Association of Endothelial Nitric Oxide Synthase Glu298Asp Gene Polymorphism with the Risk of End-Stage Renal Disease

Abstract

The association between endothelial nitric oxide synthase (eNOS) Glu298Asp gene polymorphism and end-stage renal disease (ESRD) risk is still controversial. A meta-analysis was performed to evaluate the association between eNOS Glu298Asp gene polymorphism and ESRD susceptibility. A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic database. Six articles were identified for the analysis of association between eNOS Glu298Asp gene polymorphism and ESRD risk. T allele was associated with ESRD susceptibility in overall populations and in Asians (overall populations: p = 0.01, Asian: p < 0.00001). Furthermore, GG genotype might play a protective role against ESRD onset for overall populations, Asians, and Africans. However, a link between eNOS Glu298Asp gene polymorphism and ESRD risk was not found in Caucasians and Brazil population. In conclusion, T allele might become a significant genetic molecular marker for the onset of ESRD in overall populations, and in Asians. However, more studies should be performed in the future.

• end-stage renal disease
• endothelial nitric oxide synthase
• eNOS
• Glu298Asp
• gene polymorphism
• meta-analysis

INTRODUCTION

End-stage renal disease (ESRD) represents a worldwide health problem with an irreversible loss of endogenous renal function and with an epidemic extent.^1–3 Patients with ESRD have significantly increased morbidity and mortality,⁴ and should receive a kidney transplant/live on dialysis.^5,6 Patient suffering from ESRD displays enhanced genomic damage, and DNA repair gene polymorphisms may affect DNA repair capacity and modulate susceptibility to ESRD.⁷ Some current investigations^8–10 suggested that genetic factors might play a key role in the onset of ESRD.

Nitric oxide (NO) is a ubiquitous vasodilator and an important regulator of renal sodium excretion.¹¹ Reduced NO generation induces renal injury,¹² and impairment of endothelial NO generation is considered as the major deterioration factor for progressive renal disease. NO is produced by inducible nitric oxide synthase (iNOS).¹³ Glu298Asp is an important gene mutation of eNOS, and the eNOS Glu298Asp gene polymorphism includes GG (Glu/Glu), GT (Glu/Asp), and TT (Asp/Asp) genotypes and G (Glu) and T (Asp) alleles. The present epidemiologic study shows that the eNOS Glu298Asp gene polymorphism has been implicated in the etiology of ESRD. However, the available evidence reported to date is weak, due to the sparseness of data or disagreements among studies. There was rare meta-analysis to explore the association of eNOS Glu298Asp gene polymorphism with ESRD risk. We performed this meta-analysis using data from all English published reports to investigate the relation between eNOS Glu298Asp gene polymorphism and ESRD susceptibility, with the intention to provide a much more reliable finding on the significance of the association.

MATERIALS AND METHODS

Search Strategy

The relevant studies were screened from the search engines of PubMed, Embase, and Cochrane Library on 1 March 2012. The following terms in English were used to complete the search: “end-stage renal disease,” “ESRD,” “end-stage renal failure,” “ESRF,” “endothelial nitric oxide synthase,” “eNOS,” and “gene.” We also extended search spectrum to the “related articles” and the bibliographies of all retrieved studies. If multiple publications of the same data from the same study group occurred, we recruited only the later paper for analysis.

Inclusion and Exclusion Criteria

Inclusion criteria

(1) A case–control study; (2) the outcome had to be ESRD; and (3) there had to be at least two comparison groups (ESRD group vs. control group).

Exclusion criteria

(1) Review articles, editorials, and case reports; (2) articles did not provide the detail genotype data; (3) investigating the association of other genes with ESRD; (4) investigating the role of eNOS to diseases; and (5) multiple publications of the same data from the same study group.

Data Extraction and Synthesis

The following information was extracted from each study independently by two investigators: first author’s surname, year of publication, ethnicity of study population, and the number of cases and controls for eNOS genotype. Frequencies of T allele were calculated for the case and control groups, from the corresponding genotype distribution. The results were compared, and the disagreements were resolved by discussion.

Statistical Analysis

Available data were entered into Cochrane Review Manager (RevMan, version 5, Oxford, UK) and analyzed. The pooled statistic was counted using the fixed effects model, but a random effects model was conducted when the p value of heterogeneity test was less than 0.1. Results were expressed with odds ratios (ORs) for dichotomous data, and 95% confidence intervals (CIs) were also calculated. A p value of <0.05 was required for the overall OR to be deemed statistically significant. I² was used to test the heterogeneity between the included studies. We classified the investigations into studies for Caucasians, Asians, Africans, and Brazil population, because genotype frequencies and prevalence of ESRD were different among ethnic groups. In order to avoid excessive comparisons, the OR was calculated by using three methods¹⁴: method 1, allele comparison (T allele vs. G allele); method 2, comparing TT homozygous with the other two combinations (TT vs. TG + GG); and method 3, comparing GG genotype with the other two combinations (GG vs. TG + TT). A chi-square test using a web-based program was applied to determine whether genotype distribution of the control population reported conformed to Hardy–Weinberg equilibrium (HWE) (p < 0.05 was considered significant). Sensitivity analysis was performed when studies with controls were not in HWE. Begg’s adjusted rank correlation test¹⁵ and Egger’s regression asymmetry test¹⁶ were used for exploring publication bias (p < 0.1 was considered significant) when the sample number was more than five. All descriptive data were expressed as mean ± SD.

RESULTS

Study Characteristics

The search yielded 145 references, 67 in Pub med, 38 in Embase, and 0 from Cochrane Library. According to the inclusion and exclusion criteria, six articles^17–22 were identified for the analysis of the association between eNOS Glu298Asp gene polymorphism and ESRD susceptibility in our review (Figure 1). Two studies^17,21 were conducted in Caucasians, two investigations^18,19 for Asians, one²² in Africans, and one²⁰ in Brazil population. The data of our interest were extracted and shown in Table 1. These six studies contained 675 case series and 903 controls. The average frequency of T allele was 46.85% in Caucasians ESRD patients and 31.43% in controls. For Asians, the average frequency of T allele was 22.57% in the case group and 9.54% for the control group. The frequency of T allele in Brazil population was 33.50% in cases and 30.00% for controls. When compared with that in Caucasians and Brazil population, the ratio of cases/controls for average frequency of T allele was markedly elevated in Asians (Asians: cases/controls = 2.37; Caucasians: cases/controls = 1.49; Brazil population: cases/controls = 1.12).

Figure 1.  Flow chart for our studies.

Table 1.  Characteristics of the studies evaluating the effects of eNOS Glu298Asp gene polymorphism on ESRD risk.

		ESRD				Control				T allele (%)
First author, year	Ethnicity	TT	TG	GG	Total	TT	TG	GG	Total	ESRD	Control
Noiri et al.^22	African	–	–	136	185	–	–	251	304	–	–
de Prado et al.^17	Caucasian	21	33	37	91	21	46	42	109	41.21	40.37
Nagase et al.^18	Asian	2	33	124	159	2	18	250	270	11.64	4.07
Thaha et al.^19	Asian	3	61	36	100	2	26	72	100	33.5	15
Marson et al.^20	Brazil	10	47	43	100	9	42	49	100	33.5	30
El-Din and Hamdy^21	Caucasian	12	18	10	40	1	7	12	20	52.5	22.5

Association of the eNOS Glu298Asp Gene Polymorphisms with ESRD Risk

In this meta-analysis, a significant association between T allele and ESRD risk was observed in overall populations (Figure 2 for T allele; Table 2). Furthermore, the GG genotype might be a protective factor against the risk of ESRD in overall populations (Figure 3 for GG allele; Table 2). However, the association of TT genotype with ESRD risk was not observed (Table 2).

Figure 2.  Association between T allele and ESRD risk in overall populations.

Figure 3.  Association of GG genotype with ESRD risk in overall populations.

Figure 4.  Publication bias for the analysis of association of eNOS Glu298Asp gene polymorphism and ESRD susceptibility for overall populations.

Table 2.  Meta-analysis of the association of eNOS Glu298Asp gene polymorphism with risk of ESRD.

Genetic contrasts	Group and studies subgroups	Q test	p-Value	Model selected	OR (95% CI)	p-Value
T vs. G	Overall	5	0.0003	Random	1.99 (1.18, 3.37)	0.01
	Caucasian	2	0.007	Random	1.87 (0.52, 6.69)	0.33
	Asian	2	0.83	Fixed	2.96 (2.06, 4.26)	0.00001
	Brazil	1	–	Fixed	1.18 (0.77, 1.79)	0.45
TT vs. (TG + GG)	Overall	5	0.55	Fixed	1.50 (0.92, 2.43)	0.10
	Caucasian	2	0.09	Random	2.44 (0.41, 14.57)	0.33
	Asian	2	0.93	Fixed	1.60 (0.42, 6.08)	0.49
	Brazil	1	–	Fixed	1.12 (0.44, 2.89)	0.81
GG vs. (TG + TT)	Overall	6	0.0003	Random	0.46 (0.27, 0.78)	0.004
	Caucasian	2	0.01	Random	0.53 (0.11, 2.53)	0.43
	Asian	2	0.55	Fixed	0.25 (0.16, 0.38)	0.00001
	African	1	–	Fixed	0.59 (0.38, 0.91)	0.02
	Brazil	1	–	Fixed	0.79 (0.45, 1.37)	0.39
Sensitivity analysis
T vs. G	Overall	4	0.001	Random	1.78 (1.00, 3.18)	0.05
	Caucasian	2	0.007	Random	1.87 (0.52, 6.69)	0.33
	Asian	1	–	Fixed	2.85 (1.75, 4.64)	0.0001
	Brazil	1	–	Fixed	1.18 (0.77, 1.79)	0.45
TT vs. (TG + GG)	Overall	4	0.39	Fixed	1.48 (0.90, 2.44)	0.12
	Caucasian	2	0.09	Random	2.44 (0.41, 14.57)	0.33
	Asian	1	–	Fixed	1.52 (0.25, 9.27)	0.65
	Brazil	1	–	Fixed	1.12 (0.44, 2.89)	0.81
GG vs. (TG + TT)	Overall	4	0.0003	Random	0.48(0.21, 1.10)	0.08
	Caucasian	2	0.01	Random	0.53 (0.11, 2.53)	0.43
	Asian	1	–	Fixed	0.22 (0.12, 0.40)	0.00001
	Brazil	1	–	Fixed	0.79 (0.45, 1.37)	0.39

The ethnical and geopolitical difference might affect the results of our analysis for the association of eNOS Glu298Asp gene polymorphism with ESRD susceptibility. In order to evaluate this effect, we divided the population according to the ethnicity. T allele was associated with the risk of ESRD in Asians (Table 2). Furthermore, GG genotype might play a protective role against ESRD risk in Asians and in Africans. However, there was an association between TT genotype and ESRD susceptibility in Asians (Table 2). Furthermore, there was no association of eNOS Glu298Asp gene polymorphism with ESRD susceptibility in Caucasians and Brazil population (Table 2).

Sensitivity Analysis

One study¹⁸ from Asians that the genotype distributions in the controls were significantly deviated from HWE was excluded from our sensitive analysis. Furthermore, one study²² from Africans had not provided the detailed gene distribution data for the control group for the HWE test and it was excluded from the sensitive analysis. Finally, two studies^17,21 from Caucasians, one¹⁹ from Asians, and one²⁰ from Brazil population was included in our sensitive analysis.

In the sensitivity analysis for overall populations, we found that the pooled OR for T allele was favorable to ESRD group (OR = 1.78, 95% CI: 1.00–3.18; Table 2), although the difference was not statistically significant (p = 0.05). Furthermore, the pooled OR for GG genotype seemed to play a protective role against ESRD disease (OR = 0.48, 95% CI: 0.21–1.10; Table 2), although the difference was not statistically significant (p = 0.08). The results were not consistent with those obtained from the nonsensitivity analysis. Interestingly, TT genotype was not associated with the risk of ESRD in the sensitivity analysis, and this result was consistent with that from the nonsensitivity analysis (Table 2).

In Asians, T allele was associated with the risk of ESRD in Asians (Table 2). Furthermore, GG genotype might play a protective role against ESRD risk in Asians. However, there was an association between TT genotype and ESRD susceptibility in Asians (Table 2). The results from the sensitivity analysis for Asians were consistent with those from the nonsensitivity analysis.

In Caucasians and Africans, the results from the sensitivity analysis were the same as those from the nonsensitivity analysis (Table 2).

Testing for Publication Bias for the Analysis of Association of eNOS Glu298Asp Gene Polymorphism and ESRD Susceptibility

There was no significant publication bias in overall populations (Begg: p = 1.000; Egger: p = 0.296; Figure 4).

DISCUSSION

Impairment of endothelial NO generation brought about by gene polymorphism is considered the major deterioration factor for progressive renal disease, and the present study aimed to elucidate the Glu298Asp polymorphism of eNOS in patients with ESRD.²² ESRD is a major health problem associated with very high morbidity and mortality, and data on the risk factors for the pathogenesis of ESRD were insufficient. There was rare genetic molecular marker to predict the onset of ESRD. This meta-analysis was performed to explore whether the eNOS Glu298Asp gene polymorphism could predict the susceptibility of ESRD.

In this investigation, six suitable studies were recruited into our meta-analysis, two investigations for Caucasians, two studies in Asians, one from Africans, and one from Brazil population. Our meta-analysis showed that T allele was associated with the onset of ESRD in overall populations, but TT genotype did not. Furthermore, GG genotype seemed to play a protective role against ESRD risk. There was no significant publication bias in overall populations. The results for the overall populations might be robust to some extent. However, sensitivity analysis was also performed in our meta-analysis, and we found that the results from the sensitivity analysis were not similar to those from the nonsensitivity analysis for overall populations. We speculated that this disagreement might be caused by that the number of included studies was small. As those mentioned above, more studies should be performed to investigate the association of eNOS Glu298Asp gene polymorphism with the susceptibility of ESRD in overall populations.

The geographic and ethnic differences might be an important factor to effect the association of gene polymorphism with the susceptibility of ESRD. In our study, we found that the average frequency of T allele in controls was 31.43% in Caucasians. Furthermore, the frequency of T allele in Brazil population was 30.00% for control group. However, for Asians, the average frequency of T allele for controls was 9.54%. The disequilibrium of T allele distribution in controls was observed among those different races. The subgroup analysis was conducted to explore the association of eNOS Glu298Asp gene polymorphism with the susceptibility of ESRD in different races.

In Caucasians, the association of eNOS Glu298Asp gene polymorphism with the susceptibility of ESRD was not found. The gene distributions of all the included studies were in HWE, and the results from the sensitivity analysis were the same as those from the nonsensitivity analysis in Caucasians. However, there were only two studies recruited into our meta-analysis for Caucasians, and it was difficult to draw a robust conclusion for Caucasians.

In Asians, there was an association between T allele and the risk of ESRD. The ratio of cases/controls for average frequency of T allele was elevated in Asians (cases/controls = 2.37). T allele might be a risk factor to predict the risk of ESRD in Asians. Furthermore, GG genotype seemed to play a protective role against ESRD risk in Asians. Interestingly, the results from the sensitivity analysis were consistent with those from the nonsensitivity analysis. T allele might be a risk factor for the risk of ESRD in Asians. However, more well-designed studies should be performed in the future.

For African population, GG genotype might be a protective factor against the susceptibility of ESRD. However, there was only one study recruited into meta-analysis for African population, and the gene distribution of TT and GT was not provided. The conclusion for African population might be less robust. Whether there was an association between eNOS Glu298Asp gene polymorphism and the susceptibility of ESRD, more religious studies should be performed in future.

In Brazil population, eNOS Glu298Asp gene polymorphism was not associated with the susceptibility of ESRD. The gene distributions of the included study were in HWE, and the results from the sensitivity analysis were the same as those from the nonsensitivity analysis in Brazil population. However, there only one study was included in our meta-analysis, and it was difficult to draw a robust conclusion for Brazil population. More studies in Brazil population should be conducted in the future.

In the past years, there were some meta-analyses to explore the association of eNOS Glu298Asp gene polymorphism with the susceptibility of some diseases. Casas et al.²³ performed a meta-analysis to explore the relationship between eNOS genotype and ischemic heart disease, and reported that homozygosity for the TT was associated with an increased risk of IHD. Yu et al.²⁴ conducted a meta-analysis to study the relationship between eNOS Glu298Asp polymorphism and pre-eclampsia risk, and found that the eNOS Glu298Asp polymorphism was not associated with a significant increased risk of pre-eclampsia in overall populations, Caucasians, and Asians. Su et al.²⁵ performed a meta-analysis to investigate the association of eNOS Glu298Asp gene polymorphism with recurrent pregnancy loss (RPL), and reported that eNOS Glu298Asp gene polymorphism was significantly associated with RPL. Shaik et al.²⁶ reported that eNOS Glu298Asp gene polymorphism was not associated with the risk of pre-eclampsia in women by meta-analysis method. In our meta-analysis, we explore the relationship between eNOS Glu298Asp gene polymorphism and ESRD risk. Our result was similar to Su et al.²⁵

In our investigation, we found that T allele was associated with ESRD susceptibility in overall populations and in Asians, and GG genotype might play a protective role against ESRD susceptibility in overall populations, for Asians, and in Africans. However, the association was not found in Caucasians and for Brazil population. These findings should be regarded cautiously because many other ingredients, such as heterogeneity of enrolled cases, limited statistical power, variable study designs, and different interventions, were closely related to affect the results. Furthermore, whether the eNOS Glu298Asp gene polymorphism is just linked with other discrete loci involved in the occurrence of ESRD is not clear at the moment. Undoubtedly, the limitations mentioned above might affect our final conclusions.

In conclusion, the results of our study support that T allele was associated with ESRD susceptibility in overall populations and in Asians, and GG genotype might play a protective role against ESRD susceptibility in overall populations, in Asians, and in Africans. However, more case–control association investigations on larger, stratified populations are required to further clarify the role of this eNOS Glu298Asp gene polymorphism in ESRD susceptibility in different ethnicities.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the most helpful comments on this paper received from Professor Liang Rong, Department of Pediatric-Neonatology, Baylor College of Medicine, Houston, TX, USA.

Declarartion of interest: The authors declare that they have no conflict of interest.