3.3 Types of Genetic Study Design

Fig. 3.3 (a) Each of the many restriction enzymes cleaves DNA at a speciÞc site. Four (MspI, TaqI, EcoRI, and HindIII) are illustrated here along with the loci they cleave. (b) The result of applying a restriction endonu-

clease to DNA is a set of DNA fragments that can be separated by electrophoresis. The electrophoretic pattern can distinguish different alleles (A and a) (Redrawn after Musarella61)

associations with diabetic retinopathy, this has been a major problem. It is not a problem with RVO, because the diagnosis is rarely in doubt.

3.3 Types of Genetic Study Design

A common design in association analyses is the case-control format. In that format, from the same population of similar age and gender, two groups are selected: patients with RVO and a control group without RVO. Information is collected regarding a genetic marker for each person in the population sample. Then, statistical

tests are performed to examine associations of the genetic marker polymorphisms with presence or absence of the disease. Common statistical tests include chi-square tests, odds ratios, likelihood ratios, and others. In theory, this approach could be used to screen many genetic markers at intervals across the entire genome to test for associations, but several methodological constraints limit such a wide net approach.

The genetic marker may have no causal relationship to RVO; it could just be linked with the gene that creates the risk. Many statistical tests performed in genome-wide scans lead to false-positive

different criteria for patient selection. Given the many pitfalls and divergent results among studies, conÞdence in association studies should increase in proportion to the number of studies that replicate a given set of Þndings. Meta-analyses can be useful in deriving a common thread, if one exists, in discordant genetic studies.87 They may also partially overcome the problem of inadequate power if the surveyed studies are small.

The Human Genome Project has led to the construction of a genetic map that locates all known genes and polymorphisms to their particular positions on the 23 pairs of chromosomes (22 pairs of autosomes and 1 pair of sex chromosomes Ð X and Y). Thus, there are 24 genetic submaps, one for each of 22 autosomes and a submap for X and Y. Groups of polymorphisms located in the same chromosomal region tend to be inherited together as a unit and are statistically associated. These are referred to as haplotypes. The HapMap Project is an ongoing international collaboration in which regions of linked polymorphisms are being deÞned in four international populations.40 Haplotypes are represented by socalled tag SNPs, which are SNPs that are indicators that an entire ensemble of SNPs is present. The ensemble is the haplotype. The tag SNP is the indicator label for a speciÞc haplotype.

A critical tool in genetic analysis is the polymerase chain reaction (PCR). This method involves taking a small amount of a speciÞc segment of DNA and generating a large amount of identical DNA. It provides enough material to be detectible and measurable to determine the presence or absence of a genetic marker in an individual. Using PCR, each person in a study can be genotyped for a

speciÞc allele. The genotype of the allele can be correlated with disease status to determine whether there are associations. In PCR, short synthetic pieces of single-stranded DNA called ÒprimersÓ are made. They ßank a speciÞc, small region of DNA of interest in the sample from the person. The double-stranded DNA sample is separated and the primers bind to the region of interest based on their complementary sequences. DNA polymerase and deoxynucleotide triphosphates are then added to the mixture at varying temperatures,

producing new, complementary DNA adjacent to the primers at the sequence of interest. This results in two copies of the double-stranded DNA in the region of interest from a single copy. The process is repeated many times Ð 30 times would be typical Ð producing a billion copies of the DNA sample from a single starter molecule. This larger amount of DNA can then be manipulated, separated, and measured electrophoretically and alleles distinguished. Figure 3.5 illustrates the process of PCR.

Fig 3.5 (a) Schematic representation of the process of PCR. A double-stranded molecule of DNA, called the template, is broken into two single-strand components by heating. Primers are synthesized speciÞcally to bind to DNA sequences that ßank the region of interest (the region being examined for a particular polymorphism). These primers are added to the mixture and anneal to the complementary sequences in the single separated strands of DNA (hybridization). (b) Polymerase enzymes and deoxynucleotide triphosphates (dNTP) are added to the mixture and new DNA is produced starting at the end of the bound primer. Two double-stranded DNA molecules result. The process is repeated many times to produce enough DNA to manipulate electrophoretically. (c) Schematic of the exponential ampliÞcation of DNA through PCR. After 30 cycles, an initial template molecule of DNA results in one billion molecules of identical DNA (Redrawn Della16 and Dragon19)

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2. Denature DNA at 94°

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5. Twice the DNA product

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DNA synthesis

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