There are approximately 500,000 cancer-related deaths annually in the
United States. Scientists believe as that many as 80% of those deaths could
be prevented due to the fact that most malignancies are a result of external
factors rather than inherent biological conditions. With recent advances
in molecular biology, a new field that combines highly sensitive and specific
techniques for detecting early damage associated with cancer has emerged.
By combining knowledge about external factors related to lifestyle and environmental
or occupational exposure to chemicals with knowledge of how genetic differences
cause variations in human responses to environmental pollutants, scientists
are developing a better understanding of questions such as why some smokers
get cancer but others do not, why certain groups of people have a higher
incidence of cancer after exposure to a toxicant and others do not, and
why certain women are more prone to develop breast cancer than others. Scientists
using biomarkers of susceptibility will be able to identify risks and prevent
adverse health effects through prevention and intervention strategies.
The Clues at Hand
In 1987 the National Research Council of the National Academy of Sciences
defined biological markers as "indicators signaling events in biological
systems or samples" and classified them into three types: markers of
exposure, markers of effect, and markers of susceptibility. Each of these
types of biomarkers has specific and relevant application to the assessment,
and potentially ensurance, of environmental health.
Continuum of change. Gray arrows indicate
sequence of events from exposure to clinical disease. Yellow lines indicate
the influence of individual susceptibility and other factors on steps of
the disease process.
Exposure. External exposure is the amount of a xenobiotic substance
(outside living systems) to which a person is subjected, whereas internal
dose is the amount of a substance absorbed into the body. Markers of exposure
indicate the presence of a xenobiotic compound and its interactions within
an organism. In addition to assessing exposure by mathematical modeling
and ambient monitoring by chemical or physical analysis of food, air, water,
and soil, exposure is also assessed by measuring xenobiotics in blood, urine,
saliva, cerebrospinal fluid, and other biological samples. Markers of exposure
offer a more accurate means of estimating exposure than mathematical modeling
or analyzing environmental samples.
Effect. Biological markers of effect indicate the presence of
disease, early precursors of disease, or events peripheral to a disease
process that may predict the development of impaired health. Markers of
effect represent points on a continuum of health impairment and may be measured
qualitatively or quantitatively. Early responses to exposure may include
changes in the function of target tissues or responses in organs or tissues
such as chromosomal damage, mutations of critical target genes, or altered
hormone status.
Susceptibility. A third kind of biological markers indicates differences
in individuals or populations that affect the body's response to environmental
agents. Markers of susceptibility may include genetic characteristics, preexisting
disease that results in an increase in the amount of agent absorbed or the
target tissue responses, differences in metabolism, variations in immunoglobulin
levels, or the capacity of an organ to recover from environmental insult.
Markers of susceptibility are particularly relevant to determining inherited
predisposition to risk of adverse health effects as a result of exposure
to environmental chemicals.
Environmental Exposure
People living in industrialized countries are exposed extensively to
chemicals that cause mutations, cancer, and birth defects. For example,
more than 700 organic chemicals have been identified in the drinking water
supply in the United States; 40 are possible carcinogens. There are at least
320 toxic industrial chemicals released into ambient air; 60 are possible
carcinogens. Of approximately 380 pesticides manufactured in this country
that are applied to food products, 66 have been identified as carcinogenic
or potentially cancer causing. Categories of common substances that could
increase carcinogenic risk include aromatic amines, nitrosamines, chlorinated
hydrocarbons, polycyclic aromatics, radionuclides, metal dusts, molds, and
steroid hormones.
Exposure to aromatic amines such as benzidine and 2-naphthylamine used
in dye making, rubber manufacture, and analytical laboratories may cause
bladder cancer. Nitrosamines are used as additives for food preservation,
in rubber manufacture, and are present in tobacco smoke. Chlorinated hydrocarbons
include vinyl chloride used in the manufacture of polyvinylchloride (PVC)
and organochlorine insecticides such as aldrin, dieldrin, and DDT. Polycyclic
aromatic hydrocarbons (PAHs) are products of tobacco smoke and combustion
of fossil fuels. Radionuclides such as strontium-90 and plutonium-239 are
used as medical and scientific diagnostics, used by the military, and used
as fuel in nuclear power stations. There are various uses for different
metal dusts such as arsenic, used in pesticides and drug manufacture, beryllium,
used in the production of lightweight alloys and rocket fuel, and chromates,
used in paint pigments. Food may be contaminated by molds that produce toxic
compounds. The infestation of rye by ergot (the fungus Claviceps purpurea)
causes ergotism, a disease characterized by severe mental disorder and hallucination.
Other fungal products, such as aflatoxins produced by the mold Aspergillus
that grows on peanuts, corn, rice, and sorghum, are known carcinogens.
An example of a carcinogenic steroid hormone is diethylstilbestrol, which
is added to animal feed and used as a "morning after pill." The
question for all of these substances is: How do we know if a person is likely
to develop disease or incur a heritable genetic mutation that could cause
problems, such as learning disabilities or high risk for cancer, in future
generations as a result of exposure?
Susceptibility Markers
The underlying principle of susceptibility markers is the interindividual
differences that confer sensitivity or resistance to environmentally induced
disease. The first type of susceptibility marker is based on the fact that
most chemicals are altered by enzymes, and these alterations may increase
or decrease the ability of a chemical to interact with DNA, RNA, or proteins.
The balance between enzymes that detoxify or enhance the toxicity of chemicals
differs among individuals and ethnic groups. These differences often are
inherited and can lead to pronounced differences in a person's sensitivity
to the effects of chemical exposure.
A second type of susceptibility marker reflects genetic differences in
the capacity of cells to repair DNA damage caused by environmental insult.
People deficient in DNA repair genes may exhibit more DNA damage manifest
as DNA adducts; alterations in chromosome number; structural modifications
such as chromosome breaks, rearrangements, and exchanges; micronuclei, which
are fragments of nuclear material left in the cytoplasm after replication;
activated oncogenes and their protein products; and much higher incidences
of cancer.
A kink in the chain. There are at least three
classes of inherited susceptibility to cancer with great genetic variation
among humans.
A third type of susceptibility marker is preexisting inherited genetic
defects that increase the risk of cancer. Cancer is generally understood
to be a multistage process, requiring several genetic alterations or mutations
to produce a clinically detectable tumor. If a person has inherited one
or more of the necessary genetic alterations, fewer steps are needed for
a chemical to cause cancer, putting this person at a greater risk.
Enzyme metabolism. There is considerable variation among humans
in the production of enzymes that either activate formation of electrophilic
metabolites that covalently bind to DNA or catalyze detoxification of chemical
carcinogens. A large number of enzymes involved in regulation of metabolic
pathways, many of which originally evolved in humans to handle naturally
occurring toxicants, play an important role in detoxifying compounds humans
are exposed to today as a result of environmental pollution. Within each
group of enzymes, there are many forms that have evolved to metabolize different
chemicals. For example, there are approximately 40 cytochrome P450 enzymes
that evolved from a common ancestral cytochrome gene, estimated to be 2000
million years old. Each of the 40 forms is composed of variants that have
different capacities to metabolize drugs and chemicals.
Recent findings by several research groups including those at NIEHS,
Duke University Medical Center, and the University of North Carolina at
Chapel Hill School of Medicine illustrate that the ability of individuals
to metabolize carcinogens before damage occurs varies widely. Researchers
from the three groups collaborated on an epidemiological study correlating
exposure risk and genetic susceptibility to bladder cancer. "There
is large variation in the human populations in how well individuals are
protected," says Douglas Bell, an NIEHS researcher. "Some people
have a high ability to detoxify environmental chemicals and others do not."
Douglas Bell-- Not
everyone is protected the same. |
The association between lung cancer and smoking is well established,
and there is now evidence of a link between smoking and bladder cancer.
In a study of bladder cancer patients and control subjects, researchers
found that approximately half of all subjects inherit a gene that encodes
for glutathione S-transferase M1, an enzyme that detoxifies environmental
carcinogens such as the PAHs in tobacco smoke. Because the DNA nucleotide
sequence of the GSTM1 gene is known, it was possible to use a molecular
genetic technique known as polymerase chain reaction to rapidly screen patient
blood samples and determine whether a subject had two copies of the gene,
one copy of the gene, or was missing the gene. Individuals who had at least
one functional copy of the GSTM1 gene were less likely to develop
bladder cancer than individuals without the gene. Individuals who have two
copies of the gene probably have an even greater level of protection. The
findings show that genetic predisposition combined with environmental exposure
is an important factor in development of disease.
Fred Kadlubar, a researcher at the National Center for Toxicologic Research
studying biochemical pathways associated with carcinogen-DNA adducts, has
shown how certain individuals may be susceptible to some carcinogens and
protected from others. The enzyme acetyltransferase also plays an important
role in risk for bladder cancer among smokers. Individuals who are "slow
acetylators" inherit two recessive genes from their parents, and there
is a higher incidence of bladder cancer among smokers of this genotype.
Smokers who have at least one dominant gene are fast acetylators and have
lower susceptibility to bladder cancer. Further work also showed, however,
that fast acetylators had greater susceptibility to colon and rectal cancer
when exposed to harmful by-products of cigarette smoking. In addition, other
behaviors, such as eating well-done meat, also increase cancer risk in these
individuals. Changes in lifestyle, such as quitting smoking or modifying
diet, may help reduce these risks. Scientists and the public are becoming
increasingly aware of how important diet can be in the prevention of disease,
as researchers discover that certain chemicals in foods are necessary for
proper biochemical function of metabolic pathways. According to Kadlubar,
"Enzymes that play a role in detoxification are induced by eating yellow
and green vegetables." Such foods contain inducers of enzymes that
prevent formation of the carcinogen-DNA adducts that lead to cancer, such
as vitamin C, which reduces the rate of DNA damage resulting from nitrates,
and vitamin E, which is a free-radical scavenger and prevents oxidative
damage to DNA.
Frederica Perera of the Columbia University School of Public Health is
collaborating with researchers from Sweden and Poland to study gene mutation
and oncogene activation in relation to markers of exposure, effect, and
susceptibility. These studies show increased levels in PAH-DNA adduct formation
and other biomarkers as a result of environmental and occupational exposure
to chemicals. PAHs emitted from industrial and residential burning of coal
are known to have mutagenic and carcinogenic activity. In a recent segment
of the study conducted in Silesia, Poland, a heavily polluted city with
a high rate of cancer and infant mortality, multiple biomarkers such as
carcinogen-DNA adducts, chromosomal aberrations, sister chromatid exchange,
and activation of ras oncogenes were analyzed in blood. PAH exposure
from ambient air, drinking water, and the food supply in Silesia is 10-30
times higher than in most Western countries. There were significant increases
in PAH-DNA adducts, aromatic-DNA adducts, and chromosomal mutations, as
well as a doubling in ras oncogene expression in environmentally
exposed residents compared to control subjects who lived in a rural, less
polluted area.
Frederica Perera--Biomarkers may tell
us who is likely to get cancer. |
Although there was a correlation between exposure to PAHs and chromosomal
mutation, linking a specific environmental exposure to induced genetic modifications
relevant to cancer and reproduction risks, some people were especially sensitive
and others were resistant. Therefore, the Silesia population offers an opportunity
to determine if various susceptibility markers can explain interindividual
differences in response to a given exposure level of an environmental carcinogen.
The study also has relevance for other kinds of exposures in parts of the
world where PAHs are a common pollutant, particularly those areas with numerous
coal-burning factories.
In another study conducted by Perera and her colleagues, workers in an
iron foundry in Finland who were occupationally exposed to PAHs had significantly
higher levels of carcinogen-DNA adducts than controls. Fluctuations in carcinogen-DNA
adducts, carcinogen-protein adducts, gene mutation at the HPRT and
GPA loci, and chromosomal mutation are being historically monitored.
Lowered PAH exposure due to improved industrial hygiene and reduction of
industrial output, caused by an economic recession, has resulted in a decrease
in adduct levels. To assess individual variation in susceptibility, these
workers are also being screened by researchers at NIEHS for genetic traits
that govern metabolism and detoxification of PAHs. A profile of the interrelationship
of environmental exposure with genetic or acquired factors governing individual
susceptibility to mutagenic and carcinogenic effects is being charted. Said
Perera, "These findings suggest that adducts are not only an environmentally
relevant dosimeter but that they may also indicate heightened risk of cancer."
George Lucier of NIEHS adds that the results of this study "represent
a growing consensus that gene-environment interactions must be better understood
if we are going to be able to accurately estimate risks from exposure to
environmental agents." Ultimately, this approach will enhance risk
assessment, improve prediction of individual health outcome, and facilitate
prevention.
Slippery DNA
When DNA was first discovered, it was thought to be the perfect book
of life. Each letter of every word--every amino acid sequence in a gene--directed
cells to perform. But then came junk DNA, repeated or random stretches of
base pairs that serve no apparent purpose. Now comes slippery DNA, where
repeated fragments of gene sequence change in length frequently, the result
of editing that fails to keep coding tight and to the point. In some cases,
these changes cause disease.
Researchers at the University of North Carolina at Chapel Hill and Yale
University School of Medicine have discovered why stretches of repetitive
DNA make a genome dramatically unstable. They found this out by setting
up a novel experiment: knowing that DNA is transcribed into RNA and translated
into protein 3 base pairs at a time, they inserted into the genome of yeast
cells an unstable strand of genetic lettering, a marker of 29 base pairs.
A variety of responses occurred: some cells replicated with more than 29
base pairs in the marker, some with less. But they were all unstable.
The process of genome replication uses an enzyme called DNA polymerase,
which copies DNA. But DNA polymerase tends to make mistakes at a higher
rate than can be tolerated by cells. A set of specific proteins in the cell,
dubbed the DNA mismatch repair system, acts like an editor in recognizing
these mistakes and correcting them. Specifically, the repair system corrects
mistakes the polymerase makes when it picks up the slipped DNA strand at
the wrong place on the repetitive tract, resulting in a segment that is
either longer or shorter. The repair system reduces the rate of error from
one in a thousand base pairs to one in a million. The researchers found
that slippery strands of DNA became more unstable when the mismatch repair
system was absent, leading to 100- to 700-fold increases in replication
errors.
What's more, instability in the experimental yeast cells resembles mutations
found in human cells, particularly those seen in some types of colorectal
cancer, said one of the researchers, Thomas D. Petes of UNC's Department
of Biology. "In these [colorectal cancer] patients the DNA is unstable,
suggesting exactly the sort of things we see when the mismatch repair system
is knocked out."
Petes says that similar errors may underlie other diseases, produced
when other repair systems go awry. For example, failure to repair DNA damage
caused by an environmental insult like ultraviolet radiation may result
in skin cancer. Petes said that efforts are now underway at many research
institutions to study whether other cancers could result from loss of DNA
repair systems. "The theory is that people who develop cancer are getting
high rates of mutations during cell replication. Some of these mutations
may knock out tumor-suppressor genes or turn on proto-oncogenes. It's not
clear yet."
The inventory of diseases caused by mismatch repair systems and slippery
DNA will add to a growing list of other fairly common inherited conditions
produced by similar errors of DNA replication. Fragile X syndrome, Huntington's
disease, and myotonic dystrophy are all produced when one or two amino acid
sequences in a specific gene are replicated with a stutter and become greatly
amplified.
Renee Twombly |
DNA repair. In addition to signaling exposure or susceptibility
to a mutagenic agent, biomarkers also indicate inability to repair damage.
DNA has a network of repair mechanisms to prevent injury and maintain the
integrity of the genetic material that drives normal cellular and biological
processes. Under normal circumstances, during replication of DNA, repair
enzymes travel along the molecule and excise mismatches of nucleotide base
pairs or aberrations in molecular structure, such as adducts. When there
is a deficiency in the system of repair genes of an individual, that person
is predisposed to developing cancer. Excision repair enzymes also provide
backup protection for correcting errors in the genetic code and architectural
distortions during replication. Just as metabolic responses to chemical
exposure vary from one person to another, DNA excision repair is highly
variable among individuals of any population.
The idea that recovery mechanisms could repair damaged DNA was conceived
by Philip C. Hanawalt when he was a graduate student in Richard Setlow's
laboratory at Yale University in the early 1960s. In subsequent years, Hanawalt
has been a leader in pioneering research to elucidate the complicated system
of nucleotide-excision repair (NER). Many agents, both endogenous and environmental,
damage DNA. The recovery mechanism has to be highly specific to ensure that
repair enzymes act only on damaged DNA instead of other DNA configurations
that occur during transcription (synthesis of messenger RNA). Hanawalt has
shown that NER is linked to transcription. As polymerase I, the transcription
enzyme, moves along a DNA strand and approaches a lesion, it pauses, and
a multienzyme complex recognizes and binds the damaged portion of DNA. The
interaction of the repair enzymes initiates excision of the damaged segment,
then transcription continues, and the polymerase, using the old DNA strand
as a template, generates a new piece of DNA and replaces the excised segment
with the correct nucleotide pairs, which are then bound to the DNA molecule.
Philip Hanawalt--DNA recovery mechanisms
must be highly specific. |
An example of an inherited disease characterized by defective NER is
xeroderma pigmentosum, in which people are highly sensitive to ultraviolet
light and have more than a 2000-fold increased risk of skin cancer. By studying
the genetic sequences in xeroderma pigmentosum that are involved in correcting
UV sensitivity and repairing the DNA, significant progress has recently
been made in cloning and characterizing human NER genes. Seven proteins
have been implicated in the early steps of damage recognition and nucleotide
excision. It has been shown that there is preferential repair in transcribed
DNA strands, and there is greater efficiency in recognizing and repairing
adducts formed by different chemicals within certain nucleotide sequences.
Inherited susceptibility. Although inheritance of a defective
gene is a factor in a small percentage of people who develop cancer (less
than 5%), susceptibility in such individuals is many times greater in people
who carry a defective gene. Inherited cancers provide an opportunity to
understand the genetic mechanisms of mutagenesis.
Breast cancer has dramatically increased in the last 50 years. It is
estimated that 1 out of every 10 women will develop breast cancer over the
course of her lifetime. The extent to which this increase can be attributed
to better detection versus diet or exposure to environmental pollutants
is not known. Mary Claire King and colleagues at the University of California
at Berkeley have discovered the region in the human genome where the gene
for inherited breast cancer resides. This region, on the long arm of chromosome
17, is composed of approximately 400,000 nucleotides; some code for proteins
and others are genes. Roger Wiseman of NIEHS is one of many scientists trying
to identify the genes in this region with the goal of finding the gene for
increased susceptibility to breast and ovarian cancer. Work on the gene,
named BRCA1, promises to shed new light on the etiology of breast
cancer. Wiseman says that "once the gene has been found, it will be
possible to determine whether individuals have germline mutations that would
predispose them to developing breast cancer."
Roger Wiseman--A blood test for the
breast cancer gene may be available within a year. |
It is already known that the genetic alteration in the defective gene
is confined to one amino acid. One in 200 women inherit a bad copy of the
gene, and of those, approximately 80% develop breast or ovarian cancer.
Even when an individual inherits a defective copy of the gene, other factors
come into play in the initiation of tumorigenesis. Environmental agents
may inactivate genes that are important in genetic control. According to
Wiseman, "The p53 tumor-suppressor gene has been inactivated or deleted
in one-half to two-thirds of all breast cancers."
Once the BRCA1 gene has been identified and cloned, it will provide
valuable clues for unlocking the mystery of the genetics of breast and ovarian
cancer. With that knowledge, detection, risk assessment, and opportunities
to develop new therapies and examine a causative role for environmental
agents will be greatly enhanced. Scientists believe this breakthrough is
imminent. Wiseman predicts that "within the next year a blood test
to detect mutations in the gene will be a fairly routine assay, depending
on how big the gene is and how many mutations are in it." According
to Wiseman, such an assay will make it possible to identify high-risk individuals,
develop better therapies for treating people with tumors who have a mutation
in the gene, and study what kinds of environmental factors increase risks
when a woman has inherited a bad copy of the gene.
For individuals with an inherited predisposition to colon cancer, a disease
that threatens 1 in 200 Americans, these risk identification and treatment
possibilities may come even sooner. On 3 December 1993, molecular biologists
Richard Kolodner of the Dana-Farber Cancer Institute and Richard Fishel
of the University of Vermont announced the discovery of the gene for inherited
colon cancer, barely edging out a team from Johns Hopkins University School
of Medicine, headed by Bert Vogelstein and Kenneth W. Kinzler, who reported
the same discovery two weeks later in the journal Cell. "It
was like finding a misplaced comma in a 300-volume encyclopedia,"said
Jeffrey Trent, a researcher at the National Center for Human Genome Research
(NCGHR), in an article announcing the discovery in the New York Times.
Researchers expect to have a blood test available to test for the gene,
which is blamed for the 15% of colon cancers estimated to be inherited,
as well as many inherited cancers of the uterus and ovaries in women, within
six months.
Vogelstein has shown that colon cancer generally requires the actions
of half a dozen or more oncogenes to initiate tumors. The newly discovered
gene, MSH2, located on chromosome 2, is a flaw in the system of DNA
repair and creates oncogenes. A normal copy of the gene produces a protein
that corrects for errors in DNA during replication. MSH2 lacks this
ability and allows random mutations to accumulate in cells, which eventually
activate enough oncogenes to produce a tumor.
Colon and rectal cancer strike about 158,000 people in the United States
each year, killing 60,000. Scientists estimate that people who carry the
gene have an 80-90% risk of developing colon or rectal cancer and a 60-85%
chance of developing cancer of the uterus or ovaries. With knowledge that
they carry the gene, however, individuals can take preventive steps such
as changing their diet and having frequent examinations. Says Frances Collins,
director of the NCGHR, "For most people, knowing you have this gene
will probably save your life."
The Search for Cancer-Causing Genes
Just as archaeologists study excavated artifacts to understand the origins
and declines of ancient civilizations, Curtis C. Harris and co-workers at
the National Cancer Institute are conducting "molecular archaeology"
to identify the sources and types of alterations in genes that play a key
role in the onset of many cancers. Says Harris, "Differences in patterns
from one tissue type to another are clues to etiological factors that are
mechanisms of genetic change." It is important to determine if these
molecular changes are inherited or a consequence of exposure to environmental
agents.
Curtis Harris--Searching for molecular
clues to genetic change. |
Although there are multiple types of cancers in different tissues and
organs, the discovery that there is a single gene that plays an important
role in a wide variety of tumors was surprising to scientists. The most
common cancer-related genetic change is in the p53 tumor-suppressor gene.
Harris's work has revealed a spectrum of mutations in the p53 gene associated
with a variety of tumors. The p53 gene encodes a pleiotropic nuclear phosphoprotein,
i.e., one that is involved in many functions including cell cycle control,
DNA repair, DNA synthesis, and cell differentiation. The positions of tumor
mutations in the p53 gene sequence correlate with regions of the p53 protein
that are important for its biological activities. A single base substitution
can result in loss of function or production of this protein essential to
tumor suppression. Endogenous and exogenous mutagens generate specific kinds
of base substitutions at preferred sites. There are exceptionally high numbers
of mutations clustered within certain regions of a gene, creating mutational
"hotspots" within this gene. Different types of cancer, including
colon, lung, liver, and breast cancers, lymphomas, and leukemias, are associated
with specific mutational hotspots.
A significant finding of Harris's study of the p53 gene is that certain
mutations correlate with precise exposure to carcinogens. In areas of China
and southern Africa where people are exposed to aflatoxin and hepatitis
B virus, there is a high incidence of liver cancer. Applying the technology
of polymerase chain reaction to amplify DNA from liver tissue samples of
cancer patients and then sequencing the DNA led to the discovery that the
majority of cancer patients have a high frequency of a single point mutation
at codon 249 of the p53 tumor-suppressor gene. Using polymerase chain reaction
and sequencing DNA from healthy tissue from these same patients, it was
determined the mutation was not inherited; it was a direct result of exposure
to aflatoxin. This work shows that mutations that confer a selective growth
advantage to cells occur nonrandomly and produce characteristic fingerprints
of modifications in DNA. Harris's examination of the broad range of p53
mutations in different tumors is shedding light on environmental and/or
endogenous processes that contribute to carcinogenesis.
Bermuda triangle? Disease is a result of the
interaction between genes, age, and the environment. |
Human Genome Project promises to shed new light on the human genetic
code and the mechanisms by which changes in that code produce biological
effects that contribute to the complex etiologies of many cancers. This
new knowledge will generate novel approaches to diagnosis and treatment
of disease. Therapies will include gene replacement, development of drugs
that mimic defective proteins, immunology strategies employing serum antibodies,
and development of cancer vaccines. With better methods for phenotyping
and genotyping individuals, it will be possible to develop cancer risk profiles
for any individual within the next 10 years. Such information will not only
strengthen quantitative risk assessment, it will provide valuable data for
determining susceptibility and focusing on appropriate chemopreventive strategies.
With the knowledge that individuals vary greatly in their response to
xenobiotic exposure, the basic assumption that all people respond equally
to environmental chemicals, the heretofore modus operandi of risk
assessment, needs to be refined. By using knowledge gained from molecular
epidemiology about the interaction of environmental exposure and the susceptibility
of individuals to predict biological effect, the accuracy of risk assessment
can be improved and methods of prevention for targeted individuals can be
prescribed. As advances in biomarker research continue, developments in
this field will have a dramatic impact on environmental and public health
policies and will stimulate debate on controversial ethical issues in the
years to come.
Mary Eubanks
Mapping the Human Genome: A Status Report
When the Human Genome Project began in 1988, many skeptics, and some
supporters, doubted that it could meet its ambitious goals and wondered
if the results of such a program could justify the enormous cost. Mapping
the entire human genome--somewhere around 3 billion base pairs--by the year
2005 seemed an almost impossible task, given the technology available at
the time. Yet as the project enters the fourth year of its initial five-year
phase, questions about its validity have faded and "advances in genome
research have already changed the way the research is being done,"
says Frances Collins, director of the National Center for Human Genome Research
(NCHGR), prompting the release of a new set of goals to guide the project
into its next phase.
The program represents a massive international effort to answer questions
about genetic components of illness and disease. By mapping all 24 chromosomes
of the human genome, scientists hope to be able to isolate genes responsible
for particular diseases. In 1993 alone, genes responsible for a number of
diseases have been found including Menkes syndrome; glycerol kinase deficiency;
adrenoleukodystrophy, the disease portrayed in the film Lorenzo's Oil;
Lou Gehrig's disease; and Huntington's disease. In addition to determining
who is at risk for developing heritable diseases, researchers hope the project
will lead to the ability to detect risk of genetic damage from exposure
to chemicals and the genetic basis for individual susceptibility to damage
as a result of such exposure.
DNA superhighway. The Human Genome Project
is on the fast track to genetic discovery. |
A New Set of Goals
Recognizing that advances in technology have put the Human Genome Project
on or even ahead of schedule in several areas, a group of advisors from
NIH and DOE, with input from scientists, scholars, and the public, drafted
goals to expand the scope of the project beyond the first five years. In
an October 1 article in Science, Collins and David Galas, former
associate director of the Office of Health and Environmental Research of
the Department of Energy, outlined the extended goals.
Genetic mapping. The most significant international cooperative
project thus far is the work of NCHGR and France's Centre d'Etude du Polymorphisme
Humain (Center for the Study of Human Polymorphism) to develop a genetic
map containing more than 1400 markers and covering 90% of the human genome.
More than 70 laboratories around the world cooperated in the work, with
the original five-year goal of producing a high-resolution linkage map.
The program directors anticipate that this goal will be met on time. The
new goals call for development of technology for rapid genotyping and markers
that are easier to use and to develop new mapping technologies. This would
make the genetic map more useful and more accessible, for example, by allowing
nonexperts to type families for medical research.
Physical mapping. Physical maps make cloned segments of
DNA easily usable for the "great gene hunts." Thus far, physical
maps have been assembled for two human chromosomes. For the Y chromosome,
a research group at the Whitehead Institute for Biomedical Research assembled
the entire functional portion into an organized set of clones that represent
the intact chromosome. A French group, collaborating with an international
team, developed a physical map of the long arm of chromosome 21. Among the
diseases associated with genes on chromosome 21 are Down's syndrome, a number
of neurological diseases including some forms of Alzheimer's, and amyotrophic
lateral sclerosis (Lou Gehrig's disease). Researchers predict that significant
sequence maps should be ready in the next two to three years with varying
amounts of detail. The average interval between sequence-tagged site (STS)
markers will be about 300 kilobases (kb). While a map with that kind of
interval will be of limited use to researchers searching for disease genes,
sequencing technology is progressing rapidly, and the original goal of a
physical map with markers at intervals of 100 kb, which would serve both
mappers and sequencers, is still possible within the first five years. The
expanded goal is to provide STS maps at even higher resolution and to develop
cloning systems that are well integrated with advanced sequencing technology.
DNA sequencing. Sequencing will provide a variety of information
about all human genes, including their regulation and inheritance. Work
on model organisms E. coli and C. elegans, a roundworm, is
transferable to similar efforts to make large-scale human DNA sequencing
faster and cheaper. The original goal was to have technology available to
sequence DNA at 50 cents per base pair; the current cost is about $1.00
per base pair. The goal may be reachable by 1996, but the rate at which
DNA may be sequenced won't be sufficient for sequencing the whole genome.
The new goal is to increase sequencing capability (to a rate of 50 megabases
per year by the end of the period) by increasing the number of research
groups oriented to large-scale production sequencing. Collins calls for
an immediate investment of $100 million to develop efficient approaches
to sequencing one to several megabase regions of DNA of high biological
interest, to develop technology for high through-put sequencing; and to
build up the sequencing capacity by sequencing the standard model organisms
(mouse, yeast, E. coli, C elegans, and S. cerevisiae) by 1998.
Gene identification. The goal of gene identification wasn't
specified originally, although it certainly formed an implicit element of
the project. It is an area made feasible by progress in mapping and development
of technologies. The new goal involves developing efficient methods for
identifying and placing genes on physical maps or sequenced DNA.
Technology development. Technology development in all areas,
particularly those such as automation and robotics, is vital to the completion
and success of the project and has so far been insufficiently funded. In
general, the goal is to substantially expand technological developments
to meet the needs of the Human Genome Project as a whole.
Informatics. The rapid growth of information on genome
mapping and sequencing make computer-based data management systems increasingly
important. The major goal is to create technology that will be available
to researchers across the board. Here, too, the project's original goals
are being met with some efficiency. Several NCHGR- and DOE-supported databases
are available to the research community, either at no cost or for the cost
of a telephone call to access them. The Genome Data Base at Johns Hopkins
University is the main public facility, with approximately 4000 registered
users. It contains information on maps, polymorphisms, and probes. Other
databases either in operation or under development will provide access to
information on the mouse, Drosophila, and yeast genomes. The expanded
goals are to continue creating the algorithms, software, databases, and
tools to facilitate access to the information exchange and expand the ability
to compare and interpret information.
Model organisms. Progress is being made in sequencing model
organisms and is expected to exceed original goals. The expanded goals involve
finishing a high resolution STS map of the mouse genome and to continue
or finish sequencing E. coli, S. cerevisiae, C. elegans, and Drosophila
genomes. Emphasis will be on sequencing selected areas of mouse DNA of high
biological interest side by side with corresponding human DNA.
Training. The original training goal was to eventually
support 600 pre- and postdoctoral trainees a year in programs to take advantage
of the knowledge generated by the Human Genome Project such as clinical
applications and applications to basic research. That turned out to be unrealistic,
largely because of the inability to train so many people in the relevant
interdisciplinary sciences. But now that there are more genome centers,
training opportunities should expand. The revised goal encourages training,
but doesn't specify a certain number of trainees.
Technology transfer. One of the original assumptions under
which the project has consistently worked is that knowledge will be made
available, not only to government and academic scientists, but to the private
sector, including physicians and members of private industry. Both the NIH
and DOE genome programs are required to share information with the private
sector. New data and materials have to be released within six months of
their creation by submitting the information to public databases and repositories.
In practice, much of the new information is disseminated before the 6-month
deadline. The mutual exchange of technology increases the flow of information
from the genome centers and brings relevant technologies from other fields
in. This practice is again encouraged in the new goals.
Ethical, legal, and social implications (ELSI). In the
first set of goals, four areas were identified by ELSI advisors for emphasis:
privacy of genetic information, safe and effective introduction of genetic
information into the clinical setting, fairness in the use of genetic information,
and professional and public education. The expanded goals recognize the
ever-increasing need for strong public policies based on sound science to
deal with the issues that develop as a result of the project. New goals
include continuing to identify, define, and develop policies to address
issues, developing and disseminating policy options regarding genetic testing
and services, fostering greater understanding and acceptance of human genetic
variation, and expanding public and professional education sensitive to
sociocultural and psychological issues.
Outreach. An original goal of the program that continues
to be essential to its success is to make available to the community the
products of the Human Genome Project, in all its forms. The new goals expand
outreach goals by encouraging flexible distribution systems, potentially
developed through the private sector, and rules for information sharing
that promote prompt delivery within six months of development.
Money Crunch
The Human Genome Project takes its funding from both NIH and DOE. But
the goals are extremely ambitious and expensive, and NIH funds are increasingly
tight. Originally, a committee of the National Research Council predicted
that the program would cost about $3 billion over its 15-year life span,
or about $200 million a year. The rosy estimates have suffered in the budget
crunch; today, when even the most popular programs are taking budget cuts,
the genome project's $165 million a year seems relatively generous. Adjusted
for inflation, though, that comes to about 75% of what NRC predicted the
program should cost. As a result, the priority had to go to gene mapping;
sequencing and other equally important facets of the program have suffered.
There is no guarantee that the next couple of years will be any easier.
The new plan calls for $100 million a year devoted entirely to sequencing
technologies, which would raise the total to the original $200 million.
A 1991 projection of needs for the program increased its estimation of the
yearly amount needed by 1994 and 1995 to close to $250 million. If the money
doesn't come through, Collins predicts that the timetable won't be met;
that would mean delayed medical benefits and, equally important, a loss
of U.S competitiveness in biotechnology. Even if enough money is forthcoming,
it may be hard to meet the sequencing goal. The program expected that researchers
would be able to fund new approaches to sequencing, like mass spectrometry
or atomic force microscopy. Instead, they will have to try to improve the
technologies that already exist and do as much cost cutting as possible
while trying to meet the program's goals. Still, Collins and Galas argue
that the genome project has already had a profound influence on biomedical
research and say they expect exciting discoveries in the years ahead.
Marion Zeiger |