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Endothelial nitric oxide synthase genotypes and haplotypes modify the responses to sildenafil in patients with erectile dysfunction

Abstract

Erectile dysfunction (ED) is usually treated with sildenafil. Although genetic polymorphisms in the endothelial nitric oxide synthase (eNOS) gene may impair endogenous NO formation, there is little information about how eNOS polymorphisms and haplotypes affect the responses to sildenafil. We studied 118 patients; 63 patients had ED secondary to radical prostatectomy (PED) and 55 had organic, clinical ED. eNOS genotypes for three eNOS polymorphisms (T−786C, rs2070744; a variable number of tandem repeats (VNTR) in intron 4; and Glu298Asp, rs1799983) were determined, and eNOS haplotypes were estimated using PHASE 2.1. The clinical responses to sildenafil were evaluated and the patients were classified as good responders (GR) or poor responders (PR) when their changes in five-item version of International Index for Erectile Function questionnaire were above or below the median value. The TC/CC genotypes and the C allele for the T−786C polymorphism were more common in GR, compared with PR patients with PED. However, the 4b4a/4a4a genotypes and the 4a allele for the VNTR polymorphism in intron 4 were more common in GR, compared with PR patients with clinical ED. The C-4a-Glu haplotype was more common in GR than in PR patients with PED. Conversely, the T-4b-Asp haplotype was less common in GR than in PR patients with PED. No other significant differences were found. Our findings show evidence that eNOS polymorphisms affect the responses of PED and clinical ED patients to sildenafil.

Introduction

Erectile dysfunction (ED) is a very common disorder with organic, psychological and mixed origins.1 However, there is now clear evidence supporting the notion that ED is a vascular disease affecting the endothelium,2 thus explaining how several cardiovascular risk factors increase the risk of ED.3, 4 Although ED may be due to a neurogenic cause,5 it may be a result of injury to the cavernous nerves following radical prostatectomy.6, 7

Nitric oxide (NO) is a key mediator involved in penile erection8 and this molecule is produced from L-arginine by different NO synthases (NOS) including the neuronal (nNOS), endothelial (eNOS) and inducible NOS. Importantly, impaired NO production by nNOS or eNOS may affect the start and maintenance of penile erection.1, 6, 9 Therefore, drugs that improve NO signaling have been used to treat ED. Indeed, phosphodiesterase type 5 (PDE-5) inhibitors, such as sildenafil, are usually prescribed to patients with ED.10, 11 Their use is justified by the fact that PDE-5 hydrolyses cyclic guanosine monophosphate and terminates NO signaling. Conversely, PDE-5 inhibitors increase intracellular cyclic guanosine monophosphate levels, thus improving ED.8, 11, 12 However, it is possible that genetic variations in the gene encoding eNOS may affect the responses to PDE-5 inhibitors.

Genetic polymorphisms in the eNOS gene may impair endogenous NO formation,13, 14, 15 and these findings may explain how the eNOS gene polymorphisms are associated with ED16, 17, 18, 19, 20 and with other cardiovascular diseases.21, 22, 23, 24 The most studied eNOS polymorphisms include a single-nucleotide polymorphism at the −786 position of the promoter region (T−786C, rs2070744), a variable number of tandem repeats (VNTR) in intron 4 (common alleles: 4b and 4a), and a single-nucleotide polymorphism in the exon 7 (G894T, rs1799983), which results in an amino acid change (Glu298Asp). However, although eNOS polymorphisms may modify the responses to drugs that modify NO bioavailability,25, 26 there is only one study showing significant effects of individual eNOS polymorphisms on the responses to sildenafil,27 and no previous study has examined whether eNOS haplotypes modify the responsiveness to sildenafil.

In the present study, we hypothesized that eNOS polymorphisms could affect the responses of patients with organic ED to sildenafil, especially when the effects of the eNOS gene variants are combined within the eNOS haplotypes. Moreover, we examined for the first time how eNOS polymorphisms (and haplotypes) affect the responses of patients with ED secondary to radical prostatectomy (PED) that were treated with sildenafil.

Subjects and methods

Subjects

The present work was carried out in accordance to the Helsinki declaration, and was approved by the Institutional Review Board at the Faculty of Medicine of Ribeirao Preto. Informed consent was obtained from each participant. We recruited 118 patients with a medical diagnose of ED, which was already being followed up at the Urology Outpatient Clinic of Department of Surgery. We included patients with age between 40 and 80 years that had complaints regarding sexual activity and medical diagnosis of erectile function. Erectile function was evaluated before and after the use of sildenafil, using the five-item version of International Index for Erectile Function (5-IIEF) questionnaire, the ED domain.28 The patients were divided into two study groups: 63 patients had postoperative ED, because they underwent radical prostatectomy, and 55 patients had clinical (organic) ED. We assessed the responses to sildenafil for each patient by subtracting the 5-IIEF score before treatment with sildenafil from the 5-IIEF score after treatment with sildenafil, and this difference was defined as Δ5-IIEF. The response to sildenafil was evaluated after at least eight attempts of intercourse in which sildenafil was used at 50 mg or 100 mg dose. On the basis of the responses to sildenafil, the patients were divided into two groups: good responders (GR) or poor responders (PR) if their Δ5-IIEF was higher or lower than the median Δ5-IIEF, respectively. This analysis was performed independently for postoperative ED and for clinical ED patients. We excluded from the study those patients that did not use at least eight doses of sildenafil, or that had endocrine disorders, psychiatric disorders, neurogenic bladder dysfunction, hypogonadism, penile implants, cerebrovascular accident, central nervous system trauma, follow-up of radical prostatectomy shorter than 1 year from surgery, and anatomical abnormalities such as Peyronie's disease.

Genotyping

Genotyping for the T−786C and Glu298Asp polymorphisms

Venous blood samples were collected, and genomic DNA was extracted from the cellular component of 1 ml of whole blood and stored at −20 °C until analyzed. Genotypes for the eNOS T−786C (rs2070744) and Glu298Asp (rs1799983) polymorphisms were determined by Taqman Allele Discrimination assay (Applied Biosystems, Carlsbad, CA, USA) using real-time PCR. The probes and primers used for the T−786C genotyping assay were customized as follows: forward 5′-ACCAGGGCATCAAGCTCTTC-3′, reverse 5′-GCAGGTCAGCAGAGAGACTAG-3′, and probes 5′-CAGGGTCAGCC[G/A]GCCA-3′. TaqMan PCR was performed in a total volume of 12 μl (3 ng of DNA, 1 × TaqMan master mix, 1 × assay mix) placed in 96-well PCR plates. Fluorescence from PCR amplification was detected using Chromo 4 Detector (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed with the manufacturer's software. Probes and primers used in Glu298Asp assay were designed by Applied Biosystems (ID: C__3219460-20). TaqMan PCR and fluorescence reading were performed as described above for the T−786C polymorphism.

Genotyping for the VNTR polymorphism in intron 4

Genotypes for the VNTR polymorphism in intron 4 were determined by PCR using the primers 5′-AGG CCC TAT GGT AGT GCC TTG-3′ (forward) and 5′-TCT CTT AGT GCT GTG GTC AC-3′ (reverse). PCR was performed in 20-μl reaction volume that included approximately 300 ng of template genomic DNA, 0.25 μM of each primer, 200 μM of each dNTP, 2.5 mM MgCl2, 1 × PCR buffer and 1 U of DNA Taq Polymerase (Promega, Madison, WI, USA). The PCR reactions were heated to 94°C for 4 min for initial denaturation and underwent 35 cycles at 94°C for 30 s for denaturation, 63 °C for 30 s for annealing, and 72 °C for 1 min for extension. A final step of extension was conducted at 72 °C for 4 min. PCR generated products of 393, 420 and 447 bp, which correspond to the eNOS alleles 4a, 4b and 4c, respectively. Products were separated by electrophoresis in 8% polyacrylamide gels and visualized by silver staining as previously described.29

Laboratory analyses

Serum lipid profile (total cholesterol, triglycerides, high-density lipoprotein cholesterol), glucose, testosterone, urea and creatinine concentrations were measured with commercially available kits using standardized techniques. Low-density lipoprotein concentration was calculated according to the Friedewald's formula.

Statistical analysis

Clinical features and biochemical parameters of the studied groups were compared by unpaired t-test (parametric data), by Mann–Whitney test (non-parametric data), or by χ2-test (categorical variables). Differences in allelic and genotypes distributions and deviation from the Hardy–Weinberg equilibrium were tested by χ2-test. A P-value <0.05 was considered statistically significant.

Haplotypes were estimated using the program PHASE version 2.1 (http://www.stat.washington.edu/stephens/software.html). The possible haplotypes including genetic variants of the three polymorphisms studied here in the eNOS gene (T−786C, intron 4 and Glu298Asp) were H1 (T 4b Glu), H2 (T 4a Glu), H3 (T 4b Asp), H4 (T 4a Asp), H5 (C 4b Glu), H6 (C 4a Glu), H7 (C 4b Asp) and H8 (C 4a Asp). Only the haplotypes with observed frequencies >1% were taken into consideration. Differences in haplotype frequencies among groups were further tested using χ2-test, considering P<0.00833 as statistically significant (after Bonferroni's correction: 0.05/number of observed haplotypes).

Results

Table 1 summarizes the clinical and laboratory characteristics of the patients enrolled in this study. We studied 63 patients with postoperative ED, secondary to radical prostatectomy, and 55 patients with organic (clinical) ED. Although both clinical and postoperative groups were very similar with respect to their clinical features, there were minor differences in age, testosterone levels and frequency of diabetes between groups. These differences are not relevant, because the main goal of the present study was to examine the effects of eNOS polymorphisms on the responses to sildenafil in each group of patients, and we carried out independent analysis in postoperative and in clinical ED patients.

Table 1 Clinical and laboratory characteristics of subjects

The genotypes distributions for all eNOS polymorphisms did not deviate from the Hardy–Weinberg equilibrium in both ED groups (P>0.05). The genotypes and allele distributions were similar in both postoperative and clinical ED groups (P>0.05; Table 2). The analysis of genotypes for the intron 4 VNTR polymorphism showed that the rare 4c allele (which corresponds to six repeats of the 27 bp fragment) was found in only four subjects in the postoperative ED group (Table 2). To simplify the statistical analysis, we grouped the 4c allele with the 4a allele in our statistical tests. In parallel with genotypes results, we found similar eNOS haplotypes distribution in the postoperative and in the clinical ED groups (P>0.05; Table 3).

Table 2 Genotypes and alleles frequencies distributions in patients with postoperative or clinical ED
Table 3 Estimated haplotypes frequencies distributions in patients with postoperative or clinical ED

To investigate whether eNOS polymorphisms affect the responsiveness to sildenafil, we carried out an independent analysis for each group of ED patients. We evaluated the 5-IIEF28 before and after the use of sildenafil, and the change in 5-IIEF (Δ5-IIEF) was calculated for each patient. For each ED group, the patients were classified as GR or PR when their Δ5-IIEF was above or below the median Δ5-IIEF value of the ED group, respectively.

Interestingly, the analysis of results for postoperative ED patients showed higher frequencies of the TC and CC genotypes, and of the C allele for the T−786C polymorphism in GR compared with PR (P<0.05; Table 4). However, no significant differences were found in this group of ED patients with respect to the VNTR polymorphism in intron 4, and a minor difference was found for the Glu298Asp single-nucleotide polymorphism (Table 4).

Table 4 Distribution of genotypes and alleles for eNOS polymorphisms in postoperative ED patients divided in PR and GR responders to sildenafil

The analysis of results for clinical ED patients showed higher frequencies of the 4b4a and 4a4a genotypes, and of the 4a allele for VNTR polymorphism in intron 4 in GR compared with PR (P<0.05; Table 5). However, no significant differences were found in this group of ED patients with respect to the T−786C to the Glu298Asp single-nucleotide polymorphisms (P>0.05; Table 5).

Table 5 Distribution of genotypes and alleles for eNOS polymorphisms in clinical ED patients divided in PR and GR responders to sildenafil

We evaluated whether the eNOS gene variants combined within eNOS haplotypes affect the responsiveness to sildenafil in the two groups of ED patients. Interestingly, we found that the H6 haplotype (C 4a Glu) is more commonly found in GR than in PR patients with postoperative ED (P<0.0083; Table 6). Conversely, the H3 haplotype (T 4b Asp) is less commonly found in GR than in PR patients with postoperative ED; however, this result did not resist to the Bonferroni's correction for comparisons (Table 6). Finally, no significant differences in eNOS haplotypes distributions were found when GR and PR patients with clinical ED were compared (Table 6).

Table 6 Estimated haplotypes frequencies in postoperative and clinical ED patients divided in PR and GR responders to sildenafil

Discussion

The main findings of this study were: i) the C allele for T−786C polymorphism is associated with better responses of postoperative ED patients to sildenafil, whereas the 4a allele for the intron 4 VNTR polymorphism is associated with better responses of clinical ED patients to sildenafil; ii) eNOS haplotypes may modify the responses to sildenafil in postoperative ED patients; specifically, the H6 haplotype is associated with a better response to sildenafil; iii) the Glu298Asp polymorphism had minor effects on the responses of postoperative ED patients to sildenafil. To our knowledge, this is the first study to explore the association of eNOS haplotypes and responsiveness of ED patients to sildenafil.

The functionality of the three polymorphisms studied here has been demonstrated in previous studies. The C allele for the T−786C polymorphism decreases the transcriptional activity of the eNOS gene by 50%.30 The 4a allele for the intronic VNTR polymorphism is associated with lower levels of small interference RNA, thus leading to higher eNOS expression in comparison with the 4b allele.31 The functional consequences of the Glu298Asp polymorphism result in altered ligation of eNOS to caveolin-1, which is essential for eNOS activity.32 Although molecular mechanisms have been demonstrated, clinical studies showed minor (if any) effects of individual eNOS polymorphisms on relevant markers of NO formation.13, 14, 15 Conversely, eNOS haplotypes apparently have significant effects on endogenous NO formation.13, 14, 15

Whereas the polymorphisms studied here were associated with ED in previous studies,16, 17, 18, 19, 20 there is only one previous study, suggesting that individual eNOS polymorphisms affect the responses to sildenafil.27 The authors found the 4a allele for the intronic VNTR polymorphism associated with better responses to sildenafil in patients with ED associated with multiple causes, and our results from clinical ED patients align with these previous findings.27 Although we have not addressed the molecular mechanisms underlying the improved responses to sildenafil in carriers of the 4a allele, it is possible that higher-tissue eNOS expression associated with this polymorphism31 increases tissue NO activity, and this may enhance the responses to PDE-5 inhibitors. This effect associated with the 4a allele may be especially relevant in patients with clinical ED, as this clinical condition clearly involves the endothelium.2

Our findings in clinical ED were different from those in postoperative ED. The T−786C polymorphism modified the responses to sildenafil in postoperative ED, and the C allele for this polymorphism apparently improves the responses to sildenafil in these patients as compared with the T allele. These patients have undergone prostatectomy and were exposed to variable levels of nerve injury, which impairs the initial NO wave produced by nNOS in non-cholinergic non-adrenergic nerve fibers.33 However, these patients may not have the same extent of endothelial dysfunction in their penile vessels as compared with clinical ED patients. Although unproven, it is possible that postoperative ED patients have much better endothelial function and are more similar to healthy subjects than clinical ED patients. Interestingly, the improved responses to sildenafil in carriers of the C allele versus non carriers is consistent with previous findings showing significant increases in NO bioavailability in healthy male subjects carrying the C allele (but not in subjects carrying the T allele) after treatment with atorvastatin.25 In other words, if the C allele reduces eNOS expression and NO activity,30 it is possible that apparently healthy subjects carrying this allele will respond better to drugs that activate the NO-cyclic guanosine monophosphate pathway than those carrying the T allele, as suggested by our results in postoperative ED patients and in a previous study examining the effects of atorvastatin on endogenous NO production.25 The differences between postoperative and clinical ED may well reflect major differences in the pathogenesis between these two conditions, especially with respect to the severity of vascular dysfunction.

The analysis of eNOS haplotypes may offer improved genetic information than the analysis of genetic markers one by one.22 In fact, although we found minor effects (if any) for the Glu298Asp polymorphism on the responses to sildenafil, thus supporting a previous report,27 it is possible that this polymorphism may interact with the other eNOS polymorphisms. Interestingly, we found the H6 haplotype associated with improved responses, and the H3 haplotype possibly associated with worse responses to sildenafil in postoperative ED. We do not have a precise explanation for the molecular mechanisms possibly explaining such an association, and these eNOS haplotypes were not associated with high or low endogenous NO formation.13 However, that study included only healthy subjects,13 and it is possible that eNOS polymorphism interact with other factors involved in the pathogenesis of disease conditions. It is intriguing that the H6 and the H3 haplotypes, which were associated with good and poor responses to sildenafil, respectively, share no common allele (C 4a Glu versus T 4b Asp), thus suggesting that there must be a molecular mechanism possibly related with these differences in eNOS haplotypes.

There are clear interethnic differences in eNOS genotypes and haplotypes distributions when Blacks and Whites are compared,34, 35 and therefore, ethnicity could have an effect on the responses to sildenafil. However, we found that similar improvement of erectile function in both Black and White subjects in the present study (P>0.97 for postoperative ED and P>0.39 for clinical ED patients).

Some limitations of the present study should be taken into consideration. Firstly, although we studied a reasonable number of ED patients, our study may have not detected minor effects of eNOS polymorphisms or haplotypes. However, minor effects may not be so relevant from the clinical standpoint. Secondly, it would have been interesting to study patients with ED associated with particular medical conditions. Thirdly, the number of diabetic patients was different in the two ED groups, and it is possible that diabetes mellitus may have affected the responses to sildenafil. However, this study has not been designed to address this issue. Finally, it would be interesting to study how eNOS polymorphisms may interact with polymorphisms in other genes downstream in the NO signaling pathway. In addition, it is possible that other polymorphisms (such as those found in nNOS gene) may affect the responses to sildenafil. However, our results support the idea that eNOS polymorphisms may affect therapeutic responses to drugs used in to treat cardiovascular diseases.36, 37

In conclusion, we found evidence that eNOS genetic polymorphisms affect the responses of postoperative and clinical ED patients to sildenafil. It remains to be determined whether subjects with the worst responses to sildenafil would benefit from a more aggressive therapy including other drugs that may upregulate NO formation, such as statins,25 thus possibly improving the responses to sildenafil,38 or other drugs affecting the cardiovascular system.

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Acknowledgements

This study was supported by Fundação de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Correspondence to J E Tanus-Santos.

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Muniz, J., Lacchini, R., Rinaldi, T. et al. Endothelial nitric oxide synthase genotypes and haplotypes modify the responses to sildenafil in patients with erectile dysfunction. Pharmacogenomics J 13, 189–196 (2013). https://doi.org/10.1038/tpj.2011.49

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Keywords

  • eNOS
  • erectile dysfunction
  • haplotype
  • polymorphisms
  • prostatectomy
  • sildenafil

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