The Association Between Intron 4 VNTR, E298A and IVF 23+10 G/T

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ORIGINAL ARTICLE The association between intron 4 VNTR, E298A and IVF 23 þ 10 G/T polymorphisms of ecNOS gene and sildenafil responsiveness in patients with erectile dysfunction

L Peskircioglu1, FB Atac2, SR Erdem3, S Deveci1, H Verdi2,HO¨ zkardes¸1

1Department of Urology, Baskent University, School of Medicine, Bahçelievler, Ankara, Turkey; 2Department of Medical Biology and Genetics, Baskent University, School of Medicine, Bahçelievler, Ankara, Turkey and 3Department of Pharmacology, Baskent University, School of Medicine, Bahçelievler, Ankara, Turkey

The objective of the study was to determine the association between intron 4 variable number of tandem repeats (VNTR), E298A and IVF 23 þ 10 G/T polymorphisms of ec-NOS gene and sildenafil responsiveness in patients with erectile dysfunction (ED). Ninety-six patients who were evaluated for ED between November 2003 and June 2004 and 167 healthy individuals representing the normal population as controls were included in the present study. The patients were evaluated by medical history, five-item version of International Index of Erectile Function, serum glucose, testosterone levels and lipid profiles. Sixty-seven patients received four consecutive doses of sildenafil from 25 to 100 mg according to the response. The ec-NOS gene intron 4 VNTR, E298A and IVF 23 þ 10 G/T polymorphisms were evaluated in the isolated DNA blood samples obtained from the patient group with ED (n ¼ 96), from the group received sildenafil (n ¼ 67) and from the healthy group (n ¼ 167). Genotype distributions of ec-NOS gene intron 4, E298A and IVF 23 þ 10 G/T polymorphisms in the patient group were similar to those in the healthy group. The frequency of the ec-NOS gene intron 4 genotype were found as bb ¼ 41.7%, ab ¼ 50% and aa ¼ 8.3% in the sildenafil responders and bb ¼ 93.5% and ba ¼ 6.5% in the sildenafil non-responders. This finding was statistically significant. Statistical analysis of ec-NOS gene E298A and IVF 23 þ 10 G/T polymorphisms did not reveal any significant difference between sildenafil responders and non-responders. These findings may indicate that ‘a’ allele of ec-NOS gene intron 4 VNTR polymorphism associates with a better sildenafil response. International Journal of Impotence Research (2007) 19, 149–153. doi:10.1038/sj.ijir.3901501; published online 27 July 2006

Keywords: erectile dysfunction; oral treatment; sildenafil; genetics; gene polymorphism; molecular biology

Introduction cavernosal smooth muscle during sexual stimulation.2 Penile erection has been shown to be mainly The Massachusetts Male Aging Study noted that mediated by nitric oxide (NO).3 NO is released from erectile dysfunction (ED) is a common disorder penile non-adrenergic, non-cholinergic nerve end- in men between 40 and 70 years of age with a ings and endothelial cells of corpus cavernosum and prevalence of 52%.1 Penile erection and detumes- penile blood vessels and additionally from the cence are regulated by corporeal smooth muscle smooth muscle.4–6 NO diffuses into the smooth relaxation and contraction. ED mainly results from muscle cell, and activates soluble guanylyl cyclase, the inadequate relaxation of the penile corpora which in turn gives rise to the production of cyclic guanosine monophosphate (cGMP). NO-induced increase in intracellular cGMP levels results in the relaxation of the cavernous smooth muscle by Correspondence: Dr L Peskircioglu, Department of Urol- 2 þ ogy, Baskent University, School of Medicine, 5.Sok.No:48, reducing intracellular Ca . The above-mentioned Bahc¸elievler, Ankara 06490, Turkey. pathway appears to be of major importance to E-mail: [email protected] modulate erectile function. Factors disrupting this Received 7 March 2006; revised 16 May 2006; accepted 19 pathway may impair the relaxation of corporeal June 2006; published online 27 July 2006 smooth muscle and may lead to ED.7 Association between intron 4 VNTR, E298A and IVF 23 þ 10 G/T polymorphisms L Peskircioglu et al 150 Sildenafil citrate is the first phosphodiesterase Materials and methods type 5 (PDE-5) inhibitor approved by Food and Drug Administration (1998). PDE-5 inhibition Ninety-six male patients suffering from ED and 167 causes an elevation in intracellular cGMP level healthy individuals representing normal population as cGMP is degraded intracellularly by PDE.8 in our country were included in the study. Patients Therefore, PDE-5 inhibition augments the normal were evaluated by a detailed medical history, erectile response. Sildenafil has been shown to physical examination followed by laboratory tests, be beneficial in men with ED owing to organic, including serum glucose, lipid profile, testosterone, psychogenic and mixed etiological factors. Gold- prolactine and combined intracavernous injection, stein et al.9 have reported an overall beneficial effect and stimulation test. Penile Doppler ultrasonogra- of sildenafil in 70% of patients compared with that phy was performed when indicated. Following of placebo (20–30%). As sildenafil acts through the this initial evaluation, the etiology (vasculogenic, inhibition of intracellular cGMP degradation, it is neurologic, endocrinologic, mixed and psychogenic) obvious that the physiological production of this and contributing risk factors (atherosclerosis, dia- PDE substrate in response to NO is an essential step betes mellitus, hyperlipidemia, medications, smok- in sildenafil action. Therefore, any impairment in ing and cardiovascular comorbidities) were defined. this process might result in irresponsiveness to Patient profile with regard to the risk factors is given sildenafil treatment. However, to the best of our in Table 1. Sexual function before and after treat- knowledge, there has not been any satisfactory ment were determined by the five-item version of explanation of sildenafil irresponsiveness in the International Index of Erectile Function (IIEF-5). literature. The degree of the severity of ED of the patients was NO is synthesized as a by-product during classified into five categories according to their IIEF- L-citrulline production from L-arginine, a reaction 5 scores. Success with treatment was defined catalyzed by the enzyme NO synthase (NOS).8 according to IIEF-5 categories as 4 (mild ED) and 5 Three isoforms of NOS, namely neuronal (nNOS, (no ED). Out of 96 patients, sildenafil was given to NOS 1), inducible (iNOS, NOS 2) and endo- 67 men with no contraindication for sildenafil use. thelial (eNOS, NOS 3) NOSs have been deter- The dosage ranged from 25 to a maximum 100 mg. mined.10 All three isoforms of NOS have been Those who did not respond to 100 mg were regarded shown to play a role within the penile tissues. as non-responders (Tables 2–4). eNOS is mainly expressed in endothelial cells and is involved in several important cardiovascular functions. Therefore, regulation of the expression of NOS isoforms including any polymorphism of Genotyping the gene encoding for NOS enzyme might be of All of the participant patients were requested to sign clinical importance. the written informed consent form regarding the Several previous studies have supported this protocol of the present study, including the blood suggestion by pointing out the association of sample collection for DNA analysis. The Ethics intron 4 variable number of tandem repeats (VNTR), E298A and IVF 23 þ 10 G/T polymorphisms of Table 2 Distribution of ecNOS intron 4 genotype frequency eNOS gene with diabetes-related and cardiovascular disorders.11,12 Thus, we aimed to investi- N bb (%) ab (%) aa (%) gate the possible association of eNOS gene intron 4 VNTR, E298A and IVF 23 þ 10 G/T poly- Healthy group 167 morphisms in particular, with the responsive- Patients with ED 96 80 16.7 3.1 Patients receiving sildenafil 67 65.7 29.9 3 ness to sildenafil treatment in patients with Sildenafil responders 31 41.7 50 8.3 ED. We hypothesized that ED patients with Sildenafil non-responders 36 93.5 6.5 0 eNOS gene polymorphisms respond less to sildenafil. Abbreviation: ED, erectile dysfunction.

Table 1 Etiology of ED in the patient group

N Vasculogenic Neurologic Psychogenic Endocrinologic Mixed

Patients with ED 96 50 5 20 3 18 Patients receiving sildenafil 67 42 4 6 2 13 Sildenafil responders 31 20 2 3 — 6 Sildenafil non-responders 36 22 2 3 2 7

Abbreviation: ED, erectile dysfunction.

International Journal of Impotence Research Association between intron 4 VNTR, E298A and IVF 23 þ 10 G/T polymorphisms L Peskircioglu et al 151 Table 3 Distribution of ecNOS intron 23 genotype frequency Results

N gg (%) gt (%) The mean age was 52715.2 years in the patient group. The etiological factors determined to be Healthy group 167 80.4 16.9 Patients with ED 96 90.3 9.7 responsible for ED in the patient group were as Patients receiving sildenafil 67 90.5 9.5 follows: vasculogenic (50 patients, 52.1%), endo- Sildenafil responders 31 90.9 9.1 crinologic (three patients, 3.1%), psychogenic (20 Sildenafil non-responders 36 90.2 9.8 patients, 3.1%), mixed (18 patients, 18.8%) and neurologic (five patients, 5.2%). Thirty-one of 67 Abbreviation: ED, erectile dysfunction. patients who received sildenafil responded posi- tively to the treatment. The mean age was 48.5 years (range 35–70 years) in sildenafil responders and Table 4 Distribution of ecNOS exon 7 genotype frequency 52.3 years (range 39–66 years) in non-responders. The etiological evaluation of ED in sildenafil N gg (%) gt (%) tt (%) responders was as follows: vasculogenic (20 patients, 64.5%), neurologic (two patients, 6.5%) Healthy group 167 32.5 50.8 16.7 Patients with ED 96 17.3 52 30.7 mixed (six patients, 19.4%) and psychogenic (three Patients receiving sildenafil 67 20.3 59.5 20.3 patients, 9.7%). Sildenafil responders 31 18.2 63.6 18.2 Sildenafil non-responders 36 22.0 56.1 22.0 Association of ecNOS intron 4 polymorphism with Abbreviation: ED, erectile dysfunction. response to sildenafil The ecNOS intron 4 genotype frequencies were found as bb ¼ 68.3%, ba ¼ 29.3% and aa ¼ 2.4% in Committee of our faculty approved the study the normal population, bb ¼ 80%, ba ¼ 67% and protocol. Genomic DNA was prepared from leuko- aa ¼ 3.1% in the patient group and bb ¼ 65.7%, cyte pellets by sodium dodecyl sulfate lysis, ammo- ba ¼ 29.9% and aa ¼ 3% in the group receiving nium acetate extraction and ethanol precipitation.13 sildenafil. No statistical significance was found The primers and polymerase chain reaction (PCR) between these three groups. The frequencies of conditions were performed as described pre- the genotype were found as bb ¼ 41.7%, ab ¼ 50% viously.14 and aa ¼ 8.3% in the sildenafil responders and bb ¼ 93.5% and ab ¼ 6.5% in the non-responders. The genotype distribution was significantly differ- Gel analysis and genotyping ent in sildenafil responders from non-responders. The PCR products of b and a alleles were identified by the presence of 420 and 393 bp bands on 2% Association of the ecNOS E298A polymorphism agarose gel electrophoresis, respectively, for ecNOS with sildenafil response intron 4 VNTR genotypes. ecNOS E298A genotype frequencies were found as The E298A polymorphism of ecNOS gene was GG ¼ 80.4% and G/T ¼ 19.6%) in the normal popu- detected by digesting a 152 bp amplified fragment lation and GG ¼ 90.3% and G/T 9.7% in the patient with BanII. In the case of a G-to-T substitution at group. Although the difference between the groups position 894 in exon 7 of the ecNOS gene, a BanII was statistically significant (P ¼ 0.031), the distribu- restriction site is lost. The G allele consisted of 56 tion of the groups was similar. The frequencies and 96 bp. of the genotype were GG ¼ 90.9% and GT ¼ 9.1% A 676 bp PCR product was cut with HindII for in sildenafil responders and GG ¼ 90.2% and ecNOS intron 23 G10-T polymorphism. The ampli- G/T ¼ 9.8% in non-responders. This association fied DNA was then digested by HindII into fragments failed to reach statistical significance. (577 and 99 bp). In the case of a G-to-T substitution at position 10 of intron 23 of the ecNOS gene, an additional HindII restriction site was produced and Association of ecNOS IVS 23 þ 10G/T the amplified fragments were digested into smaller polymorphism with sildenafil response fragments (374, 203 and 99 bp). The genotype distribution of ecNOS IVS 23 þ 10G/T polymorphism was GG ¼ 32.5%, GT ¼ 50.8% and TT ¼ 16.7% in the healthy group and GG ¼ 17.3%, Statistical analysis GT ¼ 52% and TT ¼ 30.7% in the patient group. The statistical comparison between the frequencies This finding was statistically significant (P ¼ 0.004). of ecNOS gene polymorphisms among groups (the Genotype frequencies were found as GG ¼ 18.2%, patient group, the sildenafil received group and the GT ¼ 63.6% and TT ¼ 18.2% in sildenafil respon- control group) was carried out by w2 test. ders and GG ¼ 22%, GT ¼ 56.1% and TT ¼ 22% in

International Journal of Impotence Research Association between intron 4 VNTR, E298A and IVF 23 þ 10 G/T polymorphisms L Peskircioglu et al 152 non-responders. However, this difference was sta- ecln23 gene and ED. However, we could not tistically insignificant. determine an association between sildenafil response and ecNOS IVS 23 þ 10G/T and E298A polymorphisms. eNOS exon 7 genotype distribution in men with Discussion ED was similar to that in healthy population. In this study, we found that patients with ‘a’ allele NO, a potent relaxant of peripheral vascular smooth of ecNOS intron 4 VNTR show a better response to muscle with an action mediated by cGMP, has been sildenafil than do ‘b’ allele carriers. We suggest that shown to be the major mediator of penile erection.3–5 ‘a’ allele carriers are more prone to erections In the penis, NO is synthesized mainly from the supported by sildenafil. On the other hand, ‘b’ allele penile nerve endings by the reaction catalyzed by carriers are expected to develop diabetes or cardio- NOS1. Nevertheless, additional sources for endo- vascular morbidities that will damage their vascular genous NO production have been shown,6 that is, in endothelium and prevent their response to sildena- addition to the above-mentioned major biosynthesis fil in the future. process, NO can also be produced in the penile In conclusion, we suggest that ecNOS intron 4 endothelium and corpora cavernosal smooth muscle VNTR polymorphism may be one of the determining by the enzymes NOS3 and NOS2. When diffused factors for sildenafil response in ED and that it might into the neighboring smooth muscle cells, NO be used as a predictive indicator while developing activates soluble guanylyl cyclase to produce cGMP, a treatment strategy for this widely seen disease. which finally mediates a reduction in intracellular Ca2 þ level leading to the relaxation of penile corpora cavernosal smooth muscle. The inability of the cavernosal smooth muscle to Acknowledgments undergo complete relaxation results in ED, which is manifested in aging and in several cardiovascular This study has been supported by a grant from and metabolic diseases, such as diabetes mellitus,15 Baskent University (KA: 04/26). myocardial infarction and atherosclerosis.16,17 It was shown previously that hypertension impairs NO availability owing to the production of cyclo- References oxygenase-derived vasoconstrictor substances and reduced endothelinB receptor-mediated NO activa- 1 Melman A, Gingell JC. The epidemiology and pathophysiol- 16 tion. Experimental studies verify impaired NOS ogy of erectile dysfunction. J Urol 1999; 161: 5–11. activity in diabetes and hyperlipidemia. 2 Murray FT, Geisser M, Murphy TC. Evaluation and treatment A bulk of evidence from animal models and from of erectile dysfunction. Am J Med Sci 1995; 309: 99–109. 3 Ignarro LJ, Bush PA, Buga GM, Wood KS, Fukuto JM, Rajfer J. clinical trials has reported the beneficial effect of 18,19 Nitric oxide and cyclic GMP formation upon electrical field NO level augmentation in ED. Two major stimulation cause relaxation of corpus cavernosum smooth therapeutic strategies may serve to increase penile muscle. Biochem Biophys Res Commun 1990; 31: 843–850. NO levels in ED, that is, the use of NO donor agents 4 Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. 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International Journal of Impotence Research