Abstract
Retinopathy of prematurity (ROP) is a neurovascular complication in preterm babies, leading to severe visual impairment, but the underlying mechanisms are yet unclear. The present study aimed at unraveling the molecular mechanisms underlying the pathogenesis of ROP. A comprehensive screening of candidate genes in preterms with ROP (n = 189) and no-ROP (n = 167) was undertaken to identify variants conferring disease susceptibility. Allele and genotype frequencies, linkage disequilibrium and haplotypes were analyzed to identify the ROP-associated variants. Variants in CFH (p = 2.94 × 10−7), CFB (p = 1.71 × 10−5), FBLN5 (p = 9.2 × 10−4), CETP (p = 2.99 × 10−5), and CXCR4 (p = 1.32 × 10−8) genes exhibited significant associations with ROP. Further, a quantitative assessment of 27 candidate proteins and cytokines in the vitreous and tear samples of babies with severe ROP (n = 30) and congenital cataract (n = 30) was undertaken by multiplex bead arrays and further validated by western blotting and zymography. Significant elevation and activation of MMP9 (p = 0.038), CFH (p = 2.24 × 10−5), C3 (p = 0.05), C4 (p = 0.001), IL-1ra (p = 0.0019), vascular endothelial growth factor (VEGF) (p = 0.0027), and G-CSF (p = 0.0099) proteins were observed in the vitreous of ROP babies suggesting an increased inflammation under hypoxic condition. Along with inflammatory markers, activated macrophage/microglia were also detected in the vitreous of ROP babies that secreted complement component C3, VEGF, IL-1ra, and MMP-9 under hypoxic stress in a cell culture model. Increased expression of the inflammatory markers like the IL-1ra (p = 0.014), MMP2 (p = 0.0085), and MMP-9 (p = 0.03) in the tears of babies at different stages of ROP further demonstrated their potential role in disease progression. Based on these findings, we conclude that increased complement activation in the retina/vitreous in turn activated microglia leading to increased inflammation. A quantitative assessment of inflammatory markers in tears could help in early prediction of ROP progression and facilitate effective management of the disease, thereby preventing visual impairment.
Highlights
Retinopathy of prematurity (ROP) is a complex disease of the retina with a multi-factorial etiology and an early intervention has been observed to prevent irreversible vision loss in some of these prematurely born infants [1]
No mutations were observed in the Norrin signaling genes in ROP in our earlier study [20], we explored genes involved in angiogenesis, growth, and development of the fetal retina, trans-endothelial migration, oxidative stress, inflammation, and neurodegenerative processes, in order to understand their role in ROP pathogenesis
Strong linkage disequilibrium (LD) was observed across all the variants in CFH and rs891141 and rs289716 in CETP gene, while moderate LD was observed between rs891141 and rs289713 in CETP and rs2268002 and rs2284340 in FBLN5 (Figure S1 in Supplementary Material)
Summary
Retinopathy of prematurity (ROP) is a complex disease of the retina with a multi-factorial etiology and an early intervention has been observed to prevent irreversible vision loss in some of these prematurely born infants [1]. Lower gestational age (GA), lower birth weight (BW), and oxygen supplementation are the primary risk factors associated with ROP [7] It is a selflimiting disease with initial symptoms of avascular retina that progresses to abnormal growth of retinal vessels causing retinal detachment [8]. Few functionally relevant genes (NDP, FZD4, LRP5, CFH, VEGF, ANGPT2, EPO, BDNF, and CETP) have been associated with ROP in a small fraction of cases. Many of these variants could not be replicated across different ethnicities [11,12,13]. Their roles in risk predictions and disease management are yet to be determined
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