Molecular Vision 2024; 30:390-398 <http://www.molvis.org/molvis/v30/390>
Received 05 October 2023 | Accepted 10 November 2024 | Published 12 November 2024

The association of endothelial nitric oxide synthase (eNOS) gene polymorphisms and diabetic retinopathy among patients with type 2 diabetes: A case-control study

Diala W. Abu-Hassan,1 Muawyah D. Al-Bdour,2, 3,4 Iman Aolymat,5 Mohammed El-Khateeb4

1Department of Physiology and Biochemistry, School of Medicine, the University of Jordan, Amman, Jordan; 2Department of Ophthalmology, School of Medicine, the University of Jordan, Amman, Jordan; 3Jordan University Hospital, Amman, Jordan; 4The National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan; 5Department of Anatomy, Physiology and Biochemistry, Faculty of Medicine, The Hashemite University, Zarqa, Jordan

Correspondence to: Diala W. Abu-Hassan, Department of Physiology and Biochemistry, School of Medicine, the University of Jordan, Amman, Jordan 11942; Phone: +962797296821; email: diala_abuhassan@yahoo.com

Abstract

Purpose: Diabetic patients experience chronic hyperglycemia that increases oxidative stress by enhancing free radical formation and nitric oxide (NO) production. Genetic mutations in the endothelial nitric oxide synthase (eNOS) enzyme gene affect the levels of NO formation. These mutations, together with chronic hyperglycemia, may increase the risk of diabetic retinopathy (DR) development and/or DR progression as a complication of diabetes. This study aimed to determine whether the eNOS polymorphisms intron 4ab, exon 7 Glu298Asp variant (G894T), and T-786C are associated with DR severity.

Methods: This case-control study involved 250 subjects (172 with type 2 diabetes mellitus (DM) with or without DR and 78 healthy controls). DR was detected by slit lamp biomicroscopy and its severity was determined with the evidence-based International Clinical Diabetic Retinopathy Disease Severity Scale. The genotyping of eNOS polymorphisms was analyzed by polymerase chain reaction (PCR) only or with restriction fragment length polymorphism. The haplotype analysis was performed using the SNPStats tool.

Results: The genotype distribution for the three polymorphisms was significantly different in patients with diabetes compared to controls (intron 4 ab: a/a, 1.7%; a/b, 20.4%; b/b, 77.9%. G894T: GG, 56.4%; GT, 34.3%; TT, 9.3%. T-786C: TT, 58.2%; TC, 33.5%; CC, 8.3%). Differences in genotype or allele frequency was non-significant between subjects with diabetes and DR compared to those without DR, except the C allele of the T-786C polymorphism was significantly less common in DR patients. DR severity was not affected by any polymorphisms. Haplotypes bTC and aTT were significantly less common or more prevalent in DR than DM patients, respectively.

Conclusions: We demonstrated that type 2 DM patients exhibited a higher prevalence of the three polymorphisms when compared to the control group. The C allele of T-786C polymorphism may have a protective effect against DR. Also, none of the mutations was correlated with a higher DR risk nor with the severity of DR. Haplotype aTT increased the risk for DR, while the bTC haplotype reduced it.

Introduction

Diabetes mellitus (DM) is a globally common metabolic disorder. It is considered a public health issue due to the high morbidity and mortality rates, as well as long-term complications, associated with it. Microvascular complications associated with diabetes are a common finding in patients. The pathogenesis of these complications has been under study for a long time. In patients with diabetes, hyperglycemia increases oxidative stress due to free radicals and nitric oxide (NO) production, and this may play a key role in the development of such complications [1,2]. Previous studies of endothelial function under diabetic conditions have yielded conflicting findings [3]. With hyperglycemia, both enhanced and reduced generation of NO have been reported, and diabetic complications have been related to NO changes in clinical and experimental conditions [4,5]. Hence, NO may have a dual role in the development of diabetic complications.

Diabetic retinopathy (DR) is a common diabetic complication associated with chronic hyperglycemia; however, the resulting DR is non-severe in most affected patients. A growing body of clinical and experimental evidence suggests that genetic factors have a potential role in DR pathogenesis [6]. Several polymorphisms that increase the ocular NO levels have been detected, including those in the endothelial nitric oxide synthase (eNOS) gene [79]. These polymorphisms have been connected to the development and/or progression of DR. Three polymorphisms in the eNOS gene have been studied. Of these, intron 4 ab polymorphism, in which a four variable number tandem repeat (VNTR) in a 27-bp consensus sequence in intron 4 of the eNOS gene (eNOS4a allele) has been found to be associated with the progression of DR [10,11]. Other polymorphisms, including the exon 7 Glu298Asp variant (G894T) and T-786C in the promotor region, have also been reported to be correlated with DR [10,11].

In this case-control study, we investigated whether these eNOS polymorphisms are associated with the development of severe DR in a sample of Jordanians with type 2 diabetes. Understanding the potential roles of eNOS polymorphisms in DR pathogenesis is crucial for enhancing our understanding of diabetes pathophysiology and the genetic factors that impact susceptibility to DR, identifying high-risk individuals, and developing individually tailored treatment plans. This increased understanding can improve early detection, ultimately contributing to better outcomes for those diagnosed with DR.

Methods

Study design and setting and subject recruitment

In this case-control study, a total of 250 subjects were recruited at the ophthalmology clinic of the National Center for Diabetes, Endocrinology and Genetics (NCDEG). Out of these 250 participants, 94 had DM without DR, 78 had DM with DR, and 78 were controls without DM. Inclusion criteria for subject collection included age ranging between 40 and 78 years, type 2 diabetes diagnosed by standard means as defined by the American Diabetes Association, diabetes duration of less than 15 years, and no retinal problems before the diagnosis of diabetes. Diagnosis of diabetes was according to the American Diabetes Association standards for diagnosing diabetes, which include the use of one of four tests to establish a firm diagnosis: (i) fasting plasma glucose (FPG) >6.9 mmol/l (>125 mg/dl), the most commonly used test; (ii) random plasma glucose ≥11.1 mmol/l (≥200 mg/dl) with diabetes symptoms such as polyuria, polydipsia, fatigue, or weight loss; (iii) two-hour post-load glucose ≥11.1 mmol/l (≥200 mg/dl) on a 75 g oral glucose tolerance test; (iv) or HbA1c ≥48 mmol/mol (≥6.5%). All of these tests require confirmation with a second test, which may be the same test or a different one.

Written informed consent was obtained from all participating subjects before collecting a blood sample. Subject names were kept confidential, and each was given a coding number. The deanship of scientific research at the University of Jordan and the institutional review board (IRB) at the NCDEG approved this study, which adhered to the tenets of the Declaration of Helsinki. The HbA1c test was only performed on patients with diabetes, but not on controls. We referred to historical medical records to exclude diabetes in control group participants. In addition to medical history of diabetes, hypertension, ischemic heart disease, and dyslipidemia, we collected personal data (age and sex) and medical information, such as height and weight. Moreover, consistent sex distribution in each group was also considered. DR was defined by the presence of characteristic changes, including hemorrhages, exudates, new vessels, and fibrous proliferation, detected by slit lamp biomicroscopy through dilated pupils by an experienced ophthalmologist. DR severity was determined based on the evidence-based International Clinical Diabetic Retinopathy Disease Severity Scale that was agreed to by the American Academy of Ophthalmology (AAO) in 2001 and the International Council of Ophthalmology (ICO) in 2002. This scale classifies DR severity as follows: no apparent retinopathy is indicated by the absence of ophthalmoscopic abnormalities; mild non-proliferative DR (NPDR) indicates the presence of microaneurysms (MA) only; moderate NPDR includes the presence of more than just MA but fewer abnormalities than the severe form; severe NPDR is the presence of more than 20 intraretinal (IR) hemorrhages in each of the four quadrants, definite venous beading in two or more quadrants or prominent intraretinal microvascular abnormality (IRMA) in one or more quadrants, and the absence of proliferative retinopathy signs; and finally, proliferative DR is indicated by the presence of neovascularization and/or vitreous/preretinal hemorrhage.

DNA extraction and genotyping

Venous blood samples were collected from all subjects in EDTA tubes for DNA extraction. Whole blood samples were used to extract DNA using QIAGEN Puregene Blood Core Kit B (QIAGEN N.V., Venlo, The Netherlands) according to the manufacturer’s instructions. Genomic DNA samples were amplified by polymerase chain reaction (PCR) using the following primers: intron 4 ab polymorphism, a 27-bp VNTR in intron 4 (chromosome 7q35-q36), forward primer 5′-AGG CCC TAT GGT AGT GCC TTT-3′ and reverse primer 5′-TCT CTT AGT GCT GTG GTC AC-3′; G894T polymorphism (rs1799983) forward primer 5′-CAT GAG GCT CAG CCC CAG AAC-3′ and reverse primer 5′- AGT CAA TCC CTT TGG TGC TCA C-3′; and T-786C polymorphism (rs2070744) forward primer 5′-ATG CTC CCA CCA GGG CAT CA-3′ and reverse primer 5′-GTC CTT GAG TCT GAC ATT AGG G-3′. PCR reactions yield sizes were 420-bp, 206-bp, and 237-bp for intron 4 ab polymorphism, G894T polymorphism, and T-786C polymorphism, respectively. The PCR conditions for intron 4 ab polymorphism were 5 min of initial denaturation at 94 °C, followed by 30 cycles at 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min, with a final extension at 72 °C for 7 min (S1000 Thermal Cycler, Bio-Rad Laboratories, Hercules, CA). PCR products were detected on a 2% agarose gel. The detected fragment sizes were 393-bp for a/a (normal), 420-bp for b/b (homozygous), and 393-bp and 420-bp bands for a/b (heterozygous).

The PCR conditions for G894T polymorphism were five minutes of initial denaturation at 94 °C, followed by 40 cycles at 94 °C for 30 s, 66 °C for 30 seconds, and 72 °C for 30 seconds, with a final extension at 72 °C for eight minutes (S1000 Thermal Cycler, Bio-Rad). A 206-bp fragment indicated the presence of the G allele, and 119-bp and 87-bp fragments indicated the T allele, after digestion with MboI for 4 h at 37 °C. PCR products were detected on a 2.5% agarose gel. The detected fragment sizes were 206-bp for GG; 206-bp, 119-bp, and 87-bp for GT; and 119-bp and 87-bp for TT.

The PCR conditions for T-786C polymorphism were five minutes of initial denaturation at 94 °C, followed by 40 cycles at 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 8 min (S1000 Thermal Cycler, Bio-Rad). The T allele was indicated by a 237-bp fragment, while 204-bp and 33-bp fragments indicated the C allele, after digestion with NaeI for four hours at 37 °C. PCR products were detected on a 2.5% agarose gel. The detected fragment sizes were 237-bp for TT; 237-bp, 204-bp, and 33-bp for TC; and 204-bp and 33-bp for CC.

Validation of PCR findings was performed by 1) running a negative control that had all PCR components except the DNA template in every PCR run and 2) repeating around 13% of all samples by other laboratory personnel.

Statistical analysis

The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 16 (IBM Corp., Armonk, NY). All values were represented as mean ± standard deviation (SD) or as counts (n) and percentages (%). The correlation between variables was evaluated by ANOVA for quantitative variables and by a chi-square (χ2) test for categorical variables. Genotype and allele frequencies were analyzed for concordance to the Hardy–Weinberg equilibrium. The recruited number of subjects in this study was enough to generate a study power of 0.9. A p-value of less than 0.05 was considered significant.

Haplotype analysis

An assessment of the interaction between genetic eNOS polymorphisms at the three studied loci was conducted by haplotype analysis. The haplotype frequencies of the three single nucleotide polymorphisms investigated in this study were analyzed for patients affected by diabetes with or without DR and were compared with those of the controls. Haplotype frequencies were calculated using the SNPStats tool (Institut Catala’ d’Oncologia, Spain) and linkage disequilibrium (LD) was represented by D prime (D′) [12].

Results

The clinical characteristics of affected patients with diabetes (n=172) versus (n=78) healthy controls are shown in Table 1. Body mass index (BMI) was significantly higher in patients with diabetes than in controls. Patients with diabetes had a significantly higher prevalence of hypertension, dyslipidemia, and ischemic heart disease compared to normal controls. This confirms the frequent combination of diabetes with these diseases and indicates that normal controls may not have undiagnosed diabetes.

eNOS genotype frequencies for the three polymorphisms among patients affected by diabetes, including subjects with or without DR, versus controls are presented in Table 2. A few samples yielded inconclusive results due to sample depletion, failure of PCR, or failure of endonuclease digestion. Significant differences in genotype frequencies for the three polymorphisms between patients with diabetes (n=172) and controls (n=78) were detected. In Table 3, allele frequencies both counts and percentages for the three polymorphisms are shown. Significant differences of the alleles of G894T and T-786C polymorphism were noticed. Table 4 displays the comparison of genotype frequency both counts and percentages in patients with diabetes but not DR (n=94) as a complication to their counterparts with DR (n=78), in which no significant differences were detected. However, the C allele frequency in T-786C polymorphism was significantly different between the two groups, as shown in Table 5. The allelic distribution of the polymorphisms was in Hardy–Weinberg equilibrium (intron 4 ab: DR [χ2: 0.3, p=0.9], DM [χ2: 0.2, p=0.9]; G894T: DR [χ2: 0.7, p=0.7], DM [χ2: 2.4, p=0.3]; T-786C: DR [χ2: 2.2, p=0.3], DM [χ2: 1.9, p=0.4]).

The correlation between each of the three eNOS polymorphisms and the severity of DR was tested by chi-square test, and the results are provided in Table 6. No significant association was detected; however, G894T showed a p-value very close to 0.05.

Haplotype analysis of the three eNOS polymorphisms in DM and DR patients are shown in Table 7. The bTC haplotype was significantly less common in DR patients than in DM patients, whereas, aTT was significantly more prevalent in DR patients. Several haplotypes were significantly less common in patients diagnosed with diabetes when compared to normal controls, including bTT, bTC, bGC, and aGC. The haplotype aTC was only present among healthy controls. Our results indicate that the three loci show LD between intron 4 with G894T in DM and DR (D′DM=0.458, D′DR=0.567) and between intron 4 with T-786C in DR (D′DM=0.280, D′DR=0.396) and a fair LD between G894T and T-786C in both DR (D′DR=0.108) and DM (D′DM=0.185).

Discussion

Chronic hyperglycemia induces DR in the majority of patients affected by a long duration of diabetes; however, it is insufficient to result in severe DR in most. This suggests a potential additional role of genetic factors, as shown by the analysis of the Diabetes Control and Complications Trial (DCCT) where a strong familial transmission of DR was detected in patients with severe forms of DR, but not in those with non-severe DR [6,13]. Several previous studies that investigated multiple polymorphisms in the eNOS gene had contradictory findings. In the current study, we investigated the association and interaction of the eNOS gene polymorphisms intron 4ab, G894T, and T-786C in DR patients with disease severity and compared their distribution to patients affected by diabetes but not DR as well as to healthy controls. We also identified DR disease haplotypes of eNOS gene polymorphisms and tested their correlation with the risk of DR development in Jordanian patients with type 2 diabetes. A sample of British Caucasian patients with type 1 diabetes showed a significant association of the polymorphism T-786C, the VNTR intron a/b, and DR [14]. A Slovenian Caucasian study also found an association between eNOS 4a/b polymorphism and the risk of proliferative DR (PDR) in patients with type 2 diabetes [15]. In a West African cohort, researchers observed a significant association of the 4a/b polymorphism of the eNOS gene and DR [16]. A Tunisian study showed that intron 4ab and promotor T-786T polymorphisms had a significantly different genotype distribution between DR and patients with type 2 diabetes [17]. It was suggested that the CC genotype of T-786C polymorphism could confer protection from type 1 DR in the Algerian population, while the T allele seemed to confer susceptibility [18]. The b/b genotype of intron 4ab polymorphism was significantly higher in severe DR in Caucasian populations [19]. No association was found in a sample of Caucasian Brazilians with type 2 diabetes between the two eNOS gene polymorphisms or haplotypes, intron 4ab or G894T, and the presence or severity of DR [20]. Other studies have not found an association of eNOS intron 4ab polymorphism and DR in patients with type 2 DM [2124], and the same lack of an association has been found with 894G/T or T-786C polymorphisms [22,23]. A meta-analysis that involved 15 studies with 3,183 type 2 diabetes cases and 3,410 controls showed no significant correlation between eNOS intron 4ab polymorphism and the risk of DR [25]. Another meta-analysis that included more than 8,000 subjects showed that the intron 4a allele of the intron 4ab polymorphism has protective effects against DR, particularly in type 2 DM patients, and the C allele of the T-786C polymorphism may act as a protective factor for proliferative DR, whereas, the G894T polymorphism may not affect DR development [26].

A wide range of advanced retinal vascular complications was detected in diabetic eNOS-knockout mice when compared with age-matched diabetic mice. These findings support that eNOS-derived NO plays a key role in retinal vascular function [27]. The mechanisms by which eNOS gene polymorphisms may affect the risk of DR are not well understood. Previous studies detected higher serum NO levels in patients with type 2 diabetes carrying a/b and a/a genotypes. A correlation of eNOS gene T-786C and G894T polymorphisms with endothelial and vascular function was detected in one study, where it influenced vascular endothelial growth factor (VEGF) serum levels in patients with type 2 diabetes [28]. The study found significantly higher levels of VEGF in homozygous patients for both polymorphisms compared to the other genotypes, indicating cross-talk between eNOS and VEGF and an effect on the vascularization of the retina in individuals affected by diabetes [28]. The elevation in NO levels may slow the progression of diabetic microvascular complications through mechanisms such as reduced vascular tone and changed angiotensin II effects [29,30]. Insulin is a normal activator of eNOS and NO production. With insulin-resistant type 2 DM, NO secretion is suppressed, leading to enhanced levels of VEGF [31]. Previous studies have found that eNOS G894T is subjected to selective proteolytic cleavage in endothelial cells and vascular tissues [32]. This may lead to reduced vascular NO production. In contrast, the T-786C variant has been shown to be associated with significantly decreased eNOS gene promoter activity [33]. Additionally, other studies have detected lower eNOS mRNA and serum nitrite/nitrate levels in individuals with the T-786C variant [3436]. However, no functional difference was observed with G894T polymorphism in expression studies by Fairchild et al. and Tesauro et al. [37,38]. More mechanistic studies should be performed to investigate the mechanisms by which NO changes the vascularization of the retina and results in the pathological changes in DR. In addition to mechanistic studies, the genetic profile of patients with diabetes should be investigated to include all documented polymorphisms based on sex and race, and then researchers should try to find the combined effect of most genetic factors on the development, risk, and/or progression of DR.

The limitations of this study include the relatively small sample size and the wide age range of subjects. Furthermore, the blood glucose of healthy subjects (controls) was not tested to confirm that they were free of diabetes or retinal diseases at the time of recruitment. DNA sequencing could have been performed to confirm results, but financial limitations interfered with performing sequencing. Our study was conducted at a central and specialized center for diabetes. Patients from different regions of the country seek medical care at this center; however, because this study was not multicentric, we did not have access to very large data sets. Our study population was in Hardy–Weinberg equilibrium. The strengths of this study include the recruitment of patients diagnosed with diabetes both with and without DR as well as healthy controls, whereas other studies mostly have recruited only patients with diabetes. Our study is one of a limited number that have investigated the three polymorphisms in the same study population. Most studies have focused on intron a/b polymorphism and DR with no investigation of other polymorphisms. To the best of our knowledge, our study is the only one performed on the Jordanian population and is one of only a few studies in Arab countries in this field. In our study, we excluded the potential effects of several factors, including age and gender, on the progression of DR by matching them among the three groups included in this study. We also compared patients with diabetes to healthy control individuals in addition to comparing patients with DR to patients with diabetes but no DR. We also subdivided the severity level of the non-proliferative type of DR into several levels to determine any correlation between severity and the study mutations. These case-control association studies can assist in the identification of disease biomarkers and the analysis of multiple potential factors for complex diseases such as DR.

In conclusion, the C allele of T-786C polymorphism may have a protective effect against DR. The aTT haplotype was associated with a higher risk of DR, whereas the bTC haplotype decreased DR risk. Additionally, none of the mutations was associated with a higher risk of DR or its severity. eNOS mutations may have dual roles in the pathogenesis of DR, which is consistent with the current controversy over the change in NO levels in DR patients. Further mechanistic studies should be directed toward understanding the role of NO in DR pathogenesis.

Acknowledgments

We would like to thank the Deanship of Scientific Research at the University of Jordan for funding this project (project number 19/2015/2521). We also would like to thank the National Center for Diabetes, Endocrinology and Genetics for facilitating patient recruitment and data collection. Conflict of interests: The authors declare that they have no conflict of interests to disclose.

References

  1. West IC. Radicals and oxidative stress in diabetes. Diabet Med. 2000; 17:171-80. [PMID: 10784220]
  2. Santilli F, Cipollone F, Mezzetti A, Chiarelli F. The role of nitric oxide in the development of diabetic angiopathy. Horm Metab Res. 2004; 36:319-35. [PMID: 15156413]
  3. Li H, Förstermann U. Nitric oxide in the pathogenesis of vascular disease. J Pathol. 2000; 190:244-54. [PMID: 10685059]
  4. Chan NN, Vallance P, Colhoun HM. Nitric oxide and vascular responses in Type I diabetes. Diabetologia. 2000; 43:137-47. [PMID: 10753034]
  5. Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci. 2000; 21:249-52. [PMID: 10979862]
  6. Klein R, Klein BE, Moss SE, Davis MD, DeMets DL. The Wisconsin epidemiologic study of diabetic retinopathy. II. Prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years. Arch Ophthalmol. 1984; 102:520-6. [PMID: 6367724]
  7. Toda N, Nakanishi-Toda M. Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy. Prog Retin Eye Res. 2007; 26:205-38. [PMID: 17337232]
  8. Leal EC, Manivannan A, Hosoya K, Terasaki T, Cunha-Vaz J, Ambrósio AF, Forrester JV. Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy. Invest Ophthalmol Vis Sci. 2007; 48:5257-65. [PMID: 17962481]
  9. Yuan AH, Mei Y, Zhou HY, Xiang T, Yang HJ. [Expression pattern of nitric oxide synthase in the retina of diabetic rats] Nan Fang Yi Ke Da Xue Xue Bao. 2007; 27:454-7. [PMID: 17545029]
  10. Ezzidi I, Mtiraoui N, Mohamed MB, Mahjoub T, Kacem M, Almawi WY. Endothelial nitric oxide synthase Glu298Asp, 4b/a, and T-786C polymorphisms in type 2 diabetic retinopathy. Clin Endocrinol (Oxf). 2008; 68:542-6. [PMID: 17973941]
  11. Uthra S, Raman R, Mukesh BN, Padmaja Kumari R, Paul PG, Lakshmipathy P, Gnanamoorthy P, Sharma T, McCarty CA, Kumaramanickavel G. Intron 4 VNTR of endothelial nitric oxide synthase (eNOS) gene and diabetic retinopathy in type 2 patients in southern India. Ophthalmic Genet. 2007; 28:77-81. [PMID: 17558849]
  12. Solé X, Guinó E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006; 22:1928-9. [PMID: 16720584]
  13. The Diabetes Control and Complications Trial Research Group. Clustering of long-term complications in families with diabetes in the diabetes control and complications trial. Diabetes. 1997; 46:1829-39. [PMID: 9356033]
  14. Bazzaz JT, Amoli MM, Pravica V, Chandrasecaran R, Boulton AJ, Larijani B, Hutchinson IV. eNOS gene polymorphism association with retinopathy in type 1 diabetes. Ophthalmic Genet. 2010; 31:103-7. [PMID: 20565248]
  15. Cilenšek I, Mankoč S, Globočnik Petrovič M, Petrovič D. The 4a/4a genotype of the VNTR polymorphism for endothelial nitric oxide synthase (eNOS) gene predicts risk for proliferative diabetic retinopathy in Slovenian patients (Caucasians) with type 2 diabetes mellitus. Mol Biol Rep. 2012; 39:7061-7. [PMID: 22311033]
  16. Chen Y, Huang H, Zhou J, Doumatey A, Lashley K, Chen G, Agyenim-Boateng K, Eghan BA, Acheampong J, Fasanmade O, Johnson T, Akinsola FB, Okafor G, Oli J, Ezepue F, Amoah A, Akafo S, Adeyemo A, Rotimi CN. Polymorphism of the endothelial nitric oxide synthase gene is associated with diabetic retinopathy in a cohort of West Africans. Mol Vis. 2007; 13:2142-7. [PMID: 18079690]
  17. Midani F, Ben Amor Z, El Afrit MA, Kallel A, Feki M, Soualmia H. The Role of Genetic Variants (rs869109213 and rs2070744) Of the eNOS Gene and BglII in the α2 Subunit of the α2β1 Integrin Gene in Diabetic Retinopathy in a Tunisian Population. Semin Ophthalmol. 2019; 34:365-74. [PMID: 31257963]
  18. Mihoubi E, Bouldjennet F, Raache R, Amroun H, Azzouz M, Benazouz N, Touil-Boukoffa C, Attal N. Polymorphisme T-786C de l’eNOS dans la rétinopathie du diabète de type 1 chez la population algérienne. J Fr Ophtalmol. 2019; 42:579-85.
    T-786C endothelial nitric oxide gene polymorphism and type 1 diabetic retinopathy in the Algerian population
    [PMID: 30962068]
  19. Taverna MJ, Sola A, Guyot-Argenton C, Pacher N, Bruzzo F, Chevalier A, Slama G, Reach G, Selam JL. eNOS4 polymorphism of the endothelial nitric oxide synthase predicts risk for severe diabetic retinopathy. Diabet Med. 2002; 19:240-5. [PMID: 11918626]
  20. Kátia G. Santos, Daisy Crispim, Luís H. Canani, Paula T. Ferrugem, Jorge L. Gross & Israel Roisenberg. Relationship of endothelial nitric oxide synthase (eNOS) gene polymorphisms with diabetic retinopathy in Caucasians with type 2 diabetes. Ophthalmic Genet. 2012; 33:23-7.
  21. She C, Yang X, Gu H, Deng Y, Xu J, Ma K, Liu N. [The association of variable number of tandem repeats polymorphism in the endothelial nitric oxide synthase gene and diabetic retinopathy] Zhonghua Yan Ke Za Zhi. 2015; 51:338-43. [PMID: 26311693]
  22. Narne P, Ponnaluri KC, Siraj M, Ishaq M. Association Analysis of Polymorphisms in Genes Related to Oxidative Stress in South Indian Type 2 Diabetic Patients with Retinopathy. Ophthalmic Genet. 2016; 37:1-8. [PMID: 24621175]
  23. de Syllos RW, Sandrim VC, Lisboa HR, Tres GS, Tanus-Santos JE. Endothelial nitric oxide synthase genotype and haplotype are not associated with diabetic retinopathy in diabetes type 2 patients. Nitric Oxide. 2006; 15:417-22. [PMID: 16581274]
  24. Bitarafan F, Khodaeian M, Tabatabaei-Malazy O, Amoli MM. Influence of antioxidants’ gene variants on risk of diabetes mellitus and its complications: a systematic review. Minerva Endocrinol. 2019; 44:310-25. [PMID: 28548478]
  25. Ma ZJ, Chen R, Ren HZ, Guo X, Guo J, Chen LM. Association between eNOS 4b/a polymorphism and the risk of diabetic retinopathy in type 2 diabetes mellitus: a meta-analysis. J Diabetes Res. 2014; 2014549747 [PMID: 24895640]
  26. Zhao S, Li T, Zheng B, Zheng Z. Nitric oxide synthase 3 (NOS3) 4b/a, T-786C and G894T polymorphisms in association with diabetic retinopathy susceptibility: a meta-analysis. Ophthalmic Genet. 2012; 33:200-7. [PMID: 22506535]
  27. Li Q, Verma A, Han PY, Nakagawa T, Johnson RJ, Grant MB, Campbell-Thompson M, Jarajapu YPR, Lei B, Hauswirth WW. Diabetic eNOS-knockout mice develop accelerated retinopathy. Invest Ophthalmol Vis Sci. 2010; 51:5240-6. [PMID: 20435587]
  28. Konsola T, Siasos G, Antonopoulos AS, Kollia C, Oikonomou E, Tentolouris N, Gouliopoulos N, Vogiatzi G, Papamikroulis GA, Kassi E, Tousoulis D. The impact of T786C and G894T polymorphisms of eNOS on vascular endothelial growth factor serum levels in type 2 diabetes patients. Int J Cardiol. 2016; 222:155-6. [PMID: 27494728]
  29. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362:801-9. [PMID: 8479518]
  30. Zanchi A, Moczulski DK, Hanna LS, Wantman M, Warram JH, Krolewski AS. Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism. Kidney Int. 2000; 57:405-13. [PMID: 10652017]
  31. Capellini VK, Celotto AC, Baldo CF, Olivon VC, Viaro F, Rodrigues AJ, Evora PR. Diabetes and vascular disease: basic concepts of nitric oxide physiology, endothelial dysfunction, oxidative stress and therapeutic possibilities. Curr Vasc Pharmacol. 2010; 8:526-44. [PMID: 19485895]
  32. Nagase S, Suzuki H, Wang Y, Kikuchi S, Hirayama A, Ueda A, Takada K, Oteki T, Obara M, Aoyagi K, Koyama A. Association of ecNOS gene polymorphisms with end stage renal diseases. Mol Cell Biochem. 2003; 244:113-8. [PMID: 12701818]
  33. Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, Nakao K. T-786–>C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999; 99:2864-70. [PMID: 10359729]
  34. Hibi K, Ishigami T, Tamura K, Mizushima S, Nyui N, Fujita T, Ochiai H, Kosuge M, Watanabe Y, Yoshii Y, Kihara M, Kimura K, Ishii M, Umemura S. Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension. 1998; 32:521-6. [PMID: 9740620]
  35. Miyamoto Y, Saito Y, Kajiyama N, Yoshimura M, Shimasaki Y, Nakayama M, Kamitani S, Harada M, Ishikawa M, Kuwahara K, Ogawa E, Hamanaka I, Takahashi N, Kaneshige T, Teraoka H, Akamizu T, Azuma N, Yoshimasa Y, Yoshimasa T, Itoh H, Masuda I, Yasue H, Nakao K. Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension. 1998; 32:3-8. [PMID: 9674630]
  36. Yoshimura T, Yoshimura M, Tabata A, Shimasaki Y, Nakayama M, Miyamoto Y, Saito Y, Nakao K, Yasue H, Okamura H. Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with severe preeclampsia. J Soc Gynecol Investig. 2000; 7:238-41. [PMID: 10964023]
  37. Fairchild TA, Fulton D, Fontana JT, Gratton J-P, McCabe TJ, Sessa WC. Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)–>Asp variant of human endothelial nitric-oxide synthase. J Biol Chem. 2001; 276:26674-9. [PMID: 11331296]
  38. Tesauro M, Thompson WC, Rogliani P, Qi L, Chaudhary PP, Moss J. Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298. Proc Natl Acad Sci U S A. 2000; 97:2832-5. [PMID: 10717002]