called alglucerase (Ceredase). Now a newer form of the enzyme, imiglucerase -- a recombinant form is starting to be used. The many forms of gaucher's disease -- infantile, juvenile and adult on-sets are continuing to be studied and the optimum dosage refined for each form. The cost remains high for the life long treatment of this condition. There continues to be ongoing developments and adjunctive ideas for those that are being followed under therapy. The success of this enzyme replacement (however imperfect) has many other families clinging to the hope that their child's disorder is next in line.

The Changing Story Of Genes
and Genetic Disorders

Williams syndrome (WS) (also called, Williams-Beuren syndrome) is a genetic condition with variable presentation. Classically the child presents with hypecalcemia, supravalvular aortic stenosis, and hypotonia and developmental delays. Many atypical presentations are well documented in the literature. Failure to thrive, severe gastro-esophageal reflux, and sometimes seizures can be major findings. The horse voice, and typical cute `pixie' or `elfin' facies may not be obvious until mid-childhood. Characteristic behaviors, good memory, and echolalia are features often seen in the older child or adult. Hypertension, renal artery stenosis, and coronary artery muscular hypertrophy may develop very late or not at all. The genetic nature of this contiguous gene syndrome is complex to say the least.

A few years ago the elastin gene was found to show an obvious mutation in 40 out of 40 children with classical William's syndrome. It appeared that the gene for Williams-Beuren syndrome was the elastin gene. However, of the next 30 patients with the clinical phenotype of William's syndrome the first child that did not show this gene deletion was a child diagnosed by myself. The child had all the non-classical findings of this condition. A number of authors have since found patients that they have followed for years as having William's syndrome, to test negative for the elastin deletion. Now the adjoining genes on chromosome #7 show a group of genes involved in the phenotype of William's syndrome. Deletions within and along side of elastin, but not involving the gene itself. The full nature of this clinical syndrome and the responsible genes will continue to evolve as more science is learned about the involved genes.

Unlocking The Intracellular Mechanisms

Cellular processing of proteins, amino acid leader-sequence signaling, intracellular communications, transport and packaging, and enzyme- enzyme interactions that result in `energy channeling', and sub-system controls on metabolism are very complex. Some sub-systems have a string of proteins—all numbered 1-15. The basic temporal interactions between and among each helps to unlock the inner working. Knowledge of these mechanisms are vital to further the understanding of disease states. Ubiquitin is one system that appears to be involved in many cellular pathways. Ubiquitin has been `known' since the 1980's. The ubiquitin-mediated proteolysis of intracellular proteins has been compared to an anti-ribosome - in that it takes apart proteins rather then assemble them. Many diseases may be involved with ubiquitin. One genetic syndrome suspected to involve this pathway is Angelman syndrome. This is a disorder in childhood where ataxia, seizures, inappropriate laughter, and profound mental retardation is seen. Ubiquitin may have potential in fighting cancer, detoxifying heavy metals, and preventing neuronal degeneration in Alzheimer's Disease. The approach of looking at intracellular `units' and how different subsystems exchange units and operate will continue to bear fruit. Still, there is much to learn about the complex `molecular soup' inside the cell.

Another area of investigation is the mechanisms of transcription and DNA management. The DNA is entwined on a scaffolding of histone proteins and arranged in a highly conserved and protected environment. Much work is needed to investigate the regulation and controls or these basic nuclear functions. Histone acetyltransferase activity (HAT) and a protein originally found in yeast called GCN5 (General Control of Nutrition 5) have key roles interacting with histone proteins. Now it seems that a previously separate system of chromatin-mediated energy pathways now has direct links to the activation of histones and transcription processes. Knowing the flow of energy and the regulation of DNA transcription is a vital link that may be useful to exploit in many disease states (i.e. cancer therapy).

The Role Of The Pediatric Geneticist And Very Rare Conditions

Family is told that their child has a severe brain anomaly by prenatal ultrasound. Couple gets opinions from pediatrician, Ob/Gyn, pastor, and neurosurgeon and goes on the Internet to join a support group. Despite encouragement to see a geneticist about their child's birth defect, they do not go. The family believes that the geneticist has nothing to offer. Since the diagnosis is already known, what does the geneticist do? "All that a geneticist is going to do is point the finger of blame at one of the parents." "Geneticists tell you to abort the child, and tell you not to have more children". So they do not go to genetics. The child is born and does not immediately die. What next?

After the birth of the child the care of the child is in question. What is normal for this condition? What do these children do? What do they not do? How do we care for this child? The pediatrician does not know. The neurosurgeon does not do surgery. A heart defect is found. The cardiologist says that if the child survives to be 4 months old they would then start to plan for surgery. Meanwhile, two heart medications are started. The geneticist is called in because he knows about the child's rare disorder. He begins to take history and become acquainted with the child and the family. The family and the geneticist talk at length about what to expect. Different supports are put into place. The family takes the baby to their pediatrician and the geneticist frequently over the next months. The family finds much of the personal knowledge the geneticist has is very beneficial. They eventually tell the genetic doctor about all the misconceptions they had, or were told by others, about what a geneticist does. They regret not coming before the birth of their child. The family asks, "Why don't others know more about what a pediatric geneticist does?"

Cloning

Cloning of animal cells has been a prize that many have been working on for some time. Plant systems are already well established with cloning techniques and the general public puts little interest into the overall process. Cloning of mammalian cells would provide researchers the means to show definitively what cell systems do in response to different agents, hormone treatments, and toxins. The list is endless of what and how identical cells or animals could provide the researcher and animal husbandry industry. In medicine, Twin Studies have always been used to define the genetic basis versus the environmental aspects of a given condition. Cloning has the potential to allow unlimited investigations into the mechanisms of disease and in measuring the response to therapy. There will clearly be continued investigations into the nature of cloning. The science and understanding of cloning will always be overshadowed by the social concerns of what this laboratory technique defines -- the vital force of life itself.

Genes For The Treatment Of Common Conditions

Enzymes are the nanotechnology of our bodily processes. DNA is the template by which cellular processes make cytoskeletons and build the tissues of the body. Blood vessels are the suppliers of oxygen, nutrients, and regulatory factors. Knowing the controls of making and inhibiting the formation of blood vessels has many potential applications. Stopping the blood vessels from forming may be beneficial to controlling the inflammatory process. This has direct application in allergic and arthritic conditions, in transplants, brain and eye diseases. Likewise, turning on blood vessel formation (angiogenesis) when critical events warrant - myocardial infarction and cerebovascular stroke are logical endeavors worth pursuing. Finding the keys to turn on the DNA templates, and start-up production of the proteins and enzymes to make new blood vessels, and to turn them off again will be one system that has a extremely wide range of potential uses. This application is a somatic therapy. Hypertension is another condition that is ripe for genetic somatic therapy.

Growth Of Genetics

The changes are happening everywhere. The field of Genetics is going from a somewhat poorly defined group of oddball doctors and pundits spouting jargon with day dreaming lab researchers to encompass the whole of medicine. We will continue to see the emergence of International Genetic Conferences for every organ system in the body. Similar to the recent Orlando Conference on Cardiovascular Genomics -- which is grammatically incorrect. While some geneticists will sit and make grammatical corrections, the doctors of the world that embrace genetics will be racing ahead with new found mechanisms and therapies for each of their respective organ systems.

Conclusions

The future of genetics and the Human Genome Project does have great promise. It's promise will yield great things well into the next millennium. The end of the sequencing is just around the corner. The application of the knowledge that comes from this will play out over our short life-spans. Our children will not understand what it was that we were doing for the past 300 years in the field of medicine.

Jacksonville Medicine / February, 1999

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