Inherited retinal degenerations account for a substantial fraction of blindness in children and young adults and represent clinically and genetically heterogeneous disorders. On one end of the genetic spectrum are retinal disease phenotypes associated with one gene. For example, ABCA4 (ABCR) is the causal gene for autosomal recessive (ar) Stargardt macular dystrophy (arSTGD). In addition to arSTGD, at least three more different retinal disease phenotypes; cone-rod dystrophy (arCRD), retinitis pigmentosa (arRP), and age-related macular degeneration (AMD) are caused by mutations in this gene. Due to the size (ABCA4 contains 50 exons) and a substantial genetic heterogeneity (>450 known mutations), this gene presents an extremely difficult target for genetic analysis and diagnostic applications.

On the other end are 'multigenic' diseases such as RP, where mutations in more than 30 genes can cause the same RP phenotype (estimated prevalence 1:3500), making it impossible to predict the specific gene underlying the disease in a patient based on a clinical examination. For example, the early-onset form of RP, Leber congenital amaurosis (LCA), can be caused by more than 300 mutations in at least six genes, which together account for less than 50% of the disease load. Therefore, it is not surprising that the current management of patients with retinal degenerations relies on clinical examination, electrophysiology and other ancillary tests, since available methodology does not allow for an efficient, comprehensive, and cost-effective genetic screening of patients, who are often left with no specific information on their genotype.

To overcome these limitations, we developed genotyping microarrays for ABCA4 ('gene array') and for LCA ('disease array'), representing comprehensive and cost-effective screening tools. Arrays were designed utilizing a method called solid-phase minisequencing or arrayed primer extension (APEX), which has been developed for high-throughput detection of nucleotide variations (1, 2). The APEX approach can be successfully applied for the detection of single nucleotide polymorphisms (SNPs), as well as any

deletions and insertions in heterozygous and homozygous patient samples. The designed arrays contain all currently known disease-associated genetic variants (mutations) in ABCA4 and in all known LCA genes for one-step screening of patients with STGD, CRD, RP and LCA. Both arrays are more than 99% effective in screening for known mutations, can be easily updated with new variants, and are used for highly efficient, accurate, and affordable screening of patients.

In the following chapter, we will summarize the application of APEX technology for genotyping large cohorts of patients with various eye diseases. We will also show how it allows a systematic detection and analysis of genetic variation, which facilitates proper diagnosis, results in more precise prognosis of the disease progression, helps in genetic counseling for family members and, eventually, allows the suggestion of emerging therapeutic options.

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