Genetics and Diabetic Retinopathy

Diabetic retinopathy (DR) is the leading cause of new cases of blindness for people between 20 and 64 years of age in the United States. While glycemic control is the chief risk factor for development and progression of diabetic retinopathy, there is increasing evidence for heritable risk factors. An increasing number of genetic linkage studies have uncovered the role that several genes have in the development and progression of DR.

Unlike sickle cell anemia or Huntington’s disease, diabetes mellitus does not demonstrate a mendelian inheritance pattern. No single gene causes diabetes. Rather, it is a complex genetic disease with interaction among genes and the environment. The alleles, or forms of genes, that are associated with complex genetic diseases such as diabetes are often common variants. Rather than being causative, these alleles contribute to the risk of disease expression affecting severity and age of onset.1 Diabetic retinopathy can be considered as a complex trait as well and may have a constellation of susceptibility and protective genes distinct from those associated with diabetes mellitus. There are many levels to the complexity of the interaction of these genes and gene products. For example, hemoglobin A1c (HbA1c) levels have a genetically determined component in type 1 diabetes that is independent of blood glucose level and are in turn associated with rates of progression of retinopathy.2

Non-genetic risk factors for diabetic retinopathy are well known, including duration of diabetes, glycemic control, and hypertension; however, the

D.G. Telander (*)

Davis Medical Center, University of California, Sacramento, CA 95817, USA

e-mail: dgtelander@ucdavis.edu

genetic risk factors for development and progression of diabetic retinopathy are only beginning to be understood.3,4 A greater than expected prevalence of diabetic retinopathy exists in siblings with diabetic retinopathy than in non-siblings.5 Differences in frequency of the disease among different ethnicities and populations also suggest a genetic component contributing to diabetic retinopathy.6,7 Differences in relative prevalence of diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) in different racial groups further suggest that components of diabetic retinopathy have independent genetic susceptibility profiles.8 Within a given population, the marked variation in onset and severity of retinopathy that cannot be explained by known risk factors indicates genetic susceptibility to DR.9 For example, in African-American type 1 diabetics, clinical risk factors could account for only 27% of the variance in DR severity.10 Because of difficulties unraveling the effects of shared environment from shared genes in family studies, these epidemiologic studies provide suggestive evidence only.7 Nevertheless, such evidence is an important precursor to molecular genetic explorations searching for specific genetic associations.7

2.1 Background for Clinical Genetics

This section is a brief review of genetic concepts for understanding the relevant literature and associated terminology. Lack of familiarity of genetic concepts by clinicians has often been identified as an obstacle in progress toward understanding disease pathogenesis.11 Our intent in this chapter is to help bridge this obstacle.

Understanding of molecular genetics begins with the study of deoxyribonucleic acid (DNA), a linear polymer comprised of two strands containing sequences of four nitrogen-containing bases – adenine (A), guanine (G), thymine (T), and cytosine (C) (Fig. 2.1).

Fig. 2.1 Each strand of DNA consists of a backbone of deoxyribose phosphate sugars with attached purine and pyrimidine bases. The bases show complementarity by forming hydrogen bonds with paired bases in the fellow strand. Adapted with permission from Della12

DNA is the template for its own replication. DNA also serves as a template for the creation of ribonucleic acid (RNA) in a process called transcription. This RNA copy of the DNA then in turn serves as the template for synthesis of proteins. In transcription, DNA is always read beginning at the 50 end and proceeding to the 30 end. The two strands of a DNA molecule are held together by hydrogen bonds between paired bases. Adenine in one strand always binds to thymine in its fellow strand and likewise for cytosine and guanine. For example, if a section of the sequence of one strand is 50-ATGAC-30, then its fellow strand at this locus reads 30-TACTG-50. Binding of complementary strands of DNA or between a single strand of DNA and its complementary RNA strand is termed hybridization.13

The central dogma of molecular biology states that DNA is transcribed to an RNA which is in turn translated into protein (Fig. 2.2). In the cell nucleus, transcription of DNA occurs first as a messenger RNA precursor (mRNA) that contains

the transcript of the protein-coding DNA sequence (exons), the non-protein-coding DNA sequence (introns), and untranslated regions adjacent to the 30 and 50 termini. The transcribed introns are spliced out to yield a mature mRNA product (Fig. 2.2). In the cell cytoplasm, mature mRNA is translated as three base segments called codons into amino acids that compose the protein product of the gene.14 This intricate process of an RNA-guided protein synthesis is called translation.

Humans have 23 pairs of chromosomes, each comprised of DNA and associated proteins. Each chromosome contains many genes, or sequences of DNA that code for proteins, as well as sequences of DNA dedicated to regulatory functions such as initiating transcription or translation. There are 3.3 billion base pairs in the human genome.1 The latest estimates on the number of genes in the human genome are 20,000–25,000.15 Ninety-nine percent of the genome does not code for proteins.16 These noncoding sections of the genome are broken into classes called introns and intergenic DNA. Introns, comprising 24% of the human genome, are segments of DNA adjacent to exons that are initially transcribed into an RNA strand but are then excised from initial RNA transcripts before the final mRNA strand leaves the nucleus for the cytoplasm on the way to protein synthesis (Fig. 2.2). Intergenic DNA, comprising 75% of the genome, remains untranscribed and has unknown func-

tion.16 The remaining 1% of DNA composes the exons that code for proteins.16,17

2.2The Role of Polymorphisms in Genetic Studies

The DNA sequences of any two human beings are 99% identical. The 1% of DNA that differs between any two individuals constitutes the genetic basis of certain diseases. In addition, factors that control the expression of the genes contribute to different phenotypes including disease. The purpose of genetic investigations is to determine which genes contribute to disease. On average, a difference in DNA sequence between two persons is found once per 1,200 base pairs. When a variation of the genetic locus is found in at least 1% of a given human population, the variation is termed as polymorphism.

Fig. 2.2 DNA is transcribed into an mRNA precursor which is refined to mature mRNA by the excision of sequences corresponding to introns and transported out of the nucleus

into the cytoplasm. Translation of mature mRNA results in a protein product. Adapted with permission from Della12

There are several categories of polymorphisms. Single nucleotide polymorphisms (SNPs) are single substitutions of one base for another at a certain position. There are many variations in

nomenclature for these, but one common way of naming an SNP is exemplified by c.74C>G. This means that at the level of the coding DNA sequence (indicated by the c. prefix), at position 74, a cytosine

is replaced by a guanine. The first letter characterizes the normal base and the second letter following the arrowhead signifies the mutant base. To add to the perplexing nature of the nomenclature, some authors refer to mutations not at the level of the coding DNA, but rather at the level of the altered amino acid sequence in the protein product of the mutant gene. In using this nomenclature, the convention is to list the normal amino acid first followed by the codon number in the sequence of the protein followed by the mutant amino acid. Thus, for example, Ala276Glu means that at codon 276 one finds glutamine replacing the normally expected alanine.18

Another important class of polymorphisms is that of short tandem repeats (STRs) or microsatellites. These are strings of repetitive base pair sequences that vary in length between persons. Thus the alleles are variations in the number of

repeats. For example, at a given location, one might find that one person shows CACA [or (CA)2], the next CACACA [or(CA)3], the next CACACACA [or (CA)4], and so on. Nucleotides are repeated in tandem a number of times. SNPs and STRs are scattered at different locations across the human genome, and encyclopedias of such polymorphisms have been compiled. They provide a fine toothed comb such that any part of the human genome can be probed by selecting an SNP or STR located nearby in the genetic map.

Yet another type of polymorphism, more often used in older genetic association studies, is the restriction fragment length polymorphism. Enzymes called restriction endonucleases cleave DNA at sites where certain DNA sequences are detected. There are many restriction endonucleases (Fig. 2.3). By analyzing the length of fragments of

Fig. 2.3 (a) Restriction enzymes cleave DNA at specific sites. There are many restriction enzymes. Four (MspI, TaqI, EcoRI, and HindIII) are illustrated here as well as the loci cleaved by them. (b) The result of applying a restriction

endonuclease to DNA is a set of DNA fragments that can be separated by electrophoresis. The electrophoretic pattern can distinguish different alleles (A and a). Adapted with permission from Musarella19