|Ahead of print publication
Association of endothelial nitric oxide synthase polymorphisms with an increased risk of erectile dysfunction
Lei Gao1, Zhifeng Zhao1, Fengfu Guo1, Yan Liu2, Jianhua Guo3, 4, Yang Zhao3, Zhong Wang3
1 Department of Urinary Surgery, Linyi People's Hospital, Linyi 276000, China
2 Department of Urinary Surgery, Linyi Cancer Hospital, Linyi 276000, China
3 Department of Urology and Andrology, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China
4 Department of Urology, Shanghai Pudong Hospital, Fudan University Pudong Medical Centre, Shanghai 200011, China
|Date of Submission||13-Jan-2015|
|Date of Decision||17-Apr-2015|
|Date of Acceptance||15-Jul-2015|
|Date of Web Publication||19-Feb-2016|
Department of Urinary Surgery, Linyi Cancer Hospital, Linyi 276000, China
Source of Support: None, Conflict of Interest: None
The purpose of our meta-analysis is to examine the associations between three single nucleotide polymorphisms of endothelial nitric oxide synthase (eNOS) gene, G894T, intron 4 and T-786C, and the risk of erectile dysfunction. An electronic database search was performed to identify case-control studies reporting the association between single nucleotide polymorphisms of eNOS gene and erectile dysfunction. Stringent inclusion and exclusion criteria were employed to select high-quality studies for this meta-analysis. Comprehensive Meta-analysis 2.0 software (Biostat Inc., Englewood, New Jersey, USA) was used for statistical analysis of the data extracted from the selected studies. From the initial 203 articles retrieved from database search, this meta-analysis finally selected 12 high-quality case-control studies that conformed to our inclusion criteria. The 12 studies contained a total of 1962 patients with erectile dysfunction and 1752 healthy controls. The results of our meta-analysis showed that G894T correlated with an increased risk erectile dysfunction under both the allele and dominant models (allele: OR = 1.556, 95% CI = 1.064-2.275, P = 0.023; dominant: OR = 1.613, 95% CI = 1.050-2.476, P = 0.029). A similar association was found between T-786C and erectile dysfunction under the allele model (OR = 1.679, 95% CI = 1.341-2.102, P < 0.001), but not under the dominant model (all P > 0.05). Our meta-analysis showed that the two single nucleotide polymorphisms in eNOS gene, G894T and T-786C, are strongly associated with the risk of erectile dysfunction.
Keywords: endothelial nitric oxide synthase; erectile dysfunction; G894T; intron 4; meta-analysis; single nucleotide polymorphism
Article in PDF
|How to cite this URL:|
Gao L, Zhao Z, Guo F, Liu Y, Guo J, Zhao Y, Wang Z. Association of endothelial nitric oxide synthase polymorphisms with an increased risk of erectile dysfunction. Asian J Androl [Epub ahead of print] [cited 2017 Mar 27]. Available from: http://www.ajandrology.com/preprintarticle.asp?id=163300
| Introduction|| |
Erectile dysfunction (ED) is defined as the inability to achieve or maintain penile erection sufficient for sexual performance.  It is most common suffering among men over 55 years old and its occurrence increases significantly with age, which seriously impacts the quality of life in patients and contributes to decreased self-confidence, panic and depression. , There is a significant number of men under 40 who experience ED and various diseases or medications contribute to ED in this age group. ED includes organic ED, characterized by a gradual onset, and psychogenic ED, which is considered as situational ED and varies in severity from mild to severe, according to the International Index of Erectile Function. , ED is a multifactorial disease involving organic, endocrine changes, smoking, and psychogenic factors, and in addition, an increased risk of ED is also associated with diseases such as diabetes mellitus, cardiovascular disease, genitourinary diseases, mental or psychological disorders, as well as other chronic diseases. ,, With respect to ED treatment, both pharmacological and psychological therapies are prevalent, and current evidence suggests that the majority of patients respond well to selective phoshodiesterase type-5 inhibitors, but at least 30%-35% of men with ED fail to respond to this therapy. ,, Considering the enormity of the negative influence of ED on the relationship between man and woman, it is important to investigate additional factors that may contribute significantly to ED risk. 
There are three major isoforms of nitric oxide synthase (NOS): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS), and each of them is encoded by separate gene.  eNOS is located in the cytoplasm of cavernous endothelium and is activated by shear stress or mechanical stress via increased arterial blood flow. , eNOS is also a major regulator of angiogenesis, and low levels of the enzyme are constitutively expressed in endothelial cells, as well as other cell types. , eNOS gene is mapped to chromosome 7q35-36 and contains 26 exons that span 21 kb. , Three important single nucleotide polymorphisms (SNPs) in the eNOS gene (G894T, intron 4, and T-786C) have received substantial attention in the last few years, especially in relation to ED. ,,, A few studies reported positive associations of G894T, T-786C, and intron 4 with the risk of ED. ,, However, other studies contend that this association of SNPs of eNOS gene appear to be a "bystander" effect for ED risk. ,, In this context, we used a meta-analysis framework to investigate the associations between G894T, intron 4, T-786C SNPs of eNOS gene, and the risk of ED.
| Materials and Methods|| |
Data sources and keywords
To identify potential relevant published studies that investigated the association between the SNPs of eNOS and ED, we systematically searched PubMed, SpringerLink, Web of Science, Wiley, Cochrane Library, CINAHL, and China National Knowledge Infrastructure (CNKI) using keywords related to ED and gene polymorphism. We applied the following keywords and MeSH terms in our literature search: ("polymorphism, genetic" or "polymorphisms" or "polymorphism" or "SNP" or "mutation" or "variants") and ("nitric oxide synthase, Type III" or "endothelial nitric oxide synthase" or "eNOS Enzyme" or "ECNOS Enzyme") and ("erectile dysfunction" or "impotence" or "male impotence" or "male sexual impotence"). Additionally, we also manually scanned the bibliographies of related articles to identify other potential relevant articles.
Study selection criteria and data extraction
Human case-control studies providing genotype data of the SNPs in eNOS gene, in both ED patients and healthy controls, were identified. We selected only those studies that contained larger sample size and extracted the required information on eNOS gene polymorphisms and other relevant data. A stringent study selection process was followed to exclude summaries, letters, meta-analyses, nonhuman studies, studies with incomplete, unavailable or irrelevant clinicopathologic data, and studies not relevant to the topic or those studies published in non-English or non-Chinese languages. In order to reduce bias and increase confidence in the data, two investigators separately extracted information from the selected articles and reach a consensus on all the items through discussion and reexamination. In case of disagreements between the two investigators, a third investigator was consulted to find a resolution. The following relevant data were extracted from each eligible study: first author, year of publication, country, ethnicity, language, number of samples, source of sample, age and SNPs.
We calculated the odds ratio (OR) with 95% confidence intervals (95% CIs), displayed by forest plots, to evaluate the differences in genotype frequencies of the SNPs of eNOS gene (G894T, intron 4 and T-786C) between cases and controls and employed Z-test to examine the significance of the overall effect size.  Heterogeneity among the enrolled studies was confirmed by Galbraith radial plot and Cochran's Q-statistic, and P < 0.05 indicated the existence of heterogeneity. , Subsequently, I2 statistics was utilized to quantify the size of heterogeneity.  The value of I2 ranged from 0% to 100%, where I2 <50% indicated no significant heterogeneity and I2 >50% suggested increasing heterogeneity. Random-effects model was applied if significant heterogeneity was detected (P < 0.05 or I2 test exhibited >50%), otherwise fixed-effects model was utilized.  Sensitivity analyses were performed to evaluate whether removal of any one single study influenced the overall outcomes. Publication bias was evaluated by visual inspection of the funnel plots, further analyzed by both classic fail-safe N and Egger's linear regression test to demonstrate the reliability of the overall results. ,, We also estimated the expected power of individual study as determined by the probability of evaluating a definitive association between eNOS SNPs and the risk of ED.  All tests were two-sided (P < 0.05 was considered significant). All statistical data were analyzed with Comprehensive Meta-analysis 2.0 (CMA 2.0) software (Biostat Inc., Englewood, New Jersey, USA).
| Results|| |
A total of 203 studies were identified from electronic database search and manual searches, and after the study selection process was completed, only 12 studies published between 2003 and 2012 qualified for our meta-analysis. The study selection process excluded 19 articles for being duplicates, 130 for being irrelevant article types (letters, reviews, or meta-analyses), nonhuman studies or unrelated to research topics. After evaluating full texts, 36 studies were excluded for containing incomplete data or data of low relevance, and 4 were eliminated for the absence of required data. Besides, 2 low-quality studies were also excluded ([Figure 1]). Finally, 12 studies were enrolled into our meta-analysis. ,,,,,,,,,,, The subjects in these 12 studies were Asians (5 studies) and Caucasians (7 studies), and the studies contained a combined total of 1962 ED patients and 1752 healthy controls. The available SNPs of eNOS gene in this meta-analysis were intron 4, T-786C, and G894T. The SNP detection methods used were polymerase chain reaction with the restriction fragment length polymorphism (PCR-RELP), allele-specific polymerase chain reaction (AS-PCR), and TaqMan assay. The baseline characteristics and power value of the studies are presented in [Table 1].
|Figure 1: Flowchart showing the detailed steps of study screening procedure and reasons for exclusion. Twelve studies were included in this meta-analysis.|
Click here to view
|Table 1: The baseline characteristics of each included literature in the present meta-analysis |
Click here to view
The association between G894T and the risk of ED
Considering the significant heterogeneity detected among the eight studies reporting the association between G894T of eNOS gene and the risk of ED, random-effects model was applied (allele: I2 = 83.703%, P < 0.01; dominant: I2 =80.971%, P < 0.01). The result of the meta-analysis suggested that G894T contribute significantly to the risk of ED (allele: OR = 1.556, 95% CI = 1.064-2.275, P = 0.023; dominant: OR = 1.613, 95% CI = 1.050-2.476, P = 0.029) ([Figure 2]a , [Figure 2]d and [Table 2]). Further subgroup analysis based on ethnicity suggested that G894T was significantly associated with risk of ED in Asians (allele: OR = 2.417, 95% CI = 1.402-4.167, P = 0.001; dominant: OR = 2.545, 95% CI = 1.374-4.712, P = 0.003), but not in Caucasians (allele: OR = 1.260, 95% CI = 0.945-1.681, P = 0.116; dominant: OR = 1.304, 95% CI = 0.896-1.899, P = 0.166) ([Figure 3]a and [Figure 3]d) .
|Figure 2: (a-f) Forest plot showing the differences of genotype frequencies among the SNPs of eNOS gene between case group and control group (G894T, intron 4, and T-786C).|
Click here to view
|Figure 3: (a-f) Forest plot showing the differences of genotype frequencies among the SNPs of eNOS gene between case group and control group based on ethnicity (G894T, intron 4, and T-786C).|
Click here to view
|Table 2: Comparisons of genotype and allele frequencies between the case and the control groups |
Click here to view
The association between intron 4 and the risk of ED
There was no significant heterogeneity among the 5 studies reporting the association between intron 4 of eNOS gene and the risk of ED, thus fixed-effects model was used (allele: I 2 =22.600%, P = 0.270; dominant: I 2 =30.779%, P = 0.216). The result of the meta-analysis revealed that intron 4 was not relevant to the risk of ED (allele: OR = 1.017, 95% CI = 0.822-1.258, P = 0.877; dominant: OR = 0.941, 95% CI = 0.737-1.201, P = 0.624) ([Figure 2]b, [Figure 2]e and [Table 2]). Further subgroup analysis based on ethnicity suggested that intron 4 was not associated with the risk of ED either in the Asians (allele: OR = 1.164, 95% CI = 0.886-1.529, P = 0.276; dominant: OR = 1.095, 95% CI = 0.800-1.500, P = 0.571) or in Caucasians (allele: OR = 0.825, 95% CI = 0.588-1.159, P = 0.267; dominant: OR = 0.748, 95% CI = 0.508-1.101, P = 0.141) ([Figure 3]b and [Figure 3]e).
The association between T-786C and the risk of ED
Four studies reported the association between T-786C of eNOS gene and the risk of ED. The heterogeneity test indicated that heterogeneity existed among these studies under the allele model (I 2 =53.691%, P = 0.091), and random-effects model was used for this analysis while heterogeneity was absent under the dominant model (I 2 =9.101%, P = 0.348), and fixed-effects model was used for analysis. The result of meta-analysis revealed that T-786C significantly contributed to the risk of ED under the allele model (OR = 1.430, 95% CI = 1.067-1.917, P = 0.017), but not under the dominant model (OR = 1.145, 95% CI = 0.900-1.456, P = 0.271) ([Figure 2]c, [Figure 2]f and [Table 2]). Further, subgroup analysis based on ethnicity suggested that T-786C was associated with a significant risk of ED in the Asians (allele: OR = 1.679, 95% CI = 1.341-2.102, P < 0.001; dominant: OR = 1.247, 95% CI = 0.905-1.718, P = 0.178), but not in Caucasians (allele: OR = 1.289, 95% CI = 0.894-1.858, P = 0.173; dominant: OR = 1.026, 95% CI = 0.713-1.476, P = 0.891) ([Figure 3]c and [Figure 3]f).
Sensitivity analysis and publication bias
Under the allele model, removal of any single study, except Lee et al. and Erol et al. (removal led to adverse results), had minimal impact on the pooled OR values of G894T and the risk of ED ([Figure 4]a). Under dominant model, removal of any single study, except Lee et al. and Safarinejad et al. (removal led to adverse results), had minimal impact on the pooled OR values of G894T and the risk of ED ([Figure 4]d). For pooled OR values of intron 4 and the risk of ED, removal of any single study exerted no influence ([Figure 4]b and [Figure 4]e). However, except Safarinejad et al. (removal led to adverse results) under allele model, removal of any single study had minimal impact on the pooled OR values of T-786C and the risk of ED ([Figure 4]c and [Figure 2]f). Symmetrical funnel plots indicated the absence of publication bias, which was further confirmed by both classic fail-safe N and Egger's linear regression test (all P > 0.05) ([Figure 5]).
|Figure 4: (a-f) Sensitivity analyses about the differences of genotype frequencies among the SNPs of eNOS gene between case group and control group (G894T, intron 4, and T-786C).|
Click here to view
|Figure 5: (a-f) Funnel plot demonstrating publication biases about the differences of genotype frequencies among the SNPs of eNOS gene between case group and control group (G894T, intron 4, and T-786C).|
Click here to view
| Discussion|| |
Using data from previous studies, we applied a meta-analysis approach to determine the associations between three SNPs of eNOS gene, G894T, T-786C, and intron 4 and ED risk. Our findings showed that G894T and T-786C are tightly associated with an increased risk of ED. Penile tumescence begins with the release of NO from either nonadrenergic noncholinergic nerve terminals or endothelial cells, and inadequate NO production during sexual stimulation can result from down-regulation of NOS expression.  Our findings demonstrated the association between T-786C and the risk of ED. Low eNOS mRNA and serum nitrite levels were observed in individuals carrying the CC allele of T-786C polymorphism and homozygosity in this polymorphism correlated with reduced eNOS expression in human endothelial cells and decreased NO-induced vasomotor function.  The G894T polymorphism results in an amino acid substitution in eNOS, altering its activity. Measurement of eNOS enzyme activity in T allele carriers revealed 20% lower activity compared to individuals homozygous for the G allele.  Erectile function depends on the relaxation and contraction of penis which is regulated by smooth muscle, and G894T polymorphism was found to interfere with this process, increasing ED predisposition.  The two SNPs (T-786C and G894T) variously affect either eNOS transcription rate or its enzyme activity to dramatically reduce NO production, resulting in decreased functioning of the eNOS/NO system to buffer blood pressure fluctuations.  Evidence shows that functional SNPs within eNOS gene interfering with normal eNOS functions down-regulate nitric oxide (NO) production and lower cyclic guanosine monophosphate (cGMP) concentrations, leading to alteration in vasoactive substances, endothelial function and erectile function. Thus, ED is closely associated with eNOS gene polymorphisms. , Based on the above analyses, we postulate that G894T and T-786C are the major genetic risk factors for ED. In support of this, Sinici et al. demonstrated that the frequency of CC genotype of T-786C SNPs was higher in ED patients, indicating that the T-786C eNOS polymorphism may be an independent risk factor in the pathogenesis of ED. 
To examine other factors that may influence the association between the of eNOS gene SNPs and the risk of ED, we conducted subgroup analysis based on ethnicity. In this analysis, G894T and T-786C were strongly associated with an increased risk of ED in Asians, but not in Caucasians. The intron 4 was not associated with the risk of ED either in Asians or in Caucasians. The possible reason could be that gene polymorphisms are not the only factors influencing ED and other environmental factors also play a very important role in the development and progression of ED. Further, genetic polymorphisms at other loci, such as in protein disulfide isomerase (PDI) or in the other members of the multi-subunit eNOS complex, geographical location, lifestyle, dietary habits, or limitations in the detection methods, could influence these findings. We intend to pursue further studies to address ethnic differences related to eNOS SNPs and the risk of ED. Therefore, our results should be interpreted taking into consideration the genetic backgrounds and the fact that the results need to be replicated in other ethnic populations.
There were limitations to the current meta-analysis: (1) although there were a total of twelve studies involved, three different SNPs were incorporated for analyses. Therefore, the sample size may not be adequate. (2) The studies employed multiple detection methods, which may have influenced our overall results. (3) Although there was no detectable publication bias in our study, the sample size was comparatively small and the outcomes were varied. (4) Our study may not have the sufficient statistical power to detect small difference between cases and controls Power values of some included literature were relatively low largely due to their small sample size or incompleteness of relevant data, which has a potential impact on the reliability of our study. These limitations may lower the validity and reliability of our overall results.
| Conclusion|| |
Our results strongly support that two SNPs of eNOS gene, T-786C and G894T, are associated with an increased risk of developing ED. Due to the multifactorial nature of ED, future studies in a larger population with diverse ethnic backgrounds are needed to confirm our results.
| Author Contributions|| |
LG carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. ZZ carried out the immunoassays. FG participated in the sequence alignment. YL and JG participated in the design of the study and performed the statistical analysis. YZ and ZW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
| Competing Interests|| |
We declared that we have no competing interests.
| References|| |
Shamloul R, Ghanem H. Erectile dysfunction. Lancet
2013; 381: 153-65.
Andersen ML, Guindalini C, Santos-Silva R, Bittencourt LR, Tufik S. Association analysis of endothelial nitric oxide synthase G894T gene polymorphism and erectile dysfunction complaints in a population-based survey. J Sex Med
2010; 7: 1229-36.
Glina S, Sharlip ID, Hellstrom WJ. Modifying risk factors to prevent and treat erectile dysfunction. J Sex Med
2013; 10: 115-9.
Miner M, Seftel AD, Nehra A, Ganz P, Kloner RA, et al.
Prognostic utility of erectile dysfunction for cardiovascular disease in younger men and those with diabetes. Am Heart J
2012; 164: 21-8.
Jackson G, Boon N, Eardley I, Kirby M, Dean J, et al.
Erectile dysfunction and coronary artery disease prediction: evidence-based guidance and consensus. Int J Clin Pract
2010; 64: 848-57.
Lewis RW, Fugl-Meyer KS, Corona G, Hayes RD, Laumann EO, et al.
Definitions/epidemiology/risk factors for sexual dysfunction. J Sex Med
2010; 7: 1598-607.
Gerber RE, Vita JA, Ganz P, Wager CG, Araujo AB, et al.
Association of peripheral microvascular dysfunction and erectile dysfunction. J Urol
2015; 193: 612-7.
Lippi G, Plebani M, Montagnana M, Cervellin G. Biochemical and genetic markers of erectile dysfunction. Adv Clin Chem
2012; 57: 139-62.
Garcia MM, Fandel TM, Lin G, Shindel AW, Banie L, et al.
Treatment of erectile dysfunction in the obese type 2 diabetic ZDF rat with adipose tissue-derived stem cells. J Sex Med
2010; 7: 89-98.
Andersson KE. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol Rev
2011; 63: 811-59.
Garcia JA, Sanchez PE, Fraile C, Escovar P. Testosterone undecanoate improves erectile dysfunction in hypogonadal men with the metabolic syndrome refractory to treatment with phosphodiesterase type 5 inhibitors alone. Andrologia
2011; 43: 293-6.
McGraw SA, Rosen RC, Althof SE, Dunn M, Cameron A, et al.
Perceptions of erectile dysfunction and phosphodiesterase type 5 inhibitor therapy in a qualitative study of men and women in affected relationships. J Sex Marital Ther
2015; 41: 203-20.
Pigott B, Bartus K, Garthwaite J. On the selectivity of neuronal NOS inhibitors. Br J Pharmacol
2013; 168: 1255-65.
Koo A, Nordsletten D, Umeton R, Yankama B, Ayyadurai S, et al. In silico
modeling of shear-stress-induced nitric oxide production in endothelial cells through systems biology. Biophys J
2013; 104: 2295-306.
Cybulsky MI, Marsden PA. Effect of disturbed blood flow on endothelial cell gene expression: a role for changes in RNA processing. Arterioscler Thromb Vasc Biol
2014; 34: 1806-8.
Forstermann U, Li H. Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling. Br J Pharmacol
2011; 164: 213-23.
Craige SM, Chen K, Pei Y, Li C, Huang X, et al.
NADPH oxidase 4 promotes endothelial angiogenesis through endothelial nitric oxide synthase activation. Circulation
2011; 124: 731-40.
Janssens SP, Shimouchi A, Quertermous T, Bloch DB, Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem
1992; 267: 14519-22.
Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, et al.
Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem
1993; 268: 17478-88.
Cilensek I, Mankoc S, Globocnik Petrovic M, Petrovic 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.
Jira M, Zavodna E, Honzikova N, Novakova Z, Vasku A, et al.
Association of eNOS gene polymorphisms T-786C and G894T with blood pressure variability in man. Physiol Res
2011; 60: 193-7.
Safarinejad MR, Safarinejad S, Shafiei N, Safarinejad S. Effects of the T-786C, G894T, and intron 4 VNTR (4a/b) polymorphisms of the endothelial nitric oxide synthase gene on the risk of prostate cancer. Urol Oncol
2013; 31: 1132-40.
Yu Q, Zhang Y, Xia Y, Yang X, Li N, et al.
Analysis of endothelial nitric oxide synthase (eNOS) G894T polymorphism and semen parameters in a Chinese Han population. Andrologia
2014; 46: 541-6.
Lopushnyan NA, Chitaley K. Genetics of erectile dysfunction. J Urol
2012; 188: 1676-83.
Safarinejad MR, Khoshdel A, Shekarchi B, Taghva A, Safarinejad S. Association of the T-786C, G894T and 4a/4b polymorphisms of the endothelial nitric oxide synthase gene with vasculogenic erectile dysfunction in Iranian subjects. BJU Int
2011; 107: 1994-2001.
Lee YC, Huang SP, Liu CC, Yang YH, Yeh HC, et al.
The association of eNOS G894T polymorphism with metabolic syndrome and erectile dysfunction. J Sex Med
2012; 9: 837-43.
Wang JL, Wang HG, Gao HQ, Zhai GX, Chang P, et al.
Endothelial nitric oxide synthase polymorphisms and erectile dysfunction: a meta-analysis. J Sex Med
2010; 7: 3889-98.
Chen H, Manning AK, Dupuis J. A method of moments estimator for random effect multivariate meta-analysis. Biometrics
2012; 68: 1278-84.
Higgins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. Stat Med
2002; 21: 1539-58.
Jackson D, White IR, Riley RD. Quantifying the impact of between-study heterogeneity in multivariate meta-analyses. Stat Med
2012; 31: 3805-20.
Peters JL, Sutton AJ, Jones DR, Abrams KR, Rushton L. Comparison of two methods to detect publication bias in meta-analysis. JAMA
2006; 295: 676-80.
Zintzaras E, Ioannidis JP. Heterogeneity testing in meta-analysis of genome searches. Genet Epidemiol
2005; 28: 123-37.
Wikstrom EA, Naik S, Lodha N, Cauraugh JH. Balance capabilities after lateral ankle trauma and intervention: a meta-analysis. Med Sci Sports Exerc
2009; 41: 1287-95.
Zintzaras E, Ioannidis JP. HEGESMA: genome search meta-analysis and heterogeneity testing. Bioinformatics
2005; 21: 3672-3.
Egger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ
1997; 315: 629-34.
Lee YH, Harley JB, Nath SK. Meta-analysis of TNF-alpha promoter-308 A/G polymorphism and SLE susceptibility. Eur J Hum Genet
2006; 14: 364-71.
Eisenhardt A, Sperling H, Hauck E, Porst H, Stief C, et al.
ACE gene I/D and NOS3 G894T polymorphisms and response to sildenafil in men with erectile dysfunction. Urology
2003; 62: 152-7.
Rosas-Vargas H, Coral-Vazquez RM, Tapia R, Borja JL, Salas RA, et al.
Glu298Asp endothelial nitric oxide synthase polymorphism is a risk factor for erectile dysfunction in the Mexican Mestizo population. J Androl
2004; 25: 728-32.
Erkan E, Muslumanoglu AY, Oktar T, Sanli O, Ozbek U, et al.
Polymorphism of endothelial nitric oxide synthase gene in patients with erectile dysfunction. J Sex Med
2006; 3: 69-75.
Peskircioglu L, Atac FB, Erdem SR, Deveci S, Verdi H, et al.
The association between intron 4 VNTR, E298A and IVF 23+10 G/T polymorphisms of ecNOS gene and sildenafil responsiveness in patients with erectile dysfunction. Int J Impot Res
2007; 19: 149-53.
Erol B, Bozdogan G, Akduman B, Dursun A, Bozdogan S, et al.
eNOS gene intron 4 VNTR and exon 7-G894T polymorphisms in Turkish men with erectile dysfunction: a case control study. J Sex Med
2009; 6: 1423-9.
Meluzin J, Vasku A, Kincl V, Panovsky R, Sramkova T. Association of coronary artery disease, erectile dysfunction, and endothelial nitric oxide synthase polymorphisms. Heart Vessels
2009; 24: 157-63.
Eisenhardt A, Stief C, Porst H, Wetterauer U, Weidner W, et al.
Genetic association study of the GNB3 C825T, the ACE I/D and the eNOS G894T polymorphisms and the risk to develop erectile dysfunction in a German ED population. Andrologia
2010; 42: 218-24.
Sinici I, Guven EO, Serefoglu E, Hayran M. T-786C polymorphism in promoter of eNOS gene as genetic risk factor in patients with erectile dysfunction in Turkish population. Urology
2010; 75: 955-60.
Zhang XR, Zhang ZJ, Zhu RX, Yuan YG, Jenkins TA, et al.
Sexual dysfunction in male schizophrenia: influence of antipsychotic drugs, prolactin and polymorphisms of the dopamine D2 receptor genes. Pharmacogenomics
2011; 12: 1127-36.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]