Endothelial nitric oxide synthase G894T,intron 4 VNTR,and T786C polymorphisms in retinopathy of prematurity

Abstract

BACKGROUND:

Our objective in this study was to assess the association between eNOS gene, that achieves synthesis of nitric oxide especially in the endothelial cells known to have an important role in angiogenesis and vasculogenesis, G894T, intron 4 VNTR (27-bp repeat) and T786C functional polymorphisms and retinopathy of prematurity (ROP), which is an important cause of morbidity in premature or low birth weight babies.

METHODS:

A total of 139 babies who were followed up in our neonatal intensive care unit because of premature birth in our hospital or admitted to our unit. 69 of them had retinopathy of prematurity and comprised the patients group. The remaining 70 babies who did not have ROP comprised the control group. An additional of 1 ml of blood samples were drawn from babies who were in the study groups during routine laboratory analysis. eNOS gene polymorphisms were determined by using polymerase chain reaction method.

RESULTS:

eNOS G894T, intron 4 VNTR and T786C gene polymorphisms did not differ between the patient and control groups (p > 0.05). Using logistic regression analysis; while gender did not differ between two groups; gestational age, birth weight, time on mechanical ventilation differ between two groups. After adjustment for variables other than eNOS gene polymorphisms, we found no significant difference in the genotype distribution of eNOS G894T, intron 4 VNTR and T786C polymorphisms (p > 0.05).

CONCLUSION:

We observed no association between ROP and eNOS gene polymorphisms but needs more investigation.

Keywords

Endothelial nitric oxide synthase polymorphism retinopathy of prematurity

1 Introduction

Retinopathy of prematurity (ROP) is a potentially blinding disorder of retinal vascular development affecting premature infants and is a leading cause of blindness in infancy. After preterm birth, the developing retina is exposed to a sudden increase in tissue oxygen tension resulting in the generation of reactive free radicals which may lead to impairment of retinal vascular development and even to loss of already developed retinal capillaries (ROP Phase 1). This insufficient vascularization results in retinal hypoxia, which, in turn induces a release of various growth factors, stimulating new and abnormal blood vessel growth (ROP Phase 2) [1].

Despite the known risk factors such as gestational age and birth weight, genetic factors also play an important role in the pathogenesis of ROP [2].

Nitric oxide (NO), is one of the most important factors known to play an important role in pathogenesis of ROP. NO is a multifunctional, free radical molecule, which serves as a key signaling molecule in physiological processes such as host defense, neuronal communication, inhibition of platelet aggregation, and regulation of vascular tone [3, 4]. NO cannot be stored due to its short half-life and NO synthesis is tightly regulated by nitric oxide synthases (NOS). NOS is an enzyme present in three isoforms: the constitutive which is differentially expressed in neuronal tissue (nNOS or NOS 1), the inducible which is demonstrated in bronchial airway epithelium, in macrophages and other cells related to NO-induced cytotoxicity generally has low or undetectable basal expression levels that are increased by various cytokines and other stimuli (iNOS or NOS 2), and the active endothelial isoform selectively but not exclusively expressed in endothelial cells (eNOS or NOS 3) [5, 6].

The eNOS is a dimer consisting of two identical monomers of 134 kD. The gene encoding for the eNOS monomer is located on chromosome 7q35-36 and contains 26 exons, spanning 21 kb. eNOS enzyme, an isoform of NO-producing enzymes, fairly specific to endothelial cells, is only fully functional in a dimeric form and has been found to play a prominent role in both angiogenesis and vasculogenesis [4, 7].

The expression of eNOS is affected by particularly, G894T in exon 7, T786C in the promoter region, and intron 4 VNTR (eNOS b/a) functional polymorphisms which have gained more attention those have already been reported to be associated with cardiovascular diseases [8, 9], essential hypertension [10], microangiopathy in type 1 diabetes [6], and diabetic retinopathy in type 2 diabetes [11].

A very limited number of studies have examined the possible association between these eNOS polymorphisms and retinopathy of prematurity. Here we compared the distribution of the three eNOS polymorphisms in Turkish premature and low birth babies with or without ROP, together with their possible association with ROP.

2 Materials and methods

2.1 Study groups

The study group comprised of 139 babies with birth weight between 670–2100 g or born between 24–32 weeks of gestational age. They were all treated in NICU of Ihsan Dogramaci Children’s Hospital, Hacettepe University, Ankara, Turkey. Local ethical committee approved our prospective study. The research followed the tenets of the Declaration of Helsinki, and an informed consent was obtained from the parents to collect an additional of 1 ml of blood samples from their children for diagnostic and scientific purposes during routine laboratory analysis. We monitored them for G894T, intron 4 VNTR, and T786C polymorphisms.

All infants went under ophthalmological examination on 4–6th postnatal age. The babies were divided into two groups based on developing ROP of any stage or not. A total of 69 of the babies had retinopathy of prematurity of any stage comprised the patient group and the remaining 70 babies who did not have ROP comprised the control group. Any babies who had a suspicion of a genetic disease, chromosomal abnormality, congenital malformation or known metabolic disease were discarded from the study.

Both the patient and the control groups were monitored for gestational week, birth weight, sex, duration of mechanical ventilation, Apgar scores (5th minute) and additional clinical problems such as respiratory distress syndrome (RDS), patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC).

2.2 eNOS genotyping

eNOS Glu298Asp and T786C genotype analysis was performed by PCR-RFLP analysis, using Mbo I and Msp I digestion, respectively. For Glu298Asp, PCR amplification of exon 7 with the primers; 5’- AAG GCA GGA GAC AGT GGA TTG A-3’ (sense) and 5’- CCC AGT CAA TCC CTT TGG TGC TCA-3’ (antisense) [12], was followed by Mbo I restriction. Digested products were separated by electrophoresis on 3%agarose gel; G/G genotype was visualized as a 248 bp band, T/T genotype was visualized as 158 and 90 bp fragments and G/T genotype was visualized as 248, 158 and 90 bp PCR products (Fig. 1).

Fig. 1

Glu298Asp gene polymorphisms visualized as PCR products of 248, 158 and 90 bp fragments.

T786C polymorphism was assessed by PCR-RFLP analysis of the 180 bp PCR product by using 5’-TGG AGA GTG CTG GTG TAC CCC A-3’ (sense) and 5’-GCC TCC ACC CCC ACC CTG TC-3’ (antisense) primers was followed by Msp I digestion [12]. Restricted fragments were separated by using electrophoresis on 3%agarose gel. T/T genotype was visualized as 140 and 40 bp while C/C genotype was visualized as 90, 50 and 40 bp bands.

The intron 4 VNTR polymorphism was detected by PCR using the following primers; 5’- AGG CCC TAT GGT AGT GCC TTT- 3’ (sense), 5’- TCT CTT AGT GCT GTG GTC AC-3’ (antisense) [12]. PCR products comprised a 420 bp band corresponding to the five 27-bp repeats (4b), and 393 bp band corresponding to the four 27-bp repeats (4a).

2.3 Statistical analysis

The statistical difference between two groups in genotype distribution and qualitative variables were determined by chi-square test. Mann Whitney-U test was used to investigate the difference between two groups for numerical variables. Logistic regression models were used to calculate the significance of risk factors for the disease. P values of 0.05 or less were considered statistically significant.

3 Results

A total of 139 babies who were followed up in our neonatal intensive care unit because of premature birth in our hospital or admitted to our unit were included in the study.

The median gestational age was 28 weeks and the median birth weight was 1060 g in group who developed ROP of any stage consist of 69 babies comprised the patient group. The second group who did not develop ROP consist of 70 babies with a median gestational age of 29, 7 weeks and a median birth weight of 1270 g comprised the control group. The demographic characteristics of the patient and control groups are shown in Table 1.

Table 1
Demographic characteristics of babies with and without ROP

Patient group Control group P

n = 69 n = 70

Sex (male/ female) 36 / 33 40 / 30 0.556

Birth weight (g) (range) 1060 (670–1800) 1270 (710–2100) 0.003

Gestational age (week) (range) 28 (24–32.5) 29,7 (25–32.0) <0.001

Premature groups

24–28 weeks 39 13

Gestational age (range) 27 (24–28.7) 27,7 (25–28.8) 0.190

Birth weight (range) 1000 (690–1800) 1090 (750–1400) 0.975

29–32 weeks 30 57

Gestational age (range) 30 (29–32.5) 30 (29–32) 0.619

Birth weight (range) 1205 (670–1800) 1300 (710–2100) 0.131

Duration on mechanical ventilation (days) (range) 9 (0–70) 5 (0–24) 0.001

Apgar score (5th minute) (range) 7 (4–10) 8 (5–10) 0.004

	Patient group	Control group	P
	n = 69	n = 70
Sex (male/ female)	36 / 33	40 / 30	0.556
Birth weight (g) (range)	1060 (670–1800)	1270 (710–2100)	0.003
Gestational age (week) (range)	28 (24–32.5)	29,7 (25–32.0)	<0.001
Premature groups
24–28 weeks	39	13
Gestational age (range)	27 (24–28.7)	27,7 (25–28.8)	0.190
Birth weight (range)	1000 (690–1800)	1090 (750–1400)	0.975
29–32 weeks	30	57
Gestational age (range)	30 (29–32.5)	30 (29–32)	0.619
Birth weight (range)	1205 (670–1800)	1300 (710–2100)	0.131
Duration on mechanical ventilation (days) (range)	9 (0–70)	5 (0–24)	0.001
Apgar score (5th minute) (range)	7 (4–10)	8 (5–10)	0.004

ROP, Retinopathy of prematurity.

There were statistically significant differences between the demographic characteristics of the two groups except for the sex. The birth weight, gestational age and 5th minute Apgar scores were significantly lower in the patient group when compared to the control group (p < 0.05). On the other hand, duration of mechanical ventilation was significantly lower in the control group (p < 0.05). However, when the patient and the control groups were split into smaller groups according to their gestational ages, the significant difference for gestational age and birth weight disappeared (p > 0.05).

Analysis of the genotype frequency distributions of the three polymorphisms did not reveal any statistically significant differences between the two groups (Table 2).

Table 2

Genotype distribution of eNOS G894T, intron 4 VNTR and T786C polymorphisms between two groups of babies with/ without ROP

Gene	Genotype	Patient group (n = 69)	Control group (n = 70)	p values
eNOS G894T	GG	41	40	0.410
	GT	23	28
	TT	5	2
intron 4 VNTR	bb	50	53	0.855
	ba	16	15
	aa	3	2
eNOS T786C	TT	29	37	0.349
	TC	34	26
	CC	6	7

ROP, Retinopathy of prematurity.

A comparison of the allele frequencies also revealed no significant difference in the allele distributions of eNOS G894T, intron 4 VNTR (27 bp repeat) ‘a‘ and eNOS T786C between the two groups (Table 3).

Table 3

Allele distribution of eNOS G894T, intron4 VNTR‘a’ and eNOS T786C polymorphisms in patient and control groups

Gene	Allele	Patient	Control	p value
eNOS G894T	G	105	108
	T	33	32	0.835
intron 4 VNTR	b	116	121
	a	22	19	0.577
eNOS T786C	T	92	100
	C	46	40	0.738

When the G894T and intron 4 VNTR gene polymorphisms were compared according to the groups’ sexes, there were no significant differences, but on the other hand when T786C gene polymorphisms were compared according to groups’ sexes, there was a slight difference about female dominancy on homozygous C/C polymorphism (p < 0,05) (Table 4).

Table 4

The distribution of G894T, intron 4 VNTR and T786C gene polymorphisms among both groups according to sex

	Sex
Polymorphism	Male (n = 76)	Female (n = 63)	Total (n = 139)	p
G/G	47	34	81
	58,0%	42,0%	100%
G/T	28	23	51	0.083
	54,9%	45,1%	100%
T/T	1	6	7
	14,3%	85,7%	100%
b/b	57	46	103
	55,3%	44,7%	100%
b/a	15	16	31
	48,4%	51,6%	100%	0.405
a/a	4	1	5
	80,0%	20,0%	100%
TT	36	30	66
	54,5%	45,5%	100%
TC	37	23	60	0.040
	61,7%	38,3%	100%
CC	3	10	13
	23,1%	76,9%	100%

p values when compared according to χ² test.

Logistic regression model was performed to analyze the risk factors for retinopathy of prematurity, such as gestational age, birth weight, duration of mechanical ventilation and eNOS G894T, intron 4 VNTR and eNOS T786C. According to the logistic regression model gestational age and duration of mechanical ventilation showed statistically significant difference. On the other hand, G/T, T/T polymorphisms; b/a, a/a and T/C and C/C polymorphisms did not reveal any significant risk for ROP (Table 5).

Table 5

Logistic regression analysis of eNOS G894T, intron 4 VNTR and T786C

Variable	Odd’s ratio (95%CI)	p
Male	0.737 (0.332–1.636)	0.454
Gestational age	0.718 (0.534–0.964)	0.028
Birth weight	0.999 (0.998–1.001)	0.463
Duration of mechanical ventilation	1.051 (1.004–1.100)	0.034
eNOS G894T (GT versus GG)	0.883 (0.403–1.936)	0.756
eNOS G894T (TT versus GG)	1.926 (0.263–14.113)	0.519
Intron 4VNTR (aa versus bb)	1.529 (0.596–3.924)	0.378
Intron 4 VNTR (ba versus bb)	1.699 0.217–13.285)	0.614
eNOS T786C (TC versus TT)	1.884 (0.848–4.189)	0.120
eNOS T786C (CC versus TT)	0.618 (0.149–2.559)	0.507

When all G894T, intron 4 VNTR and T786C gene polymorphisms were compared according to splitted groups by gestational ages, there were no statistically significant differences between patient and control groups (Table 6).

Table 6

G894T, Intron 4 VNTR, T786C gene polymorphisms in patient and control groups according to their gestational age groups

Gestational age	Gene polymorphisms	Patient	Control	p
	GG	25 (64.10%)	5 (38.50%)
	GT	10 (25.6%)	7 (53.8%)	0.16
	TT	4 (10.30%)	1 (7.70%)
	Bb	29 (74.40%)	9 (69.20%)
24–28 weeks	Ba	8 (20.50%)	4 (30.80%)	0.84
	Aa	2 (5.10%)	0 (0%)
	TT	17 (43.60%)	5 (38.50%)
	TC	19 (48.70%)	4 (30.80%)	0.1
	CC	3 (7.70%)	4 (30.80%)
	GG	16 (53.30%)	35 (61.40%)
	GT	13 (43.30%)	21 (36.80%)	0.62
	TT	1 (3.30%)	1 (1.80%)
	Bb	21 (70.00%)	44 (77.20%)
29–32 weeks	Ba	8 (26.70%)	11 (19.30%)	0.74
	Aa	1 (3.30%)	2 (3.50%)
	TT	12 (40.00%)	32 (56.10%)
	TC	15 (50.00%)	22 (38.60%)	0.31
	CC	3 (10.00%)	3 (85.30%)

4 Discussion

Nitric oxide, a potent vasodilator, has been reported to control the retinal blood flow under normal as well as after ischemic conditions. An isoform of NO producing enzymes, eNOS, is an endothelial specific enzyme which has an important role in both vasculogenesis and angiogenesis. Altered eNOS expression or activity changes endothelial integrity and function resulting in pathological events. Impaired eNOS activity and endothelial dysfunction can be caused by hypoxia, stress, hormones or gene polymorphisms, can also be related to primary endothelial damage [4].

Nitric oxide plays an important role in the control of ocular vascular tone and blood flow of the newborn and adult. NO is a potent signaling molecule in blood vessels, where a continuous formation from endothelial cells acts on the underlying smooth muscle to maintain vasodilatation and blood flow. eNOS and nNOS are present in the retina and the choroid which have expression and activity increased in choroidal and neuroretinal tissue of the perinatal subject. Increased NO formation in the newborn exerts an important effect on vasomotor tone which masks those of constrictors implicated in autoregulatory responses. This improved autoregulation exerted by NOS inhibition stabilizes O₂ delivery. Thus, a reduction in NO synthesis in the newborn could improve the control of O₂ delivery to the retina without apparently compromising the lower limit autoregulation [13]. Ando et al. [5] used eNOS knockout mice with ischemic retinopathy and detected a significant decrease in neovascularization.

Apart from having been associated with other systemic diseases such as myocardial infarction [14], essential hypertension [8], diabetic nephropathy [15], coronary artery disease [16], there are also studies which have investigated the role of eNOS gene polymorphisms in connection with ophthalmological diseases like diabetic retinopathy in type 1 and type 2 diabetes [6 , 17–20]. The data are, however, controversial. Few studies have investigated eNOS gene polymorphisms with retinopathy of prematurity up to date which have controversial data as well [3 , 22].

This study is one the first studies to evaluate a potential association between retinopathy of prematurity and all three eNOS gene polymorphisms. In our study, we found no association between ROP and individual eNOS gene polymorphisms, thus suggesting that variations in the eNOS gene do not affect the risk of development of ROP.

eNOS G894T polymorphism and association with essential hypertension, coronary artery disease, ischemic heart disease, myocardial infarction, end stage renal disease and diabetic retinopathy has been widely investigated and found to have controversial results. Some of the experimental studies have shown increased activity of NOS in retina of diabetic rats. Studies on eNOS knock out mouse models suggest that eNOS is essential for growth factor mediated angiogenesis [23]. Chen et al. [20] investigated eNOS G894T polymorphism for its association with type 2 diabetes and diabetes related phenotypes and found no association between the genotypes or alleles of G894T polymorphism with diabetic retinopathy, hypertension or nephropathy. In the same study they also investigated 4a/b polymorphism of eNOS gene and observed that bb genotype of the 4a/b polymorphism was associated with increased risk of diabetic retinopathy in type 2 diabetes [20]. In another study, deSyllos et al. [17] studied eNOS gene, T786C in the promoter region, G894T in exon 7 and VNTR in intron4. They compared the distribution of genetic variants of the three mentioned polymorphisms in healthy volunteers and type 2 diabetic patients, with or without diabetic retinopathy. They found no association between diabetic retinopathy and individual eNOS haplotypes. In our study, which is the first study to investigate the association between eNOS G894T gene polymorphism and retinopathy of prematurity, we found no significant difference in the genotype distribution nor in the frequency of alleles between the patient and the control groups. There was no significant difference in sex distribution between patient and control groups and also when G894T polymorphisms were compared between sexes, we found no statistically significant difference in terms of G/G, G/T and T/T. Logistic regression model was performed to analyze the risk factors for retinopathy of prematurity, such as gestational age, birth weight, duration of mechanical ventilation and eNOS G894T, intron4VNTR. According to the logistic regression model gestational age and duration of mechanical ventilation showed statistically significant difference. On the other hand, G/T, T/T polymorphisms, b/a, a/a polymorphisms and T/C and C/C polymorphisms did not reveal any significant risk for ROP.

Functional intron 4 VNTR polymorphism has also an important role in eNOS production. Tsukada et al. [24] found a strong correlation between eNOS intron 4 VNTR gene polymorphism and the level of plasma NO metabolites in healthy subjects, and the mean plasma NO level of subjects who were homozygous for the a allele was nearly 20%lower than the subjects with the b allele. Intron 4 VNTR gene polymorphism has been widely studied in diabetic retinopathy in type 1 and type 2 diabetic patients. The findings of the study of Mamoulakis et al. [6] suggest that a/b polymorphism of the intron 4 eNOS gene is not associated with early onset diabetic microangiopathy. They found no significant relationship demonstrated between eNOS gene intron 4 polymorphisms and microalbuminuria, hypertension or retinopathy in young individuals with type 1 diabetes. But they also emphasized that microvascular complications of type 1 diabetes usually occur 15–20 years after the onset of the disease.

Uthra et al. [18] studied intron 4 of eNOS gene and diabetic retinopathy in type 2 diabetic patients, but found that the frequencies of both a/a and b/b genotypes of the eNOS did not show any statistical difference in their distribution between patients who have diabetic retinopathy or who do not have diabetic retinopathy in Indian population.

In the study of Rusai et al. [3], they found the genotype distribution of intron 4 VNTR at was significantly different between the study groups of retinopathy of prematurity who were treated for severe proliferating ROP when compared to preterm infants with stage 1 or 2 ROP that did not require treatment, but found no difference in the frequency of the ‘a‘ allele. They also found that intron 4 VNTR aa genotype presented an independent risk factor for ROP requiring treatment. Our results for intron 4 VNTR between ROP and without ROP groups did not confirm their results. In our study we also found no significant difference in the allele distribution between the patient and the control groups.

Rusai et al. [3] also found no significant difference in the genotype distribution of eNOS T786C polymorphisms nor in the frequency of –786C allele between the treated and untreated groups of ROP. Our results confirm this study; that we also found no association between eNOS T786C polymorphisms and allele frequency and risk of development for ROP.

5 Conclusion

In conclusion, our findings suggest no association between retinopathy of prematurity and individual eNOS gene polymorphisms of G894T, intron 4 VNTR, and T786C in premature babies who have ROP and who do not have ROP. A limitation of this study was that it was limited to Turkish population, thus necessitating follow up studies in different ethnic groups. So further investigations are certainly necessary to determine the genetic predisposing factors for ROP; so that early interventions for treatment for high-risk babies could be done to avoid blindness.

Footnotes

Acknowledgments

No conflicts of interests declared.

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