FLT1 genetic variation predisposes to neovascular AMD in ethnically diverse populations and alters systemic FLT1 expression

Abstract

Purpose: Current understanding of the genetic risk factors for age-related macular degeneration (AMD) is not sufficiently predictive of the clinical course. The VEGF pathway is a key therapeutic target for treatment of neovascular AMD; however, risk attributable to genetic variation within pathway genes is unclear. We sought to identify single nucleotide polymorphisms (SNPs) associated with AMD within the VEGF pathway.

Methods: Using a tagSNP, direct sequencing and meta-analysis approach within four ethnically diverse cohorts, we identified genetic risk present in FLT1, though not within other VEGF pathway genes KDR, VEGFA, or VASH1. We used ChIP and ELISA in functional analysis.

Results: The FLT1 SNPs rs9943922, rs9508034, rs2281827, rs7324510, and rs9513115 were significantly associated with increased risk of neovascular AMD. Each association was more significant after meta-analysis than in any one of the four cohorts. All associations were novel, within noncoding regions of FLT1 that do not tag for coding variants in linkage disequilibrium. Analysis of soluble FLT1 demonstrated higher expression in unaffected individuals homozygous for the FLT1 risk alleles rs9943922 (P = 0.0086) and rs7324510 (P = 0.0057). In silico analysis suggests that these variants change predicted splice sites and RNA secondary structure, and have been identified in other neovascular pathologies. These data were supported further by murine chromatin immunoprecipitation demonstrating that FLT1 is a target of Nr2e3, a nuclear receptor gene implicated in regulating an AMD pathway.

Conclusions: Although exact variant functions are not known, these data demonstrate relevancy across ethnically diverse genetic backgrounds within our study and, therefore, hold potential for global efficacy.

Keywords: FLT1; VEGF; age-related macular degeneration; angiogenesis.

Figure 1

sFLT1 interaction with FLT1 SNPs and Neovascular AMD. Serum was obtained from the discovery cohort and soluble FLT1 levels were determined using ELISA. Statistical analysis was performed for the interaction between each FLT1 SNP, sFLT1, and neovascular AMD. The interaction P for SNPs rs7324510 (A) and rs9943922 (B) demonstrate significance between these three variables. In patients with these SNPs, serum sFLT1 were elevated in patients with two risk alleles.

Figure 2

Nr2e3 binds Flt1 regulatory sequence. (A) Transcription factor binding sites for murine Flt1. Region represent nucleotides 148,527,456 to 148,557,564 on mouse chromosome 5. Red arrow indicates Flt1 transcriptional start site. Bars indicate approximate location of binding sites. Red asterisk indicates Nr2e3 RE site identified by Chromatin immunoprecipitation (ChIP). (B) ChIP-qPCR showing the nuclear hormone receptor Nr2e3 binds Flt1 regulatory sequence.