ROP represents one of the major challenges of pediatric ophthalmology because: 1) it has a difficult to predict course - so far it has not been possible to identify the factors that cause the progression of the disease to the most severe stages threatening retinal detachment and loss of vision; 2) it affects an increasing number of patients, due to the growing number of rescued premature infants with lower and lower birth weight and younger GA; 3) currently there are no known ways to prevent this disease. To achieve better monitoring and treatment, it seems important to understand the pathogenesis and etiology of this disease through genetic studies and correlate the information obtained with clinical data. To date, however, the contribution of specific genetic risk factors to ROP remains unclear.
In this study, we focused on three genes associated with the presence of IH and two with broader functions in the pathogenesis of ROP (Supplementary Table S3). Our analyses did not confirm the involvement of IH-related genes in the pathogenesis of ROP. They did, however, indicate the importance of the ADRB2 gene, which may modulate the pharmacotherapy of IH. We found that the 79G allele was a risk factor for ROP, especially for the form requiring treatment. This allele was also associated with NEC and ELGA. The highest frequency of this allele was present in ROP requiring treatment (0.621) and NEC (0.615), followed by ELGA (0.580) and spontaneously resolving ROP (0.507). Its frequency in infants without ROP was 0.421, which corresponds to the frequency (0.436) in the healthy adult Polish population (blood donors)33. The effect of the ADRB2 79G allele on ROP was more substantial than in ELGA and NEC, which were significant only for homozygotes. Co-occurrence of NEC or ELGA with the ADRB2 79G allele did not further increase the risk of ROP, but factors associated with neonatal respiratory failure (including surfactant administration, postnatal resuscitation, and mechanical ventilation), as well as the presence of RDS (a multiplicative interaction) did. Interestingly, the same variant in a previous Polish population study showed an interaction with cigarette smoking increasing the risk of coronary artery disease33. In the present study, we also observed a suggestive trend for the IGF1R 3174A allele to be associated with DWMI.
The ADRB2 gene encodes the beta-2-adrenergic receptor. The rs1042714 (79C > G) variant is a common SNV located in the protein-coding region. This variant causes an amino acid change in the protein (Gln27Glu), which has previously been associated with receptor desensitization34. This change in receptor activity ultimately leads to its reduced responsiveness to its ligands, which are catecholamines. The ADRB2 rs1042714 variant has a relatively high prevalence in the Caucasian population and is one of the most common ADRB2 variants with functional activity, making it a good candidate for association studies. It has been studied in the context of several common human diseases, including myocardial infarction, coronary artery disease, polycystic ovary syndrome, primary open-angle glaucoma, cancer, and preterm birth35,36,37,38.
Increasing evidence from experimental and clinical studies indicates that beta-adrenergic receptors allow retinal vessels to respond to hypoxia and may therefore further influence the development of ROP. Studies in a mouse model have shown that VEGF, IGF1, and HIF1A mRNA levels increase after hypoxia, but beta-adrenergic receptor blockade normalizes the levels of these transcripts39. In a subsequent clinical study, Filippi et al. tested the hypothesis that beta-blockade plays a role in inhibiting ROP. They used topical administration of the beta-blocker propranolol in eye drops. This drug is approved as a pharmacotherapy for cardiovascular conditions such as hypertension or tachycardia, but is also effective in the treatment of IH. The results indicated that propranolol may reduce the risk of progression from early-stage ROP to more severe forms24. They emphasize the safety of using propranolol drops, but point to its insufficient effectiveness in more advanced ROP and recommend conducting further clinical trials, both with propranolol and other types of drugs.
The effect of the ADRB2 79G gene variant on ROP was further strengthened by a multiplicative interaction with RDS and factors related to respiratory failure. The strongest interaction effect was observed for the advanced ROP phenotype and the combination of the ADRB2 genotype with surfactant administration or mechanical ventilation (risk multiplied by a factor of 4). Respiratory failure is the main risk factor responsible for hypoxia in preterm infants, which additionally induces pathogenic pathways leading to neonatal oxidative stress and the development of ROP.
This specific interaction between the rs1042714 variant and the respiratory system may also be due to the shared function of beta-adrenergic receptors in the lungs and eyes. These receptors are expressed in tissues important for the cardiorespiratory system, including bronchial and arterial smooth muscle. Beta-adrenergic receptors enable bronchial and vascular dilation, including the retinal arteries40. Genetically determined changes in beta-adrenergic receptor activity may therefore affect both respiratory function and diseases associated with pathological neovascularization. Previous studies have shown associations between ADRB2 gene variants and the development of congenital respiratory diseases in preterm infants, supporting a partial shared role of beta-adrenergic receptor pathways in the pathogenesis of respiratory disorders and ROP in preterm infants41.
PPI analysis revealed six major pathways involved in the pathogenesis of ROP, but only one of them, linking CXCL12 and DPP4, has a product of a gene identified in GWAS. In this cluster, CXCL12 is involved in hypoxia-induced angiogenesis, whereas DPP4 is an enzyme present in the fetal colon that disappears after birth. This analysis showed that a hypothesis-free study can be difficult to interpret, even if genes are identified. PPI analysis shows that ADRB2 is a protein in the RAAS, which also includes AGTR1, AGT, ACE, IL1B, and TLR4, that may directly interact with the vascular endothelial growth factor signaling pathway (via VEGFB)42. Furthermore, both ADRB2 and VEGFB have been mentioned in studies related to cardiovascular disease, vessel growth, and tumor angiogenesis43,44. They are also involved in the regulation of immune cells and play a role in the interaction with VEGF receptors. ADRB2 is one of the VEGF receptors that binds to VEGFB produced by nucleus pulposus cells, as shown by Tu et al.45. Because we have previously analyzed two other RASS components in ROP, AGTR1 and ACE32, we performed a G x G interaction analysis for ADRB and AGRT1 or ACE SNV (Supplementary Table S2). In this analysis, we observed that the cooccurrence of the ADRB2 rs1042714G (79G) allele with the AGRT1 rs5186C allele exceeds by a factor of 3.4 the effect expected from the sum of the separate impacts of these alleles (multiple interaction; Supplementary Table S2a). A similar interaction was not observed for the ACE gene variant (Supplementary Table S2b). PPI analysis confirms that AGRT1 can directly interact with ADRB2 (Fig. 1).
In our study, the ADRB2 79G allele was also associated with ELGA. This association may contribute to an increased risk of ROP, NEC, or IH in preterm infants and may partly represent a downstream effect of this allele on pregnancy complications. A study in a Korean population of 166 women showed an association between this allele and preterm birth46. The increased frequency of the 79G allele in children may therefore be a consequence of its frequency in mothers. Although our study did not analyze the maternal genotype, it should be noted that the frequency of the 79G allele observed in children with ELGA in this study (0.580) is 1.8 times higher than that observed in the healthy Polish population (0.436; OR = 1.8; 95%CI (1.2–2.7); P = .006)33.
In addition, we observed a suggestive association between the IGF1R 3174G > A variant and DWMI. This disorder results from disruption of the myelination process of neurons in brain tissue and has potentially fatal consequences, preventing normal motor and cognitive development47. Prematurity is a risk factor for DWMI, increasing the likelihood of insufficient myelination of neurons at birth and rendering cells and surrounding glial tissue susceptible to oxidative stress48. The variant studied has previously been associated with melanoma, hypertension, as well as intrauterine growth retardation due to insulin-like growth factor I resistance and preterm birth49. It is a growth factor produced, among others, in the placenta that promotes fetal tissue development. Insufficient levels of this factor in utero have been correlated with fetal growth restriction, vascular abnormalities (including ROP and encephalopathy of prematurity), and BPD in preterm infants50. This factor is essential for brain tissue development because it controls glucose metabolism in neural tissue. Lower levels of insulin-like growth factor I are correlated with small brain volume in very preterm infants51.
A better understanding of the impact of individual variants on the course of ROP, i.e. their contribution to determining a mild course ending in spontaneous regression or a severe one leading to progression requiring treatment, which may end in retinal detachment and blindness, could help to identify a subgroup of high-risk newborns. This would allow for intensified ophthalmological monitoring of at-risk newborns and ensuring treatment at the most appropriate time, which is key to preventing progression. In the future, the proposed study could also play a key role in proposing new methods for targeted treatment or even prevention of ROP, which seems to be particularly important in low- and middle-income countries, where this disease accounts for up to 40% of blindness cases. It also seems important to personalize drug therapy for ROP according to genetic predisposition, including those resulting from the presence of ADRB2 variants, because the variant analyzed in this study was previously associated with an insufficient response to treatment of heart disease with the beta-blocker metoprolol52.
STRING analysis has shown that by influencing VEGFB activity, it is possible to influence other proteins involved in the VEGF signaling pathway. Evaluation of gene variants that influence the vascular or angiogenic response in ROP, in particular further study of RAAS components, may provide valuable information on the pathogenesis of the disease and identify treatment strategies. ADRB2 acts as a natural regulator of vascular function and has physiological interactions that influence various cellular processes involved in the development of ROP.
Limitations. Several limitations of our study should be highlighted. The first limitation is the lack of follow-up of infants tested for ROP for symptoms of IH, because IH usually does not occur immediately after birth. Second, it would be interesting to analyze the influence of the studied genes, especially ADRB2 and IGFR1, on intrauterine growth restriction, but the number of cases with intrauterine hypotrophy identified in this study (n = 14, Table 1) was too small to perform such an analysis. This area is a promising direction for further research. Since the mothers of preterm infants were not genotyped, the third drawback of this study is the lack of information regarding the role of the studied variants in the occurrence of pregnancy complications leading to preterm birth. The fourth limitation is the relatively small sample size in the genetic analyses. However, post-hoc statistical power analysis confirmed that the obtained results for most analyses were highly reliable. For example, when assessing the effect of the ADRB2 79G allele on the occurrence of ROP requiring treatment, statistical power analysis showed power of 97%, 75%, and 92% for recessive, dominant, and additive models. For the G x E interaction, the statistical power of the interaction between ADRB2 and mechanical ventilation was 75%, whereas for ADRB2 and RDS, surfactant administration, or resuscitation, it ranged from 60–65%. For the G x G interaction, the statistical power of the observed interaction between ADRB2 and AGTR1 was 84%.