https://orcid.org/0000-0002-5430-6561
ABSTRACT
Several studies have focused on the impaired role of endothelial nitric oxide synthase (NOS3) gene polymorphism and its association to erectile dysfunction (ED). However, currently controversial results have been reported due to their significant heterogeneity. The present study aimed to assess the genotypic distribution and the allelic frequency of Intron 4 VNTR and Glu298Asp gene polymorphisms in vasculogenic ED patients compared to healthy controls of a specific region of Turkey. A total of 75 patients with ED (median age: 56, IQR:10.5) and 75 healthy controls (median age: 56, IQR:10.5) were prospectively analyzed. All subjects were equally evaluated by the same physician with detailed history-taking, physical examination, International Index of Erectile Function (IIEF) questionnaire, and blood tests (incl. glucose, testosterone, triglyceride and total cholesterol level). Those with an IIEF score under 26 were considered to have ED, by classifying them according to their scores as mild (22–25), moderate (11–21) and severe (1–10) ED. Color doppler ultrasonography was carried out in patients with an IIEF score <22. Hypertension, diabetes mellitus, coronary artery disease, and smoking status were significantly associated with the ED group compared to control subjects with p values of <0.001, <0.001, 0.002 and <0.001, respectively. Overall genotype frequencies was 47 (31%) a/a, 22 (15%) a/b, 82 (55%) b/b for Intron 4 VNTR and 56 (37%) GG, 78 (52%) GT, 16 (11%) TT for the Glu298Asp polymorphism. The frequencies of Intron 4 VNTR a/a allele and Glu298Asp GT allele were associated with severe ED, while a/b and TT were associated with moderate or mild, and b/b and GG were associated with no ED. In contrast to Glu298Asp, statistically significant differences in genotypic frequencies of Intron 4 VNTR gene polymorphism between ED and control subjects was established.
Abbreviations: NO: nitric oxide, NOS: nitric oxide synthase, NOS3: endothelial nitric oxide synthase, NOS2: inducible nitric oxide synthase, NOS1: neuronal nitric oxide synthase, HT: hypertension, DM: diabetes mellitus, CAD: coronary artery disease, ED: erectile dysfunction, IIEF: international index of erectile function, VNTR: variable number of tandem repeats, CDU: color doppler ultrasonography, PCR: polymerase chain reaction.
Introduction
Erectile dysfunction (ED) is a crucial urological disease, involving a male polpulation, that is especially increasing with age. Basically, it can be allocated as organic, psychogenic, or mixed ED according to its etiology.
A great part of organic ED cases, are developed through vascular problems like arterial insufficiency, or disrupted veno-occlusion, that is commonly an inadequate effect of corporal smooth muscle relaxation. Cardiovascular-related diseases such as atherosclerosis, hypertension (HT), diabetes mellitus (DM), dyslipidemia, and smoking are frequently associated with ED. Actually, in the past few years, within the background of common risk factors ED has been claimed as an indicator of silent vascular diseases (Lee et al. Citation2007).
Contraction and relief of cavernosal smooth muscles is the main regulating process of penile erection. Nitric oxide (NO), the compound catalyzed by NO synthase (NOS) through L-arginine, is the major agent crucial for corpora cavernosal smooth muscle cell relaxation.
There have been three NOSs detected playing a role within the penile tissue, named inducible (NOS1), neuronal (NOS2) and endothelial (NOS3) NOS (Hurt et al. Citation2002). Calcium-independent output of NOS3 is liable for the accomplishment and sustenance of adequate erection, whereas calcium-reliant mechanisms activate penile erection (Musicki and Burnett Citation2006). Hence, adjustment of the NOS subtype containing any polymorphism of the encrypted gene for NOS enzyme could be clinically crucial. Previously conducted studies supported this suggestion by demonstrating the relation between NOS3 gene polymorphisms and systemic diseases like DM and coronary heart disorders (Qian-Qian et al. Citation2014; Kumar et al. Citation2016). It is well known that physiological and functional alterations in endothelial cells play a major role in the evolution and progression of atherosclerosis. Researchers have shown that imbalances in NOS3 expression may alter NOS3 activity or regulation associated with endothelial dysfunction, microvascular complications of DM, and the pathogenesis of several cardiovascular diseases (Qian-Qian et al. Citation2014; Kumar et al. Citation2016).
To date, studies evaluating NOS3 gene polymorphisms and ED are quite few and reporting controversial results due to their significant heterogeneity (Eisenhardt et al. Citation2003; Rosas-Vargas et al. Citation2004; Erkan et al. Citation2006; Lee et al. Citation2007, Citation2009, Citation2012; Peskircioglu et al. Citation2007; Erol et al. Citation2009; Meluzín et al. Citation2009; Eisenhardt et al. Citation2010; Sinici et al. Citation2010; Safarinejad et al. Citation2011; Hermans et al. Citation2012; Muniz et al. Citation2013; Gao et al. Citation2017). This can occur as a result of the inclusion/exclusion criteria, sample size and the ethnic-genetic discrepancies of the population.
Thus, the aim of the present study was to evaluate possible associations of Intron 4 VNTR (variable number of tandem repeats) and Glu298Asp (G894T polymorphism in exon 7) polymorphisms with vasculogenic ED patients of a specific region of Turkey and to correlate these genetic findings with the severity of ED.
Results
summarizes the demographic properties of patients with ED and control subjects enrolled in this study. In addition to the exclusion criteria, 12 patients refusing color doppler ultrasonography (CDU) and 3 patients with missing or incomplete data were excluded from the study. At the end, a total of 75 patients fulfilling inclusion/exclusion criteria, and 75 volunteering age-matched controls with no complaint of sexual dysfunction were included in the study.
Table 1. Demographic properties of patients with ED and control group.
Patients with ED had significantly higher cholesterol, glucose and triglyceride levels than the control group (167 vs. 130, p < 0.001; 102 vs. 87, p < 0.001; 140 vs. 90, p < 0.001, respectively). Patients group IIEF score was significantly lower than the control group (9 vs. 26, p < 0.001).
Hypertension, DM, coronary artery disease (CAD) and smoking status were significantly associated with the ED group compared to control subjects with p values of <0.001, <0.001, 0.002 and <0.001, respectively. The proportion of patients with HT, DM, CAD was significantly higher in the ED group than the control group (45% vs. 16%; 41% vs. 13%; 20% vs. 3%, respectively). The proportion of smokers was also, significantly higher in the patients group compared to the control group (57% vs. 25%).
The distribution of Intron 4 VNTR and Glu298Asp in patients and non-ED controls are shown in . The overall genotype frequencies of the population was 47 (31%) a/a, 22 (15%) a/b, 82 (55%) b/b for the Intron 4 VNTR and 56 (37%) GG, 78 (52%) GT, 16 (11%) TT for the Glu298Asp polymorphism. Statistically significant association of Intron 4 VNTR a/a allele was found between ED patients and controls, whereas no association was found between the two groups according to Glu298Asp allele's.
Table 2. The genotypical distribution of Intron 4 VNTR and Glu298 Asp between groups.
The distribution of Intron 4 VNTR and Glu298Asp allele frequencies according to ED severity are demonstrated in . Both gene polymorphisms showed a statistically significant relation with the severity of ED. We found that a/a and GT allele’s are associated with severe ED, while a/b and TT are associated with moderate or mild and b/b and GG are associated with no ED. The strongest association with ED severity showed a/a allele of Intron 4 VNTR. (, )
Table 3. The genotypical distribution of Intron 4 VNTR and Glu298 Asp in terms of ED severity according to IIEF score.
Figure 1. A. Correspondence plot of severity of erectile dysfunction (ED) and Intron 4 variable number of tandem repeats (VNTR): Endothelial nitric oxide synthetase (NOS3) gene polymorphism of Intron 4 VNTR showed a statistically significant relation with the severity of erectile dysfunction (ED). It has been shown that a/a allele is associated with severe ED, while a/b allele is associated with moderate or mild and b/b allele is associated with no ED. The strongest association with ED severity showed a/a allele of Intron 4 VNTR. B. Correspondence plot of severity of erectile dysfunction (ED) and Glu298 Asp: Endothelial nitric oxide synthetase (NOS3) gene polymorphism of Glu298Asp showed a statistically significant relation with the severity of erectile dysfunction (ED). It has been shown that GT allele is associated with severe ED, while TT allele is associated with moderate or mild and GG allele is associated with no ED.
Demographic properties like cholesterol-glucose-triglyceride level, IIEF score and smoking status had a statistically significant relationship with the distribution of Intron 4 VNTR allele frequencies shown in , whereas Glu298Asp allele frequencies showed only a significant relationship with age. Post hoc tests of pairwise comparison of Intron 4 VNTR alleles revealed that a/a allelic group had a significantly higher cholesterol, glucose, triglyceride and IIEF score than a/b allelic group and a significantly higher cholesterol, triglyceride and IIEF score than b/b allelic group. Additionally, a/b allelic group had a significantly higher IIEF score than b/b allelic group.
Table 4. The distribution of Intron 4 VNTR genotypes according to demographic properties.
Discussion
The major findings of the present study are; i) ED patients compared to control subjects demonstrated a statistically significant relationship with CAD, HT, DM, glucose-triglyceride-cholesterol levels and smoking status; ii) Frequencies of a/a Intron 4 VNTR heterozygotes were significantly higher among patients with ED whereas a/b and b/b heterozygotes were significantly higher among control subjects; iii) Intron 4 VNTR a/a allele frequency showed a very strong and significant association with ED severity; iv) No difference between ED and control subjects according to Glu298Asp polymorphism was detected, however GT, TT and GG allele frequencies were associated with severe, moderate or mild, and no ED, respectively.
To date, it is known that, ED shares unmodifiable (genetic) and modifiable risk factors with CAD, HT, DM, obesity, dyslipidemia and smoking status. Therefore, in patients of a particular region like Trakya which has a distinct ethnic background, the determination of a genetic predisposition for ED, might possibly affect men to take modifiable risk factors more serious. Thus, the deceleration of microvascular injuries, by changing their daily lifestyle and/or providing proper medical assistance, could prevent early stage or severe ED. Results of the present study highlight the significance of studying the biological pathways of gene polymorphisms in understanding factors related to ED. The role of genetic risk factors in ED is attracting more attention; however, our knowledge on the relevance of genetic polymorphisms in affecting the exposure to, and severity of ED persists inadequate, yet.
Guanylate cyclase is stimulated by NO, delivered from nerve endings and endothelial cells, resulting with a calcium discharge from the cytosolic space and an increase of cyclic guanosine-3′,5′-monophosphate. Consequently, this leads to the relaxation of the cavernosal smooth muscles. Impaired and/or decreased NO activity appears to play an important role in the pathogenesis of ED (Erol et al. Citation2009).
Currently, limited published data indicating controversial results, between the relation of NOS3 gene polymorphism and ED are available (Eisenhardt et al. Citation2003; Rosas-Vargas et al. Citation2004; Erkan et al. Citation2006; Lee et al. Citation2007, Citation2009, Citation2012; Peskircioglu et al. Citation2007; Erol et al. Citation2009; Meluzín et al. Citation2009; Eisenhardt et al. Citation2010; Sinici et al. Citation2010; Safarinejad et al. Citation2011; Hermans et al. Citation2012; Muniz et al. Citation2013; Gao et al. Citation2017; Yang et al. Citation2017). Important reasons for this discrepancy are thought to occur due to inclusion/exclusion criteria (e.g. type of ED, systemic disorders, etc.), patient sample size and ethnic-genetic background differences between the studies. In a large proportion of previous articles, the inclusion of ED patients according to their etiology was not specifically considered. Mostly the subjective IIEF questionnaire was used to reveal ED, whereas penile CDU for accurate diagnose was only performed in some of them (Lee et al. Citation2007, Citation2009, Citation2012; Meluzín et al. Citation2009; Hermans et al. Citation2012; Muniz et al. Citation2013; Gao et al. Citation2017).
We enrolled patients with CDU documented vasculogenic ED and age-matched controls related to the same Region of Turkey, with the purpose to minimize possible bias arised due to the etiology and ethnic-genetic background of ED. There are only three published articles, considering associations between NOS3 gene polymorphisms and ED according to its etiology (Erkan et al. Citation2006; Sinici et al. Citation2010; Safarinejad et al. Citation2011).
In the study by Erkan et al. (Erkan et al. Citation2006), investigating Intron 4 VNTR among Turkish ED patients and control individuals, no statistical difference was found neither between the two groups nor among ED severity and allele frequencies, which is in total disagreement with our findings. This polymorphism was previously described and proposed as a smoking habitual risk factor for CAD by Wang et al. (Wang et al. Citation1996), encouraging our findings due to the statistically significant correlation of Intron 4 VNTR among ED patients who were smokers and/or had CAD. Therefore, our disagreement with Erkan et. al, likely reflects their small patient cohort, as well as the insignificance between their groups according to the smoking status.
In a more recently published article evaluating Intron 4 VNTR and Promoter T-786C polymorphisms among Turkish ED and control patients, Sinici et al. (Citation2010) determined that only the CC genotype of T-786C polymorphism in the promoter region of NOS3 gene was associated with an increased risk of ED. Although the groups of both studies were equal with regard to their sample size, ethnicity and age, the discrepancy that occurred might demonstrate the influence of the regional genetic background.
In another study Safarinejad et al. (Citation2011) evaluated NOS3 gene polymorphisms including T-786C, G894T and intron 4 VNTR among Iranian individuals with a large sample size. They did not show any statistical difference according to GG and GT Glu298Asp (G894T) allele frequencies between the groups which was inconsistent with our results. However, TT allele frequency was found statistically higher in the ED group, different to ours. In addition, although they could not obtain a statistically significant difference, intron 4 VNTR a/a allele frequencies of ED patients were rationally higher, which was in agreement to our findings. As mentioned before our patients were included from a particular region of Turkey that has a distinct genetic background, whereas both studies were conducted in metropol capital cities in which people from different ethnic origins are living together for decades. This diversity was believed to cause an additional and important complexity to the understanding of ED’s nature. Therefore, we support the suggestion of Safarinejad et al., that the genetic background and ethnic diversity could help to explain the difference for G894T polymorphism among the studies.
When previously conducted studies were analyzed according to their ethnic background, which we believe to have a big impact on ED (direct and indirectly), only four published articles regarding Turkish individuals were found (Erkan et al. Citation2006; Peskircioglu et al. Citation2007; Erol et al. Citation2009; Sinici et al. Citation2010). In the study performed by Peskircioglu et al. (Citation2007) with 96 ED and 167 healthy control subjects, it was reported that Glu298Asp (ecNoOS E298A) polymorphism did not show any statistical difference between the groups which is in agreement to our results. However, no difference was found between their groups according to Intron 4 VNTR polymorphism, that showed a very different allelic heterogeneity between ED patients, compared to ours.
In comparison, the study conducted by Erol et al. (Erol et al. Citation2009) reported that Glu298Asp (eNOS 894G/T) gene polymorphism was significantly higher among ED patients whereas Intron 4 VNTR did not show any difference between the groups. Considering both studies with the same ethnicity, these controversial results were believed to demonstrate the importance of the regional genetic background and to define inclusion/exclusion criteria more specifically.
Some limitations need to be considered when interpreting our findings. First, studies observing genetic variants in different individuals to identify associations between genetic regions (loci) are called genome-wide association studies and have addressed urologic topics like ED, spermatogenesis and fertility (Arpanahi et al. Citation2015; Bovijn et al. Citation2019). They have shown that, genes can be located near other genes; therefore close associations with other gene mutations might effect the detected outcomes. Second, despite the acceptable number of ED patients, detection of negligible outcomes according to the NOS3 polymorphisms could be missed. However, their impact on our results is thought to be inconsiderable. Lastly, including a third group without systemic diseases would help us provide more accurate and specific results on the role of each gene polymorphism to develop our hypothesis and outcomes, which has been planned in further research.
In conclusion, statistically significant differences in genotypic frequencies of Intron 4 VNTR gene polymorphism between ED and control subjects was established. To provide the role and function of e NOS3 gene polymorphisms in ED pathophysiology, further studies designating a specific ethnic-genetic background with comparable inclusion/exclusion criteria are needed.
Material and methods
A total of 150 Turkish men related to the Region of Trakya, according to their familial background, were collected prospectively. People in Trakya are mainly descendants of a mix between Bulgarian, Greek and several Balkanic immigrants, having an obviously distinctive genetic origin compared to the other Regions of Turkey.
Inclusion criteria were vasculogenic ED documented with CDU, normal testosterone levels (>3.5 ng/mL), sexually active life in the past year and being from the Trakya Region. Patients with lack of sexual lust, ED caused due to penile structural deformities or spinal cord injury, postoperative ED, and any medical or substance abuse afflictions were excluded.
Before the determination of vasculogenic ED, all participants medical profile (incl. surgical and psychologic history) following a complete physical examination were recorded by the same physician. Sample collection, for biochemical evaluation and hormonal analysis (incl. testosterone level, total cholesterol and triglyceride), was performed and carried out, every morning between 8:00 and 10:00 am. A 15-item International Index of Erectile Function (IIEF) questionnaire was fulfilled by each individual. Those with an IIEF score under 26 were considered to have ED, by classifying them according to their scores as mild (22–25), moderate (11–21) and severe (1–10) ED. Penile CDU, before and after intracavernosal injection with 50 mg papaverin by the same technician, was performed in patients complaining of ED with an IIEF score <22 to reveal vascular deficiency. Hemodynamic measurements of right and left cavernosal arteries were peak systolic velocity (PSV), end-diastolic velocity (EDV) and resistivity index (RI). Definitions for vascular status was described as follows: Arterial insufficiency, veno-occlusive dysfunction and mixed vascular disorder with cut-off levels of PSV<30cm/s, PSV>30cm/s + EDV>5cm/s + RI<0.8, PSV<30cm/s + EDV>5cm/s, respectively.
Participants were described as cigarette smokers if they had smoke ≥10 cigarettes weekly and have maintained until 6 months before the study. Definition of HT, DM and hyperlipidemia were; a systolic pressure of ≥140mmHg or diastolic pressure of ≥90 mmHg, a fasting blood glucose level ≥126 mg/dL and a total cholesterol level ≥200 mg/dL or triglycerides level ≥200 mg/dL, respectively. Subjects with a history of medically regulated HT, DM, or hyperlipidemia, were also included.
The present work was carried out in accordance with the Helsinki declaration and was approved by the Institutional Review Board of our Faculty. All of the individuals were asked to sign a written informed consent describing the regulation of the present study.
Genotyping
The DNAs of patient and control subjects were isolated from peripheral blood samples containing ethylenediaminetetraacetic acid (EDTA) using an isolation kit (Invitrogen).
The purity and quality of the isolated DNAs were determined spectrophotometrically at 260 and 280-nanometer wavelengths and were checked by 0.8% agarose gel electrophoresis.
While polymerase chain reaction (PCR) method was used to determine the Intron 4 VNTR gene polymorphism, PCR and Restriction Fragment Length Polymorphism (RFLP) methods were used to determine the Glu298Asp gene polymorphism. For Intron 4 VNTR gene polymorphism; 15 µl PCR mixture containing 200 ng of isolated DNA, 0.2 mM dNTP for each, 0.5 nM forward and reverse primers, 1xPCR Buffer, 2.5 mM MgCl2, 0.75 unit Taq DNA polymerase, were prepared. For Glu298Asp gene polymorphism; 200 ng of isolated DNA was prepared the mixture 25 μl of PCR containing 0.2 mM dNTP, 0.5 nM forward and reverse primers, 1xPCR buffer, 2.5 mM MgCl2 and 1.25U Taq DNA polymerase (Thermo Fisher Scientific) for each.
For Intron 4 VNTR gene polymorphism; 5’-AAG CCC TAT GGT AGT GCC TTT-3 forward primer and 5’-TCT CTT AGT GCT GTG GTC AC-3 reverse primer were used. For Glu298Asp gene polymorphism; 5‘-AAG GCA GGA GAC AGT GGA TGGA-3 ‘forward primer and 5‘-CCC AGT CAA TCC CTT TGG TGC TCA-3‘ reverse primer were used.
For Intron 4 VNTR gene polymorphism; the PCR conditions were one cycle at 94ºC for 1 min followed by 25 cycles of denaturation at 95ºC for 35 s, annealing at 56ºC for 35 s and extension at 72ºC for 40 s, 5 min at 72ºC for termination. For the Glu298Asp gene polymorphism; PCR conditions were applied for 5 min at 95°C for 5 min followed by 35 cycles of 1-min denaturation at 94°C, 1 min at 59°C and 1 min at 72°C followed by 5 min at 72°C for termination. The resultant PCR products were observed on 2% agarose gel electrophoresis for both gene polymorphisms.
Intron 4 VNTR gene polymorphism genotypic distributions were determined as 393 base pair, 393, 420 base pair and 420 base pair for a/a, a/b and b/b genotypes, respectively. The PCR product length was observed as 248 base pairs for Glu298Asp gene polymorphism. The resulting PCR products were fractioned at 37°C for 3 h to determine genotype distributions of Glu298Asp gene polymorphism. For the cutting process, a mixture consisting of 1xBuffer Tango, PCR reaction products, dH2O and 5U restriction enzyme (BanII) (Thermo Fisher Scientific) was used. Glu298Asp gene polymorphism genotypic distributions were determined as 85, 163 base pair, 85, 163, 248 base pair and 248 base pair for GG, GT and TT, respectively.
Statistical analysis
The continuous variables did not show normal distribution, as was documented by the use of the Kolmogorov–Smirnov test, PP and QQ plots. Summary statistics were constructed with the use of median and interquartile ranges for continuous data and frequencies and percentages for categorical data. The two-tailed Wilcoxon rank sum test was used to evaluate the differences in continuous variables (age, cholesterol, glucose, triglyceride, IIEF score) between control and patient groups. Kruskal–Wallis analysis of variance test was used to compare the two groups (Intron 4 VNTR and Glu298 Asp) with respect to continuous variables. Dunn test with Bonferroni correction was used with pairwise comparisons.
Fisher’s exact test of association was used to evaluate the association between categorical variables. All tests are two-sided, and statistical significance was set at p < 0.05. All analyses were carried out using the R statistical program.
Power analysis
It was of interest to test whether the gene polymorphisms (three categories) and severity erectile dysfunction (four categories) were associated. Thus, either the chi-squared test of association or Fisher exact test were suitable tests. ‘pwr.chisq.test’ function of the ‘Pwr’ package of R was used to calculate the required sample size. For the study assuming an alpha level 0.05, power 0.80, medium effect size (0.3) and degrees of freedom 6, the required sample size was 150.
Authors' contributions
Contributed to the design, drafting of manuscript, and critical revision of the manuscript: E Arda; contributed to the design, analysis and interpretation of data: AA; contributed to the critical revision of the manuscript: HA; contributed both to statistics and acquisition of data: E Akdeniz.
Disclosure statement
No potential conflict of interest was reported by the authors.
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