We demonstrated significant association with ROBO1
SNPs showing increased risk of wet and early/intermediate dry AMD in a combined cohort of sibling pairs, cases and controls from Central Greece, as well as a prospective case control study from the NHS/HPFS. Moreover, we discovered variants from different LD blocks that could explain the separate association signals for wet and early/intermediate dry AMD. This suggests that different regions of this gene may be responsible for risk of the different subtypes of AMD or possibly indicate who may progress to wet or neovascular AMD, which would have implications for therapeutic targets. Previous genetic association studies reported that ROBO1
polymorphisms are associated with other diseases of complex etiology. For example, variation in ROBO1
is associated with language ability
and shared genetic factors between asthma and obesity in children.
A prior study shows that ROBO1
is also associated with autism, showing that mRNA expression is significantly down-regulated in those with autism.
Furthermore, a role for Robo1 expression in retinal angiogenesis has been demonstrated in a rabbit model of proliferative retinopathy as well as in vitro studies of epiretinal and subretinal membranes from patients with proliferative retinopathies.
In addition to establishing association of ROBO1
with AMD, we were able to document a statistically significant interaction between ROBO1
and another AMD-associated gene, RORA
a gene that is known to be involved in wet AMD based on retrospective
studies, regulates expression of genes in the mammalian clock mechanism
and in lipid metabolism by changing levels of total plasma cholesterol, triglycerides, and apolipoprotein.
DNA response elements of RORA
comprise a 5’ AT-rich sequence and along with coactivators, change constitutive activation of target gene transcription.
Interestingly, analogous to ROBO1
, the protein product of the RORA
gene is also significantly reduced in the autistic brain.
Statistical association and interaction with ROBO1
genes were validated by a bioinformatic search for response elements residing on ROBO1
sequences followed by experimental confirmation using chromatin immunoprecipitation assays and qRT-PCR in normal mouse maculae. Using the known Rora response elements as a positive control, we established similar quantity of precipitation with the regulatory region of the ROBO1
gene as a potential Rora binding site. This gave further evidence underlying a biological interaction between a Rora product and the regulatory element of the ROBO1
gene. We are currently extending the findings in mouse using chromatin immunoprecipitation assays to direct sequencing in human donor eyes and immortalized patient cell lines.
Our previous observation of down-regulation for ROBO1
in immortalized cell lines
was validated in human donor RPE-choroid and retina in the current study. Similar to the reduced expression of this gene in autism, expression levels for ROBO1
in AMD macular and peripheral retinas compared with normal maculas and peripheral retinas were significantly reduced. This also lends support for the hypothesis that AMD is a systemic disease with a localized manifestation, as significant differences in expression of ROBO1
on a systemic level, in cell lines,
was confirmed on the DNA level although no differences in expression of ROBO1
were seen between AMD eyes and non-AMD eyes. Our findings along previous reports suggest that pathogenesis of ROBO1
in complex diseases is potentially shared by down-regulation of expression in response to neurodegeneration and these findings could have significant implications for therapeutic interventions and drug delivery.
Our statistical findings along with molecular verification have improved our understanding on the potential synergetic effect of ROBO1 and RORA in the early/intermediate AMD stages as well as the severe advanced neovascular form of AMD. In addition to the discovery of multiple variants in ROBO1 that may differentiate wet and early/intermediate dry AMD, SNPs in ROBO1 were found to interact with RORA in the early/intermediate dry form of AMD in meta-analysis that were not found to significantly interact in the neovascular AMD subtype as shown in .
The study design is unique from others such that we separated two subtypes of AMD from all AMD or advanced AMD, to investigate multiple variants that may be involved in the early/intermediate or advanced/severe neovascular AMD subtype. This approach is supported by an illustration of a review
that three different components of AMD, drusen formation, neovascularization, and RPE atrophy, have seen in many different complex diseases, implying that there may be independent underlying mechanisms to develop each of these components. A previous study also demonstrated that drusen formation may have both unique and shared underlying genetic mechanisms with intermediate or advanced AMD development.
Specifically, this study showed that drusen formation as an intermediate stage of advanced AMD types identified previously known linkage signals for advanced AMD as well as novel peaks. One of the unique peaks for large drusen size is on chromosome 19q13.21 that is accounted for the genotype of APOE
gene. These further support our results on differential association signals for wet and early/intermediate dry AMD. Our hypothesis-driven, genomic convergent approach based on prior biological plausibility provided collective evidence from statistical tests and molecular experiments demonstrating potentially yet another pathway underlying AMD pathogenesis. Thus, our results, together with statistical findings and molecular verification, warrant further investigation for both diagnostics and therapeutics implications by taking both genes into consideration, as they appear to work together.