Analysis of association between the 5-HTTLPR and STin2 polymorphisms in the serotonin-transporter gene and clinical response to a selective serotonin reuptake inhibitor (sertraline) in patients with premature ejaculation

Authors

Errata

This article is corrected by:

  1. Errata: Retraction statement: Analysis of association between the 5-HTTLPR and STin2 polymorphisms in the serotonin-transporter gene and clinical response to a selective serotonin reuptake inhibitor (sertraline) in patients with premature ejaculation Volume 115, Issue 3, E9, Article first published online: 15 February 2015

Mohammad R. Safarinejad, PO Box 19395-1849, Tehran, Iran.
e-mail: safarinejad@unrc.ir

Abstract

Study Type – Therapy (cohort)
Level of Evidence 2b

OBJECTIVE

To test the hypothesis that polymorphism within the gene-linked polymorphic region (5-HTTLPR) and second intron of SLC6A4 gene (STin2) is associated with selective serotonin-reuptake inhibitors (SSRIs) response in subjects with premature ejaculation (PE).

PATIENTS AND METHODS

In all, 246 men with PE were recruited in this study. They were asked to take sertraline 50 mg daily for 2 weeks, and thereafter 100 mg daily, for a 12-week treatment period. Pretreatment evaluation included history and physical examination, intravaginal ejaculatory latency time (IELT), and International Index of Erectile Function (IIEF). The efficacy of treatment was assessed using responses to IIEF, and geometric mean IELT evaluation. 5-HTTLPR was genotyped using polymerase chain reaction techniques. A repeated-measures analysis of variance of geometric mean IELT was done to test a genotype effect on treatment outcome with SSRI (sertraline).

RESULTS

Of 227 participants who completed the study, 175 (77.1%) responded to sertraline (IELT >1 min). Overall the patients had a 3.7-fold (95% confidence interval, CI, 1.72–5.46) increase of the geometric mean IELT (P = 0.001). The results showed that responses were significantly better for the LA/LA genotype of the 5-HTTLPR polymorphism than for S-allele carriers (P = 0.001). The STin2 12/12 genotype was found more often in those responding to sertraline than in those not responding (P = 0.001). The probability of patients responding sufficiently to sertraline with an LA/LA genotype was highest (odds ratio 4.66, 95% CI, 2.48–6.14).

CONCLUSIONS

These findings indicate that the genotype of 5-HTT contributes in unique ways to the variation in the outcome of PE treatment with SSRIs.

Abbreviations
PE

premature ejaculation

5-HT

5-hydroxytryptamine (serotonin)

SSRI

selective serotonin-reuptake inhibitor

5-HTTLPR

serotonin transporter promoter region length

VNTR

variable number of tandem repeats

IELT

intravaginal ejaculatory latency time

IIEF

International Index of Erectile Function

STin2

second intron of SLC6A4 gene.

INTRODUCTION

Premature ejaculation (PE) is the most prevalent male sexual dysfunction [1]. The prevalence of PE varies widely among societies. From primary-care settings the reported prevalence of PE is 31% in the USA [2] and 66% in Germany [3]. The current most popular pharmacotherapeutic approach to treat PE is ‘off-label’ administration of selective serotonin (5-hydroxytryptamine, 5-HT)-reuptake inhibitors (SSRIs, e.g. paroxetine, fluoxetine, sertraline, citalopram, and escitalopram), which are reported to be effective for treating PE [4–6]. However, treatment of PE with SSRIs is not universally successful [7].

It was previously suggested that the response to SSRIs is partly under genetic control [8]. The serotonin transporter (5-HTT) gene is a major regulator of serotonergic neurotransmission in different regions of the brain [9]. 5-HTT is responsible for the active clearance of synaptic 5-HT and thus regulation of presynaptic and postsynaptic 5-HT receptor stimulation. Human 5-HTT is encoded by a single gene (SLC6A4) that has been cloned and mapped on chromosome 17q11.1–q12 [10].

The expression rate of 5-HTT is controlled by the 5-HTT promoter region length (5-HTTLPR) polymorphism [11]. A deletion/insertion in the 5-HTTLPR creates a short (S) allele and a long (L) allele, i.e. 14- and 16-repeat alleles, which alter the promoter activity [12]. The S allele induces lower 5HTT expression and 5HT reuptake activity than the L allele [13]. These two alleles more correctly were designated as C14 and C16, respectively, to denote the identification system and the number of repeat units [14]. There is a single nucleotide polymorphism within the L allele, and as a result it can be further subdivided into LA or LG variants [15].

It was reported that 5-HTT mRNA expression levels of the gene containing the S allele or LG allele are equivalent [16]. The second polymorphism that has been described, STin2, is a variable number of tandem repeats (VNTR) in the second intron of SLC6A4[17]. The three most common alleles of this polymorphism are designated 9, 10 and 12, based on the relative number of VNTR elements. The frequency of the most common variant, the 12 allele, is 0.54–0.66 in the general population [18]. STin2-polymorphism acts as a transcriptional regulator. It has been suggested that the 12/12 allele has a higher transcriptional activity than the 10/10 allele [19]. Polymorphisms in this gene with two genetic variants (5-HTTLPR and STin2) that code for the serotonin transporter (SLC6A4) have been proposed as possible explanations for observed inter-individual and inter-ethnic different responses to SSRI medication [8,20,21]. In other words, variations in expression and/or regulation of SLC6A4 could lead to individual and ethnic differences in treatment response among patients using SSRIs.

Several studies have reported a positive association with the L allele [21], and some studies have reported a negative association between the L allele and treatment response [22]. This might be due to individual and ethnic differences in the 5HTTLPR polymorphism. In the future, the definition of a genetic liability profile will offer considerable help in the early detection of effective therapy in psychiatric disorders [23,24].

Sertraline is highly potent and has a relatively long half-life of 26 h, allowing once-daily dosing, and achieves peak plasma levels within 4–6 h [25]. In addition it has a higher selectivity than fluoxetine and paroxetine for blocking the reuptake of 5-HT [26]. The useful effect of sertraline on PE has been reported in previous studies [27,28]. To the best of the author’s knowledge, this is the first study to investigate the influence of the 5-HTTLPR and STin2 genotype in response to an SSRI (sertraline), an established strategy for treatment of PE.

PATIENTS AND METHODS

From March 2006 to February 2008, 246 married men (mean age 31.2 years, range, 20–42) with lifelong definite PE, and their wives, were recruited in this study. An intravaginal ejaculatory latency time (IELT) of <1 min that occurred in >90% of sexual intercourse episodes was defined as PE. The IELT was the interval between the start of vaginal intromission and the start of intravaginal ejaculation [29].

Only married men in a stable relationship with their spouses for at least the previous 6 months, and possible sexual intercourse attempted once or more per week were recruited. Wives also agreed to participate in the study. The whole study protocol and possible side-effects were explained to patients before entering the study. Patients who agreed to proceed with the study procedures gave their informed consent. The study was conducted in accordance with the Declaration of Helsinki. The local Medical Ethical Committee approved the study. The recruited patients and their wives were interviewed separately on their first visit to determine an independent estimation of the IELT.

During the study, the patients were not permitted to use condoms or topical anaesthetic cream or spray. Interrupted intromission or change the speed of intercourse were not allowed. Moreover, couples were requested that if intercourse occurred more than once in a single session, only the first incident was measured. To measure the IELT a stopwatch was provided and female partners were also given instructions on how to use a stopwatch at home.

To establish the true diagnosis, the medical and sexual history were reviewed and physical examination, including structured interviews for diagnostic of mental and physical disorders, and self-administration of the International Index of Erectile Function (IIEF) (Appendix A) was done [30].

The couples were requested to attempt sexual intercourse at least four times, during which the pretreatment IELT was measured by the wives. To identify any underlying organic sexual dysfunction, standard biochemistry, haematological laboratory tests, and measurement of sex hormones and prolactin levels were also done. A Meares-Stamey test was also done to exclude genital tract infection when necessary. The mean weekly frequency of sexual attempts in the past 4 weeks was used to calculate pretreatment frequency of sexual intercourse.

Included in the study were only men with possible sexual intercourse attempts of once or more per week but without any obvious organic cause of PE. They were seeking medical help for their problem. Exclusion criteria included: patients with erectile dysfunction according to the IIEF; an organic cause of PE, including anatomical abnormalities, genital infection and neurological disorder; any sexual dysfunction other than PE; chronic psychiatric or physical illness; alcohol or substance abuse; and men who were receiving concomitant psychotropic, and antidepressant medication. Patients were excluded from the study if they had organic illness causing a limitation in SSRI use, and serious relationship problems. Medically, they were also excluded for gastrointestinal, liver, kidney or other known conditions that would possibly interfere with the absorption, distribution, metabolism or excretion of sertraline.

Blood samples (12–20 h after the last dose) were collected at baseline and at 1, 6 and 12 weeks. The sampling time and dosage of medication were documented. Plasma levels of sertraline were quantified using HPLC.

Whole blood originally collected for the sertraline assay was used to genotyping, conducted whilst unaware of the clinical data, and in duplicate, with patients responding or not to sertraline analysed simultaneously to eliminate errors in genotyping. Genomic DNA was extracted from whole blood and 5-HTTLPR L and S alleles were analysed by PCR (GC-Rich PCR System; Roche Molecular Biochemicals, Basel, Switzerland). The forward (5′-GGCGTTGCCGCTCTGAATGC-3′) and reverse (5′-AGGGGACTGAGCTGGACAAC-3′) oligonucleotides were used to amplify the 5-HTTLPR region. The reaction included 50-µL solution with 20 ng of genomic DNA, 10 mm dNTPs, 2 µm per primer, 1.5 mm MgCl2, and GC-Rich enzyme mix. The PCR consisted of 3-min preheating of the samples at 95.5 °C, followed by 1-min of 35 cycles of denaturation at 95.5 °C, 1 min annealing at 60 °C, 1 min extension at 72 °C, and a final hold of 7 min at 72 °C. The PCR products were then resolved by electrophoresis on 2% agarose gels and stained with ethidium bromide for visualization.

To further distinguish between the LA (common type; high production of the 5HTT) and LG allele (single-nucleotide variant replaced an adenine to guanine; low production of the 5HTT), 7 µL of the above-mentioned PCR product was digested with HpaII (New England Biolabs, Ipswich, MA, USA). The STin2 genotype was determined using the primer 5′-GTCAGTATCACAGGCTGCG AG-3′ with the reverse primer 5′-TGTTCCTAGT CTTACGCCAGTG-3′. The PCR was carried out in 96-well microplates on a Biometra T1 thermocycler (Westburg, Leusden, the Netherlands). Genomic DNA, 10–100 ng, was used in a 25-µL reaction mixture containing 1 × PCR buffer, 0.2 mmol/L dNTPs, 0.4 µmol/L of each primer, 0.75 mmol/L MgCl2 and 1 U Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA, USA). The PCR conditions comprised a cycle of pre-denaturation at 95 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 45 s, and extension at 72 °C for 8 min. PCR products were pooled (1 µL each) for each sample, and then size-resolved on an ABI3100 Genetic Analyser (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands), using 36 cm capillaries filled with POP6 polymer. The peaks corresponding to the various alleles were STin2.9: 248 bp, STin2.10: 265 bp, and STin2.12: 299 bp. The Genescan Analysis software version 3.7 (Applied Biosystems) was used to identify these alleles. To test for typing accuracy, 20% of the samples were repeated, with no discrepancy in genotype between assays.

Patients were asked to take sertraline 50 mg daily for 2 weeks, and thereafter 100 mg daily, for a 12-week treatment period. Any drugs, over-the-counter medications or supplements that might be affecting PE were to be discontinued 12 weeks before enrolment and were not allowed in the study period. Patients were asked not to consume alcoholic beverages within 6 h of sexual activity. The primary outcome measure was the change in the geometric mean IELT from baseline to 12 weeks. The time to ejaculation as reported by patients was based on stopwatch measurement. Secondary outcomes included changes in the intercourse satisfaction domain values of the IIEF and mean weekly intercourse episodes. Efficacy was assessed every week after the initial dose of sertraline on the first day and at the end of the 12-week treatment period. Patients and their wives were interviewed individually and the IELT noted. Safety and tolerability were evaluated on the basis of spontaneously reported adverse events, and clinical laboratory tests.

The sample size of 246 patients with PE provided enough power to detect an association between the 5-httpLR and STin2 polymorphisms and clinical response to SSRI. Indeed, it was calculated that a sample size of 214 subjects would be required to achieve a power of 80% with a 5% type I error. Taking into account withdrawal rates, 32 additional participants (15%) were included to ensure sufficient power, resulting in 246 participants in all. The geometric mean IELT was measured instead of the mean IELT, because the IELT in individual patients usually follows a skewed distribution. Thus, the IELT values were logarithmically transformed before statistical analysis, and the results are reported as fold-increases from baseline with associated 95% CI. The effects of treatment on IELT and weekly intercourse attempts were assessed using a paired t-test. Baseline clinical characteristics were analysed with a one-way anova. Qualitative variables were compared with the chi-squared test with Yates’ correction, or Fisher’s exact test, when necessary. Plasma levels of sertraline were analysed using an unpaired t-test. Genotype distribution and allele frequency were analysed with using a chi-squared test. To evaluate the association between 5-HTTLPR and STin2 polymorphisms and treatment effects, changes of mean geometric IELT from baseline during treatment were analysed with two-way repeated-measures anova. To analyse the effect of genotype on treatment response to sertraline, a multilevel modelling approach, with restricted maximum likelihood estimation procedures, was used. Genotype and allele frequencies of all polymorphisms, as well as Hardy–Weinberg equilibrium, were tested with the chi-squared test. As there is evidence of equivalent expression of S and LG alleles, with respect to 5-HTTLPR, analyses were conducted under three models: dominant S model (S/S + S/LA + S/LG + LG/LG + LA/LG vs LA/LA), dominant L model (LA/LA + LA/LG + S/LA vs S/S + S/LG + LG/LG), and genotype model (S/S + LG/LG + S/LG vs S/LA + LA/LG vs LA/LA) [16]. A two-tailed independent samples t-test (α = 0.05) was used to test for an effect of genotype on PE.

RESULTS

Participants and their wives were seen by the investigator and interviewed separately to evaluate their sexual activity and patient’s ejaculation function. In all, 227 (92.3%) patients completed the 12-week treatment period; 19 (7.7%) did not complete the study, seven because of a lack of effects, four because of adverse effects, and five because they were lost to follow-up. Three of the patients in the 4-week study were excluded from the present study because at least one of their duplicate plasma samples showed no detectable sertraline and their compliance was considered to be extremely poor. Therefore, there were 62 (27.3%) patients who did not respond to sertraline. Of 227 participants, 165 (72.7%) responded to sertraline (IELT >1 min). During the study, from week 1 onward and at the study endpoint (12 weeks), there were significant increases in geometric mean IELT. At baseline, the mean pretreatment IELT was 34 s; at the end of 12-week treatment period, overall the patients had a 3.7-fold (95% CI 1.72–5.46) increase in the geometric mean IELT (P = 0.001). The geometric mean IELT had a 1.2-, 1.6-, 2.8- and 3.7-fold increase after 1, 2, 3 and 4 weeks of treatment with sertraline, respectively. Thereafter, throughout the remainder study period, the geometric mean IELT remained unchanged. The patients were also divided into four groups according to the fold increase in the geometric mean IELT including, i.e. <1, 2–3, 4–5 or >5. Of 227 patients who completed the 12-week treatment period, 62 (27.3%), 78 (34.4%), 52 (22.9%), and 35 (15.4%) had a <1, 2–3, 4–5 and >5 geometric mean fold increase in their baseline IELT, respectively. The mean pretreatment intercourse frequency was 1.6 weekly; the mean weekly intercourse frequency was 2.7 at 12 weeks (P = 0.01). The baseline and 12-week mean intercourse satisfaction domain values of the IIEF were 10 and 16, respectively (P = 0.01). Anejaculation did not occur in these patients.

Genotype frequencies for 5-HTTLPR and STin2 for the total group showed no significant deviation from Hardy–Weinberg equilibrium. The distribution of genotypes was 61 (26.8%) LA/LA, 71 (31.3%) S/LA, four (1.8%) LA/LG, 19 (8.4%) S/LG, 10 (4.4%) LG/LG, and 62 (27.3%) S/S.

The results showed that responses were significantly better for the LA/LA genotype of the 5-HTTLPR polymorphism than for S-allele carriers (P = 0.001) (Table 1). The STin2 12/12 genotype was found more often in sertraline responders than nonresponders (P = 0.001). Sertraline nonresponders more often had an S/S or STin210/10 genotype than responders. The probability of patients responding sufficiently to sertraline was highest with an LA/LA genotype (odds ratio 4.66, 95% CI 2.48–6.14). Post hoc tests showed that the most pronounced improvement of PE was in patients homozygous for the LA-allele of the 5-HTTLPR (P = 0.001), followed by heterozygous patients (P = 0.01), while ≈60% of patients homozygous for the S allele did not improve at all. The odds ratios for 5-HTTLPR suggest a slightly increased risk for nonresponders to sertraline, with odds for LA/LG of 1.84 (1.22–2.21, P= 0.04), and for S/LA of 1.44 (1.02–1.81, P = 0.04).

Table 1.  Genotype distribution and allele frequencies in patients responding or not to sertraline, and the relationship between the fold increase in geometric mean IELT and genotype
GenotypeResponseNo responseFold increase in IELT
<12–34–5>5
No. of patients16562    
n (%):
5-HTTLPR genotype      
 LA/LA61 (37)0012 (5.3)14 (6.2)35 (15.4)
 LG/LG4 (2.4)6 (9.7)6 (2.6)4 (1.8)00
 LA/LG3 (1.8)1 (1.6)1 (0.4)3 (1.3)00
 S/S24 (14.5)38 (61.3)38 (16.7)24 (10.6)00
 S/LA66 (40)5 (8.1)5 (2.2)28 (12.3)38 (16.7)0
 S/LG7 (4.2)12 (19.4)12 (5.3)7 (3.1)00
STin2 genotype      
 10/1012 (7.3)45 (72.6)45 (19.8)12 (5.3)00
 10/1224 (14.5)14 (22.6)14 (6.2)17 (7.5)7 (3.1)0
 12/12129 (78.2)3 (4.8)3 (1.3)31 (13.7)42 (18.5)56 (24.6)
Allele frequencies      
 LA130 (78.8)6 (9.7)6 (2.6)43 (18.9)52 (22.9)35 (15.4)
 LG14 (8.5)19 (30.6)19 (8.4)14 (6.2)00
 S97 (58.8)55 (88.7)55 (24.2)59 (26)38 (16.7)0

The results from the logistic regression analyses on the separate alleles were similar to the results of the genotype analyses. Nonresponders more often had the S-allele than had responders. The odds ratio for nonresponders for the S-allele vs the LA-allele was 3.22 (1.68–4.87; P = 0.001).

When comparing patients for improvements in PE over the 12 weeks of treatment there was a significant difference between the geometric mean IELT fold-increase among genotypes (P = 0.01). The dominant 5-HTTLPR genotype among patients who had a <1-, 2–3-, 4–5- and >5-fold increase in geometric mean IELT was S/S, S/LA, S/LA and LA/LA, respectively (Table 1). When S carriers were pooled together and compared with LA/LA homozygotes, they had a significantly worse treatment outcome (P = 0.001). Patients with LA/LA genotype also showed a rapid improvement in PE (P = 0.004). Of all patients, 166 (73.1%), 136 (59.9%), and 91 (40.1%) had a dominant S model, dominant L model, and genotype model, respectively (Table 2). Patients with a dominant L model had a significantly higher response rate than those with a dominant S model (P = 0.01) or genotype model (P = 0.001).

Table 2.  Dominant genotype model frequencies in patients responding or not to sertraline
Dominant genotype model (n)ResponseNo response
L dominant (136)130 (95.6) 6 (4.4)
S dominant (166)104 (62.6)62 (37.4)
Genotype dominant (91) 35 (38.5)56 (61.5)

DISCUSSION

This is the first study to investigate the influence of the 5-HTTLPR and STin2 polymorphism genotypes for the clinical response to an SSRI (sertraline) in patients with PE. It was reported that the 5HTTLPR polymorphisms alter serotonin transporter mRNA synthesis, leading to changes in 5HTT expression and 5HT cellular uptake [11]. An effect of genotype was detected at 1 week after starting treatment. SSRIs act directly on the 5HTT, and a different treatment result might be due to a difference in locus [17]. 5HTT determines the duration and magnitude of 5-HT responses. In this study logistic regression analysis indicated that the overall effect of 5-HTTLPR on the outcome of treatment with an SSRI is high. Clinicians dealing with psychopharmacology often face variability in clinical response together with a lack of clinical or biological predictors of response. Variations in the inter-individual and ethnic frequencies of the 5HTTLPR polymorphisms might affect the genotype influence on the response to an SSRI. The present study showed that sertraline could be more effective in patients carrying the LA allele than those carrying the S allele. All patients of the LA/LA genotype were responders. The effect is similar to that observed in depressed patients treated with fluvoxamine [31], paroxetine [32], and fluoxetine [21]. There are similar results in Asian populations; in a study of 121 Chinese patients diagnosed with major depression, patients with the L/L genotype had a significantly better response to fluoxetine [33]. The present results are in general agreement with those of previous studies from Western [31,32,34] and Eastern [35] populations. In a meta-analysis, Serretti et al.[36] pooled subjects from three pharmacogenetic studies, and the better response in depressed subjects carrying the 5HTT L/L genotype was replicated (P = 0.007). This could be consistent with the L allele having more transcriptional efficiency, and the promoter region insertion being more adaptive to pharmacological change. The S allele of the 5HTTLPR polymorphism is associated with lower 5HTT availability.

A complex integration of actions between the central and peripheral nervous systems is required to produce normal ejaculation. Ejaculation-related neural activation is present in following regions of the brain: the posteromedial bed nucleus of the stria terminalis, a lateral subarea of the posterodorsal medial amygdala, and the medial parvicellular subparafascicular nucleus of the thalamus [37,38]. Hariri et al.[39] reported that subjects with at least one copy of the S allele of the 5-HTTLPR had greater activity of the amygdala than had subjects homozygous for the L allele.

In addition, STin2 also influenced the outcome of SSRI treatment. In sertraline nonresponders the frequency of the STin2 10/10 genotype was higher, and of the STin2 12/12 genotype lower. There is evidence that the STin2-polymorphism acts as a transcriptional regulator of the 5-HTT gene in an allele-dependent manner. The 12/12 allele has higher enhancer-like properties than the 10/10 allele [40].

Subjects with the S/S genotype are at increased risk of developing PE [41]. It is also possible that the treatment response in patients with the S/S genotype is more dependent on context than on responsiveness to SSRI. In the present study, all possible covariates that could influence the findings were carefully controlled. Patients with any concomitant psychiatric and/or neurological diagnosis were excluded from the sample to avoid biases linked to these diseases. A major advantage of the present study is the large sample size. Indeed, for genetic studies, usually larger samples are needed to have sufficient statistical power [42]. Another advantage is that the patients were assessed 12 weeks after starting the treatment, therefore possible improvements would definitely have occurred in this period. In general, it seems that patients with PE and 5-HTTLPR S variants would be treated with a drug other than an SSRI.

In conclusion, the present findings suggest the possibility of a genetic influence on individual differences in the response to SSRI treatment for patients with PE. Pharmacogenetics appears to be the future hope of pharmacotherapy for PE. Further studies are required to clarify the complex interactions that might involve 5-HTTLPR, the functioning of brain regions, and PE.

CONFLICT OF INTEREST

None declared.

Appendix A

The IIEF:

  • 1How often were you able to get an erection during sexual activity?
  • 2When you had erection with sexual stimulation, how were your erections hard enough for penetration?
  • 3When you attempted sexual intercourse, how often you were able to penetrate your partner?
  • 4During sexual intercourse, how often you were able to maintain your erection to completion of intercourse?
  • 5During sexual intercourse, how difficult was it to maintain your erection to completion of intercourse?
  • 6How many times have you attempted sexual intercourse?
  • 7When you attempted sexual intercourse, how often was it satisfactory for you?
  • 8How much have you enjoyed sexual intercourse?
  • 9When you had sexual intercourse, how often did you ejaculate?
  • 10When you had sexual intercourse, how often did you have the feeling of orgasm or climax?
  • 11How often have you felt sexual desire?
  • 12How would you rate your level of sexual desire?
  • 13How satisfied have you been with your overall sex life?
  • 14How satisfied have you been with your sexual relationship?
  • 15How do you rate your confidence that you could get and keep an erection?

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