The Roundabout, Axon Guidance Receptor, Homolog 1 (ROBO1), a Potential Modifier and Therapeutic Target for Early and Intermediate Dry Age-Related Macular Degeneration

Abstract

Purpose: : To examine the ROBO1 gene - -identified using a systems biology based approach that included data from gene expression data, its hypothetical anti-angiogenic network function and its genomic location -- as a candidate for, and possible contributor to the pathophysiology of AMD.

Methods: : We ascertained 254 families comprised of 585 siblings that included individuals with normal maculas, and all subtypes of AMD (AREDS category 2, 3 and neovascular AMD). Fifteen tagging SNPs were genotyped by employing the Sequenom iPLEX technology. Using AMD categories as a quantitative trait, single SNP analysis was conducted using FBAT after adjusting for age and smoking status under an additive model. Haplotypes were constructed in a moving window approach using SNPs with p ≤ 0.1 in FBAT under an additive model. Gene-gene interaction between variation in ROBO1 and CFH and s ARMS2/HTRA1 was tested in Unphased 3.1.

Results: : Under an additive genetic model we identified SNPs and haplotypes in ROBO1 that were associated with risk of dry AMD (AREDS category 2 and 3) when compared to normal siblings. The most significant haplotype was comprised of the minor alleles from the two most significantly associated AMD risk SNPs (rs7640053 and rs7626242), p = 2.5 x 10 ^-3 after permutation testing correction. Significant interactions between variants in ROBO1 and variation in ARMS2/HTRA1 were identified (p value ranged from 2.5 x 10 ^-5 to 8.0 x 10 ^-3). This finding is complemented by our gene expression microarray studies and linkage analysis that show that ROBO1 mRNA is statistically significantly down-regulated in AMD patients and located in a region that harbors AMD susceptibility genes (3p12).

Conclusions: : A significant haplotype of ROBO1, a previously unreported dry AMD-associated susceptibility gene coupled with significant interaction with ARMS2/HTRA1 has given us a refined region to identify causal variants by direct sequencing in our family based cohorts as well as subsequent replication and validation in larger unrelated case control populations.

Keywords: age-related macular degeneration • gene screening • clinical (human) or epidemiologic studies: risk factor assessment