[From CASE Reports, Vol. 12, No. 2, 1997]


Connecticut Scientists Play Key Roles in Massive International Effort to Map and Sequence Human Genes

The Human Genome Project began in 1990 with the ambitious goal of mapping and sequencing all human genes by the year 2005. It has been compared to efforts in the nineteenth century to produce the periodic table, which systematically arranged the 100 known atoms in an array that captured their similarities and differences.

The magnitude of the Human Genome Project is far greater, however: mapping the human genome (all of the genes in an individual), one of the goals of the project, involves physically locating each of the roughly 100,000 human genes to its exact position on one of the 23 human chromosomes. Genes are composed of DNA, which is a linear molecule made up of four bases: adenine, thymine, guanine, and cytosine. Each DNA molecule consists of two strands of DNA, with each base on one strand connected by a hydrogen bond with a base on the other strand (called a base pair), so that the molecule twists into the famous double helix. Sequencing, the other major goal, involves determining the order of the bases in each gene.

The Human Genome Project is a massive scientific undertaking-there are three billion base pairs in the human genome-and could not be accomplished by any single group. Instead, it is a collaborative effort involving researchers in the United States and Europe, with 258 laboratories in the United States alone currently funded by the National Human Genome Research Institute (NHGRI), which is part of the National Institutes of Health (NIH); some research is also funded by the federal Department of Energy. The NHGRI was founded in 1989 to administer the Human Genome Project. Francis Collins, director of the NHGRI, was educated in Connecticut: he obtained a master's degree in philosophy and a PhD in physical chemistry at Yale University, then later returned after his medical training to do a fellowship in human genetics and pediatrics at Yale.

This article will describe some of the exciting work on the Human Genome Project that is being performed in Connecticut, and some of the progress that has been made in understanding diseases and developing new drug therapies as a result of the information that has come from the project.

Gene Mapping Nearly Complete; Sequencing is Next Major Goal

By the end of 1996, researchers had nearly completed the first goal, that of mapping the human genome. There now exist virtually complete and dense genetic maps, which relate the location of genes to each other but do not pinpoint a location on a chromosome, and good first generation physical maps, which have specifically located about 20,000 of the 100,000 human genes on chromosomes.

Richard Lifton, a professor in the Departments of Medicine and Genetics at the Yale University School of Medicine, is on a subcommittee for funding sequencing projects. According to Professor Lifton, the NIH plans to spend $60 million per year over the next seven years, with the goal of sequencing approximately 60% of the human genome; the hope is that the remaining 40% of the work will be performed in other countries or with private funding. About 1% of the human genome has been sequenced, and a real push on the work will begin in 1998. With new techniques that will speed the sequencing, it appears that the project will achieve its goal of sequencing the entire human genome by 2005.

David Ward, acting chair of the Department of Genetics at the Yale University School of Medicine, is one of the researchers who received a large grant from the NHGRI to map part of the human genome. "We worked in collaboration with about five other labs, at Yale and at Albert Einstein in New York, to produce a physical map of the human chromosome 12," explains Professor Ward. "This chromosome has genes that are involved in leukemia, lymphoma, and other types of cancer, as well as those [genes] for the production of structural proteins like collagen, so we feel that this was fairly important work."

Integrating the Information

In another aspect of the Human Genome Project, researchers in Professor Ward's laboratory used a technique called fluorescence in situ hybridization (FISH) to integrate information on the physical and genetic maps. With FISH, a piece of DNA can be labeled with a fluorescent marker and then mixed in with chromosomes and viewed under a microscope. The chromosomes are clearly visible under magnification, and the labeled DNA will hybridize (attach) with the chromosome where the analogous piece of DNA is located. The location of the fluorescence indicates where on which chromosome that piece of DNA is located. "This technique allows you to walk between the three types of maps (physical and genetic maps and chromosome smears)," explains Professor Ward. "It's like a translator for different languages."

Professor Ward's group is not working on the Human Genome Project right now. "Our expertise is in mapping, and that work is nearly completed," says Professor Ward. "It was exciting to be part of this massive undertaking and it's fun to see how things fall together. We found a number of interesting genes while we were working on the project, which was a sideline for the project but great for us, so my laboratory benefited greatly from this work."

According to Professor Ward, one of the main benefits of the Human Genome Project has been an increased understanding of how genes are associated with inherited and acquired diseases. "This knowledge base is expanding exponentially," he says. "We've learned ten times more about the association of gene mutations with disease in the last six months than in the previous ten years." The next step is to work on cures, with either genetic or replacement therapies.

Inherited Susceptibilities

Researchers in Professor Lifton's laboratory at the Hughes Medical Institute in the Boyer Center for Molecular Medicine at Yale are using information derived from the Human Genome Project to identify inherited susceptibilities to cardiovascular and renal diseases. They are specifically working on genes that change blood pressure. According to Professor Lifton, high blood pressure affects approximately 50 million people in the United States, and management of patients with high blood pressure is one of the largest single expenditures in health care. "We need to understand the factors that contribute to high blood pressure to treat these patients more effectively," says Professor Lifton. "Right now our treatment is empiric-we tell them to change their diet, to lose weight, because those things seem to work, but we don't know why-we need a more rational approach."

Professor Lifton's group has identified eight genes that, when mutated, have significant effects on blood pressure; four of the mutated genes cause extreme increases and four cause decreases in blood pressure. "We're working with genes that cause extreme differences because we wanted to see if these genes all have common pathways that might also be involved in the more prevalent forms of high blood pressure," explains Professor Lifton.

The eight mutations identified by Lifton's group do all have a single, common final pathway, which regulates how the kidneys handle salt. All of these genes act either to change the signal to the kidney, telling it to retain more or less salt and water, or on ion channels that mediate salt and water reabsorption. "This link establishes for the first time a direct, mechanistic link between salt and blood pressure in humans," says Professor Lifton. "Epidemiologic evidence has long suggested a relationship, but exactly how this is mediated is only now being elucidated in precise mechanistic detail."

These eight mutations occur rarely and have severe consequences, but still have important bearing on diseases associated with high blood pressure. "Now that we've identified this common pathway," says Professor Lifton, "we're interested in finding out whether more frequently occurring mutations may cause less drastic alterations in blood pressure-the garden variety high blood pressure."

Genetic epidemiological studies also suggest that patients with high blood pressure may be more susceptible than patients with normal or low blood pressure to other diseases, such as stroke, end-stage renal disease, and heart attacks. Professor Lifton's group has tried to identify genes that might contribute to susceptibility to these other diseases. "We believe that high blood pressure interacting with other genes may cause these outcomes," explains Professor Lifton. "And we have identified one chromosome region that contains a gene responsible for an inherited form of stroke."

"From the clinical perspective, the Human Genome Project has given us remarkable tools with which to begin dissecting causes of or susceptibility to diseases like heart attack, stroke, asthma, diabetes, obesity, Alzheimer's, schizophrenia, and all forms of cancer," says Professor Lifton. "For the first time we can unravel the causes of these diseases. There are also important environmental contributions to many of these diseases, and a complete picture of both the genetic and environmental components is crucial in developing preventative strategies or therapies."

Advanced Analysis, Computation Methods Critical to Success

CuraGen Corporation, located in Branford and New Haven, started as a technology development company developing instruments for analyzing DNA, new molecular biology techniques, and computational methods to keep track of all of the data from the company's work on the Human Genome Project. "With three billion nucleotides to be sequenced, there was a need for an efficient bioinformatics system, so we designed one, which is called GeneScape," explains Joel Bader, group leader in instrument development and therapeutic development at CuraGen. "We collaborate with universities and pharmaceutical companies, providing our technology as a research service," adds Dr. Bader. The equipment and techniques developed at CuraGen, including GeneScape and the company's method for analyzing gene expression, Quantitative Expression Analysis, are currently being used in several laboratories across the country working on the Human Genome Project.

Researchers at CuraGen have more recently started to identify disease-related genes, by analyzing genes that are turned on in people with certain diseases and not in healthy people. "Once we identify a gene associated with a disease, we're pursuing two approaches," says Dr. Bader. "We can use this information as a diagnostic tool for the disease, and we can use it to help develop drugs to remedy the aberrant process."

Researchers at Genaissance Pharma-ceuticals (formerly Bios Laboratories) in New Haven are developing new methods to be used in gene discovery, mapping, and sequencing, and are also using the information derived from the Human Genome Project to develop better drug therapies.

Bios Laboratories was founded on a DNA sequencing technique called Coupled Amplification and Sequencing (CAS), which is an enzyme-based method for sequencing DNA. When the name of the company was changed to Genaissance, the research rights to this technology were licensed to Amersham International PLC, and the system is currently being tested in a number of the Human Genome Project research centers. "With three billion base pairs to sequence, speed is of the essence," says Kevin Bentley, senior staff scientist and director of laboratory operations at Genaissance. "CAS is a more rapid sequencing method than most of those currently available, and we think it will supersede other methods."

Another product currently under development at Genaissance is a microdissection "library" (a collection of DNA fragments) for gene-rich regions of the human genome. "Genes are not evenly distributed across the genome," explains Dr. Bentley. "Some regions of the chromosome have a higher proportion of the bases guanine and cytosine and are gene-rich-about sixteen times more gene-rich than those regions with a high content of adenine and thymine." Researchers at Genaissance have identified 24 gene-rich regions and have made about half of the libraries for those regions. "These pieces of DNA have coding regions embedded in them. They can be used for gene discovery and mapping, or they can be sequenced," says Dr. Bentley. One possible application for these libraries is to use them as chromosome "paint" to identify a particular band on a chromosome. For example, a researcher might look at several tumor tissues and notice that a portion of a certain chromosome is deleted or rearranged. The researcher could label all the pieces of this library and map them back to the chromosome spread of the tumors, using the library as a diagnostic tool for that type of cancer. Reseach products resulting from this technology have been licensed to Quantum Pharmaceuticals.

Using Genetic Variations for Customized Drug Design

Having been deeply involved with developing research tools for the Human Genome Project, and realizing the vast potential of the information being generated, management at Genaissance decided to use their expertise to strike out in a new direction. "We have looked at the information coming out of the Human Genome Project and asked ourselves 'What can we do with all of this information?'" explains Dr. Bentley. "We decided to apply the science of gene mapping and sequencing (genomics) in other areas throughout the drug development process, to use genetic variation as the basis for directed drug discovery and design."

The Human Genome Project will provide a backbone of genetic information, but in reality, the genome of each person is different. "We can identify variations in the population, based on age, gender, ethnicity, and could use those variations to improve therapy, to tailor drugs to certain population groups," explains Dr. Bentley. "We could have a battery of three, four, or five slightly different drug molecules that could be used individually or in combination in different patient populations." Patients may respond better to this type of customized therapy than to a one-drug-fits-all approach, and the side effects associated with the drugs may also be decreased. "We're excited by the possibilities provided by this approach," says Dr. Bentley. "It's been validated by other sources, and we think we're on the way to providing improved drug therapy for several diseases."

Researchers at Genaissance are working in collaboration with Alexandros Makriyannis, professor of medicinal chemistry, molecular and cellular biology at the University of Connecticut Health Science Center. Professor Makriyannis is using the information on genes that is discovered at Genaissance to design new drugs.

Research conducted through the Human Genome Project identifies genes that produce defective or disease-causing proteins, and researchers like Professor Makriyannis can use that information to design drugs to act specifically on that protein, rather than having more widespread effects throughout the body. "Genes contain information that allow the body to manufacture proteins-each gene makes one protein," explains Professor Makriyannis. "We make drugs that fit each protein."

The research and drug development projects underway in Connecticut laboratories exemplify the vast potential of the Human Genome Project for finding the causes of and developing treatments for many human diseases. The use of information coming out of the project also raises many ethical questions. If you had a gene that increased your chances of having a debilitating or fatal disease, would you want to know? Especially if the disease currently cannot be treated? Ten years ago that question was purely theoretical; today it is possible, and the possibility has far-reaching effects on health insurance and job security for people who might have disease-causing genes.--Lisa Christenson, freelance science writer

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