Figure 7.1  Number of mapped and identified retinal disease genes from 1980 to 2008. Reproduced from RetNet, the Retinal

Information Network, http://www.sph.uth.tmc.edu/RetNet/, copyright Stephen P Daiger, PhD and The University of Texas Health Science Center, Houston, Texas, with permission.

KEY CONCEPTS AND FUNDAMENTS OF GENETIC METHODS IN THE STUDY OF RETINAL DISEASE

A glossary is provided to clarify terms used in genetic studies (Table 7.1).

GENETICS: ILLUMINATING MECHANISMS OF PATHOGENESIS, REVEALING COMPLEXITY

Developing effective, targeted therapy requires an understanding of the mechanisms of disease pathogenesis. Defining diseases on a molecular level has revealed the staggering genetic and pathogenic heterogeneity of retinal dystrophies, and perhaps explains why until now there has been a failure to develop such therapy.

GWAS of such a scale allow a huge amount of genomic information to be derived without the gold standard of needing complete genome sequencing for all, which, although rapidly approaching, is still just out of reach for most researchers for practical and economic reasons. Resequencing of a gene or locus of interest is now an accessible method to identify specific or novel changes in a gene that have been previously identified in disease.5,17

From the first complete sequencing of the human genome in 2001 up until 2004, SNPs were thought to be the major cause of genetic variation among individuals. Most attention had turned from structure to function. However the identification of genome-wide copy number variation (CNVs) has changed this belief. Analogous to chromosomal changes seen in cytogenetics, yet found in phenotypically normal individuals, these submicroscopic structural variants include deletions, duplications, inversions, and translocations which range in scale from a few kilobase pairs (kbp) up to 130 kbp.18 They are randomly distributed throughout the genome and collectively they are thought to account for a large amount, if not most, of genomic variation. In view of their ubiquity, it is not surprising that the contribution to diseases by CNVs has become increasingly apparent. CNVs (deletions) in CFHR genes 1 and 3 have been shown to reduce the risk of developingAMD,16 and deletions in PRPF31 have been found to account for at least 5% of autosomal-dominant RP.3 Data regarding the fine-scale architecture and complexity of CNVs are still being compiled; this information is necessary to create an accurate baseline of genomic variation and to develop specific methods to examine further their disease associations. A step towards this is the first high-resolution map of structural variation, published in recent months.19 Future GWAS with high densities of SNP markers and the ability to probe simultaneously for CNVs are likely to shed considerably more light on many diseases, including AMD.

Genetic testing improves diagnostic accuracy, provides prognostic information, is used in prenatal screening, and guides therapy, but the ultimate goal is to provide effective genotype-specific treatment, of which defective gene replacement through gene therapy holds the most promise. The precursors to techniques used in human gene therapy emerged in the 1970s and early 1980s. Mammalian genes were subcloned into prokaryotic plasmids and bacteriophages, retroviral vectors were developed and were in common laboratory use by the mid-1980s as a means to transfer genes into mammalian cells. By the late 1980s the stage was set for the first human trials of gene transfer in systemic disease. There have since been encouraging results from animal models and mixed, but promising, results from clinical trials in a range of retinal disease from retinoblastoma to AMD, many of which are still under way. There have also been exciting developments in pharmacotherapy as a direct result of our growing understanding of the pathogenesis of diseases such as AMD. These topics will be examined more closely later in the chapter.

RP: A “COMPLEX” MONOGENIC DISEASE

A spectacular example of such heterogeneity is RP, which is now recognized as not one disease, but a group of neurodegenerative retinal diseases that cause photoreceptor cell death that leads to progressive visual loss and blindness. They are clinically broadly similar despite many different causative genetic mutations and diverse pathogenic pathways; RP has been said to be no more a single disease than “fever of unknown origin.” Fifty-three genes have been implicated in the pathogenesis of nonsyndromic RP and the related LCA, and many different mutations have been reported in these genes. There is a great functional diversity in the types of genes that have been implicated in RP; some are eye-specific (e.g., visual transduction cascade, structural proteins, retinoid cycle) or widely expressed (e.g., splicing factors, nucleotide metabolism).3,4

Even once a specific gene encoding a particular protein is known, it is not completely straightforward to link genotype and phenotype. An example is the gene encoding peripherin/retinal degeneration slow (RDS) protein. This is a multifunctional protein, important in disc morphogenesis, maintenance of proper disc alignment, and disc shedding. Peripherin/RDS mutations are an important cause of autosomaldominant (ad) RP, accounting for approximately 9% of cases in northernEuropeanpopulations.Todate,34differentmutationsofperipherin/ RDS have been implicated in adRP. Transgenic mouse models and in vitro experiments have allowed detailed study of the variations in protein products from these different mutations and the resulting phenotypes; it appears that both haploinsufficiency and negative dominance play a role in pathogenesis. All mutations cause photoreceptor and RPE cell death, although the exact mechanisms remain unclear. Some animal studies have shown that distinct mutations affect rods and cones differently. As mentioned above, peripherin/RDS mutations can cause both RP and a range of macular dystrophies, but what is particularly interesting is that an identical mutation can cause completely different phenotypes within the same family20 (Figure 7.2). These may range widely, from an apparently normal fundus to various macular dystrophies, cone–rod dystrophy as well as adRP, despite an identical causative mutation. Incomplete penetrance has been reported in other families. No satisfactory explanation yet exists for this spectrum in phenotype and variability in penetrance, but it is apparent that there are other factors which exert their influence on the phenotypic outcome: genetic background, environmental influences, and modifier genes have been suggested as plausible candidates.21

One such gene suspected of being a modifier gene was ROM1. Three different mutations in the ROM1 gene each cause RP only when coinherited with a specific missense mutation (Leu185Pro) of peripherin/ RDS; this is therefore termed digenic RP. However, mutations in ROM1 have not been found in two families with intrafamilial phenotypic variation,22 suggesting that other modifier genes are likely to exist.

Interest in modifier genes has grown with the discovery of a number of families with identical mutations producing distinct phenotypes. In one family with a peripherin/RDS mutation, rod–cone dystrophy was seen in affected male members, whereas female members had a macular disease; this raised the possibility of an X-linked modifier gene. Discordant phenotypes in fraternal (dizygotic) twins with a mutation in the RP GTPase regulator gene (RPGR) have also recently been reported.23 Modifier genes thus add another layer of complexity to the pathogenic mechanisms of retinal dystrophies. As their effect is small, modifier genes are difficult to identify; GWAS and eventually routine sequencing are likely to further our knowledge in this area. It is possible that modifier genes may prove to be another potential target for gene therapy.

Environmental factors, which have been traditionally considered to exert little or no effect on monogenic disease (and therefore on which little research has been conducted), may also warrant further examination in future studies; some factors, such as light exposure, vitamin A, and dietary fats, are thought to influence macular dystrophies. Our

expanding knowledge of the genetics of mendelian diseases and complex traits increasingly blurs the boundaries between them: mendelian traits are not so “cut and dry” in being simply inherited, and complex diseases such as AMD have been found to have a strong genetic component, with genetics unraveling many of its complexities.

SHEDDING LIGHT ON AMD

AMD is a multifactorial disease: in addition to a genetic component, age, diet and other environmental influences such as smoking affect phenotype. Late complications, which can lead to severe visual loss, include choroidal neovascularization (CNV), known as “wet AMD,” and geographic atrophy, also known as “dry AMD.” The immune system was first implicated in the pathogenesis of AMD in 1999 as a result of some elegant immunocytochemical analyses performed by Hageman et al.24 on drusen, the hallmark of early AMD. These were