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The Promise of Pharmacogenomics
It is well established that individuals with identical clinical symptoms
may respond very differently to the same drug therapy. In current clinical
practice, the suitability of a drug for a given individual is determined
by trial and error. A patient is prescribed a standard drug and the physician
then monitors the patient to determine if the drug is providing any benefit
and/or whether there are any adverse side effects. If the patient’s
condition does not improve, the dose of the drug may be altered or an
entirely different drug may be prescribed.
Pharmacogenomics is the study of the genetic factors that mediate an individual’s
drug response. It is thought that the same genetic differences that make
us all the unique individuals we are in terms of appearance, behavior,
etc. are also the same factors that determine whether or not a drug will
work against our diseases and whether they will give us side effects.
Pharmacogenomics promises to "get the right drug to the right patient"
and tailor therapy to the individual. The objective is to assess an individual’s
genetic background and use that information to make informed treatment
decisions and subsequently increase the odds of effective treatment the
first time (see figure below).
Roses, AD Pharmacogenetics and the practice of medicine. (2000) Nature
405:857
The above figure depicts a group of SNPs on DNA, and that these SNPs can
be identified for a group of patients. In the example, some of these
patients respond to the drug during the trial (with efficacy) and some
patients don't (without efficacy). By comparing the SNP patterns
of the two groups one can find the differences that correlate to whether
the patients will respond to the drug or not (indicated by red lines).
Thus, any patient who has SNPs that are of the "blue" type will
respond to the drug, whereas any patient with the "red" type
of SNPs will not respond and the drug will not be given to them.
This will allow doctors to predict the effectiveness of a drug for treating
a patient without having to test it through trial and error.
Although diet and physiological factors are also important, much of the
heterogeneity observed in drug response is thought to be due to the underlying
genetic variation of the human population. Genes encode proteins and thus
genetic variation could alter the structure of proteins important in drug
response. Proteins likely to be critical would include those involved
in metabolism or absorption of the specific drug as well as the molecular
drug target (often a receptor) and other components of the biochemical
pathway targeted by the intervention.
The advancing field of pharmacogenomics will benefit a number of sectors
of our society. Patients will benefit from pharmacogenomics through a
reduction in the incidence of morbidity and mortality due to adverse drug
reaction. It has been estimated that death due to adverse drug reactions
is the fourth to sixth leading cause of death in the US. Further, knowing
beforehand which treatment regimen will be most effective in a given individual
will save time that would have been lost using drugs that would provide
no relief. This issue becomes particularly important for individuals with
rapidly progressing disease, such as cancer, where it is imperative to
begin effective treatment as soon as possible.
Drug companies are touted as the biggest winners from pharmacogenomics.
It is predicted that pharmacogenomics will mainly identify patients that
do no respond to therapy. At first glance, it is not clear how identification
of unresponsive patients would benefit pharmaceutical companies. Clearly,
a number of patients take prescription drugs for some time before realizing
that the treatment is not effective. If these non-responders were identified
a priori, then the monetary profits to the drug companies from the drugs
taken during the trial period would be lost. However, this profit loss
is insignificant compared to the costs of not using pharmacogenomics.
A priori identification of non-responders would be beneficial
to drug-developers for two reasons. First, when a newly discovered drug
finally reaches late-stage clinical trials, a significant amount of money
has been spent developing that drug. A drug that fails at this point is
a huge financial loss for a pharmaceutical company. Thus, if one were
to identify genetic variation that affected the drug response during Phase
I and Phase II clinical trials, the Phase III efficacy trial could be
restricted to those individuals that have a higher likelihood of responding
well to the new drug. Consequently, the likelihood of conducting a successful
Phase III trial demonstrating clear drug efficacy would be increased and
ultimately result in a savings in cost and time to market. Second, although
the use of genetic information to divide patient subgroups would reduce
the target market for a drug, the drug would be a better drug and would
have maximum efficacy. With a better product with clear benefit, patients
and doctors would be much less likely to turn to competing products. Clearly,
drugs that provide consistent benefit with minimal adverse effects will
be more widely used and accepted. Finally, this may open up new drug targets
to companies if current drugs work only on a subset of targets.
It is estimated that there are approximately 1.5 million positions in
the genome that vary among individual humans. To advance pharmacogenomics,
the specific DNA variations important for drug response must be identified
from among the 1.5 million. In order to accomplish this task, high-throughput
methods to detect genetic variation (genotype) are being developed. In
parallel, enormous numbers of healthy and diseased individuals are being
asked to participate in large population studies and clinical trials aimed
at unraveling genetic determinants of drug response. Companies interested
in pharmacogenomics research can be broadly divided into two types, those
that provide the technology to detect genetic variation and those that
have assembled DNA samples and health data on a large number of individuals.
Although the number of published clinical trials with pharmacogenomic
elements to the trial are limited, high profile partnerships between genotyping
companies and clinical research/pharmacutical companies have been reported
and are briefly mentioned below. The remainder of this article will summarize
the current and developing technologies being used for high-throughput
genotyping.
Genotyping Technology
Individual differences at a single nucleotide of DNA, otherwise known
as single nucleotide polymorphisms (SNP) are the most abundant source
of genetic variation in humans. The human genome consists of strings of
nucleotides that can be either adenine (A), cytosine (C), thymine (T),
or guanine (G). As an example, at a SNP location, 70% of all human chromosomes
may have an A at a certain position whereas the remaining 30% have a G.
This genotypic difference can cause a phenotypic difference in hair color,
height, or drug response depending on the gene.
Conveniently, SNPs have certain technical properties that make detection
amenable to numerous high-throughput approaches. The flood of sequence
information from the Human Genotype Project has enabled organizations
to mine for SNP variation in the human population. The potential value
of SNP information is underscored by the willingness of 11 pharmaceutical
and technological companies to ally themselves with 4 academic institutions
and a medical research charity to form the SNP Consortium. The companies
provided the capital while the academic centers provided the technical
expertise necessary to identify and map SNPs throughout the human genome.
The SNP Consortium has exceeded its original goal to identify and map
300,000 SNPs and currently provides information on 856,666 mapped SNPs
in the human genome.
Numerous clever and sophisticated techniques have been developed to measure
the genotypes of SNPs. High-throughput (HT) genotyping technology can
be sorted based on three characteristics. The first is the molecular mechanism
or biochemistry used to discriminate between the two genetic variants
otherwise known as alleles. These molecular mechanisms include differential
hybridization, allele-specific nucleotide incorporation (also called primer
extension and single-base extension (SBE)) and allele-specific DNA cleavage.
The second feature distinguishing HT genotyping technologies is the assay
environment. Assays can be performed on solid supports (such as oligonucleotide
chips), in homogenous solution or using a combination of the two environments.
The last feature of HT genotyping assays is the detection method used.
Methods of allele detection implement light absorbance, fluorescence,
fluorescence polarization, luminescence and mass spectrometry.
A common feature of HT genotyping techniques is the use of the polymerase
chain reaction (PCR). See figure below. Nearly all techniques
start with PCR amplified DNA containing the SNP of interest. In brief,
short pieces of DNA (oligonucleotides) which recognize the position surrounding
the SNP location are generated. These pieces of DNA are called primers
and initiate DNA synthesis in the presence of DNA polymerase and deoxyribonucleotides.
In PCR, numerous cycles of DNA synthesis using thermostable DNA polymerases
results in the exponential amplification of a very specific region of
the genome from nanogram quantities of template genomic DNA isolated from
blood or cells. Although PCR is an enormously powerful technique it does
have certain disadvantages. First, a single PCR reaction generally amplifies
a single or limited number of SNP regions. Efforts have been made to use
multiplex PCR techniques where several SNP locations are amplified at
once, but these multiplex techniques are still not generally used and
can only be applied to certain combinations of SNPs. Thus, future improvements
in genotyping technology will likely involve better multiplex PCR protocols
or perhaps techniques that do not rely on PCR reactions to provide starting
material.
from http://www.accessexcellence.com/AB/IE/PCR_Xeroxing_DNA.html
SNP Detection by Primer Extension
Orchid Bioscience is one of several companies that provides technology,
equipment and reagents for genotyping. Orchid has a variety of platforms,
most of which utilize their proprietary primer extension biochemistry,
SNP-IT. In primer extension PCR amplified target sequence is annealed
to a primer complementary to the region adjacent to the SNP site. Dideoxyribonucleotides
(ddNTPS) and DNA polymerase are added to the mixture and the primer is
extended by a single nucleotide. The single nucleotide added is dependent
on the allele of the amplified DNA. Primer extension biochemistry can
be coupled with a variety of detection schemes and Orchid is developing
many methods for detection of SNP-IT reaction products one of which is
mass spectrometry. Mass spectrometry (MS) measure the mass of different
molecules and can discriminate two different oligonucleotides based not
only on nucleotide number but also on nucleotide composition. And thus,
MS can be used to distinguish allele-specific products resulting from
primer extension.
Alternatively, allele-specific primer extension products
can be discriminated based on fluorescence polarization (FP). In this
strategy, the nucleotides added in the primer extension reaction are labeled
with two different fluorescent tags (TAMRA or ROX) corresponding to each
of the two alleles. By measuring which fluorescent marker is attached,
one can determine which allele is present (A or G in this example) See
below:
Using FP, one is able to detect whether one, the other or both nucleotides
have been incorporated. LJL BioSystems is a prominent provider of high-throughput
instrumentation used to make FP measurements.
A third method of detecting primer extension reactions is analogous to
the detection method used for DNA sequencing reaction. Applied Biosystems
(formerly, ABI/PE whose parent company is Applera) manufactures instrumentation
for DNA sequencing and the same instruments can be used to discriminate
between allele-specific primer extension products by the size and fluorescence
of the product. Indeed, PE Biosystems and Orchid Biosciences, have announced
a collaborative agreement that enables PE Biosystems to develop a product
based on Orchid’s SNP-IT reaction chemistry and a detection platform
using PE Biosystems’ instrumentation and detection technology.
Affymetrix, Inc., the leading provider of DNA oligonucleotide chips (used
primarily for mRNA expression analysis) has also entered into collaboration
with Orchid. Orchid’s primer extension technology will be coupled
to Affymetrix’s solid chip format and fluorescence detection system.
This is an example of a technique that utilizes both a homogeneous liquid
reaction and a solid support assay environment. In this system a bipartite
oligonucleotide is designed for each SNP to be interrogated (see below).
One part of the oligonucleotide is a traditional primer for primer extension
and corresponds to the region adjacent to the SNP. The other segment of
the oligonucleotide consists of a nucleotide "tag" which serves
as a capture probe and is unique to each SNP being assessed. Thus, if
there are 100 SNPs being assessed there are 100 different capture probes.
Following primer extension and the incorporation of a fluorescently labeled
nucleotide numerous reactions are captured on a DNA chip by way of the
capture probes. In this format, numerous primerextension reactions can
be performed simultaneously and each individual SNP reaction can be captured
and localized to a particular position on the solid oligonucleotide chip.
The genotype of each SNP is determined by measurement of fluorescence.
Genome Res 2000 Jun;10(6):853-60
Mini-sequencing is a technique related to primer extension. Unlike primer
extension where a primer is extended by a single nucleotide, mini-sequencing
extends a primer by several nucleotides. The company Pyrosequencing, Inc.
has developed a HT format for mini-sequencing. In traditional DNA sequencing,
the reaction products are separated according to size on a polyacrylamide
gel and both size and fluorescence is used to identify the nucleotide
at a given position.
To obviate the need for gel electrophoresis, Pyrosequencing technology
is performed in a microtiter plate format, which is more amenable to HT
genotyping. In brief the identity of the nucleotides surrounding and including
the SNP position is determined by adding known nucleotides sequentially
to a DNA sequencing reaction. If the nucleotide is incorporated into the
primer (i.e. it is complementary to the DNA at that position) a cascade
of reactions involving the enzymes sulfuryase and luciferase as well as
the pyrophosphate released by nucleotide incorporation occur and ultimately
produce detectable light, which indicates the nucleotide identity. The
enzyme apyrase degrades the excess nucleotides (which could interfere
with the subsequent reaction) and enables another cycle of Pyrosequencing.
www.pyrosequencing.com/pages/technology.html
SNP Detection by Differential Hybridization
QIAGEN Genomics, Inc. has developed a HT SNP detection assay using their
30 different Masscode tags. The Masscode tags are defined chemical structures
of various sizes that can be attached to nucleotides by way of a photolabile
linker and are thus referred to as cleavable mass spectrometry tags (CMSTs).
The allele discrimination assay consists of using a standard PCR product
as a template for an allele-specific PCR reaction. Allele-specific PCR
amplification occurs when a primer is designed such that it perfectly
matches and hybridizes to one allele but contains a mismatch for the alternative
allele. In the QIAGEN system two allele-specific primers each possess
a differing covalently-attached Masscode tag. PCR product is only produced
from the primer matching the allele perfectly. The identity of the primer
that generated the PCR product is determined by cleavage and identification
by MS of the photolabile Masscode tag. Genotype is determined by measuring
the relative proportion of the two tags.
Another genotyping technique that uses differential hybridization is PE
Biosystems’ Taqman real-time PCR. In addition to manufacturing DNA
sequencers, developing kits to genotype based on primer extension, PE
Biosystems also manufactures the ABI Prismâ 7700 which uses allele-specific
hybridization of a reporter probe to distinguish between two genetic variants
during real-time PCR reactions.
from the ABI Prism 7700 Sequence Detector User Manual
The technique, Taqman , involves two reporter probes, which hybridize
specifically to one of the two alleles. One end of each reporter probe
is labeled with a fluorescent marker. A quencher molecule is attached
to the other end of the reporter probe. PCR primers are designed to amplify
the DNA region surrounding the SNP and the reporter probe hybridization
site. During the PCR reaction the inherent 5’-nuclease activity
of PCR polymerase cleaves the reporter probe that hybridizes perfectly
to the genomic DNA. Cleavage of the reporter probe releases the fluorescence
marker and separates it from the quencher molecule. Thus, when the fluorescent
dye and the quencher molecule are in proximity of one another (i.e. in
an intact reporter probe) there is no fluorescent signal but when the
reporter probe is cleaved the fluorescent dye is no longer quenched and
fluorescence is detected. Cleavage of the reporter probe occurs only if
it matches the template perfectly and thus the presence or absence of
each allele can be interrogated.
SNP Detection by Allele-Specific Enzyme Cleavage
Third Wave Technologies, Inc. has developed the Invader genotyping assay,
which exploits the exquisite specificity of Cleavaseâ enzymes. Cleavase
enzymes recognize certain flap-like DNA structures and these structures
can be formed using PCR products and added oligonucleotides. Oligonuclotides
can be designed such that flap cleavage by the Cleavase is allele-specific.
Cleavage of the probe is detected using a fluorescent reporter. If the
reporter is cleaved, the fluorescent reporter is released by the Cleavaseâ
from the proximity of a quencher molecule and is able to emit signal.
Thus, single base differences can be discriminated between two alleles.
See figure, from www.twt.com/invader/invader.html
Variagenics, Inc. has developed a genotyping assay that also exploits
enzyme specificity. A class of enzymes termed resolvases cleave very specifically
at mismatches in DNA. Oligonucleotide probes corresponding to each allele
are complexed to PCR amplified product representing the genomic DNA. If
the probe perfectly matches the allele, there will be no cleavage of the
probe. If, however there is a mismatch between the probe and the allele,
the probe will be cleaved by the resolvase enzymes. In the Variagenics
assay, cleavage is detected by measuring the change in mass of the probe
using MS. Variagenics, Inc. has developed this assay in collaboration
with Bruker, Inc. the maker of MS instrumentation.
Corporate Collaborations and Agreements
Armed with effective HT genotyping tools, the field of pharmacogenomics
is poised to move beyond the next hurdle, namely the identification of
genetic variants that are determinants of drug response. To accomplish
this task collaboration between companies that enable HT genotyping with
companies having expertise in clinical trials/population studies is critical.
The field is moving too fast to document all the collaborations that have
been announced but a few recent announcements involving some of the technologies
described here are listed below.
* Jan. 11, 2001 Third Wave Technologies, Inc. And Leading Japanese Clinical
Reference Laboratory Enter Into Agreement To Address Large-Scale Pharmacogenomic
And Clinical Opportunities In Japan
* Feb. 13, 2001 Orchid Biosciences, Inc. And AstraZeneca Announce Broad
SNP Collaboration
* Jan. 4, 2001 Third Wave Technologies' Invader(R) Operating System Chosen
For Largest NIH-Sponsored Genotyping Study
* Jan. 4, 2001 Variagenics signs pharmacogenomics agreement with Amgen,
Inc.
* Dec. 20, 2001 Thirdwave Technologies, Inc., Juvenile Diabetes Research
Foundation, Wellcome Trust Diabetes And Inflammation Laboratory At Cambridge
University To Collaborate On High Throughput Discovery Of Genes
* Oct. 11, 2000 Orchid BioSciences, Inc. Announces Commercial SNP Scoring
Agreement With Eli Lilly and Company
* Oct. 2, 2000 Variagenics Achieves Second Milestone In Pharmacogenomics
Collaboration With Covance, one of the largest clinical research organizations
* Sept. 21, 2000 Variagenics Announces Pharmacogenomics Agreement With
Boehringer Ingelheim Pharmaceuticals, Inc.
* Nov 21, 2000 Harvard Center For Cancer Prevention Purchases Pyrosequencing's
PSQ(TM) 96 System For DNA Analysis
* Oct. 31, 2000 Pyrosequencing, Inc.'s PSQ(TM) 96 System DNA Sequencing
Technology To Accelerate UC Davis Population Genetics Study
* Oct. 5, 2000 World-Renowned Karolinska Institute Adopts Pyrosequencing,
Inc.'s Technology For Disease-Related Research
* Feb. 9, 2000 Columbia University And Virginia Commonwealth University
Release Favorable Results From LJL Biosystems’ SNP Platform
* Jan. 10, 2001 Qiagen and Agilent Technologies, Inc. Enter Into SNP Genotyping
Agreement
* Jan. 10, 2001 Qiagen Enters Masscode(TM) System Technology Access And
Purchase Agreement With Daiichi Pure Chemical Co. Ltd.
* Jan. 10, 2001 Qiagen Enters Into SNP Genotyping Research Agreement With
University of Washington
* Jan. 8, 2001 Genomics Collaborative And Qiagen Announce Joint Research
Agreement
* Aug. 10, 2000 Affymetrix And deCode Genetics Sign GeneChip(R) BiotechAccess(TM)
Agreement
* Oct. 6, 2000 Applied Biosystems Donates Sequence Detection System To
Enhance Pediatric Leukemia Research
* Jan. 10, 2000 Orchid Biosciences, Inc. And Perkin Elmer, Inc Establish
Licensing Agreement For Orchid's SNP-IT Technology In DNA SequencingNote:
Full press releases can be viewed on www.biospace.com
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