patients with proliferative diabetic retinopathy compared with samples from patients without diabetes and with nonproliferative diabetic retinopathy.50

Polymorphisms in the VEGF regulatory regions seem to be significant. In British Caucasians with either type 1 or type 2 diabetes, the VEGF -460C allele increases the risk of proliferative diabetic retinopathy (Table 2.3).51 In Japanese type 2 diabetics, the C- 634G polymorphism in the 50-untranslated region of the VEGF gene was associated with presence of any diabetic retinopathy, and subjects who were homozygous for the C-634C allele had higher fasting serum VEGF levels than those with other genotypes.33 The association was not found when PDR rather than any DR was the phenotype examined.52 In Brazilians of European descent, homozygosity for the C-634C

allele increases the risk of PDR, but the same result was not found in British Caucasians.34,51 For PDR in

a Japanese population with type 2 diabetes, the - 2578C/A polymorphism in the promoter region of the VEGF gene was informative. The A/A genotype was associated with higher risk for PDR.52

Besides determining genetic markers for increased or decreased risk of diabetic retinopathy, we would be interested to know how such genetic variations confer these properties. With respect to the VEGF gene, some information is known. VEGF has a number of isoforms. In the eye, the VEGF-165 isoform seems to be the most important one, which in turn is found in a and b subtypes. VEGF165a is pro-angiogenic, whereas VEGF165b is anti-angio- genic. VEGF165b is the predominant form in the healthy vitreous. Diabetes results in an increase in insulin-like growth factor which downregulates VEGF165b and thus changes the VEGF165a/ VEGF165b ratio. Studies have also looked at the vitreous levels of VEGF stratified by genotype.52

2.4.6 IGF-1

Insulin-like growth factor 1 (IGF-1) is a known regulator of VEGF expression, and Simo et al.

found a significantly higher level of IGF-1 in the vitreous of patients with PDR.53,54 Therefore poly-

morphisms in the IGF-1 gene have been investigated. In patients with impaired glucose tolerance (IGT) or type 2 diabetes, the presence of a variant

IGF-I gene polymorphism was associated with an increased risk of retinopathy. The odds ratio for the risk of retinopathy for variant carriers was 1.8 (95% CI 1.0–3.2, P ¼ 0.04).55

2.5 Genes in or Near the HLA Locus

The HLA locus found on chromosome 6p21 has been studied for genetic linkage to proliferative retinopathy in both type 1 and type 2 diabetes.6 HLA alleles DRB1*0301, DQA1*0501, and DQB1*0201 have been reported to be associated with severe retinopathy in patients with type 1 DM.56 In African-American type 1 diabetics, HLA-B was associated with severe DR and progression of DR.10 In a subgroup of the Wisconsin Epidemiologic Study of Diabetic Retinopathy, persons with HLA-DR4 who were negative for DR3 were more likely to have PDR than those negative for both antigens (odds ratio 5.43, 95% CI 1.04, 28.3).57 In a Finnish study of adolescents with type 1 DM, HLA DR1 was associated with presence of any DR.58 Other studies have not found influence of major histocompatibility complex genes on diabetic retinopathy.6

2.6Receptor for Advanced Glycation End Products (RAGE) Genes

Advanced glycation end products of proteins (AGEs) are produced when proteins are chronically exposed to hyperglycemia and have been found in the plasma and tissues of diabetic patients.59–61 The main effects caused by AGEs are thought to occur through the RAGE receptor including the development of diabetic retinopathy and other complications. The RAGE gene is located in the HLA region on chromosome 6.62 Studies have been inconsistent on the association of polymorphisms close to this gene and DR. Numerous studies have found no association in Caucasian and Chinese populations.9,63 Kumaramnickavel et al. found a negative association in an Asian-Indian population.64 Hudson, using a different SNP, found a positive association in a Caucasian population.65

2.7 Endothelial NOS2 and NOS3 Genes

Nitric oxide (endothelial-derived relaxing factor, S- nitrosocysteine) regulates vascular tone and inhibits platelet aggregation and monocyte adhesion to endothelial cells. Its production is under control of the nitric oxide synthases coded by distinct endothelial nitric oxide synthase genes. The NOS3 gene is expressed constitutively in retinal vascular endothelium and governs normal dilator tone. The NOS2A gene is not normally transcribed, but exposure to certain cytokines can lead to its abnormal expression. Warpeha and colleagues found that NOS3 expression is decreased in hyperglycemia, and that a 14 repeat allele of a pentanucleotide polymorphism in the 50 untranslated region of the NOS2A gene results in peak transcription of NOS2A under hyperglycemic conditions. This polymorphism is associated with absence of retinopathy in British Caucasians, thus appearing to be a protective allele. The 4b/b genotype of the 4a/b polymorphism for NOS3 has been associated with DR in West Africans. No association of the 4a or 4b alleles was found with DR in Japanese type 2 diabetics.67

2.8Renin–Angiotensin SystemAssociated Genes

A meta-analysis of seven studies exploring a possible association of an insertion–deletion polymorphism of the angiotensin-converting enzyme gene found no association. Based on a pooled sample of 1008 subjects with retinopathy and 1002 without retinopathy, the summary odds ratio for the D allele was 0.91, 95% CI 0.73–1.13.68 In a review by Uhlmann et al., 13 of 15 studies were negative for any association.9 Counterexamples exist to the general theme of lack of an association. Three studies of Korean, Japanese, and Iranian type 2 diabetics showed an association of

the D allele or genotype with any DR, advanced DR, and PDR, respectively.24,69,70

The angiotensin II (type 1) receptor gene is located on chromosome 3q2125. Genome-wide association scanning detected a tentative linkage for risk of DR with a site close ( 20 cm) to this gene.71

2.9Solute Carrier Family 2 (Facilitated Glucose Transporter), Member 1 Gene (SLC2A1)

GLUT1 is the main glucose transporter across endothelial cells in capillaries of the retina and is

also found in retinal glial cells, ganglion cells, and photoreceptors.10,72,73 Hyperglycemia is the stron-

gest clinical risk factor for prevalence, incidence, and progression of DR, thus the SLC2A1 gene on chromosome 1 that codes for GLUT1 is a candidate for influencing DR.10 In African-American type 1 diabetics, SLC2A1 was significantly associated with severe DR (i.e., severe NPDR or PDR) and with progression of retinopathy.10 However, three other studies, two in Caucasian populations and one in an Asian population, have not found associations.6

2.10 Gene–Environment Interaction

Genetic studies are beginning to uncover examples of interaction of genetic and environmental effects on DR. For example, the -677TT genotype for the methylenetetrahydrofolate reductase gene polymorphism has been found to be associated with NPDR in patients with higher HbA1c but not in patients with lower HbA1c. Other examples are certain to follow.74

2.11Potential Value of Identifying Genetic Associations with Diabetic Retinopathy

Screening for diabetic retinopathy is an inefficient but currently necessary endeavor. The early phases of PDR and DME may be asymptomatic, which requires that large numbers of asymptomatic people must be examined with dilated fundoscopy to detect those who possess treatable early forms of these complications. Genetic profiling offers the potential to narrow the group predisposed to these complications and preferentially assign screening resources in these people.86 Likewise, there are likely to exist protective genetic haplotypes that allow health-care

Hardy–Weinberg Equilibrium a state in which the gene pool of a population is not changing from generation to generation

Heritability the proportion of phenotypic variation in a population attributable to genetic variation. Heritability estimates the relative contributions of genetic and environmental factors to phenotypic variation. It describes the population, not something about an individual. If the heritability of trait P is 0.7, it is incorrect to say the 70% of X comes from genetics and 30% comes from the environment. Rather, 70% of the variation of X comes from variation in genotypes in the population and 30% comes from variation in environmental factors in the population

Heterozygous the situation in which the members of a pair of alleles are different

Homozygous the situation in which both members of a pair of alleles are identical

Intergenic DNA untranscribed DNA of unknown function

Intron a sequence of DNA that does not code for a protein; it is excised from the initial RNA transcript

Linkage cosegregation of a gene or DNA marker with another gene or DNA marker close by

Linkage Disequilibrium nonrandom association of alleles at two or more loci; in general, the closer the two loci on a chromosome, the greater the linkage disequilibrium

Locus the position of a gene or genetic marker on the genetic map

Locus Heterogeneity several different mutations causing a similar phenotype, for example, many different genetic mutations cause retinitis pigmentosa

Meiosis sex cell division which produces daughter cells with half the number of chromosomes as the parent cell. The assortment of chromosomes from parent to daughter cells is random. During meiosis, recombination occurs

Mendelian an adjective that implies that only one gene is involved

Mutation a single-letter change to the DNA sequence. A single-letter change that causes the specified codon to change (i.e., under the mutation

specifying the wrong amino acid) is called a missense mutation. One that causes a codon change that leads to a stop codon is called a nonsense mutation. One that causes a codon change that leads a stop codon to become a codon for an amino acid is called a sense mutation. Finally, one that causes a codon change but that does not change the specified amino acid (because there are multiple codons for each amino acid) is called a silent mutation.

Penetrance presence or absence of an effect of a gene. Penetrance is dichotomous. Expressivity is graded. Penetrance is defined as the ratio of the prevalence of the expressed trait to the prevalence of the underlying mutation. A high penetrance, i.e., close to 1, implies that nearly everyone who poses the mutation will express the trait.

Polymerase Chain Reaction (PCR) a technique for amplifying quantities of specific genetic material. Involves three steps repeated 30–40 times. The steps are denaturation of double-stranded DNA by heat, annealing of DNA primers to the DNA sequence of interest, and extension of each annealed primer by DNA polymerase producing a new complementary strand of DNA. One gets geometric expansion of the number of DNA fragments of interest and these can be stained and the products separated by size using polyacrylamide gel electrophoresis.

Polymorphism a variation in DNA sequence found in at least 1% of a given human population.

Odds Ratio a measure of the effect size of possessing one or two copies of an allele. For example, suppose possessing allele X is associated with a probability p of having diabetic retinopathy and possessing allele Y is associated with a probability q of having diabetic retinopathy. The odds of having retinopathy are p/(1–p) and q/(1–q) in the two cases, respectively, and the odds ratio is p/(1–p)/ [q/1–q)]. An odds ratio greater than 1 implies that having diabetic retinopathy is more likely if one has an allele X than if one has an allele Y. An odds ratio less than 1 implies that having diabetic retinopathy is more likely if one has an allele Y than if one has an allele X.

Proband the first affected family member seeking medical attention for a genetic disorder