Genetics of retinal disease

INTRODUCTION

Genetics plays an increasingly important role in our understanding of retinal disease, aiding in diagnosis, furthering our knowledge of pathogenesis, and now proving instrumental in developing specific disease treatments.

Molecular genetics has allowed us to progress beyond merely classifying inherited retinal disease in humans on the basis of their phenotypes, to now being able to define disease at the molecular level. The identification of disease mutations permits the development and testing of functional animal models, with the aim of elucidating disease mechanisms as well as developing new and improved therapeutic agents, including the replacement of the defective gene through gene therapy. Recently two independent clinical trials provided a glimpse of the potential therapeutic power gene therapy may wield in retinal disease. These reported the first successful application of gene therapy to three young adult patients using subretinal injections of recombinant adenoassociated virus vector expressing complementary DNA (cDNA) of the retinal gene RPE65. Partial restoration of vision was achieved in these individuals possessing a rare form of inherited retinal disease, Leber’s congenital amaurosis (LCA).1,2

Gene discovery in retinal diseases initially centered on those with a mendelian mode of inheritance, and these approaches were very successful in terms of numbers of genes and disease-causative mutations identified. Most recently, major advances in the study of complex retinal diseases such as diabetic retinopathy and age-related macular degeneration (AMD) have also been made, with both of these diseases showing a significant genetic component and a number of diseaseassociated variants identified. This information together with the ensuing improved knowledge of biological pathways involved in disease pathogenesis has provided a key impetus to the development of new treatment modalities.

This chapter will provide a historical perspective of gene discovery in retinal disease, using illustrative examples to look at the effect genetics has had on our understanding of retinal disease pathogenesis and consequently options for pharmacotherapy. Clearly, space restrictions prevent an indepth analysis, but it is hoped that it will serve to highlight a few aspects of this fascinating and rapidly changing field.

HISTORY OF RETINAL GENE DISCOVERY

Determining the underlying DNA sequence that constitutes a genotype and correlating this with disease phenotype is one of the fundamental aims of genetics. In the last two decades over 190 retinal disease-associated genes and loci have been identified, with hundreds of sequence variants as well as novel variants reported, and the numbers continue to increase (Figure 7.1). There is a startling contrast between the laborious, expensive methods used to unearth the first genes associated with retinitis pigmentosa (RP) in the early 1990s and the current clinic-based gene-specific microarray chips in use today. The latter are typically plated for every reported sequence variant in a number of genes involved in retinal disease. Such retinal gene chips

are now in regular use for the study of hereditary retinal disease, based on genes such as RPE65 gene in LCA disease, the ABCA4 gene in Stargardt’s disease, and multiple RP genes.3–5 Using such technology, it is possible to identify known causative mutations within days rather than months and provide readily available clinic-based testing at a diagnostic or research level for a minimal charge. Future advances in this field will invariably include the scaling-up through the use of many more genes on a chip as well as individual whole-genome sequencing once costs are reduced. One can only probe for sequence variants that are already known or characterized for their involvement in retinal disease. Recent reviews of retinal dystrophy screening report, on average, a 50% success in identifying the causative mutation. However, there are still retinal disease pedigrees for which genetic linkage analysis has excluded all currently known loci and for which no known genes are known.3,4

The vast majority of hereditary retinal disease genes, including those for RP, LCA, and Stargardt’s disease, have been identified through genetic linkage analysis where these genes have been shown to segregate with disease in a mendelian pattern of inheritance in large families or pedigrees. The mapping techniques first developed in the 1980s utilized restriction fragment length polymorphisms (RFLPs) as molecular markers, which were then superseded by distinctive sequences called microsatellite markers. However, prior to completion of the first linkage map, Botstein and Lander realized that most human traits and diseases followed complex modes of inheritance where there was no clearly definable inheritance pattern.6,7 In addition, as in the case of AMD, linkage analysis may be hampered by the limited availability of multigenerational families due to the late onset of disease. Thus alternative approaches for studying such traits were sought and rapid advances have now been made in the study of complex retinal diseases through the use of single nucleotide polymorphisms (SNPs). These represent biallelic genetic variants randomly spaced across the genome at an average spacing of 1 every 300–400 nucleotides. These markers provide the basis for association studies, which identify the effect a genetic variant has on disease by examining whether there is an increased or decreased frequency of alleles or genotypes in cases when compared to that expected by random distribution, and compared to control individuals who present with no disease trait.6

Genome-wide association studies (GWAS) have hugely accelerated the pace of gene discovery in complex traits. GWAS using microarrays or “gene chips” can efficiently probe the genome to detect genomic differences associated with disease or the phenotypic trait of interest. A pioneering GWAS executed by Klein et al.8 identified a strong association between the Y402H variant in the complement factor H (CFH) gene and AMD, similar results were obtained concurrently by other groups using distinct approaches.9–12 Although GWAS was instrumental in the discovery of CFH at 1q31, several other genes linked or associated with AMD have also been identified using various genetic techniques. including the LOC387715/HTRA1 genes at 10q26, as well as the CFH-related (CFHR), C2/BF C3, and APOE genes.13–16 Through the use of better-defined clinical phenotypes and larger cohort size, more powerful genetic studies are now being undertaken to allow genes of moderate effect size to be identified. The GWAS used by Hoh had a mere 100 000 SNPs, which was a figure initially postulated to be sufficient to map the genome effectively. The figure was then revised to 500 000; current gene chips are now in the range of 1 million SNPs.