DNA produced by the action of restriction endonucleases, one can deduce the presence or absence of polymorphisms that result in cleavage sites.

Figure 2.4 shows an example of a map of genetic markers, in this case short tandem repeats, corresponding to chromosome 11 and their positional relationship relative to a gene, PAX6, that is critical in ocular embryogenesis. From a study of this figure, it will become evident that one can judiciously choose a polymorphism and examine if it is associated with a disease such as diabetic retinopathy. If there is an association between the polymorphism and the disease phenotype, then neighboring genes to this polymorphism may be candidates for investigation regarding pathogenesis of the disease. Neighboring genes become candidates because segments of DNA tend to be inherited together as a unit. Conversely, if one suspects that a certain gene is important in causing a diabetic retinopathy, one

could choose to study a polymorphism found in close proximity to that gene based on the genetic map and then look for associations of that polymorphism with presence or absence of the disease. In the case of diabetic retinopathy studies, markers for nonproliferative retinopathy (NPDR), proliferative retinopathy (PDR), diabetic macular edema (DME), or presence of any DR have all been studied. The success of such studies depends critically on how reproducible the classification of patients is with regard to disease status. For example, if patients are misclassified as having PDR, a study may spuriously fail to find an association with a genetic marker. Genes conferring susceptibility for NPDR, PDR, DME, and possibly other diabetic retinopathy phenotypes may be distinct.6,20

Fig. 2.4 Genetic markers called polymorphisms have been mapped throughout the human genome. In this example, short tandem repeat polymorphisms have been mapped on chromosome 11 and are shown in relation to the position of the PAX6 gene. Adapted with permission from Damji et al.1

One common genetic study design is the case-control format. In such a study, a group of patients with diabetic retinopathy or one of its subtypes and a control group with diabetes but no retinopathy are collected. Information is collected regarding some genetic marker for each person in the population sample. Statistical tests are then performed to examine associations of the genetic marker polymorphisms with presence or absence of the disease state. These may be Chi-square tests, odds ratios, likelihood ratios, or 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 diabetic retinopathy, but only manifest linkage with the gene that in fact confers risk. If patients with disease and controls are drawn from genetically different populations, a false-positive association may result. Large numbers of statistical tests performed in genome-wide scans can lead to falsepositive associations. In some studies, comparisons of gene frequencies are made between patients with diabetic retinopathy and non-diabetic controls. This can lead to identification of genetic variants

associated with vulnerability to diabetes rather than to diabetic retinopathy. To determine genetic variations associated with vulnerability to diabetic retinopathy, it is important to use patients with diabetes but no retinopathy as the control population.21 The results of this type of genetic association study frequently differ.22,23 The reasons offered include different ethnic groups studied and different

criteria for patient selection such as criteria involving renal function.24,25 Thus, given the numerous

pitfalls and discordant results among studies, it is a general rule that confidence in association studies increases in proportion to the number of studies that replicate a given set of findings. Meta-analyses can be useful in deriving a common thread if one exists in frequently discordant genetic studies.22

The Human Genome Project has led to the construction of a genetic map which locates all the known genes and polymorphisms to their particular loci on the 23 pairs of chromosomes (22 pairs of autosomes and 1 pair of sex chromosomes – 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 on 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 defined in four international populations.7 Haplotypes are represented by so-called 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 specific haplotype.

A critical tool in the ability to perform linkage analysis is the polymerase chain reaction. This method of taking a small amount of a specific segment of DNA and generating a large amount of identical DNA provides enough material to be detectable and measurable in order to identify the status of a person with regard to presence or absence of a genetic marker. Thus using PCR, each person in a study can be genotyped for a specific allele. The genotype of the allele can be correlated with disease status to determine associations. In PCR, short synthetic pieces of singlestranded DNA called primers are made which flank a specific 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, resulting in production of new complementary DNA adjacent to the primers at the sequence of interest. Thus two copies of the double-stranded DNA in the region of interest now exist where one had a single copy. The process is repeated many times – 30 times would be typical – 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 2.5 illustrates the process of PCR.

Case–control association studies differ from linkage analysis studies, another type of study commonly employed in the investigation of diseases with mendelian inheritance. In a linkage analysis study, a large pedigree with numerous affected members is analyzed using similar molecular genetic techniques. The status of each member of the pedigree with regard to the genetic marker under study is determined. Likewise, the disease status of each member of the family is ascertained. By evaluating the segregation of an allele of the genetic marker with the disease in a pedigree, it is possible to determine the probability that particular alleles are inherited with the disease. Figure 2.6 illustrates the process. To summarize, case–control association analyses examine unrelated population samples. Linkage analyses examine families.

An important method of analysis used in genetics studies of diabetic retinopathy is linkage analysis. In linkage analysis, the expectation is that if an allele makes an important contribution toward disease causation, then the allele should be present in a higher frequency among affected individuals than in the unaffected. Linkage analysis is based on the phenomenon of recombination that occurs during meiosis, or the process of producing sex cells. During meiosis, homologous chromosomes exchange short segments of DNA. The probability that any two small segments of DNA on a chromosome will be separated by this process depends on how far apart they are on the chromosome. The closer the two segments are, the less likely that the segments will be separated by crossing over. Even when there is no known gene directly tied to a disease, this

Fig. 2.5 (a) Schematic representation of the process of polymerase chain reaction. A double-stranded molecule of DNA called the template from a subject under investigation is broken into two single-stranded components by heating. Primers are specifically synthesized which bind to DNA sequences that flank the region of interest (the region in which one is looking 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 amplification of DNA through PCR. After 30 cycles, an initial template molecule of DNA results in 1 billion molecules of identical DNA. Adapted with permission from Della12 and Dragon26

concept is useful. One can quantitate how closely a particular genetic marker is to the presence or absence of the disease by examining large numbers of patients and looking for cosegregation of the genetic marker and the presence or absence of the disease. A metric for quantitating how closely a genetic marker is linked to a disease-causing gene is

the LOD score. The LOD score is the logarithm of the odds ratio of linkage of the genetic marker to the disease-causing gene compared to absence of linkage. A LOD score greater than 3 implies an odds ratio exceeding 1000 to 1 in favor of linkage and by convention is taken as evidence of linkage. A LOD score more negative than –2.0 implies odds of 100:1 or

Fig. 2.6 Schematic of the approach to associating polymorphisms with disease. At the top, the DNA of patients is amplified with the polymerase chain reaction to produce enough material to allow the investigator to perform electrophoresis. Electrophoresis allows the investigator to determine which allele a given person in a pedigree possesses, because the number of CA repeats affects the position of the DNA on the electrophoretic gel. Thus any given person can be identified genetically (e.g., a person may have alleles A and B or A and C). Without knowledge of the genetic information, clinicians have independently categorized the patient

as having diabetic retinopathy or not. Having diabetic retinopathy is indicated by having the person’s symbol filled in the pedigree. In the illustration, an allele A appears to be inherited with the disease because every affected person in the pedigree possesses an allele A and none of the unaffected persons possess it. However, formal analyses need to be performed which take into account other issues such as type of genetic model (parametric, such as autosomal dominant) used or not (non-parametric or model independent) and frequency of all alleles of the marker compared to controls. Adapted with permission from Damji et al.1