Methods
for Assessing Genotypes in Human Genome Epidemiology Studies
Karen Steinberg and Margaret Gallagher
Tables | References
Introduction
As the Human Genome Project provides
the foundation for understanding the genetic basis of common disease
(1), population-based genetic studies will provide
the information needed for the practical application of genetic risk
factors to clinical and public health practice. To this end, researchers
have begun collecting specimens for molecular analyses in epidemiologic
studies and surveys in order to identify genetic risk factors for disease
(2). Genomic markers including restriction fragment
length polymorphisms (RFLPs), short tandem repeats (STRs, also called
microsatellites), insertion-deletion polymorphisms, single nucleotide
polymorphisms (SNPs) and groups of markers inherited together on one
chromosome as haplotypes are being used to locate disease-associated
genetic loci, and studies of the association between these loci and
disease are elucidating the genetic basis for disease. Once risk-associated
genotypes are identified, the validity of genetic testing for screening
and clinical practice must be assessed. This includes analytical and
clinical validity of genotyping methods. Here we address factors to
be considered in choosing appropriate specimens for epidemiologic studies,
some quality assurance issues, and analytical validity. Clinical utility,
which is the ultimate standard by which to evaluate case management
on the basis of the result of a laboratory assay, is addressed elsewhere
in this volume.
Specimen Selection:
Factors to be considered in choosing appropriate specimens for epidemiologic
studies, include cost, convenience of collection and storage, quantity
and quality of DNA, and the ability to accommodate future needs for
genotyping. Four types of specimens are commonly collected in epidemiologic
studies with a genetics component: 1) dried blood spots, 2) whole blood
from which genomic DNA is stored or extracted using either anticoagulated
or clotted blood or buffy coats, 3) whole blood from which lymphocytes
are isolated and immediately immortalized or cryopreserved for later
immortalization, and 4) buccal epithelial cells.
Blood spots are a stable, inexpensive source of DNA, useful for genotyping
polymorphisms for association studies. (2) The stability
of stored blood spots makes them a potential specimen source for population-based
studies. Although specimens collected in newborn screening programs
can serve as samples from which to determine population gene frequencies,
use of these specimens in any way other than anonymously is problematic
because the specimens may not have been collected with adequate informed
consent (3,4). Blood spots can be
collected without a phlebotomist and safely transported by regular mail.
In general, genotyping one locus requires from about 10 ng to as little
as 2.5 ng per single nucleotide polymorphism (SNP) given current technology,
so that scores to hundreds of genotypes could be obtained from one blood
spot (Table 5-1). With the advent of multiplex
testing (genotyping several loci in one assay) , these numbers can be
increased (15).
Whole blood provides high quality genomic DNA in microgram quantities
sufficient for current applications including genome scans using SNPs
or STRs, polymorphism discovery using methods such as denaturing gel
electrophoresis, single strand conformational polymorphisms (SSCP),
or sequencing, and for genotyping loci using methods such as allele
specific oligonucleotides, RFLPs, or sequencing. Quantities of DNA ranging
from 100 - 400 μg can be obtained from 10 mL of whole blood, and approximately
200 μg from 1 mL of buffy coat. Blood is most often collected using
ethylenediaminetetraacetic acid (EDTA), although anticoagulants including
heparin and acid citrate dextrose (ACD) have also been used. Cells can
be stored in anticoagulated whole blood, in clots, or in buffy coats.
Guidelines for obtaining these specimens are available (16).
olypropylene rather than glass containers should be used to store frozen
blood, and blood should be divided into aliquots to prevent freeze-thaw
cycles. Although evidence exists to suggest that lymphocytes can be
transformed with EBV after cryopreservation, (17)
and optimization of transformation of small numbers of cryopreserved
lymphocytes is an active area of research, at the time of writing, there
is insufficient evidence to be confident that lymphocytes stored in
whole blood stored for years can be consistently transformed.
EBV-transformed lymphocytes provide an unlimited source of high-quality
genomic DNA for genotyping large numbers of polymorphisms requiring
microgram quantities of DNA. Although transformed lymphocytes may provide
specimens for functional studies, properties of EBV-transformed lymphocytes
may be different from those of untransformed lymphocytes, and lymphocyte
gene expression may not be representative of the expression patterns
in the target tissue of interest. Because of the expense associated
with establishing and maintaining immortalized cell lines, many investigators
are attempting to cryopreserve lymphocytes for later immortalization
of selected specimens in nested-case control studies. However, the expense
of establishing and maintaining cell cultures has resulted in a trend
toward storing whole blood for obtaining large quantities of genomic
DNA. When lymphocytes are held in culture, they should be monitored
for contamination with mycoplasma, bacteria, and fungi, and original
specimens (e.g. including blood spots, whole blood, or extracted DNA)
should be maintained for identity checks. From 5 to 10 μg of DNA can
be obtained from 1 - 2 x106 cells.
Buccal cells can be obtained for DNA isolation using cytobrushes, swabs,
or oral lavage. Although there are few systematic studies that compare
yield of human DNA (hDNA) from buccal cells (excluding bacterial contamination)
using different collection methods, there is a growing consensus that
the use of mouthwash used to obtain cells gives yields more and higher
quality DNA (in the range of 5 μg to 100 μg) than swabs or
cytobrushes which yield DNA in the range of 1 μg to 2 μg per
cytobrush or swab. However, swabs or cytobrushes are necessary for collecting
specimens from infants and small children (Table 5-1).
Quality Control for Molecular Methods
We define quality control as the inclusion of characterized specimens
in analytical runs to ensure the correct performance of a method and
the quality of the resulting data. This discussion does not include
the broader issue of quality assurance which subsumes quality control
and includes standards of professional qualifications for personnel
performing and interpreting genetic tests as well as standards for interpretation
in the clinical context. Quality control generally entails the inclusion
of positive and negative controls, reagent blanks, and duplicates in
analytical runs to assess the precision of a method within a laboratory.
Proficiency testing (PT), or external quality assessment (EQA) as it
is also known, includes the external component of quality control in
which unknown specimens from either a commercial source or outside laboratory
are analyzed to assess consistency and accuracy among laboratories.
We first discuss several ongoing programs to standardize and assure
the quality of genetic testing through published recommendations or
regulations. We then give specific recommendations for some of the more
commonly used molecular methods.
Guidelines and Regulations
In practice, research studies that will report clinically relevant results
should use laboratories that are held to the highest standard of practice.
In the United Kingdom the common practice is not to report results of
diagnostic relevance generated as a part of research but to have the
test repeated by a diagnostic laboratory on a fresh specimen. The UK
studies that are clinically-based are expected to assure that laboratories
performing tests are diagnostic laboratories or have equivalent standards
of practice.
In the United States, a distinction is made between the quality control
requirements for laboratories that perform tests for which results are
reported for clinical use and those that perform tests as a part of
research and for which results are not reported. Laboratories performing
the former tests are regulated under the Clinical Laboratory Improvement
Amendments of 1988 (http://www.fda.gov/cdrh/ode/guidance/1147.pdf)
and the latter are not (18). But because results of
genetic testing done as a part of clinical or epidemiologic research
are sometimes reported to participants, this distinction cannot always
be easily made. Further, CLIA may usefully provide guidelines for genetic
testing done purely for research and not reported for clinical use.
In addition to CLIA guidelines, guidelines are made available by states,
such as New York (http://www.wadsworth.org/labcert/clep/Survey/standards.pdf),
and private organizations such as the College of American Pathologists
(CAP) (http://www.cap.org/html/ftpdirectory/checklistftp.html),
American College of Medical Genetics (ACMG) (http://www.kumc.edu/gec/prof/acmg.html).
Manuals which provide detailed discussions of quality control for molecular
methods include NCCLS (19) and that of Saunders and
Parkes. (20)
Distinctions between requirements for quality assurance for laboratories
that report results and those that do not, notwithstanding, the quality
of research data that will be the foundation for clinical practice depends
on implementation of quality control in research as well as clinical
laboratories. Further, quality standards are usually promulgated for
laboratories that report results rather than those that do not. Therefore,
any discussion of quality control should include regulations and recommendations
intended for clinical laboratories. In this regard, most developed countries
have systems for accrediting laboratories on the basis of government
regulations or professional guidelines. However, most are still in the
process of developing specific standards for molecular genetic laboratories.
The Centers for Disease Control and Prevention (CDC), as a part of
its mandate to implement CLIA, has funded contract studies to produce
recommendations for performance evaluation and quality assurance. An
example is available of these recommendations for quality control strategies
for laboratory genetic tests, including nucleic acid amplification by
PCR, DNA sequencing, Southern blot analysis, and fluorescence in situ
hybridization (FISH) (Table 5-2), (http://www.phppo.cdc.gov/dls/genetics/qapt.asp).
The ACMG Laboratory Practice Committee has also published practices
standards for clinical genetics laboratories that prescribe general
guidelines for laboratories and specific guidelines for molecular genetics,
as well as cytogenetics, including fluorescence in situ hybridization
(FISH), and biochemical genetics, which is in most respects the same
as for clinical chemistry laboratories except in the more extensive
interpretation that is required for results of biochemical genetic testing
(http://www.faseb.org/genetics/acmg/stds/copyrite.htm).
ACMG guidelines for molecular genetic methods include details on quality
control for DNA preparation, probe/primer/locus documentation, assay
validation, southern blot analysis, and PCR methods including containment
and amplification conditions, product detection and analysis, and use
of controls and standards.
Proficiency testing should be a component of all laboratory quality
control programs. The College of American Pathologists provides a voluntary
Laboratory Accreditation Program, and CAP and the ACMG jointly operate
PT programs in genetics that provide materials for approximately 17
different mutations that cause single-gene disorders including cystic
fibrosis, factor V Leiden deficiency, Duchenne muscular dystrophy (DMD)/Becker,
rhesus monkey antigen D (RhD), Prader-Willi/Angelman syndrome, Huntington
disease, fragile X syndrome, hereditary hemochromatosis, hemoglobin
S/C (sickle cell disease), myotonic dystrophy, type 1 (DM1), Friedrich
ataxia, prothrombin, spinocerebellar atrophy, spinal muscular atrophy,
methylene tetrahydrofolate reductase, BRCA1 and BRCA2, and multiple
endocrine neoplasia (MEN)2 (21).
CLIA requires laboratories performing tests that are not included in
available PT programs to have a system for verifying the accuracy of
the test results at least twice a year. Although laboratory participation
in the CAP Molecular Genetics survey is currently voluntary, many laboratories
performing DNA-based genetic testing elect to participate in CAP surveys
to meet the CLIA quality assurance requirement. The National Institute
of Standards and Technology provides human DNA standard reference materials
for forensic as well as clinical applications which include standards
for RFLPs, STRs, and amplification and sequencing of mitochondrial DNA
(22).
In the absence of PT materials for gene variants of interest, which
is the rule rather than the exception in the research setting, exchange
of specimens among laboratories is an acceptable means to test consistency
among laboratories. (23-25). Accuracy can also be
assessed in this way when the PT materials have been well-characterized
by a reference method.
In conjunction with the quality assurance efforts of individual nations
including those of Europe, Australia, Japan, Korea, Mexico, New Zealand,
the US and others, the Organisation for Economic Co-operation and Development
(OECD) held a workshop in Vienna in 2001 “to consider whether
the approaches of OECD member countries for dealing with new genetic
tests are appropriate and mutually compatible.” (http://www1.oecd.org/dsti/sti/s_t/biotech/act/gentest.pdf).
One of the main considerations of the workshop was the development of
international best practice policies for analytical and clinical validation
of genetic tests. The EQA/PT component of quality assurance provides
a means to measure laboratory results against an external gold standard.
Because EQA includes the laboratory’s ability to interpret results
in a clinical context as well as accurate test performance, EQA has
been developed in a disease-specific fashion. In the United Kingdom
EQA includes workshops held by representatives of participating laboratories
to develop and publish best-practice guidelines which are made available
by the Clinical Molecular Genetics Society (CMGS) (http://www.cmgs.org).
Guidelines for 10 disorders were available in 2001 including breast
cancer, Huntington disease, fragile x syndrome, Prader-Willi/Angelman
syndrome, Charcot-Marie tooth disease, retinoblastoma, Duchenne muscular
dystrophy, cystic fibrosis, Friedrich ataxia, and y-chromosome microdeletions.
These guidelines serve as the nucleus for guidelines funded by the European
Commission and published by the European Molecular Genetics Quality
Network (EMQN) (http://www.emqn.org/emqn.htm).
In Europe, compliance with guidelines is still voluntary but could ultimately
be required for accreditation for service as is true in the US through
CLIA and in the UK through Clinical Pathology Accreditation (http://www.cpa-uk.co.uk/)(OCED
document pp 41- 42).
Because most of the quality assurance schemes in the above references
are designed for clinical genetic tests, they are often disease-specific.
They do, however, provide many of the necessary generic components of
quality control for molecular laboratories (e.g. guidelines for PCR)
making them useful for the validation phase of test development before
clinical testing is available and for genetic tests for rare disorders
that are done in only a few laboratories.
Specific Recommendations
Genetic material is analyzed as part of epidemiologic studies for generally
two purposes: 1) to test the significance of an association between
a gene variant and a disease and 2) to use gene variants as markers
for mapping other gene variants that are causal in disease. Methods
most commonly used to localize gene variants associated with disease
take advantage of the sequence variation (or polymorphisms) in populations.
The most commonly used polymorphic markers include microsatellites and
SNPs. Because of the large number of individuals who must be genotyped
for a large number of polymorphisms in these studies (Kruglyak and Nickerson
26), new methods are being developed to accommodate
high throughput analyses, to facilitate assay design, and to reduce
costs. The newer methods often include array technology sometimes coupled
to a mass spectrophotometric detection system.
Because of the rapid and continuing proliferation of molecular methods
used in the research setting, and because others have furnished more
detailed guidelines for quality control in genetic testing (19,20,27),
we focus the remainder of our discussion on DNA extraction and characterization
and analytic validity because both are fundamental to all DNA-based
methods. In most cases, DNA must be extracted and amplified before automated
sequencing or polymorphism identification is done.
With regard to DNA amplification and genotyping, ideally, each step
in the analysis should be performed in duplicate, from extraction and
PCR to genotyping in order to determine the precision of the method.
Reagent blanks should be included in all runs to identify the presence
of contamination and obviate false positive results. When possible,
DNA that has the sequence of interest should be added to control specimens
to assure efficiency of the methods. This approach would be problematic
when using arrays to genotype multiple polymorphisms on one person.
In all cases, other than the so-called closed systems in which amplification
and genotyping occur in one vessel, pre- and post- amplification of
DNA must be carried out in separate work areas to prevent contamination
of specimens which can cause false positive results. Movement of specimens
should be in one direction, from specimen preparation to PCR to genotyping
with careful physical separation of sample preparation from extracted
DNA and PCR reactions. (28). Reagents should be made
from molecular biology grade chemicals and reagent-quality water. Before
they are judged acceptable, new reagents should be tested in the same
assays with reagents currently in use that have been validated. (29).
DNA Extraction and Characterization
Visvikis et al, (9)
have divided issues related to DNA extraction into three steps comprising
whole blood preservation, extraction procedures, and storage of DNA.
DNA is stable in whole blood at room temperature for about 24 hours
with only slight decreases in stability within 72 hours. Specimens held
from 4 to 8 days before DNA extraction should be held at +4°C. Optimal
yield is obtained from whole blood specimens that are processed before
freezing. Extraction methods include use of 1) enzymes (including proteinase
K and RNAse) (30), 2) organic solvents or organic
solvents with enzymes (31), 3) salt percipitation
(32,33), and 4) resins or affinity
gels which are the basis for many commercial kits. After extraction,
DNA is re-suspended in a buffer such as Tris buffer. The quality of
extracted DNA is assessed by its yield, molecular weight, purity, and
the ability to serve as a substrate for PCR and restriction enzymes
(Table 5-2).
Yield and purity are most commonly estimated using the ratio of optical
absorbance at 260 nm to absorbance at 280 nm. Although convenient and
usually sufficiently accurate for most applications, this method does
not distinguish double-stranded DNA (ds-DNA) from single-stranded DNA,
does not distinguish between DNA and RNA, is relatively insensitive,
and contaminants may cause interference. If interfering substances are
present, DNA can be more precisely quantified using one of the ds-DNA-binding
dye methods such as Picogreen. (PicogreenR ) which is not affected by
ssDNA, RNA, or protein (http://www.probes.com/resources/sitemap.html)
(Molecular Probes); Cybr-greenR ABI; Hoechst- http://www1.amershambiosciences.com/).
Molecular weight is most commonly determined using electrophoresis.
Electrophoresis employs an electric current and a sieving matrix to
separate molecules on the basis of their charge and size. Agarose is
used for separation and sizing of large DNA fragments, and polyacrylamide
gel can be used for smaller DNA fragments. In either case, the gel matrix
acts as a molecular sieve that causes the separation of DNA fragments
on the basis of size. DNA fragments of standard molecular weight, obtained
from commercial sources or developed and characterized in the laboratory
performing the tests, are included for comparison.
The DNA should be tested to confirm that it can serve as a substrate
for restriction enzymes such as EcoR1 or Hind III.. In the case of cell
lines, the source of the DNA should be confirmed by comparing patterns
of microsatellites between the processed specimen and an aliquot that
was saved, for example as a blood spot, for identification purposes.
Long-term storage of extracted DNA should be done at temperatures of
-20°C or -70°C, although DNA may be stable in suitable buffers at 4°C
for years. </p>
<p><span class="bold14">Analytical Validity:</span><br>
As is the case for all laboratory methods, analytical validity for DNA-based
tests is the probability that a test will be positive when a target
sequence is present (sensitivity) and that the test will be negative
when that target sequence is absent (specificity) and that the results
using the same target sequence will be consistently reproduced (Precision,
reproducibility, or reliability.). These characteristics should be determined
for each method used for genotyping or sequencing.<br>
Analytical sensitivity can also be measured as the lowest concentration
of the target sequence that can be distinguished from background signal
or noise and defines the assay’s limit of detection. In the case
of genotyping, detection limits approaching a single molecule are possible.
The more sensitive an assay, the less likely false negative results
will be obtained.<br>
Analytical specificity is the probability of a positive result will
occur only in the presence of the target sequence being measured. The
more specific an assay, the less likely false positive results will
be obtained.</p>
<p><span class="bold14">Summary:</span><br>
The type of specimen collected in epidemiologic studies will depend
on the costs, study needs, and the laboratory experience and technology
available to the investigators. Given that current technology can analyze
a SNP in as little as 2.5 ng of DNA, all of the specimens described
above should allow hundreds to thousands of analyses (<a href="#2">2</a>).
</p>
<p>Genomic DNA extracted from whole blood for immediate use or storage
assures that sufficient material will be available for most current
and future molecular applications at a cost that is sustainable. Blood
spots are appropriate when protocols call for easier collection and
room temperature, low-cost storage. Buccal cells allow non-invasive
collection that can be self-administered, and specimens can be mailed.
Even though these specimens provide limited amounts of DNA with wide
inter-individual variation when buccal cells are collected, they can
provide material sufficient for genotyping scores to thousands of loci.
If an unlimited source of DNA is needed for repeated or collaborative
studies, or if studies of gene expression using RNA or protein are needed,
however, there may be issues with alteration in normal gene expression
in transformed cells, and funding is sufficient, then lymphocytes should
be transformed. Although cryopreservation and later transformation of
selected specimens could reduce the number of specimens to be transformed,
the high costs of maintaining the cell lines that are created later
is still a factor, and there are too little data to confirm that this
strategy would ensure viable cell cultures upon transformation.</p>
<p>Although the emphasis on innovation in molecular genetic research makes
quality control a moving target, basic rules of quality control can
help assure quality results. Quality control measures generally include
use of control materials, duplicate specimens, blanks, and proficiency
testing. Most developed countries have guidelines or regulations for
laboratory accreditation which include recommendations for technical
proficiency, but most are still working to develop specific guidelines
for genetic testing. These guidelines are generally the same for clinical
and research laboratories. Proficiency testing is also an essential
component of laboratory quality control. Currently available proficiency
testing materials are designed for the more commonly performed clinical
DNA-based tests. Comparable materials are usually not available for
DNA-based tests being performed in epidemiologic studies of association
between gene polymorphisms and disease. In this case, laboratories performing
the tests can exchange material for external quality assurance. Nonetheless,
quality control of molecular genetic methods is essential whether tests
are performed for clinical decision-making or to serve as the basis
for hypothesis testing in research.</p>
<table width="100%" border="0" cellpadding="0" cellspacing="0" id="tables">
<tr>
<td class="hugebkheader"><a name="tables" id="tables"></a> Tables</td>
</tr>
<tr>
<td><ul>
<li class="textheight2"><a href="javascript:;" onClick="MM_openBrWindow('tables/chap5_TAB1.htm','','scrollbars=yes,width=710,height=400')">Table
5-1</a></li>
<li class="textheight2"><a href="javascript:;" onClick="MM_openBrWindow('tables/chap5_TAB2.htm','','scrollbars=yes,width=630,height=400')">Table
5-2</a></li>
</ul></td>
</tr>
</table>
<br>
<table width="100%" border="0" cellpadding="0" cellspacing="0" id="references">
<tr>
<td class="hugebkheader"><a name="ref" id="ref"></a> References</td>
</tr>
</table>
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