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Clinical Chemistry Forum |
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104-4283. Fax 215-662-7529;
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Abstract |
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 Top Abstract IntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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Introduction |
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TopAbstractIntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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The Human Genome |
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TopAbstractIntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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Progress on the publicly available DNA sequence has been exponential over the past few years, and the rate is expected to increase in the future. Approximately 195 million base pairs, or about 6.5%, of the human genome currently is available. However, identification of the DNA sequence does not necessarily mean the genes in that sequence have been identified. Currently, ~30 000 genes have been identified, but not all of these genes have been sequenced. Even if a gene sequence is complete, establishing the function of even a single gene can take a lifetime of research for an investigator and his or her laboratory to identify where and when a gene is expressed, the role of the encoded protein in cellular function and disease, and the interaction of that protein with other proteins, mRNAs, and genes in the cell. Then remember that this needs to be done for ~70 000 genes, one at a time. It is clear, and is one of the goals of the Human Genome Project, that more rapid methods are needed for elucidating gene product function and interactions. Fortunately, this information will be developed over time, and as clinically relevant mutations are discovered, molecular laboratories will rapidly translate the information into diagnostic tests for genetic assessment.
human genetic variation
Once the human sequence is complete and all the genes are
identified, the question remains: Whose sequence do we have?
Single-nucleotide polymorphisms (SNPs), or one-base differences found
among human populations, are estimated to occur about every 1000 bases
in the human genome. Other types of variations are estimated to occur
less frequently than every 1000 bp. This genetic variability between
individuals is basically the raw material for evolution; it is what
makes one person different from another. It makes one protein function
slightly better than another protein, and this is what provides the
selective advantage or disadvantage for subsets of humans over
evolutionary time. The availability of this genetic variability
information also raises many ethical, legal, and social issues
concerning how this genetic difference information is used. We will
have the potential to identify, or even predict, such human
characteristics as intelligence, physical features, and personality
characteristics as well as the risk of a vast array of medical
conditions. In many cases, these characterizations may have
implications for specific races or subgroups of the human race. The
worst possible outcome of this human genome information could be the
justification of prejudice. Therefore, it is essential that guidelines,
understanding, and even laws be established in a proactive effort to
prevent the misuse of this genetic information.
The identification of SNPs and other differences within gene coding sequences and the understanding of how these differences influence the function of the gene product will be the basis for defining the genetic causes of a vast majority of medical conditions, from classical genetic diseases to multigenic medical conditions such as cardiovascular disease, arthritis, hypercoagulability, schizophrenia, and almost any other medical condition known. Each of these conditions eventually will be characterized by a set of gene mutations or polymorphisms that interact with environmental factors to create a risk for that specific disease. The ability to define an individual's genetic risk for specific diseases, probably directed by family history rather than general screening, will provide the basis for preventive medicine in the future.
One example of genetic heterogeneity in one gene is described in a recent article by Nickerson et al. (2), published in Nature Genetics. These investigators identified the differences in exons 4 through 9 of the lipoprotein lipase (LPL) gene between three different groups: 27 African Americans, 23 people of European ancestry from Minnesota, and 24 Finns. The LPL gene plays an important role in lipid metabolism, and the genetic variability in this gene may be useful for cardiovascular disease risk assessment. The investigators sequenced exons 4 through 9 of the LPL gene in the 71 individuals and identified 88 sequence differences: 79 SNPs and 9 insertion-deletion changes. Interestingly, 34 of the 88 differences were found only in one of the three populations studied, defining one of the genetic differences between these populations. Only 7 of the 88 differences were present in the coding region of the LPL gene, and only 4 of those 7 alter the amino acid sequence of the protein. One of these four coding sequence polymorphisms has already been associated with premature atherosclerosis, but the other three polymorphisms are new and need to be investigated for their influence on cardiovascular disease or other characteristics. This is one study of one region of one gene, yielding information that requires further study to understand the implications of these differences. The amount of work that will be required to characterize the entire human genome across all individuals is immense.
medical impact of genetic information
What impact will the Human Genome Project and this vast amount of
genetic information have on medicine? Certainly, this genetic
information will be translated into an improved ability to diagnose
almost all genetic or genetically influenced diseases and the risk for
such diseases. The mutations or polymorphisms that cause single gene
diseases probably will be defined most readily, to be followed by
expansion of the genetic understanding of multigenic diseases over
time. The genetic understanding of disease will allow more rapid and
accurate diagnosis and risk assessment for a wide range of diseases. In
addition to the genetic basis of disease, the genetic basis for almost
any human difference probably will also be defined. The use, and
potential misuse, of individual genetic information needs to be
monitored carefully and controlled to prevent discrimination and other
types of misuse, so that the benefits of this information can be
maximized.
Improvement in our ability to diagnose and predict risk for disease is an excellent advancement, but the ultimate goal of medicine is to cure or even prevent disease. What impact will genetic information have on therapeutics? Genetic information already is being used to design more specific drugs. Once we understand how a specific change in a protein alters the function of that protein to cause disease, drugs can be designed that compensate for the altered protein function and correct the disease-causing defect. In addition to drug design, biotechnology companies already exist that assist pharmaceutical companies in using genetic information to design drugs and to predict the types of toxicity that may occur with the use of these drugs, before they are even tried in animals. Genetic information also will allow investigators to implement more effective drug trials or clinical therapeutic trials either by enrolling patients who would be predicted to respond to a specific therapy based on genetic differences or by defining the genetic differences between the responders and non-responders for a specific therapy. For example, if a new drug treats only 30% of patients effectively, that drug may not be considered very effective. However, if the 30% of patients who responded to the drug all have a specific polymorphism in a specific gene, then the polymorphism can be used to guide a physician in the choice of treatment for a specific patient. Such "boutique" drugs will provide pharmaceutical companies with a niche in a particular market. Therefore, in the future, the right drug will be used in the right patient, which will be defined by genetic testing. At the present time, there is a gap between what we can diagnose and what we can treat. This gap will most likely widen before it begins to narrow, as therapeutics catch up with diagnostics. And beyond therapeutics is the development of preventive interventions, such as diet, exercise, or even drugs to prevent the onset of disease (3)(4)(5).
gene therapy
Knowledge of the human genome combined with the ability to
manipulate that genome, as is already routine with the mouse and other
organisms, is the basis for treatment of disease by gene therapy. To
date, when speaking of gene therapy, one is referring to the
manipulation of the genome of somatic cells, differentiated cells of
the human body. Somatic gene therapy basically uses a gene as a drug. A
gene encoding a normal or therapeutic protein is put into cells via in
vitro manipulation or by use of a viral vector in vivo. The protein is
expressed by the altered cells in the patient, and that protein product
either replaces a dysfunctional protein for treatment of genetic
diseases or is otherwise therapeutic, as for gene therapy treatment of
cancers. Gene therapy requires proper cell targeting and physiologic
expression of the gene product when used as therapy for a genetic
disease. Given the complexity of gene therapy, treatment with the
protein as a drug is an alternative, e.g., the use of insulin for
treatment of diabetes. Problems with using the protein include
achieving physiological concentrations over time as well as
patient compliance with taking the drug. For these reasons,
investigators are working to devise methods for effective gene therapy
for many genetic diseases (6)(7).
A much more controversial topic is germline gene therapy, which basically is active evolution—the potential to correct genetic defects in germline cells or embryos to correct these defects permanently. At this point, germline gene therapy is universally banned, although several countries are beginning to discuss the scientific potential of human germline gene therapy. It is well developed and regularly applied to mice; therefore, the controversy is not with technical issues but with ethical issues. Do we want to start actively participating in our own evolution? With the elimination of mutations or polymorphisms that we define as harmful to humans, we would create genetic homogeneity, which is disastrous from an evolutionary perspective. With the encounter of new environmental factors, such as a new virus, there is the potential that no or very few people in a homogeneous population would be resistant. In addition, whose genome would be upheld as the ideal, and what genetic manipulations would be "allowed"? Although germline gene therapy currently is unacceptable, the discussions about the ethical, social, legal, and medical issues surrounding germline gene therapy should continue.
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Technological Advances |
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TopAbstractIntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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dna chips
All DNA chips are not the same. In general, three types of DNA
chips are currently available or being developed, each with a different
function. One type of DNA chip is used for automated DNA sequencing.
These chips have an array of specific oligonucleotides to which the DNA
to be sequenced is hybridized. Analysis of the hybridization patterns
allows determination of the sequence of the specimen DNA. The advantage
of this methodology in a clinical laboratory is enormous for turnaround
time and for testing and technologist time required, if compared with
gel-based DNA sequencing (8)(9).
The second type of DNA chip is used to determine gene expression profiles for cells or tissues, either as differential expression between two cell types or expression for a single cell type. The research potential for this technology is almost endless, whether comparing two different tissue types, comparing a naive cell to a drug-treated cell to look at drug effects and potential drug toxicities, or comparing a cancer cell to its normal counterpart. These gene expression chips hold great potential for the characterization of cancer cells for diagnostic applications, as well as for the definition of new chemotherapeutic targets. Ideally, in the future, gene expression analysis initially will be performed using all the human genes to identify those that are differentially expressed and potentially important diagnostically or therapeutically. Only after this initial analysis will the diagnostically relevant subset of genes be used routinely in the clinical laboratory to differentiate different types of cancers, to provide prognostic information, or to predict therapeutic response to different treatments. Of course, it is a major leap from the original differential gene expression analysis to understanding the importance of the differentially expressed mRNAs and applying this information to diagnosis, prognosis, and therapy. Certainly, the diagnostic applications will be the ones most readily developed, with the prognostic and therapeutic applications following. Although the focus here is genetic analysis, it is essential to also mention the concept of proteomics. Proteomics analyzes cellular protein concentrations for the same purpose as gene expression analysis, and some would argue, with greater correlation with cell function. mRNA concentrations do not always reflect protein concentrations, and protein expression profiles may be a better reflection of cellular function.
The third type of DNA chip is the "lab-on-a-chip", microfluidic systems that perform all the steps of molecular genetic analysis on a chip. This type of DNA chip will use blood as input, and then automatically perform many molecular processes, including nucleic acid isolation, amplification, restriction endonuclease digestion, hybridization, and detection. These are the DNA chips that will allow molecular genetic testing to progress to an automated system with minimal technologist time requirements. Except for genetic tests based on commercially available test kits, labor costs are the most expensive component of molecular tests. Therefore, automation has the potential to reduce costs by decreasing the technologist time for testing. In addition, these DNA chips use very small reagent volumes, which will reduce reagent costs.
mass spectrometry
Mass spectrometry is being used for the analysis of DNA molecules,
with applications for mutation screening, DNA sequencing, or pathogen
detection. The advantage of mass spectrometry is that each analysis
takes only 1 min or less. The most common type of mass spectrometry
being adapted for molecular analysis is matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
Several technical hurdles are being worked on to allow analysis of
larger DNA fragments by this methodology, including prevention of
fragmentation of the DNA by the laser irradiance and adduct formation,
which lowers resolution (10). A recent report describes the
use of MALDI-TOF mass spectrometry for sequence analysis of exons 5 to
8 of the p53 gene (11). A total of 21 primers
were required to sequence 670 bp of the p53 gene, with an
average of 35 bases determined per reaction. Although this may
seem like a lot of work, consider that no sequencing gels are poured,
that each reaction takes <1 min to complete, and that the
methodology is highly compatible with automation.
hplc analysis
HPLC is another familiar method being adapted for the analysis of
DNA fragments, by separating mixtures of DNA fragments based on size
alone or based on size and sequence. This method can be used for
mutation screening of large genes, similar to conformation-sensitive
methods of mutation screening such as single-strand conformational
polymorphism. The advantage with HPLC is that the fragment size can be
1000–1500 bp, compared with 300–400 bp for most gel-based
conformation sensitive methods. In addition, it is not necessary to
pour, load, and run a gel.
capillary electrophoresis
Capillary electrophoresis (CE) instruments allow sequence analysis
of DNA fragments without the time and expense of pouring sequencing
gels. In a clinical laboratory, CE is an excellent option for DNA
sequencing or accurate DNA fragment sizing, with application for many
genetic tests. If a single sample in the assay does not work well, it
can easily be reanalyzed with different loading conditions in an
additional 20–30 min without having to process an entirely new gel.
One of the limitations of CE is that the instrument performs sequential
analysis of specimens and requires 20–30 min per sample. However, new
versions of CE instruments with 96 capillaries that can simultaneously
analyze 96 samples will soon be available. This is comparable in
capacity to the largest gel-based automated DNA sequencers currently
available, and much more rapid because there is no gel to pour, load,
and run. The system also allows flexibility for the clinical
laboratory, which may have 1 or 50 specimen batches for a genetic test.
robotics
Although DNA chips and other methods have great potential for
automation of nucleic acid analysis, robotics will be useful for the
clinical laboratory to automate various steps of assays when completely
automated testing is not available. Robotic instruments can perform
nucleic acid extractions and set up reactions. This will greatly reduce
technologist hands-on time, which is one of the more costly aspects of
molecular genetic testing. As the molecular testing capabilities expand
with our increasing knowledge of the human genome, robotics may allow
genetic laboratories to continue to expand their testing repertoire
without having to continually expand the laboratory staff.
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Ethical, Legal, and Social Issues |
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TopAbstractIntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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genetic counseling
As genetic testing becomes standard practice for the diagnosis of
many "medical" diseases, such as hypercoagulability or
cardiovascular disease, who will provide adequate counseling to
patients and their family members? Genetic counselors will be required
to work with physicians to help patients understand the genetic test;
the risks, benefits, and accuracy of the test; and the implications for
family members. The other option is to ensure that physicians are
properly trained to understand genetic testing and the importance of
proper counseling. Clinical laboratories can assist in providing
appropriate counseling information to physicians and patients along
with the test results, but even this will not ensure that the
information is properly communicated to the patient. Genetic testing
also has implications for the doctor–patient relationship because
these test results have implications for the family members of the
patient who were not a traditional part of the doctor–patient
interaction. Who will be responsible for communication with the family,
and how does the privacy of medical information apply to genetic
testing?
gene patents
If the entire human genome sequence were patented in pieces by
hundreds or thousands of base pairs at a time, development of genetic
tests for multigenic diseases would be difficult because of
cross-licensing issues. Indeed, the human genome is being patented, and
the cross-licensing issues are affecting the development of
applications for the genetic information. Because of gene patents and
the exclusive licensing of patent rights to a single laboratory, one
possible future is that the molecular genetic laboratory will be a
send-out laboratory, simply directing specimens to the appropriate
exclusive laboratory for each genetic test. This is already the case
for Charcot-Marie-Toothe testing and apolipoprotein E genotyping for
Alzheimer disease, and is under negotiation for other disease gene
patents, including hereditary hemochromatosis, spinocerebellar ataxia,
and Canavan disease. Single-provider genetic testing is inadequate for
many reasons, including the lack of competition for pricing and quality
of testing, the inability to confirm a test result by a second
laboratory, and limited access to specimens for further investigation.
Unless the gene patent laws are changed to mandate broad licensing of
gene patents at a reasonable royalty as established by the courts, the
future of genetic testing may be very limited.
economic issues
Several economic issues will influence the future of genetic
testing. The equipment that will make genetic testing more rapid,
automated, and less expensive is changing rapidly as the technology
develops and is very expensive. Laboratories must choose carefully
among competing technologies, trying to determine which have the
broadest range of applications for the least amount of money. Capital
equipment budgets for most clinical laboratories currently are rather
limited, and new equipment purchases require extensive justification.
Although some companies have options for reagent rental agreements for
obtaining costly equipment, many companies are familiar only with
research laboratories and do not understand the clinical market. A
second economic issue is that the reimbursement for genetic testing by
third-party payers does not reflect the cost of performing the tests.
The Medicare reimbursement for the molecular CPT4 codes is far below
the cost of performing genetic tests and needs to be corrected.
Finally, outcomes studies are needed to determine whether expensive
genetic testing is cost-effective in the context of the cost of
complete medical care. Are diagnoses provided more rapidly and with
fewer invasive procedures by genetic testing, leading to shortened
hospital stays and decreased overall medical care costs? As with most
any aspect of life, economic issues have the potential to influence the
future of genetic testing.
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Conclusion |
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TopAbstractIntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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What is the future of genetic testing? In the future, we will be able to perform genetic analysis for any genetically encoded feature of a person to diagnose current illness, predict future disease risk, and to define other less medically relevant traits. The testing will be directed at specific diseases, probably based on family history, rather than general screening of the entire genome. This testing will be highly automated for the more common diseases or disease risks; however, rare genetic disease tests may still be performed manually. Over time, the genetic diagnostic tests will become available most rapidly, followed by the genetically directed therapies, and eventually, the preventive interventions. Because many of the ethical, legal, and social issues surrounding genetic testing already are being addressed, the potential for misuse of genetic information in the future will be limited. The challenge for the clinical genetic laboratory experiencing and participating in this information and technological revolution is to keep pace with the rapid advances in our understanding and testing capabilities to provide state-of-the-art genetic testing using high professional standards and to ensure that the genetic test results are used in a morally, ethically, and socially responsible way.
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References |
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TopAbstractIntroductionThe Human GenomeTechnological AdvancesEthical, Legal, and Social...ConclusionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  D. G.B. Leonard Improved Method for Diagnosis of Charcot-Marie-Tooth Type 1A: Patent Pending? Clin. Chem., May 1, 2001; 47(5): 807 - 808. [Full Text] [PDF]
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 M. H. Farrell, L. K. Certain, and P. M. Farrell Genetic Counseling and Risk Communication Services of Newborn Screening Programs Arch Pediatr Adolesc Med, February 1, 2001; 155(2): 120 - 126. [Abstract] [Full Text] [PDF]
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D. S. Young and D. G.B. Leonard Issues in Genetic Testing Clin. Chem., June 1, 1999; 45(6): 915 - 926. [Full Text] [PDF]
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D. S. Young Issues in Genetic Testing Clin. Chem., May 1, 1999; 45(5): 725 - 725. [Full Text] [PDF]
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