Harnessing the HGP for Public Health
The enormous amount of genetic data from the Human Genome Project (HGP) has
benefited researchers studying gene-environment interactions, but also
presents challenges in experimental design, data management, and the ethical,
legal, and social implications of gene-environment research. These challenges
were the topic of the symposium "The Human Genome Project and Public Health:
Gene-Environment Interactions," part of the 2004 annual meeting of the
American Association for the Advancement of Science in Seattle, Washington.
Presenters discussed strategies to help researchers decide where to concentrate
their research dollars and energy among the more than 7.2 million genetic variations
and single-nucleotide polymorphisms (SNPs) cataloged by the HGP. One strategy
is to focus on genes that are linked by function in pathways, said Deborah
Nickerson, a professor in the University of Washington (UW) Department of Genome
Sciences. She discussed progress on SNP discovery in gene pathways for the
Environmental Genome Project, especially the development of new algorithms
to explore associations between SNPs in human genes and environmental exposures.
One program--Hotspotter, developed by UW assistant statistics professor Matthew
Stephens--helps researchers explore recombination in human genes over time,
a history that affects SNP associations in the human genome.
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Too much information? Knowledge of genetic susceptibility to smoking-related
disease could actually decrease a person’s motivation to quit.
image
credit: Clockwise from top left: Photodisc; Photodisc; Digital Vision;
Photodisc
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Focusing on genes involved in key cellular processes such as DNA repair is
critical, according to symposium co-organizer David Eaton, director of the
NIEHS-UW Center for Ecogenetics and Environmental Health. Every day, each
cell in the human body withstands 10,000-20,000 oxidative assaults to
DNA, and the vast majority are repaired. Increased understanding of how this
DNA repair happens and other critical biochemical pathways can lead to better
methods for disease prevention and more effective drugs with fewer side effects,
said Eaton.
Better understanding of gene-environment interactions can also lead
to more accurate dosing of existing medications. This is especially crucial
for powerful drugs where the margin between effective and toxic doses is narrow,
said Kenneth Thummel, associate dean for research and new initiatives of the
UW School of Pharmacy. One example is warfarin, an anticoagulant used to prevent
recurrent myocardial infarction and other thromboembolic events. Variations
in a single gene (CYP2C9) can cause some people to be five times as
sensitive to the drug and make them susceptible to overdosing, which can cause
severe internal bleeding. According to Thummel, genetic testing could be cost-effective
if it could replace some of the blood tests now used to monitor warfarin dosing--especially
if it reduces hospitalizations by determining which patients need lower doses
of the drug and closer monitoring. Other recent research may lead to improved
dosing for the immunosuppressants cyclosporine and tacrolimus, which can cause
kidney damage and failure.
Susceptibility to heart disease has also been linked to numerous genes, including APOE.
Steve Humphries, a professor of cardiovascular genetics at University College
London Medical School, reported that variants of APOE are well known
to raise or lower blood levels of low-density lipoprotein ("bad") cholesterol,
but only have a significant impact on an individual's risk of heart disease
when the person smokes. Smoking increases the risk of heart disease of people
with any of three common APOE variants, but the risk is greatest (about
threefold) among carriers of the *4 variant, which is found in about 25% of
the population. Humphries and colleagues are working with a smoking cessation
clinic in London to determine whether smokers will be motivated to quit if
they learn that they have a high genetic risk of disease.
One possible complication is that information about APOE4 status could
lead to fatalism rather than a determination to quit smoking, said symposium
co-organizer Wylie Burke, chair of the UW Department of Medical History and
Ethics. The issue is further complicated by the fact that the APOE4
polymorphism has also been linked to a higher risk of Alzheimer disease. People
may view death by heart disease as a blessing compared to contracting Alzheimer
disease, for which there is as yet no cure and no clear-cut means of prevention.
Therefore, knowing one's own APOE4 status could decrease, rather than
increase, a person's motivation to quit smoking. In addition, according to
Burke, focusing on genetic susceptibility to smoking-related disease may draw
attention from more important environmental factors, such as advertising, that
encourage people of all genotypes to begin smoking in the first place.
Many other findings in gene-environment interactions raise similar ethical,
legal, and social issues about whether society should focus on labeling individuals
as susceptible to a given disease or simply reduce environmental exposures
for everyone. "We need to be very careful to create the right environment in
this era of rapidly accumulating genetic information," said Burke. "An overemphasis
on the effects of genes relative to the effects of the environment can lead
to oversimplification of the problem and distract attention from needed environmental
change."
Kris Freeman
The Bleeding Edge of Technology
In any given proteomics experiment, a cell or tissue can express hundreds
or thousands of proteins at a time. Unlike the static genome, the proteome
can change quickly, and key responses to toxicants and disease may involve
small amounts of rare proteins. As an added complication, gene-protein
and protein-protein interactions often are not linear. So what's a researcher
to do? At the seminar "Proteomics and Systems Biology," held at the 2004 annual
meeting of the American Association for the Advancement of Science in Seattle,
Washington, presenters discussed advances at the "bleeding edge" of proteomics
research and their use in the study of complex biochemical interactions within
and among cells--advances that may help overcome some of the challenges posed
by proteomics.
Seminar presenters discussed techniques to both measure protein signals and
analyze the enormous amounts of data that result from such experiments. In
most of the experiments reported, new techniques were tested on biological
systems that had already been partially characterized, such as blood plasma.
This allowed researchers to validate their systems with the bonus of potentially
adding to knowledge about the biological systems in question.
In one analysis of blood plasma conducted at Pacific Northwest National Laboratory
(PNNL), scientists claim to have identified about 3,700 proteins (not counting
immunoglobulins) from human plasma, results that are an order of magnitude
greater than those described only 18 months ago, according to Richard Smith,
director of the NIH Proteomics Research Resource Center at PNNL. The newly
detected proteins include many found at very low levels, some of which could
be used as biomarkers of toxic exposure or disease progression, said Smith.
The plasma analysis used high-sensitivity, high-throughput instrumentation
and techniques developed at PNNL, including Fourier transform ion cyclotron
resonance (an advanced form of mass spectrometry). The PNNL researchers also
separated out the most abundant proteins, allowing measurements to focus on
less abundant proteins and increasing the number of proteins found in their
plasma samples from about 1,000 to about 3,700. In addition, using electrospray
ionization and low flow rates of solutions into the mass spectrometer facilitated
detection of proteins in amounts as small as 10 zeptomoles, a level of sensitivity
that makes it possible to analyze many proteins expressed by a single cell,
said Smith.
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Big picture, tiny molecules. A PNNL researcher performs an experiment
on the Fourier transform ion cyclotron resonance mass spectrometer, which
is used to characterize proteins such as those in blood plasma (inset).
image credit: PNNL; inset: Photodisc
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Leroy Hood, president of the nonprofit Institute for Systems Biology (ISB),
too, discussed the potential for analyzing single cells. Researchers at the
ISB and the California Institute of Technology are developing nanochips measuring
100 microns on a side that will assess the behavior of individual cells and
gauge the concentrations of the mRNAs and proteins from a single cell. The
ISB researchers have applied microfluidics--the study of how fluids behave
at the nano level--to successfully conduct biological assays on single cells.
ISB and Caltech researchers are now working on nanochips that can analyze several
cells simultaneously. "We'll be able to interrogate a T cell and then an antigen-presenting
cell separately. Then we will let the cells interact and interrogate their
combined behaviors," said Hood.
Some presenters noted cautions about the prospects for analysis of single,
living cells. Smith, for example, said that much more progress is needed in
areas such as the construction of nanochips and microfluidics in order to make
these methods of measurement "truly useful and not just a stunt."
Presenter Matthias Mann, a professor of biochemistry and molecular biology
at the University of Southern Denmark, emphasized the need to distinguish between
at least three states to track changes in protein expression. He displayed
preliminary data from cells that were treated with a growth factor and sampled
at five points. Analysis detected changes in levels of about 400 phosphoproteins
over time. "The activation of some proteins decayed faster, and some proteins
were activated later," Mann said.
Beyond the proteome is the metabolome--the sugars, amino acids, and other
molecules that are created by or combine to create proteins. Masaru Tomita
and colleagues at Keio University and the bioventure firm Human Metabolome
Technologies are combining capillary electrophoresis and mass spectrometry
to analyze the metabolites from rice, Escherichia coli, and Bacillus
subtilis. For the purposes of these experiments, the team defined metabolites
as molecules with a molecular weight of less than 1,000. In one experiment,
the team detected more than 1,700 possible metabolites. The team is now constructing
a model of entire metabolic pathways with several thousand reactions.
It may never be possible to fully describe the proteome of any species. Unlike
the genome, which Hood characterized as "digital" and so "ultimately knowable," the
proteome is affected by a multitude of external factors. In its absolute sense,
according to Mann, the proteome is "as unreachable as the horizon."
Kris
Freeman
Diet and DNA
The emerging field of nutrigenomics explores how nutrients in foods interact
with genes that contribute to chronic diseases. The goal of nutrigenomics is
to understand individual nutrient genotypes to design dietary interventions
that restore health or prevent disease, eventually improving the health of
the population at large as well as that of specific subpopulations. The fledgling
field is packed with promise, and two new research initiatives aim to help
deliver on that promise.
A Center of Excellence for Nutritional Genomics was established in 2003 at
the University of California (UC), Davis, to coordinate nutrigenomics studies
among participating institutes. A five-year, $6.5 million grant from the NIH
National Center on Minority Health and Health Disparities funds the project.
Genetics professor Raymond Rodriguez directs the new center, which unites 25
experts in nutrition, molecular biology, bioinformatics, and related fields
from UC Davis, the Children's Hospital Oakland Research Institute, the U.S.
Department of Agriculture Western Human Nutrition Research Center, and the
Ethnic Health Institute at Alta Bates Summit Medical Center. Center members
will explore how different foods interact with genes to increase the risk of
type 2 diabetes mellitus, obesity, heart disease, and cancer.
Across the Atlantic, the European Nutrigenomics Organisation (NuGO) was launched
in February 2004. This network of 22 scientists from 10 European countries
will receive ¤17.3 million from the European Union over six years to develop
new technologies, improve model systems, and advance nutritional bioinformatics. "Particular
attention will be given to studies of human volunteers, and both biomarkers
and new methods will be developed and validated," says Siân Astley, NuGO's
communications manager.
"Nutritional genomics connects the Human Genome Project to human health in
the most personal ways--through the foods we eat several times a day," says
Rodriguez. "A better understanding of how diet and genes interact will enable
us to better manage our own health and possibly prevent, mitigate, or delay
the onset of chronic and age-related diseases."
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Lessons from lunch. The field of nutrigenomics is examining the nexus
between diet and genetic susceptibility to disease.
image credit: Photodisc
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People react to certain nutrients differently, depending on their genetic
makeup. Lactose intolerance, a well-known example of nutrigenomics, afflicts
largely Asians and Africans, and far fewer people of northern European descent.
That's because a single base pair change in DNA occurred in northern Europe
about 6,500-12,000 years ago, which allowed people there to digest lactose--in
an environment with a short growing season, access to the additional nutritious
food source of milk was helpful for survival, says Jim Kaput, a pioneer of
nutrigenomics and founder of the diagnostics company NutraGenomics.
Today's nutrigenomics researchers hope to find gene variants that explain
why, for instance, some people can lower their blood pressure through dietary
changes, while others need drugs. Other variations might explain why some people
are more susceptible to gastrointestinal cancers, inflammatory diseases, and
osteoporosis.
Scientists are finding that biologically active components of foods can alter
gene expression. For example, a deficiency of folic acid may lead to breaks
in DNA that mimic radiation damage. Other nutrients are involved in molecular
processes related to DNA structure, gene expression, and metabolism, which
contribute to the development of chronic illnesses.
The nutrigenomics approach resembles pharmacogenomics, which looks at the
relationship between single-nucleotide polymorphisms in genes and patients'
responses to drugs to personalize medicine. Although progress in pharmacogenomics
currently surpasses that in nutrigenomics, the two are closely linked. "Without
nutrigenomics, pharmacogenomic data cannot be interpreted correctly, because
diet may affect the expression of genes involved in drug metabolism," says
Kaput. He proposes that pharmaceutical companies should include nutrigenomics
in the design of new drugs because, he says, "what you eat affects a drug's
efficacy."
Carol Potera
How Viruses Sabotage Silencing
The discovery and description of RNA silencing less than a decade ago has
spawned a flood of research, revolutionizing the practice of functional genomics
and leading to intensive exploration of its potential application to treat
numerous diseases. RNA silencing was first noticed in plants when attempts
to create transgenic plants that overexpressed a natural gene often had the
opposite effect; later it was found to be an evolutionarily conserved defense
mechanism against plant RNA viruses and other molecular parasites. Viruses,
in turn, have evolved their own counterdefense mechanisms: proteins that suppress
RNA silencing, allowing the virus to maintain its invasion of a plant. Until
recently, the mechanism behind this suppression of silencing was a mystery,
but researchers at the NIEHS and the Agricultural Biotechnology Center in Gödöllõ,
Hungary, have begun to unravel how some viruses neutralize silencing, shedding
important new light on a complex molecular interaction.
In the 26 December 2003 issue of Cell, NIEHS investigator Traci M.
Tanaka Hall, postdoctoral researcher Jeffrey Vargason, and Hungarian colleagues
József Burgyán and György Szittly elucidate the nature of
viral counterdefense by solving the crystal structure of a known silencing
suppressor, the tombusvirus Carnation Italian ringspot virus (CIRV)
p19 protein, in complex with a 21-nucleotide small interfering RNA (siRNA),
the workhorse bit of nucleic acid that drives the silencing process. The structure
of a similar p19 protein found in another tombusvirus was published by Keqiong
Ye, Lucy Malinina, and Dinshaw Patel, all of the Memorial Sloan-Kettering Cancer
Center, in the 18/25 December 2003 issue of Nature. The slight differences
in the structures have allowed researchers to draw further inferences about
how a virus can interfere with RNA silencing.
There are two classes of plant siRNAs. The shorter ones, measuring 21-22
nucleotides, are responsible for detecting and destroying molecular invaders.
The longer ones, measuring 24-26 nucleotides, are suspected to be more
involved with regulating retrotransposons and DNA methylation. The structure
of p19 reveals that the protein selectively recognizes silencing siRNAs by
measuring their length. Tryptophan residues (Trp39 and Trp42) on the protein
act like molecular calipers, forming a so-called stacking interaction with
the ends of the end base pairs of the shorter siRNAs. By binding to the silencing
siRNAs, the protein in effect sequesters them, rendering them incapable of
carrying out their silencing mission and allowing the virus to run rampant
within the plant.
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Potent punch. The CIRV p19 protein (blue and green ribbons) plus a 21-nucleotide
siRNA (beige balls) adds up to a lethal combination that enables viruses
to counter a plant’s attempt at RNA silencing.
image credit: Jeffrey Vargason
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The protein can also bind to the longer siRNAs, but much more weakly. "Viruses
are smart," says structural biologist Tanaka Hall. "They don't want to kill
their hosts too quickly. They want to have time to replicate. So it makes sense
that the virus spares processes that might be essential to the plant's survival
and not directed against virus invasion."
The team also studied the importance of tryptophan residues to p19's silencing
suppression effects by introducing mutations into the p19 coding sequence of
the CIRV genome to change one or both of the tryptophan residues. A protein
database search had shown that both tryptophans were not absolutely conserved
in all identified viral p19 sequences, although all sequences conserved at
least one of the tryptophans, with the other residue usually capable of forming
a stacking interaction. Their findings suggest that while the virus can at
least partially succeed in suppressing silencing with two residues capable
of stacking, substituting one with a glycine residue lacking a side chain results
in failure of the suppression and recovery of the plant from infection.
p19 has emerged as an important research tool in the quest to refine scientific
understanding of how siRNAs and other small RNAs, such as microRNAs, work in
planta, in vitro, and in vivo. Unlike silencing siRNAs themselves,
which rely on sequence specificity to accomplish their effects, p19 appears
to simply measure the siRNAs and neutralize those designed for silencing. This
mechanistic revelation will enhance the value of the protein as researchers
continue their efforts to clarify the biological roles of small RNAs in gene
silencing and other cellular regulatory and developmental processes.
According to RNA silencing research pioneer Phillip Zamore, an associate
professor in the Department of Biochemistry and Molecular Pharmacology at the
University of Massachusetts Medical School, Tanaka Hall's structure of the
p19 protein also provides great insight into the RNA silencing pathway itself. "All
future models of the RNA interference pathway must incorporate a step at which
they are vulnerable to siRNA sequestration by p19," he says. "This conclusion
is inescapable once one has seen the structure."
Tanaka Hall's group plans to continue to use p19 in its investigations. "We're
particularly looking at its role in being able to inhibit microRNA-initiated
processes, looking biochemically at how p19 combines with microRNAs," she says.
Structural imaging of that relationship could aid in explaining the still poorly
understood mechanisms by which microRNAs accomplish their cellular tasks.
Ernie
Hood
International Sequencing
Consortium
The sequencing of multiple species' genomes by the Human Genome Project,
including those of the human, the worm Caenorhabditis elegans,
and the fruit fly Drosophila melanogaster, has laid the foundation
for the field of comparative genomics. Experts believe this field of
study represents the next step in genomic exploration, and that sequencing
of more organisms will be critical to answering cross- and multispecies
genomic questions. The International Sequencing Consortium (ISC) was
established in 2002 to provide a worldwide forum for genomic sequencing
groups and their funding agencies to share information, coordinate
research efforts, and address common issues raised by genomic sequencing,
such as data quality and release. The ISC has established a free website
at http://www.intlgenome.org/ where
scientists and the public can get the latest information on the status
of sequencing projects for the genomes of animals, plants, and other
eukaryotic organisms.
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image credit: ISC
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The ISC website consists essentially of a database of sequencing
projects. The database can be searched and sorted by organism, sequencing
group, or funding agency. In addition, information is provided for
each organism on the region of the genome being studied, strategies
being used, the purpose of the study, collaborating groups, and the
timetable for the project, if known.
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image credit: Sinclair Stammers/WHO TDR
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In most cases, links in the database connect users directly to individual
websites for the project, sequencing group, or funding agency. For
example, the website lists a project for sequencing the mosquito species Anopheles
gambiae, the principal vector of malaria. The project was conducted
by Genoscope (the national center for sequencing in France) with funding
by the U.S. Agency for International Development, the World Health
Organization Special Programme for Research and Training in Tropical
Diseases, and the French Ministry of Research. Another link on the
ISC site connects to the completed project's website, where information
is provided on the scope and importance of this work.
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image credit:
Sinclair Stammers/WHO TDR
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The ISC site also contains a listing of links to other resources
categorized under the headings of microbial websites, genome browsers,
trace archives, and other genome-related sites including other public
databases where DNA sequence data are deposited.
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image credit: A.W. Taylor-Robinson/WHO TDR
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Members of the ISC include large-scale, high-throughput sequencing
centers and their funding agencies, all of whom have agreed to
continue generating publicly available sequence data for unrestricted use by
the research community. Most of the sequencing projects included in
the ISC database adhere to the policy of rapid release of prepublication
data that has been established by the National Human Genome Research
Institute and The Wellcome Trust for efforts designated as "community
resource projects."
Kimberly G. Thigpen |
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Last Updated: May 12, 2003