Part II: Methods and Approaches 1: Assessing Disease Associations and Interactions Chapter 5

“The findings and conclusions in this book are those of the author(s) and do not
necessarily represent the views of the funding agency.”
These chapters were published with modifications by Oxford University Pressexternal icon (2004)

Human Genome Epidemiology: A Scientific Foundation for Using Genetic Information to Improve Health and Prevent Disease


Methods for Assessing Genotypes in Human Genome Epidemiology Studies

Karen Steinberg and Margaret Gallagher

Tables | References


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).

Polypropylene 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 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 ( icon), and private organizations such as the College of American Pathologists (CAP), American College of Medical Genetics (ACMG). 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), ( 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. 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.” ( 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). 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) ( icon. 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 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 ( icon) (Molecular Probes); Cybr-greenR ABI; Hoechst-

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.

Analytical Validity:

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.
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.
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.


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 (2).

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.

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.


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Address correspondence to Dr. Khoury at
Office of Genomics and Disease Prevention
Centers for Disease Control and Prevention
6 Executive Park, Mail Stop E-82
Atlanta, Georgia 30329

Page last reviewed: January 1, 2004