Ophthalmic Genetics

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iopg20

The relationship of retinopathy of prematurity

with brain-derivated neurotrophic factor, vascular
endotelial growth factor-A, endothelial PAD
domain protein 1 and nitric oxide synthase 3 gene
polymorphisms

Serdar Ilguy, Oguz Cilingir, Mustafa Deger Bilgec, Onur Ozalp, Ebru
Erzurumluoglu Gokalp, Serap Arslan, Neslihan Tekin, Ozge Aydemir,
Nazmiye Erol, Ertugrul Colak & Huseyin Gursoy

To cite this article: Serdar Ilguy, Oguz Cilingir, Mustafa Deger Bilgec, Onur Ozalp, Ebru
Erzurumluoglu Gokalp, Serap Arslan, Neslihan Tekin, Ozge Aydemir, Nazmiye Erol, Ertugrul
Colak & Huseyin Gursoy (2021) The relationship of retinopathy of prematurity with brain-derivated
neurotrophic factor, vascular endotelial growth factor-A, endothelial PAD domain protein 1
and nitric oxide synthase 3 gene polymorphisms, Ophthalmic Genetics, 42:6, 725-731, DOI:
10.1080/13816810.2021.1961279

To link to this article: https://doi.org/10.1080/13816810.2021.1961279

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OPHTHALMIC GENETICS
2021, VOL. 42, NO. 6, 725–731
https://doi.org/10.1080/13816810.2021.1961279

RESEARCH REPORT

The relationship of retinopathy of prematurity with brain-derivated neurotrophic

factor, vascular endotelial growth factor-A, endothelial PAD domain protein 1 and
nitric oxide synthase 3 gene polymorphisms
Serdar Ilguya, Oguz Cilingirb, Mustafa Deger Bilgecc, Onur Ozalpd, Ebru Erzurumluoglu Gokalpb, Serap Arslanb,
Neslihan Tekine, Ozge Aydemire, Nazmiye Erolc, Ertugrul Colakf, and Huseyin Gursoy c
a
Department of Ophthalmology, Eskişehir Yunus Emre State Hospital, Eskişehir, Turkey; bDepartment of Medical Genetics, Eskişehir Osmangazi
University Medical Faculty, Eskişehir, Turkey; cDepartment of Ophthalmology, Eskişehir Osmangazi University Medical School, Eskişehir, Turkey;
d
Department of Ophthalmology, Devrek State Hospital, Zonguldak, Turkey; eDepartment of Pediatrics, Division of Neonatology, Eskişehir Osmangazi
University Medical School, Eskişehir, Turkey; fDepartment of Biostatistics, Eskişehir Osmangazi University Medical School, Eskişehir, Turkey

ABSTRACT ARTICLE HISTORY

Background: In addition to risk factors such as low birth weight and uncontrolled oxygen therapy, Received March 18, 2021
genetic predisposition is also thought to play a role in the development of retinopathy of prematurity Revised June 27, 2021
(ROP). In our study, we aimed to analyze single-nucleotide polymorphisms (SNPs) in VEGFA, EPAS1, BDNF Accepted July 25, 2021
and NOS3 genes in infants who develop ROP. KEYWORDS
Materials and Methods: Seventy-five mild-moderate and 73 severe ROP cases were included in this Retinopathy of prematurity;
study. Eleven different SNPs regions that located in VEGFA, EPAS1, BDNF and NOS3 genes were analysed by genetic analysis; single
SnapShot technique and compared between two groups by the multiple logistic regression analysis. nucleotid polymorphisms
Results: Statistically significant results were obtained in 8 of the 11 SNPs. It was observed that the excess (SNPS); VEGFA; EPAS1; BDNF;
of mutant alleles in four (VEGFA rs2010963 and rs3025039, EPAS1 rs13419896, NOS3 rs2070744) of these NOS3
regions increased ROP severity and treatment requirement (p < .001, p < .001, p = .022, p = .004,
respectively) while the excess of mutant alleles in the other four regions (VEGFA rs833061, BDNF
rs7929344, EPAS1 rs1867785 and rs1868085) showed that ROP severtiy was milder and eliminated the
need for treatment (p < .001, p = .019, p = .017, p = .017, respectively).
Conclusions: Considering the results of our study, it was seen that besides the known environmental and
demographic factors in ROP pathogenesis, genetic predisposition also had an effect on the clinic and
course of ROP. Polymorphisms of VEGFA rs2010963 and rs3025039, EPAS1 rs13419896, NOS3 rs2070744
were found to be associated with severe ROP. More studies involving different populations cases are
needed to confirm these findings and enlighten the etiology of ROP.

Introduction
Retinopathy of prematurity (ROP) is a disease that develops in
the retinas of premature babies on the basis of the vascular
structure that has not completed its development. ROP is
conventionally known to develop in infants whose gestational
week is below 32 weeks and birth weight is less than 1500
grams (1). However, ROP has also been reported in infants
older than 32 weeks of gestation (2). Significant proportion of
ROP cases (stage 2, sometimes stage 3 ROP disease) regress in
follow-up (3).
It has been suggested that apart from the known risk factors
such as uncontrolled oxygen therapy and accompanying systemic
diseases etc., genetic factors also affect the development and
severity of ROP disease. Retinal findings similar to ROP can be
seen in X-linked familial exudative vitreoretinopathy (FEVR),
Norrie’s disease, and Coats disease (4,5). Although these cases
have unique genetic backgrounds, there are studies showed that
the same gene loci may increase the susceptibility to ROP disease
(6). There are a limited number of experimental and polymorph­
ism studies that supported genetic predisposition in ROP. Genetic
studies have focused on 4 different pathways. These are neuronal
development, angiogenesis, inflammatory and oxidative pathways
(7). Brain-derived neurotrophic factor (BDNF) is associated with
nerve development in the retina and brain tissue. Studies on the
neuronal development pathway have shown significant relation­
ships between some polymorphisms in the BDNF gene and the
development of severe ROP (7). It has been suggested that poly­
morphisms in genes belonging to factors involved in angiogenesis,
inflammatory and oxidative pathways such as Indian hedgehog
(IHH), angiotensin II receptor type 1 (AGTR1), T-Box 5 (TBX5),
cholesterol ester transfer protein (CETP), glycoprotein Ib (plate­
let) alpha polypeptide (GP1BA), Nitric oxide synthase (NOS),
endothelial PAD domain protein 1 (EPAS1) and vascular
endothelial growth factor (VEGF) may also be effective in the
development of ROP disease (8). Apart from these 4 pathways
(neuronal development, angiogenesis, inflammatory and oxida­
tive pathways) that are frequently evaluated, genetic abnormalities
in the coagulation pathway have also been evaluated in ROP
patients and FVL polymorphism was found to be more common
in ROP patients (9).

CONTACT Huseyin Gursoy hhgursoy@hotmail.com Eskişehir Osmangazi University Medical School, Department of Ophthalmology, Meşelik Kampüsü
Odunpazarı, Eskişehir, Turkey
© 2021 Taylor & Francis Group, LLC
726 S. ILGUY ET AL.
In our study, we aimed to analyze 11 SNPs that located in
BDNF, VEGFA, EPAS1 and NOS3 genes by using SnapShot
technique in ROP cases and to determine their effect on disease
severity.
Material and methods
Participants
Subjects, who were followed up by Eskişehir Osmangazi
University Faculty of Medicine, Department of
Ophthalmology and Pediatrics Neonatology between
January 2018 and January 2020, who developed ROP but
whose vascularization was completed without treatment were
determined as the control group (group 1), and the patients
who received laser photocoagulation, intravitreal anti-VEGF or
surgical treatment during their follow-up according to ETROP
(The Early Treatment for Retinopathy Of Prematurity) study
criteria were determined as the treatment group (group 2) (10).
All preterm infants before 32 weeks of gestation and birth
weight below 1500 grams, whose retinal vascularization was
completed and/or followed up regularly after treatment were
included in the study. Infants with metabolic and/or genetic
diseases and those who received blood transfusion in the last
month were excluded from the study. Demographic data, birth
weights and weeks, first examination weeks, duration of stay in
infant incubator and time of receiving oxygen therapy were
noted in all subjects.
This study was conducted according to the Declaration of
Helsinki guidelines and approved by the Clinical Practice
Ethics Committee of Eskisehir Osmangazi University,
Medical Faculty. Parents of all subjects were agreed to partici­
pate in the study and biological samples were obtained after
written informed consent obtained.
Single-nucleotide polymorphisms (SNPs) genotyping
We have selected eleven SNPs that are associated with ROP or
have conflicting suggestions about these SNPs in the literature.
Figure 1. A schematic representation of the position of the SNPs identified along the VEGFA, BDNF, EPAS1, and NOS3 genes.
Some studies were showing that rs2010963, rs3025039, and
rs833061 in the VEGFA gene (6,11–13), rs7934165,
rs2049046, and rs7929344 in the BDNF gene (14), rs1867785,
rs1868085, and rs13419896 in the EPAS1 gene (8) and
rs1799983 and rs2070744 in the NOS3 gene (6) may be used
as a biomarker in ROP screening. Ali et al. (13) found that the
distribution of VEGF rs2010963 C/G polymorphism differed
significantly between patients with and without ROP. Results
of the study revealed that the VEGF rs2010963 GG genotype
may be associated with disease severity (13). Yagi et al. (15)
found that when T alleles of VEGFA rs3025039 polymorphism
are heterozygous or homozygous carriers, the risk of develop­
ing threshold ROP increases approximately 5 times. Hartnett
et al. (14) found that the EPAS1 rs13419896 polymorphism
increased the risk of serious ROP approximately 2.4 times.
Poggi et al. (6) have shown that the rs2070744 polymorphism
in the NOS3 gene has a significant effect on ROP development.
In the study by Hartnett et al., the rs7929344 polymorphism in
the BDNF gene was found to be statistically significant in
infants with ROP (5). Considering the results of all these
studies in the literature, it was aimed to investigate the effect
of eleven candidate polymorphisms in Turkish ROP cases.
A schematic representation of the position of the SNPs identi­
fied along the VEGFA, BDNF, EPAS1, and NOS3 genes is
reported in the Figure 1.
Genomic DNA was isolated from peripheral lymphocytes
using PureLink Genomic DNA Mini Kit (Invitrogen Life
Technologies, CA, USA), according to the manufacturer’s
recommendations.
Single nucleotide polymorphisms in VEGFA, BDNF,
EPAS1 and NOS3 genes were studied by SnapShot technique.
This technique based on unlabeled oligonucleotide primers
and the elongation of a single ddNTP. The primary extension
reaction begins after the amplified target site is purified.
Specific mini-sequence primers are designed for each mutation
to be investigated in a single base next to the mutation sepa­
rately. Primers have nonspecific poly (dA) or poly (dGACT)
tails at the 5 ‘end. These poly tails are of different lengths and
are used to prevent the formation of nonspecific background

Table 1. Demographic and environmental factors by groups.
Variables
Gender (Girl)
Birth weights (gram)
Birth weeks
First examination weeks
Duration of stay in infant incubator (days)
Time of receiving oxygen therapy (days)
Q1 = 25th percentile, Q2 = 50th percentile, Q3 = 75th percentile. #Chi-square test, *Mann Whitney-U test. The statistically significant variables were
indicated in bold.
peaks and lengthen the sequence. Primers bind to the comple­
mentary DNA region in the presence of Taq DNA polymerase
and fluorescently labeled ddNTPs. The polymerase enzyme
adds a single ddNTP to the 3 ‘end of each primer complemen­
tary to the target sequence. The sequence ends when ddNTPs
are added to the sequence during the reaction. DNA fragments
of different lengths and colors are formed. These fragments are
analyzed in a capillary electrophoresis device. The peaks
formed during analysis in the device appear in different colors.
There is a possibility of seeing three different peaks in the
relevant region: homozygous wild peak, homozygous mutant
peak, and heterozygous wild-mutant peak (16).
SnapShot reactions were performed as recommended by the
manufacturer. Electrophoresis of amplified PCR samples
related to these polymorphism regions was performed on
ABI 3130 Genetic Analyzer and the data were analyzed by
using GeneMapper 4.0 Software (Applied Biosystems, Life
Technologies, CA, USA).
Statistical analysis
Statistical analysis was performed with SPSS for Windows 23.0
program (SPSS Inc. Chicago, USA) and Stata/MP 14.1
(StataCorp LP, College Station, TX). Demographic and envir­
onmental factors were compared between groups using Mann-
Whitney U and chi-square tests. Distribution of genotype and
risk allele frequency of SNPs analyzed by Hardy–Weinberg
equilibrium (HWE) test and multiple logistic regression.
Haplotype frequencies of SNPs in VEGFA, EPAS1, BDNF and
NOS3 genes were analyzed Haplotype-effects logistic regres­
sion with genetic distribution Hardy-Weinberg equilibrium.
The obtained p value less than 0.05 was accepted as significant.
Results
148 patients who were followed up for ROP in Eskişehir
Osmangazi University Faculty of Medicine, Department of
Ophthalmology and Pediatrics Neonatology between
January 2018 and January 2020 were evaluated. There were
75 patients in the control group and 73 patients in the treat­
ment group. Here, 56% (n = 42) and 47.9% (n = 35) of the
patients were girls in the control and treatment group, respec­
tively (p = .33)(Table 1). The mean birth week of the subjects in
the control group was 28.79 ± 2.57 weeks and in the treatment
OPHTHALMIC GENETICS 727
Group 1 Group 2
Mean ± SD Q2 (Q1-Q3) Mean ± SD Q2 (Q1-Q3) p value
42 (56%) 35 (47.9%) 0.327#
1110 ± 300.9 866.9 ± 317.9 0.001*
1100 (860–1435) 805 (630–1035)
28.8 ± 2.6 26.2 ± 2.4 0.002*
29 (26–32) 26 (24–27.5)
32.8 ± 2.1 31.2 ± 1.6 <0.001*
33,00 (31–35) 31 (30–31.5)
67.7 ± 42.8 100.2 ± 62 <0.001*
60 (39.3–89.3) 94 (66–120)
62.1 ± 34.7 65.8 ± 34.3 <0.001*
28 (9.5–45.5) 60 (27.5–82.5)
group it was 26.22 ± 2.36 weeks, and there was statistically
significant difference between the two groups (p = .002). The
average birth weight of the infants in the control group was
1110 ± 300.9 gr, and 866.9 ± 317.9 g in the treatment group,
and it was found to be statistically significant (p = .001). The
mean first examination time of the control group was
31.2 ± 1.6 weeks, while it was 32.8 ± 2.1 weeks in the treatment
group (p < .001). The duration of stay in infant incubator in the
treatment group (100.2 ± 62 days) was significantly higher than
the control group (67.7 ± 42.8 days), (p < .001). The duration of
receiving oxygen therapy was significantly longer in the treat­
ment group (65.8 ± 62.1 days) compared to the control group
(34.3 ± 34.7 days), (p < .001).
Hardy–Weinberg equilibrium (HWE) test showed devia­
tion, in the group 1 cases for rs2010963, rs3025039,
rs7934165, rs2049046, rs1868085, rs2070744 polymorphisms
and in the group 2 cases for rs833061, rs7934165, rs2049046,
rs7929344, rs1868085, rs1799983 polymorphisms (Table 2).
Both groups were consisted of individuals with ROP different
severity in our study. For this reason, we could not test Hardy–
Weinberg equilibrium in healthy individuals. Since HWE may
be violated in individuals with the disease, we did not seek
conformity with HWE in our groups (17,18).
The genotype distribution of the rs2010963, rs3025039
and rs833061 polymorphisms were statistically significant
between two groups. In addition, a statistically significant
difference was found in allele frequency of rs2010963,
rs13419896, rs2070744, rs833061, rs7929344, rs1867785
and rs1868085 polymorphisms (Table 2). This significant
difference showed that the presence of the G allele and GG
genotype in the rs2010963 polymorphism, the CT genotype
in the rs3025039 polymorphism, the A allele in the
rs13419896 polymorphism, and the T allele in the
rs2070744 polymorphism increased in ROP severity and
treatment require. Also, that difference showed that the
T allele and CT, TT genotype in the rs833061 polymorph­
ism, the T allele in the rs7929344 polymorphism, the
G allele in the rs1867785 polymorphism, and the A allele
in the rs1868085 polymorphism the ROP severity was
milder and eliminated the need for treatment (Table 2).
A statistically significant difference was not observed
between two groups for rs7934165, rs2049046, rs1799983
SNPs in terms of genotype and distribution of the allele
frequencies.
728 S. ILGUY ET AL.

Table 2. Distribution of genotype and risk allele frequency of SNPs in ROP.

Location/ Gene/rs ID Genotype/allele Group 1 (n = 75) Group 2 (n = 73) OR (95% CI) p value
6:43770613 CC 17 (22.6%) 4 (5.5%) Reference
VEGF-A CG 49 (65.4%) 26 (35.6%) 2.26 (0.69–7.40) 0.180
rs2010963 GG 9 (12%) 43 (58.9%) 20.31 (5.51–74.87) <0.001
G allele 67/150 (44.7%) 112/146 (76.7%) <0.001
-HWE-p value 0.005 0.978
6:43784799 CC 49 (65.4%) 29 (39.8%) Reference
VEGF-A CT 7 (9.3%) 39 (53.4%) 9.41 (3.73–23.773) <0.001
rs3025039 TT 19 (25.3%) 5 (6.8%) 0.45 (0.15–1.32) 0.144
T allele 45/150 (30%) 49/146 (33.6%) 0.510
-HWE-p value <0.001 0.09
6:43769749 CC 14 (18.6%) 49 (67.1%) Reference
VEGF-A CT 35 (46.7%) 6 (8.2%) 0.05 (0.017–0.136) <0.001
rs833061 TT 26 (34.6%) 18 (24.7%) 0.20 (0.088–0.481) <0.001
T allele 87/150 (58%) 42/146 (28.8%) <0.001
-HWE-p value 0.715 <0.001
11:27710436 GG 69 (92%) 63 (86.3%) Reference
BDNF GA 1 (1.3%) 3 (4.1%) 3.29 (0.33–32.407) 0.31
rs7934165 AA 5 (6.7%) 7 (9.6%) 1.53 (0.49–5.08) 0.48
A allele 11/150 (7.3%) 17/146 (11.6%) 0.205
-HWE-p value <0.001 <0.001
11:27702228 AA 60 (65.4%) 58 (79.5%) Reference
BDNF AT 12 (9.3%) 11 (15%) 0.95 (0.39–2.319) 0.90
rs2049046 TT 3 (25.3%) 4 (5.5%) 1.38 (0.29–6.43) 0.68
T allele 18/150 (12%) 19/146 (13%) 0.792
-HWE-p value 0.035 0.004
11:27721948 CC 1 (1.3%) 5 (6.9%) Reference
BDNF CT 21 (28%) 16 (21.9%) 0.15 (0.16–1.43) 0.100
rs7929344 TT 53 (70.7%) 52 (71.2%) 0.19 (0.02–1.73) 0.143
T allele 137/150 (91.3%) 120/146 (82.2%) 0.019
-HWE-p value 0.497 0.031
2:46307199 AA 1 (1.3%) 4 (5.5%) Reference
EPAS1 AG 10 (13.3%) 17 (23.3%) 0.42 (0.42–4.35) 0.471
rs1867785 GG 64 (85.4%) 52 (71.2%) 0.20 (0.22–1.87) 0.160
G allele 138/150 (92%) 121/146 (82.9%) 0.017
-HWE-p value 0.414 0.125
2:46329206 GG 62 (82.7%) 50 (68.5%) Reference
EPAS1 GA 12 (16%) 19 (26%) 1.96 (0.87–4.42) 0.104
rs13419896 AA 1 (1.3%) 4 (5.5%) 4.96 (0.54–45.79) 0.158
A allele 14/150(9.3%) 27/146 (18.5%) 0.022
-HWE-p value 0.636 0.243
2:46352519 GG 3 (4%) 9 (12.3%) Reference
EPAS1 GA 6 (8%) 7 (9.6%) 0.39 (0.07–2.13) 0.277
rs1868085 AA 66 (88%) 57 (78.1%) 0.29 (0.07–1.11) 0.071
A allele 138/150 (92%) 121/146 (82.9%) 0.017
-HWE-p value <0.001 <0.001
7:150992991 CC 10 (13.3%) 0 Reference
NOS3 CT 4 (5.3%) 9 (12.3%) incalculable
rs2070744 TT 61 (81.4%) 64 (87.7%) incalculable
T allele 125/150 (83.3%) 137/146 (93.8%) 0.004
-HWE-p value <0.001 0.574
7:150999023 TT 2 (2.7%) 4 (5.5%) Reference
NOS3 TG 10 (13.3%) 10 (13.7%) 0.50 (0.07–3.38) 0.477
rs1799983 GG 63 (84%) 59 (80.8%) 0.47 (0.08–2.65) 0.391
G allele 136/150 (90.7%) 128/146 (87.7%) 0.407
-HWE-p value 0.066 0.001
HWE-P: Hardy-Weinberg equilibrium P value. The statistically significant variables were indicated in bold.

We also found that VEGFA haplotypes, C-C-T (OR: 3.33,
[95% CI 1.16–9.59], p = .026); G-C-C (OR:5.81, [95% CI 1.95–
17.31], p = .002); and G-C-T (OR:3.23, [95% CI 1.04–9.99],
p = .042) were statistically significant. But no statistically sig­
nificant differences were observed in haplotypes of BDNF,
NOS3 and EPAS1 (Table 3).
Discussion
Retinopathy of prematurity is one of the preventable causes of
childhood vision loss. In addition to certain risk factors such as
low birth weight and prematurity, it has been suggested that
genetic predisposition may also affect the development and
severity of ROP disease. Polymorphisms in IHH, AGTR1,
TBX5, CETP, GP1BA, NOS, EPAS1, BDNF and VEGF genes
have been shown to be possible risk factors for ROP disease in
previous studies (8,19,20).
We performed analysis of 11 polymorphisms in VEGFA,
BDNF, EPAS1, NOS3 genes in patients with ROP in order to
evaluate genetic factors in disease etiopathogenesis and we
obtained statistically significant results in 8 SNPs.
VEGF plays a role in the development of ROP by increasing
vascular permeability and formation of new vessels in both
phases 1 and 2 of ROP (21). Some polymorphisms in VEGF

Table 3. Haplotype frequencies of SNPs in VEGFA, EPAS1, BDNF and NOS3 genes in
severe and mild retinopathy of prematurity (ROP).
Genes (single nucleotide
polymorphisms) Haplotypes OR (95% CI) p value
VEGFA C-C-T 3.33 (1.16–9.59) 0.026
(rs2010963, rs3025039, rs833061) C-T-C 1.43 (0.37–5.49) 0.603
C-T-T 2.64 (0.46–15.37) 0.279
G-C-C 5.81 (1.95–17.31) 0.002
G-C-T 3.23 (1.04–9.99) 0.042
BDNF G-A-T 1.32 (0.78–2.24) 0.302
(rs7934165, rs2049046, rs7929344) A-A-C incalculable 0.988
EPAS1 A-G-A 1.55 (0.68–3.57) 0.298
(rs1867785, rs13419896, rs1868085) G-G-G 1.53 (0.58–4.01) 0.390
NOS3 C-G 0.22 (0.03–1.50) 0.122
(rs2070744, rs1799983) T-T 1.12 (0.19–6.70) 0.905
T-G 0.90 (0.18–4.50) 0.901
The haplotype frequencies below 0.01 are not included in the table.
may affect serum VEGF levels and may have a place in treat­
ment choice and determination of prognosis (11,13). Kaya and
colleagues (22) suggested that there is no association between
VEGFA rs2010963 and progression or spontaneous regression
of ROP in preterm infants and this polymorphism has exam­
ined in 3 groups as infants without ROP, spontaneously regres­
sing with ROP and infants with ROP requiring laser treatment,
and no statistically significant difference was found (12). In the
study conducted by Vannay et al. with ROP cases followed up
without treatment and severe ROP cases requiring treatment,
the rs2010963 polymorphism was found to be significantly
higher in the treatment group (23). And also study results of
Ghadyani et al. showed that rs2010963 may serve as a risk
factor for ROP development (24). Our study supports that
GG genotype of rs2010963 was increased the severity of ROP
(p < .001) and G allele may predispose to increasing disease
severity by altering VEGF expression.
The VEGFA rs3025039 polymorphism is located in the 3
‘UTR of the VEGF gene, an important regulatory region that
controls mRNA stability and translation (25–27). The effect of
polymorphism on plasma VEGF levels in infants with ROP has
not been fully elucidated in the current literature. VEGFA
rs3025039 polymorphism has been reported to affect plasma
VEGF levels and the T allele in VEGFA rs3025039 significantly
reduced plasma VEGF levels in adult carriers (28–30), whereas
it was observed that VEGFA gene polymorphisms were not
associated with VEGFA plasma levels in a study conducted on
peripheral maternal blood of preterm women (31). In the
studies that only evaluated the relationship polymorphisms
and ROP; although Kalmeh et al (12). and Zhu et al (32). did
not identify VEGFA rs3025039 polymorphism as a risk factor
for ROP, Yagi et al (15). reported that when T alleles of VEGFA
rs3025039 are heterozygous or homozygous state, the risk of
developing ROP increases 5-fold. In our study, the rs3025039
polymorphism was found at a higher rate in the treatment
group, and being a carrier (heterozygous) of this polymorph­
ism was found to increase the risk of severe ROP 9 times.
However, plasma VEGF levels were not evaluated in our
study and therefore, we could not comment on the effect of
polymorphism in this respect.
Kwinta et al (11). detected a higher rate of rs833061 poly­
morphism in the severe ROP group requiring treatment com­
pared to those without ROP. But results of some studies did not
OPHTHALMIC GENETICS 729
show an association between VEGF rs833061 polymorphism
and the risk of ROP (15,33), besides that, in the meta-analysis
was supported that rs833061 was not associated with ROP risk
(34). In our study, we did not analyze VEGFA rs833061 poly­
morphism in non-ROP cases but the T allele was present at
a higher rate in ROP cases that do not require treatment than
severe ROP cases. We presumed that T allele might be
a protective allele for the development severe ROP (p < .001).
But our data should be supported by studies within larger
cohorts with different ethnicity.
The BDNF level, which is responsible for the development of
ganglion cells, was found to be low in mice raised in the dark
(35). Vascular pathologies similar to ROP have been demon­
strated in mice raised in the dark or lacking the gene encoding
melanopsin (36). These studies show that BDNF may play a role
in the maturation of some ganglion cells that have a role in
retinal angiogenesis and may be affected by stresses related to
prematurity. EPAS1 provides the transactivation of VEGF pro­
moter by stabilizing in hypoxic environment (8). In a study
evaluating ROP development in newborn mice using the hyper­
oxia/normoxia model, it was observed that retinal neovascular­
ization did not develop in HIF 2 alpha (the murine equivalent of
EPAS1) knockdown mice (37). Hartnett et al. investigated genes
associated with ROP risk by screening 1324 SNPs in 964 new­
borns. In this study; BDNF rs7929344 polymorphism was
detected less in ROP patients at any stage (0.5 times) compared
to non-ROP cases. When non-ROP and mild ROP cases were
evaluated together, there was no difference between this group
and severe ROP cases for the detection rates of BDNF rs7934165
and rs2049046 polymorphisms; however, it has been reported
that severe ROP cases have been detected at higher rates com­
pared to non-severe ROP cases (14). The same study found that
the EPAS1 rs13419896 polymorphism also increased the risk of
severe ROP by about 2.4 times (14). In our study, it was found
that the presence of the T allele in BDNF gene rs7929344 poly­
morphism decreased the need for treatment in ROP cases
(p = .019), while the BDNF rs7934165 and rs2049046 poly­
morphisms were not associated with the disease severity in
ROP cases (p = .205 and 0.792). In addition, it was observed
that the A allele in EPAS1 rs13419896 polymorphism increased
the risk of severe ROP (p = .022).
Mohamed et al (8). screened 455 SNPs in 153 genes to
evaluate the genetic relationship with ROP. In the analysis
comparing stage 0-I ROP (no disease—mild disease) with
stage II–III ROP (more severe disease, disease requiring treat­
ment), the EPAS1 gene in rs1868085 A/G polymorphism, the
higher number of A alleles was found to be an increased risk
for ROP (8). The rs1867785 polymorphism in the EPAS1 gene
was also found to be associated with ROP in the family-based
model (8). In our study, we found that the high number of the
G allele in the rs1867785 polymorphism and the A allele in
the rs1868085 polymorphism in the EPAS1 gene decreased the
need for treatment in ROP cases (p = .017 and p = .017). The
possible reason for our different results with Mohamed et al.
was due to the absence of stage 4–5 ROP cases in the study of
Mohamed et al. and the differences between the groups com­
pared. Studies should be increased for the effect mechanisms
and genetic contributions of BDNF and EPAS1 in the develop­
ment of ROP.
730 S. ILGUY ET AL.
Differences in the NOS gene, which play a role in angiogen­
esis, may affect the development of ROP by affecting the retinal
response to oxygen therapy in infants, and this effect can be
protective or triggering (20). Rusai et al (38). compared the
rs2070744 polymorphism in NOS gene in ROP cases with and
without treatment, and this polymorphism was not found to be
associated with ROP severity. In the systematic review and meta-
analysis study of Gohari et al (39). it was found that rs2070744
and rs1799983 polymorphisms in NOS gene do not increase the
risk of ROP. Our study contradicts the findings of these studies.
We found that the rs2070744 polymorphism was associated with
severe ROP, wild genotype (CC) was not observed, and the
T allele was higher in the treatment group (p = .004). The fact
that CC genotype was not detected in the treatment group
suggested that the polymorphism in this region may related to
the ROP severity but further studies are needed.
We observed that haplotypes C-C-T, G-C-C and G-C-T in
VEGFA gene were correlated with disease severity in ROP.
C-C-T and G-C-T haplotypes have been detected to increase
the risk of disease severity by 3-fold. In the G-C-C haplotype,
this rate was 5-fold. We have shown that VEGFA haplotypes
conferred significant risk of ROP severity, but our data need to
be replicated in large cohorts.
In our study, we showed that VEGFA rs2010963 and
rs3025039, EPAS1 rs13419896, NOS3 rs2070744 polymorph­
isms may be associated with disease severity. But polymorph­
ism studies may differ due to genetic diversity between
populations and studies involving different populations cases
are needed to confirm these findings. In addition, in our study,
mild ROP cases that do not require treatment and treated ROP
cases were compared, and the addition of genetic analysis of
infants without ROP will increase the reliability of the study.
Although we have determined the relationship between some
polymorphisms and ROP severity, it is also among our limita­
tions that none of these polymorphisms are evaluated to have
a direct effect on gene function that may affect ROP severity.
In summary, genetic component is thought to have an impor­
tant role in ROP pathogenesis besides known risk factors. In
addition to routine eye screening from birth in infants with ROP,
genetic screening can be used to predict disease severity.
Declaration of interest
The authors report no conflicts of interest. The authors alone are respon­
sible for the content and writing of this article.
Funding
This work was supported by the Scientific Research Projects Fund of
Eskişehir Osmangazi University under Grant [number 2019-1233].
ORCID
Huseyin Gursoy http://orcid.org/0000-0002-9254-4114
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