Objective
The purpose of this study was to verify the association between the Trp 64 Arg polymorphism and idiopathic overactive bladder (OAB) syndrome.
Study Design
A case-control study was conducted with 218 women. The case group consisted of 49 patients with OAB symptoms; the control group included 169 women without urinary symptoms. The studied polymorphism was detected by restriction fragment length polymorphism analysis. The χ 2 test was used to compare categoric data, with a significance level of 5%. Numeric data were compared with the use of the parametric t test or nonparametric Mann-Whitney U test.
Results
The distribution of the polymorphism in the investigated women was digested homozygous T allele 69.75%, heterozygotes 29.8%, and homozygous A allele 0.45%. A comparison between the groups showed higher prevalence of the digested homozygous T allele genotype in women with OAB syndrome ( P = .001). Multiple logistic regression analysis identified that a family history of OAB syndrome was an independent risk factor for OAB syndrome.
Conclusion
The Trp 64 Arg polymorphism was associated with OAB syndrome in the Brazilian population.
Overactive bladder (OAB) is a highly prevalent disorder that is characterized as a syndrome that consists of urinary urgency, usually accompanied by frequency and nocturia, with or without urgency urinary incontinence. The disorder afflicts both men and women at similar rates and has an estimated overall prevalence of nearly 12%, which increases with age.
The process of micturition is critically dependent on the operation of a spinal bulbospinal reflex, which comprises afferent (sensory) and efferent (motor) pathways that are integrated and coordinated at spinal and supraspinal centers. The lumbosacral parasympathetic outflow provides the excitatory motor input to the urinary bladder smooth muscle (detrusor). Activation of the sympathetic nerves contributes to urine storage by relaxing the detrusor muscle by activating β-adrenoceptors (β-ARs).
β-ARs currently are classified into β1-, β2-, and β3-subtypes. Of these 3 subtypes, human detrusor tissue predominantly expresses β3-AR messenger RNA, which suggests that the relaxation that is induced by β-adrenergic stimulation of the human detrusor is mediated mainly through β3-AR activation. Thus, this subtype is probably the most important subtype for bladder relaxation during the storage phase of the micturition cycle.
Activation of the β1-, β2-, and β3-AR subtypes all couple to adenylyl cyclase, which leads to an increase in intracellular adenosine 3′:5′-cyclic monophosphate (cAMP) levels and a subsequent activation of cAMP-dependent protein kinase A. Then protein kinase phosphorylates myosin light chain kinase, which suppresses the calcium-calmodulin–dependent interaction of myosin with actin. The increase in cAMP production also attenuates the cytoplasmic calcium ion concentration by removing calcium from the cytoplasm. Thus, cAMP is a key player in eliciting βAR-mediated smooth muscle relaxation.
A missense mutation in codon 64 of the β3-AR gene, which changes the tryptophan to arginine, occurs with an approximate frequency of 8-10% in the white population, 20% in the Japanese population, and 40% in Alaskan Eskimos. The tryptophan 64 arginine (Trp 64 Arg) polymorphism has been associated with abdominal obesity, insulin resistance, difficulty in losing weight, and high body mass index. The aforementioned studies showed that maximal cAMP accumulation was reduced significantly in response to various β3-adrenergic agonists in cells that express a mutated receptor, which suggests that the Trp 64 Arg polymorphism is less capable of stimulating adenylyl cyclase than the wild-type version.
Because activation of the β3-AR relaxes the detrusor muscle during bladder filling, a polymorphism in the β3-AR gene may impair the relaxation of the bladder muscle during urine storage and therefore may be involved in idiopathic detrusor overactivity. Based on these facts, we hypothesized that the Trp 64 Arg polymorphism is a risk factor for OAB syndrome.
Materials and Methods
The local research and ethics committee approved the study protocol. The patients who were included in this study provided informed consent. The case-control study was performed from December 2007 to July 2009. Two hundred eighteen women were recruited from the outpatient clinic of General Gynecology Ambulatory of São Paulo Federal University. The patients were submitted to a gynecologic evaluation and pelvic organ prolapse quantification assessment and were separated into 2 groups.
The case group consisted of 49 women with OAB symptoms: urinary urgency with or without urge incontinence, increased urinary frequency, and nocturia. These patients had never undergone vaginal surgery and had a genital prolapse at stage ≤2 on the pelvic organ prolapse quantification assessment, with the most distal part of the prolapse situated above the hymen. We excluded patients with mixed urinary incontinence with predominant symptoms of stress urinary incontinence. Inclusion criteria were based on the evaluation of a self-report 5-day bladder diary and the validated questionnaire OAB-V8. The OAB-V8 is a patient self-identification screening and awareness tool that consists of the first 8 questions of the OAB questionnaire and was designed to determine how bothered a patient is by bladder symptoms. Eligible subjects in the case group were women with a mean micturition frequency of ≥8 micturitions per 24 hours and mean urgency episodes of ≥2 per 24 hours in a 5-day bladder diary. The odds ratio for OAB syndrome was 95.7 (95% confidence interval [CI], 29.3–312.4) with scores ≥8 ( Table 1 ). All patients from the case group were assessed with a urodynamic test. We excluded the patients who exhibited severe stress urinary incontinence (Valsalva leak point pressure, ≤60 cm H 2 O), outflow obstruction (detrusor pressure at maximum flow, ≥20 cm H 2 O; maximum flow, ≤12 cm H 2 O) and residual urine ≥100 mL ( Table 2 ). The control group was composed of 169 women without any urinary symptoms who were ≥60 years old and who had not received hormonal replacement therapy for at least 1 year. Additional major exclusion criteria for both groups included chronic neurologic or muscular diseases.
| Characteristic | Control group (n = 169) | Study group (n = 49) | P value |
|---|---|---|---|
| Mean age, y a | 64.27 ± 3.46 | 62.27 ± 10.48 | .22 |
| Mean body mass index, kg/m 2 a | 27.78 ± 4.81 | 29.17 ± 4.47 | .01 |
| Race, n (%) | .10 | ||
| White | 83 (49.1) | 17 (34.7) | |
| Nonwhite | 86 (50.9) | 32 (65.3) | |
| Mean parity, n a | 3.68 ± 2.59 | 3.71 ± 2.59 | .97 |
| Mean age at menopause, y a | 48.40 ± 5.53 | 47.09 ± 5.48 | .10 |
| Family history for urge incontinence, n (%) | 12 (7.1) | 11 (22.4) | .01 |
| Overactive bladder-V8 score a | 0.85 ± 1.54 | 26.39 ± 6.87 | .001 |
| Frequency, 24 h a | 5.1 ± 1.76 | 8.21 ± 1.06 | .001 |
| Nocturia, 24 h a | 0.67 ± 8.86 | 3.31 ± 2.35 | .001 |
| Urgency, 24 h a | 0 | 3.23 ± 1.00 | .001 |
| Urodynamic parameter | Measure |
|---|---|
| Residual urine, mL a | 5.45 ± 2.2 |
| First sensation, mL a | 95.82 ± 9.8 |
| Maximum cystometric capacity, mL a | 380.10 ± 23.1 |
| Maximum flow rate: Qmax, mL/sec a | 74.2 ± 4.1 |
| Detrusor pressure, cm H 2 O a | 12.4 ± 5.6 |
| Average flow rate, mL/sec a | 31.6 ± 2.8 |
| Valsalva leak point pressure, cm H 2 O a | 92.7 ± 10.4 |
| Involuntary detrusor contraction, n (%) | 32 (65) |
Regarding the race of the patients, the subjects were classified as white or nonwhite (a group comprising blacks and mulatto women). The term nonwhite refers to the African inheritance present in the composition of the Brazilian population and the intense racial miscegenation characteristic of our country that hinders a precise differentiation among racial groups based on physical features.
Genomic DNA was extracted and purified from ethylenediaminetetraacetic acid-treated blood samples with the use of a commercially available kit (GFX; Amersham Biosciences UK Ltd, Buckinghamshire, UK). The Trp 64 Arg polymorphism was identified by polymerase chain reaction (PCR; Biometra T personal Thermocycler; Whatman Biometra, Goettingen, Germany) with 400 ng DNA as a template. For amplification, Taq DNA polymerase (Promega, Madison, WI) and commercially obtained oligonucleotide primers were used. PCR was carried out with the following primer set: P1, 5′-CGCCCAATACCGCCAACAC -3′ (forward) and P2, 5′-CCACCAGGAGTCCCATCACC -3′ (reverse). The 40 amplification cycles were preceded by a 5-minute denaturation at 95°C. Annealing was carried out at 57°C for 1 minute, elongation at 72°C for 1 minute, and denaturation at 95°C for 1 minute.
The Trp 64 Arg polymorphism was categorized on the basis of the PCR fragments that were generated during the BstNI (DRL-Block BD-2D; Techne Ltd, Cambridge, UK) digestion reaction and were analyzed by 3% agarose gel electrophoresis. A nondigested fragment with bands of 161 bases pairs (bp), 30 bp, 12 bp and 7 bp (homozygous A allele: AA) was detected. The digested homozygous T allele yielded 99 bp, 62 bp, 30 bp, 12 bp, and 7 bp fragments (TT), and heterozygotes (TA) showed 6 fragments of 161 bp, 99 bp, 62 bp, 30 bp, 12 bp, and 7 bp ( Figure ).
