BDNF rs6265 polymorphism is not associated with the occurrence of overactive bladder and its response to intradetrusor injection of botulinum neurotoxin type A in female patients

Abstract

Reports suggest increased urinary excretion of Brain-Derived Neurotrophic Factor in patients with Overactive Bladder (OAB). The rs6265 (c.196G > A, p.Val66Met) polymorphism in the BDNF gene was previously associated with impaired secretion of this neurotrophin. The aims of the present study were to analyze the possible association of the BDNF: rs6265 polymorphism with OAB risk and with the response to a single intradetrusor injection of botulinum neurotoxin type A (BoNT/A) in female patients. The study group comprised 160 OAB females, and the control group comprised 359 healthy females. Urodynamic parameters, frequencies of OAB symptoms, and scores from ICIQ-OAB and ICIQ-LUTS-QoL questionnaires were recorded before BoNT/A injection and six months after. Genotyping of BDNF: rs6265 polymorphism was performed using TaqMan real-time polymerase chain reaction. No statistically significant differences in the distribution of BDNF genotypes or alleles between OAB patients and healthy controls were found. No significant differences among OAB subjects regarding BDNF polymorphism were found in terms of clinical symptoms, scores of both questionnaires, and urodynamic parameters. These results do not support the hypothesis that BDNF: rs6265 polymorphism in Polish females with OAB is associated with susceptibility to this condition and with response to BoNT/A treatment.

Introduction

According to the International Continence Society (ICS), overactive bladder (OAB) is a syndrome giving an urgent need to urinate, usually with frequent urination and nocturia (increased night-time urination), with or without urge incontinence. The term OAB is restricted to cases without infection and/or without other causative pathology^1,2. OAB is not a homogeneous syndrome and includes cases with neurological causes (nOAB; neurogenic OAB), mostly from multiple sclerosis, as well as cases without any evident cause (iOAB; idiopathic OAB)³.

Results from a recent meta-analysis showed a global OAB prevalence of 20% with an increasing trend over the last 20 years. The OAB frequency in females was higher than in males (21.9% and 16.1%, respectively). In addition, a higher OAB prevalence was associated with overweight and obesity⁴. Other OAB risk factors apart from sex include aging, bladder outlet obstruction, psychological stress, income, parity, diabetes, and genetic predisposition^4–8. The evidence that genetic predisposition is a risk factor is based on both the familial occurrence of OAB^7–9 and genome-wide association studies, which have identified genetic polymorphisms associated with urinary incontinence^10–12.

Despite many years of research, OAB pathogenesis is still not completely understood. Results from animal studies suggest that Brain-Derived Neurotrophic Factor (BDNF; UNIPROT: Neurotrophic factor BDNF precursor form), a key central nervous system neurotrophin, acts as a ligand, via its high-affinity TrkB receptor, in inflammation-related bladder overactivity and cystitis pain^13,14. Furthermore, some authors have demonstrated that inhibition of the Trk receptor reduced bladder hypersensitivity in animal models of chronic cystitis and prostatitis^15,16.

Indeed, some clinical studies demonstrated that patients with OAB, whether adults or children, may present with significantly higher urinary levels of BDNF, as expressed by increased BDNF/creatinine ratios^17–25. Additionally, a correlation was found between urinary BDNF and OAB symptom severity^18,20, and in some studies, urinary BDNF was shown to decrease significantly in response to pharmacological treatment of OAB^17,23–25. However, other studies found no link between the occurrence of OAB, treatment responses, and urinary BDNF^26–28, and some authors reported a substantial individual variability in urinary levels of this neurotrophin¹⁷. Intradetrusor injection of botulinum neurotoxin type A (BoNT/A) emerged as a promising treatment for OAB. Similar to other treatments for OAB, the data on the link between intradetrusor BoNT/A injection and urinary BDNF are inconclusive, with some studies showing a post-treatment decrease in this parameter^29,30 and others not³¹.

While the evidence from animal models suggests that a link might exist between BDNF and OAB pathophysiology^13,14, the results of clinical studies mentioned above are not straightforward. The discrepancies in published results might be associated with several factors. A proportion of BDNF is secreted to the urine as an immature precursor (proBDNF), from which a propeptide must be cleaved to give the mature form. One study demonstrated that the proBDNF/BDNF ratio in aging women with OAB was significantly lower, implying that age might be a contributor to BDNF variability²⁷. Further, urinary BDNF appears to be OAB-type-specific, with higher values observed in patients with idiopathic detrusor overactivity than in those with neurogenic overactivity³².

However, this is genetically driven individual variability in BDNF synthesis, which seems to be the most obvious reason behind the discrepancies mentioned above. BDNF is encoded by the BDNF gene located on chromosome 11p14.1. The BDNF: rs6265 (c.196G > A) polymorphism, which replaces valine (Val) with methionine (Met) at amino acid position 66 (p.Val66Met), has been associated with lower serum BDNF concentrations in both healthy subjects as well as in patients with Parkinson’s disease³³. A plausible cause of this phenomenon, in carriers of a c.196 A allele, might be decreased activity-dependent release of BDNF through reduced vesicular packaging³⁴. However, a question arises whether the changes in serum BDNF associated with the presence of the functional BDNF: rs6265 polymorphism are sufficient to influence urinary levels of this ligand, and thus, whether this polymorphism could actually be associated with a predisposition to OAB and/or with response to BoNT/A treatment. We did not find any published studies dealing with the role of the BDNF: rs6265 polymorphism in OAB pathophysiology. The only published study dealing with the role of this BDNF polymorphism in a bladder control syndrome, conducted in children with primary monosymptomatic nocturnal enuresis (PMNE), yielded negative results³⁵.

Therefore, the aim of the present study was to analyze possible associations of the BDNF: rs6265 polymorphism with the risk of OAB, as well as with urodynamic changes and patient-perceived changes, as assessed by frequencies of OAB symptoms and by ICIQ-OAB and ICIQ-LUTS-QoL questionnaires, in Polish OAB females six months after a single intradetrusor injection of BoNT/A. To the best of our knowledge, this is the first study to examine the BDNF: rs6265 polymorphism in adult patients with OAB.

Materials and methods

Ethics

The study protocol for patients was approved by the Local Bioethics Committee at the Pomeranian Medical University in Szczecin (decision no. KB-0012/125/17; November 16, 2017), and all patients provided written informed consent to participate in the study. All study protocols conformed to the latest version of the Declaration of Helsinki (2024).

OAB patients

The study included 160 consecutive female patients with OAB, aged between 22 and 86 years. This group comprised 100 patients with iOAB, i.e., urinary urgency, accompanied by increased daytime frequency and/or nocturia, with urinary incontinence or without, in the absence of urinary tract infection or other detectable disease, and 60 patients with nOAB in whom the symptoms mentioned above occurred in the setting of a clinically relevant neurologic disease^1–3. All patients were of Polish nationality and European descent and lived in the Polish Western Pomeranian region. The patients were eligible for the study if they presented with detrusor overactivity confirmed by urodynamic testing, showed intolerance or did not respond adequately to a previous pharmacological treatment of OAB. Exclusion criteria of the study comprised previous use of BoNT/A, mixed urinary incontinence, presence of urinary tract infection, bladder stones, bladder cancer, urinary retention, and/or previous urogynecological surgeries. The list of collected patients’ characteristics included age, body height, body mass, body mass index (BMI), calculated as body mass (kg)/height (m²), number of pregnancies, number of deliveries, and number of cesarean sections^36,37.

Intradetrusor injection of BoNT/A

One hundred units of BoNT/A (Botox^®, Allergan Inc., Irvine, CA, USA) were diluted in 10 ml of normal saline (0.9% NaCl). All injection procedures were performed by a single urogynecologist (S.C.) under general anesthesia. A rigid cystoscope and needle (injeTak^®, Laborie, Portsmouth, NH, USA) were used to inject BoNT/A into the detrusor muscle at 20 sites (5 units of BoNT/A per site) spaced approximately evenly over the bladder, including the dome but sparing the trigone.

Pre- and post-treatment evaluation

All patients reported frequencies for urgency, urgency incontinence, frequency, and nocturia, and completed a survey with the ICIQ-OAB questionnaire and the International Consultation on Incontinence Questionnaire-Lower Urinary Tract Symptoms-Quality of Life (ICIQ-LUTS-QoL) questionnaire, before the intradetrusor injection of BoNT/A and six months thereafter. In both questionnaires, part A assessed symptom severity, and part B assessed cumulative anxiety in the patient. High scores in the questionnaires suggest worse symptom profiles (ICIQ-OAB) and/or a negative impact on the quality of life (ICIQ-LUTS-QoL). With ICIQ-OAB, the total raw scores in part A ranged from 0–16 points, and in part B, from 0–40 points. With ICIQ-LUTS-QoL, the total raw scores in part A ranged from 0 to 76 points, and in part B, from 0 to 190 points.

Before the BoNT/A injection and six months thereafter, urodynamic parameters were determined according to the ICS standards in all OAB patients. Urodynamic testing was performed by the same investigator (S.C.) after confirming a normal result from urinalysis. Two types of Menfis bioMedica catheters (9 Fr and 12 Fr) were used. One 9 Fr catheter was inserted into the urinary bladder to perform flow cystometry and measure flow pressure. A second 12 Fr catheter was inserted into the rectum to measure intra-abdominal pressure. The urodynamic examination was performed with the patient in the gynecological position. During the examination, 0.9% physiological saline was administered to the urinary bladder at a flow rate of 50–75 ml/min. A cough stress test and Valsalva maneuver were performed after administering the first 50 ml of fluid and repeated after every additional 100 ml. The micturition phase started after maximum filling of the bladder. The patient voided to a container placed on a scale, and the uroflow curve and residual urine volume were determined. Urodynamic parameters, maximum cystometric capacity (MCC), post-void residual (PVR), first sensation of bladder filling (FSBF), maximum flow rate (Qmax), average flow rate (Qave), detrusor pressure at maximum flow (Pdet at Qmax), and maximum detrusor pressure (Pdet-max) were determined using the Libra + system (Medical Measurement Systems B.V., Amsterdam, Netherlands).

Genotyping

Before the injection of BoNT/A, a buccal swab was obtained from every OAB female. Genomic DNA was extracted from buccal epithelial cells using a commercially available DNA isolation kit (PrepFiler^® Express Forensic DNA Extraction Kit, Applied Biosystems, Life Technologies Polska, Warsaw, Poland) according to the manufacturer’s instructions. The BDNF: rs6265 polymorphism was identified using an allelic discrimination assay (on a CFX96 Touch Real-Time PCR Detection System; Bio-Rad, Feldkirchen, Germany) with TaqMan^® probes as described before³⁸. To discriminate BDNF: rs6265 alleles, single-nucleotide polymorphism (SNP) assays (TaqMan^® Pre-Designed SNP Genotyping Assay; Thermo Fisher Scientific, Waltham, MA, USA) were used. The SNP assay identity (Thermo Fisher Scientific) was C_11592758_10, including primers and fluorescently labelled (FAM and VIC dye labels and minor groove binders; MGB) probes to detect alleles.

Controls

The historical control group comprised 359 females (aged between 11 and 14 years) of Polish descent recruited in the Polish Western Pomeranian region between 2019 and 2022 for “Task 05”, part of the Program “Regional Initiative of Excellence” from the Polish Ministry of Science and Higher Education (Project no. 002/RID/2018/19). “Task 05” aimed to carry out molecular analyses of oral microbiomes and DNA microarray-based genome polymorphisms from saliva samples and to evaluate their distributions with respect to predisposition to caries of permanent dentition in children. The study protocol for controls was approved by the local bioethics committee of the Pomeranian Medical University in Szczecin (decision No. KB-0012/16/19 on 14 January 2019), and written informed consent was obtained from all subjects and/or parents of all children involved in the study. The eligibility criteria for controls: the recruitment process for children; the collection of saliva samples; the extraction of genomic DNA from saliva samples; as well as DNA sample genotyping for 567 218 single nucleotide polymorphisms (including the BDNF: rs6265 polymorphism) using beadchip microarrays (Infinium CoreExome-24 v1-4; Illumina, San Diego, CA, USA); have been described in detail previously³⁹.

Statistical analyses

Normality of the quantitative variables was assessed by the Shapiro-Wilk test. As most quantitative variables were not normally distributed, all were presented as medians with minimum and maximum values. Quantitative data were compared using Mann–Whitney tests. The divergence of BDNF: rs6265 genotype frequencies from the Hardy-Weinberg equilibrium, and categorical data, were analyzed using χ2 tests for tables larger than 2 × 2, or Fisher’s exact tests. Between-genotype-group differences in the values of quantitative variables were assessed using logistic regression with the adjustment for OAB type. In the logistic regression analysis, the genotype group was treated as the outcome, and quantitative parameters were entered as continuous predictors, with odds ratios (ORs) reported per unit increase. Statistical significance for all tests was defined as p < 0.05, with Bonferroni correction for multiple comparisons applied whenever necessary. All analyses were carried out with commercial software (Dell Statistica, version 13, 2016; software.dell.com, accessed on 2 April 2025; Dell Software, Round Rock, TX, USA). Diploid SNP bases are abbreviated in this article using consecutive letters, e.g., homozygote [G]; [G], heterozygote [G]; [A] and homozygote [A]; [A] as GG, GA (= AG), and AA, respectively.

Results

The BDNF: rs6265 genotype distributions in the control group, in the iOAB group, in the nOAB group or in the combined OAB group (iOAB + nOAB) all conformed to the expected Hardy-Weinberg equilibrium (p = 0.442, p = 0.425, p = 0.625 or p = 0.360, respectively).

There were no statistically significant (here referred to as significant) differences in the frequency distributions of BDNF: rs6265 alleles or genotypes between control subjects and iOAB or nOAB patients (Table 1). As there were also no significant differences between iOAB and nOAB patients in the distributions of BDNF: rs6265 alleles and genotypes (p = 0.654 or p = 0.879, respectively), we created the combined OAB group consisting of both iOAB and nOAB subjects. There were no statistically significant differences in the frequency distributions of BDNF: rs6265 variants between control subjects and women in combined OAB group (Table 1).

Table 1.

Frequency distributions of BDNF:rs6265 alleles and genotypes in control females and in overactive bladder (OAB) women.

Group	Alleles, n (%)		p	Genotypes, n (%)			p	p d	p r	p ha
	G	A		GG	GA	AA
Control females (n = 359)	598 (83.3)	120 (16.7)	–	247 (68.8)	104 (29.0)	8 (2.2)	–	–	–	–
iOAB (n = 100)	172 (86.0)	28 (14.0)	0.356	73 (73.0)	26 (26.0)	1 (1.0)	0.594	0.419	0.433	0.560
nOAB (n = 60)	101 (84.2)	19 (15.8)	0.811	42 (70.0)	17 (28.3)	1 (1.7)	0.954	0.853	0.781	0.920
OAB (iOAB+nOAB) (n = 100 + 60 = 160)	273 (85.3)	47 (14.7)	0.412	115 (71.9)	43 (26.9)	2 (1.2)	0.647	0.482	0.454	0.625

iOAB, idiopathic overactive bladder; nOAB, neurogenic overactive bladder.

p – significance values for chi² 2 × 2 table (alleles) or for chi² 2 × 3 table (genotypes).

p_d, p_r or p_ha—significance values for dominant (p_d) and recessive (p_r) modes of inheritance for the minor allele (c.196 A) or for heterozygote advantage (p_ha; overdominance), respectively. % given to one decimal place; p-values to three decimal places.

Females with iOAB did not differ significantly from those with nOAB in terms of age, body mass, BMI, number of pregnancies, deliveries, and cesarean sections. Body height was the only baseline parameter that differed between the two groups, being lower in the iOAB than in the nOAB group (Table 2).

Table 2.

Basic characteristics of female patients with idiopathic (iOAB) or neurogenic (nOAB) overactive bladder syndrome.

Variable	All patients	iOAB	nOAB	p
	(n = 160)	(n = 100)	(n = 60)
Age (years)	60 (50:67)	60 (51:68)	59 (46:66)	0.087
BMI (kg/m2)	26.9 (24.2:30.5)	27.3 (24.6:30.7)	26.5 (23.9:30.3)	0.206
Pregnancies, n	2 (1:3)	2 (2:3)	2 (1:3)	0.479
Deliveries, n	2 (1:2)	2 (1:2)	2 (1:2)	0.689
Cesarean sections, n	0 (0:0)	0 (0:0)	0 (0:0)	0.681

BMI, body mass index (to three significant figures). Data presented as medians (lower quartile: upper quartile). P = p-values for Mann-Whitney tests with a null hypothesis of no difference between iOAB and nOAB, to three decimal places.

The iOAB and nOAB groups did not differ significantly in terms of urodynamic parameters, apart from pretreatment PVR and Pdet at Qmax. The pretreatment values of both these parameters were significantly lower in iOAB women than in those with nOAB (Table 3).

Table 3.

Urodynamic assessment of female patients, before and six months after intradetrusor injection of botulinum neurotoxin type A, with idiopathic (iOAB) or neurogenic (nOAB) overactive bladder syndrome.

Variables	Time code	All patients	iOAB	nOAB	p
		(n = 160)	(n = 100)	(n = 60)
MCC (ml)	0	216 (166:244)	219 (168:246)	210 (160:240)	0.421
	6	345 (312:367)	356 (312:367)	345 (301:367)	0.348
	∆0–6	− 128 (− 178:− 88)	− 128 (− 185:− 92)	− 128 (− 178:− 86)	0.693
PVR (ml)	0	4 (0:32)	0 (0:21)	18 (0:44)	0.003
	6	6 (0:23)	3 (0:10)	8 (0:23)	0.094
	∆0–6	0 (− 4:17)	0 (− 4:10)	1 (− 5:30)	0.118
FSBF (ml)	0	56 (28:98)	54 (28:99)	58 (34:97)	0.568
	6	137 (134:157)	137 (134:157)	156 (134:157)	0.431
	∆0–6	− 85 (− 116:− 35)	− 88 (− 117:− 35)	− 76 (− 110:− 34)	0.794
Qmax (ml/s)	0	17 (16:18)	18 (16:19)	17 (16:18)	0.203
	6	14 (13:17)	14 (13:18)	14 (13:17)	0.132
	∆0–6	2 (0:5)	3 (0:5)	2 (1:4)	0.663
Qave (ml/s)	0	8 (8:9)	8 (8:9)	8 (8:9)	0.952
	6	8 (8:11)	8 (8:11)	8 (8:9)	0.087
	∆0–6	0 (− 2:1)	0 (− 2:1)	0 (− 1:1)	0.348
Pdet at Qmax (cm H2O)	0	37 (35:46)	36 (34:43)	39 (36:47)	0.013
	6	20 (19:21)	20 (19:21)	20 (19:21)	0.236
	∆0–6	17 (15:26)	16 (13:24)	18 (16:27)	0.093
Pdet-max (cm H2O)	0	51 (37:68)	48 (37:66)	56 (71:80)	0.104
	6	27 (26:28)	27 (26:28)	27 (26:34)	0.430
	∆0–6	23 (10:41)	22 (10:40)	26 (12:45)	0.231

Time codes: 0 = before injection; 6 = six months after injection; ∆_0–6 = absolute difference (∆_% = relative difference): value at six months after injection minus value before injection;

MCC, maximum cystometric capacity; PVR, post void residual; FSBF, First sensation of bladder filling; Qmax, maximum flow rate; Qave, average flow rate; Pdet at Qmax, detrusor pressure at maximum flow; Pdet-max, maximum detrusor pressure.

Data presented as medians (lower quartile: upper quartile). P = p-values for Mann-Whitney tests with a null hypothesis of no difference between iOAB and nOAB, to three decimal places.

In addition, no significant differences were noted between the iOAB and nOAB subgroups in terms of frequency, nocturia, urgency, and urgency incontinence, as well as in terms of the scores for ICIQ-OAB part A, ICIQ-OAB part B, ICIQ-LUTS-QoL part A and ICIQ-LUTS-QoL part B; before the treatment and six months after BoNT/A injection. The subgroups also did not differ significantly regarding the absolute or relative differences in all symptom and questionnaire scores (Tables 4 and 5, respectively).

Table 4.

Overactive bladder symptoms in female patients, before and six months after intradetrusor injection of botulinum neurotoxin type A, with idiopathic (iOAB) or neurogenic (nOAB) overactive bladder syndrome.

Symptoms	Time code	All patients	iOAB	nOAB	p
		(n = 160)	(n = 100)	(n = 60)
Frequency	0	3 (2:4)	3 (2:3)	3 (2:4)	0.157
(range: 0–4)	6	1 (0:2)	1 (1:2)	1 (0:3)	0.494
	∆0–6	1 (1:2)	1 (1:2)	1 (1:2)	0.593
Nocturia	0	3 (2:4)	3 (2:4)	3 (2:4)	0.750
(range: 0–4)	6	1 (1:2)	1 (1:2)	1 (1:3)	0.649
	∆0–6	1 (1:2)	1 (1:2)	1 (0:2)	0.382
Urgency	0	4 (3:4)	4 (3:4)	4 (3:4)	0.609
(range: 0–4)	6	2 (1:2)	2 (1:2)	2 (1:3)	0.298
	∆0–6	2 (1:2)	2 (1:2)	2 (1:2)	0.914
Urgency incontinence	0	3 (2:3)	3 (2:3)	3 (2:3)	0.614
(range: 0–4)	6	1 (1:2)	1 (1:2)	1 (1:2)	0.391
	∆0–6	1 (0:2)	1 (0:2)	1 (0:2)	0.346

For abbreviations and data presentation, see Table 3.

Table 5.

ICIQ-OAB and ICIQ-LUTSqol questionnaire responses from female patients, before and six months after intradetrusor injection of botulinum neurotoxin type A, with idiopathic (iOAB) or neurogenic (nOAB) overactive bladder syndrome.

Symptoms	Time code	All patients	iOAB	nOAB	p
		(n = 160)	(n = 100)	(n = 60)
ICIQ-OAB, part A	0	11 (10:13)	11 (10:13)	12 (10:14)	0.458
(range: 0–16)	6	6 (4:8)	5 (4:7)	6 (3:9)	0.476
	∆0–6	6 (4:7)	6 (4:7)	5 (3:8)	0.634
ICIQ-OAB, part B	0	29 (25:31)	29 (25:31)	29 (25:31)	0.885
(range: 0–40)	6	14 (5:24)	13 (5:22)	15 (6:29)	0.111
	∆0–6	12 (3:21)	13 (4:22)	9 (1:19)	0.084
ICIQ-LUTS-QoL, part A	0	57 (47:65)	57 (47:65)	57 (47:65)	0.907
(range: 0–76)	6	35 (25:49)	34 (25:47)	38 (26:52)	0.448
	∆0–6	20 (6:30)	25 (6:30)	18 (6:26)	0.328
ICIQ-LUTS-QoL, part B	0	140 (106:165)	140 (11:165)	140 (99:165)	0.782
(range: 0–190)	6	38 (8:110)	32 (8:101)	59 (9:127)	0.300
	∆0–6	85 (24:123)	90 (28:126)	65 (19:110)	0.210

ICIQ-OAB, international consultation on incontinence questionnaire-overactive bladder; ICIQ-LUTS-QoL, international consultation incontinence questionnaire-lower urinary tract symptoms-quality of life. For other abbreviations and data presentation, see Table 3.

To analyze the possible relationship between the response to BoNT/A treatment and BDNF polymorphism, the two subgroups of patients with the different OAB types were pooled into a single group. Given the low frequency of AA homozygotes, in further analyses, both women with the BDNF: rs6265 AA genotype were also pooled with the 43 GA heterozygotes. After this, a logistic regression analysis was conducted within the combined OAB group (iOAB+nOAB group), with a correction for OAB type.

There were no significant differences between GG homozygous women and women carrying at least one BDNF A allele (GA heterozygotes and AA homozygotes) in terms of all analyzed urodynamic parameters (Table 6). There were also no significant differences between OAB women homozygous for c.196G allele and patients carrying at least one BDNF mutated allele in the frequency of complete bladder emptying (PVR < 50 ml) before and 6 months after BoNT/A injection (55/115 = 47.8% versus 18/45 = 40.0%, p = 0.372 and 49/115 = 42.6% versus 17/45 = 37.8%, p = 0.577, respectively).

Table 6.

Urodynamic assessment of female patients, before and six months after intradetrusor injection of botulinum neurotoxin type A, in regard to BDNF: rs6265 genotype.

Variables	Time code	BDNF:rs6265 genotype		p
		GG (n = 115)	GA + AA (n = 43 + 2)
MCC (ml)	0	214 (160:244)	228 (171:244)	0.407
	6	345 (312:367)	356 (345:367)	0.073
	∆0–6	− 128 (− 191:− 80)	− 128 (− 178:− 107)	0.500
PVR (ml)	0	1 (0:31)	10 (0:36)	0.310
	6	3 (0:23)	8 (0:23)	0.670
	∆0–6	0 (− 3:15)	0 (− 8:28)	0.449
FSBF (ml)	0	54 (28:98)	56 (34:95)	0.836
	6	156 (134:157)	137 (134:157)	0.995
	∆0–6	− 85 (− 119:− 22)	− 81 (− 106:− 37)	0.856
Qmax (ml/s)	0	17 (16:18)	18 (16:19)	0.458
	6	14 (13:17)	14 (14:17)	0.730
	∆0–6	3 (0:5)	2 (1:4)	0.418
Qave (ml/s)	0	8 (8:9)	8 (8:9)	0.572
	6	8 (8:11)	8 (8:11)	0.840
	∆0–6	0 (− 1:1)	0 (− 2:1)	0.105
Pdet at Qmax (cm H2O)	0	37 (34:43)	39 (36:46)	0.274
	6	20 (19:21)	20 (19:20)	0.095
	∆0–6	17 (13:24)	18 (16:27)	0.105
Pdet− max (cm H2O)	0	51 (37:68)	48 (37:68)	0.673
	6	27 (26:28)	27 (26:28)	0.135
	∆0–6	23 (10:40)	22 (10:41)	0.989

P = p-values for OAB-type-adjusted logistic regression analyses with a null hypothesis of no difference between GG homozygotes and females with at least one A allele for the BDNF:rs6265 (GA or AA) polymorphism. For other abbreviations and data presentation, see Table 3.

GG homozygous women did not differ from women with at least one A allele in terms of overactive bladder symptoms (Table 7). In addition, there were no significant differences between BDNF GG homozygotes and women carrying at least one A allele in terms of the scores for ICIQ-OAB part A, ICIQ-OAB part B, ICIQ-LUTS-QoL part A, and ICIQ-LUTS-QoL part B; before treatment and six months after BoNT/A injection, as well as in absolute or relative differences in all questionnaire scores (Table 8).

Table 7.

Overactive bladder symptoms in female patients, before and six months after intradetrusor injection of botulinum neurotoxin type A, in regard to BDNF: rs6265 genotype.

Symptoms	Time code	BDNF:rs6265 genotype		p
		GG (n = 115)	GA + AA (n = 43 + 2)
Frequency	0	3 (2:4)	3 (2:4)	0.380
(range: 0–4)	6	1 (1:2)	1 (0:1)	0.043*
	∆0–6	1 (1:2)	2 (1:2)	0.294
Nocturia	0	3 (2:4)	3 (2:4)	0.712
(range: 0–4)	6	2 (1:2)	1 (1:2)	0.123
	∆0–6	1 (0:2)	1 (1:2)	0.242
Urgency	0	4 (3:4)	4 (3:4)	0.438
(range: 0–4)	6	2 (1:3)	2 (1:2)	0.133
	∆0–6	2 (1:2)	2 (1:2)	0.462
Urgency incontinence	0	3 (2:3)	3 (2:3)	0.791
(range: 0–4)	6	1 (1:2)	1 (1:2)	0.188
	∆0–6	1 (0:2)	1 (1:2)	0.364

*The difference lost statistical significance after applying correction for multiple comparisons. P = p-values for OAB-type-adjusted logistic regression analyses with a null hypothesis of no difference between GG homozygotes and females with at least one A allele for the BDNF:rs6265 (GA or AA) polymorphism. For other abbreviations and data presentation, see Table 3.

Table 8.

ICIQ-OAB and ICIQ-LUTSqol questionnaire scores in female patients with overactive bladder, before and six months after detrusor injection of botulinum neurotoxin type A, in regard to BDNF:rs6265 genotype.

Symptoms	Time code	BDNF:rs6265 genotype		p
		GG (n = 115)	GA + AA (n = 43 + 2)
ICIQ-OAB, part A	0	12 (10:13)	11 (10:13)	0.357
(range: 0–16)	6	6 (4:8)	5 (3:7)	0.054
	∆0–6	5 (3:7)	6 (4:8)	0.199
ICIQ-OAB, part B	0	30 (25:31)	28 (24:31)	0.334
(range: 0–40)	6	15 (5:27)	12 (6:20)	0.124
	∆0–6	12 (2:21)	13 (6:20)	0.241
ICIQ-LUTS-QoL, part A	0	57 (47:66)	57 (45:62)	0.357
(range: 0 to 76)	6	34 (23:50)	38 (28:45)	0.985
	∆0–6	19 (6:30)	21 (3:27)	0.519
ICIQ-LUTS-QoL, part B	0	143 (106:175)	139 (100:160)	0.406
(range: 0 to 190)	6	38 (8:116)	38 (16:73)	0.743
	∆0–6	81 (22:124)	89 (25:119)	0.815

P =  p-values for OAB type-adjusted logistic regression analyses with a null hypothesis of no difference between GG homozygotes and females with at least one A allele for the BDNF:rs6265 (GA or AA) polymorphism. For other abbreviations and data presentation, see Table 5.

Discussion

This report has focused on an association analysis between the BDNF: rs6265 polymorphism and the predisposition to OAB. The results of our study do not support the presence of a relationship between either BDNF: rs6265 genotypes or alleles in Polish women and the susceptibility to OAB, as well as to iOAB and nOAB when analyzed separately. In the present study, a historical pediatric cohort was used as the control group because of ethical considerations and the cost of genotyping. While this could be considered a potential limitation due to age difference between the OAB group and the controls, it should be emphasized that the frequencies of the minor BDNF: c.196 A allele, 14.7% in OAB patients and 16.7% in control females, were not only similar to the prevalence reported in other studies (14.2–20.2%) carried out in Poles^40–46 but also to the prevalence of 19.7% in Europeans from the 1000 Genomes Project⁴⁷. The BDNF: rs6265 genotype distributions, both in OAB women and in control females, also conformed to the expected Hardy-Weinberg equilibria. This further minimized the risk of population error and stratification. However, despite the analysis of 160 OAB patients and 359 controls, we assume that major limitations of our study might still have been a too small sample size and too small differences in the prevalence of the mutated c.196 A BDNF allele shown between our studied group and controls (14.7% versus 16.7%, respectively). Nevertheless, our findings are consistent with those reported by Ece et al. who also found no association between the BDNF: rs6265 polymorphism and the risk of primary monosymptomatic nocturnal enuresis in an analysis of 104 Turkish PMNE children and 140 control children (the frequency of BDNF: c.196 A allele being 11.0% or 15.0%, respectively) in an adequately powered sample (study power between 79% and 86%)³⁵.

Previous research on OAB genetics centered primarily around the control of bladder muscle activity. The results of studies dealing with the polymorphism of the ADRB3 gene, encoding the beta-3 adrenergic receptor protein and thus involved in the control of detrusor relaxation, are inconclusive, with most authors reporting negative findings^48,49. Similarly, the research on genes involved in the control of bladder smooth muscle contraction (ROCK2, GEF) yielded negative results⁵⁰. Reports on the pharmacogenetics of BoNT/A treatment in OAB are scarce, and studies of a few candidate genes have yielded mostly inconclusive results^{36,37,51–53}. Carle et al.⁵¹ and Pirazzini et al.⁵² did not find an association between primary resistance to BoNT/A and mutations in genes encoding synaptic vesicle glycoprotein 2 (SV2) or synaptosome-associated protein 25 (SNAP25). Meanwhile, Moreno-Mayordomo et al. demonstrated that the high efficacy of treatment with BoNT/A in patients with chronic migraine was related to the rs3781719 polymorphism in the CALCA gene (encoding calcitonin gene-related peptide 1) and to the rs222749 polymorphism in the TRPV1 gene (encoding transient receptor potential cation channel subfamily V member 1)⁵³. In our previous studies, we found no association between the rs4994 polymorphism in the ADRB3 gene and response to a single intradetrusor injection of BoNT/A in Polish women with OAB^36,37.

Our present study confirmed that BoNT/A injection is an effective and safe method for OAB treatment. Six months after intradetrusor injection of BoNT/A the quality of life in our patients had improved considerably, which is consistent with the evidence from previous studies^54–56. In addition, our study showed that a single intradetrusor injection of BoNT/A was equally effective in females with iOAB as in those with nOAB. Apart from pretreatment values of PVR and Pdet at Qmax, iOAB patients did not differ from those with nOAB in terms of baseline characteristics. The pretreatment values of PVR and Pdet at Qmax in our patients with nOAB were significantly higher than in women with iOAB, which is consistent with the results obtained by Ghalayini and Al-Ghazo in a prospective study of 14 patients with neurogenic detrusor overactivity and 16 patients with idiopathic detrusor overactivity, all resistant to anticholinergic treatment⁵⁷. According to Sahai et al., baseline Pdet-max > 110 cmH₂O might be a predictor of poor response to BoNT/A injection in OAB patients⁵⁸. In the present study, we found no significant differences in baseline Pdet-max values in patients with different OAB types (iOAB versus nOAB) and different BDNF genotype carriers (GG versus GA + AA). However, these groups also did not differ significantly in terms of treatment outcomes, and baseline Pdet-max > 110 cmH2O was found in only 5 out of 115 GG homozygotes (3 nOAB women and 2 women with iOAB).

As patients with iOAB and nOAB did not differ in the frequency distribution of either BDNF: rs6265 genotypes or alleles, the analysis for the association of this BDNF polymorphism with the response to treatment with BoNT/A was performed in a single pooled group, using a logistic regression model with a correction for OAB type. The analysis revealed no significant differences between patients with the reference BDNF genotype (GG) and those with at least one A allele (GA or AA) in terms of pre- and post-treatment values of all analyzed urodynamic variables or frequencies of OAB symptoms or scores in ICIQ-OAB and ICIQ-LUTSqol questionnaires.

Ghalayini and Al-Ghazo⁵⁷ and Sahay et al.^58–60 clearly indicated that a good response to intradetrusor injection of BoNT/A in OAB patients was related to a significant increase in maximum cystometric capacity (MCC) and to a significant decrease in Detrusor Pressure at Maximum Flow and Maximum Detrusor Pressure (Pdet at Qmax or Pdet-max, respectively). However, the latter authors also reported a significant increase in median Post Void Residuals (PVR), both in patients with idiopathic detrusor overactivity (from 15 to 80 ml) and in patients with neurogenic detrusor overactivity (from 15.0 to 62.5 ml) at 6 weeks after BoNT/A administration⁵⁷. In contrast, we did not record such significant changes in PVR volumes in either iOAB or nOAB patients (PVR median changes: iOAB: from 1 to 3 ml; nOAB: from 10 to 8 ml). In addition, no significant differences in PVR volumes prior to treatment and 6 months after BoNT/A injection were found in regard to BDNF polymorphism. Importantly, our study included a substantially larger sample of OAB patients (n = 160) than that conducted by the authors mentioned above (a total of 16 women and 14 men) and the response to BoNT/A was evaluated at 6 months rather than at 6 weeks post BoNT/A injection.

Several potential explanations exist for the lack of significant associations between the BDNF: rs6265 polymorphism and OAB pathophysiology or response to BoNT/A administration documented in the present study. First, it should be remembered that BDNF is one of many factors involved in OAB pathogenesis, and therefore, the genotypic effects might be too subtle or compensated for by other factors. This explanation is highly likely given that our findings are consistent with the results published previously by Ece et al. in a pediatric population³⁵. Second, considering low penetration of the analyzed polymorphism, our sample might be too small to capture significant between-genotype differences, if any. Thus, further adequately powered studies of this relationship are warranted, along with meta-analyses of pooled data. It also needs to be stressed that our hereby presented analysis did not include urinary BDNF level. Thus, it cannot be excluded that the BDNF: rs6265 polymorphism is involved in the pathogenesis of OAB indirectly, modulating urinary levels of BDNF that, as shown in some previous studies, might be a marker of OAB risk or therapeutic response^{17–25,29,30}. Therefore, another direction of future research could be an analysis of potential BDNF polymorphism – urinary BDNF – OAB pathogenesis cascade. Finally, one might consider verifying a potential role of the BDNF: rs6265 polymorphism as a predictor of response to other novel treatments for lower urinary tract dysfunction, such as sacral neuromodulation^61–63.

From a clinical standpoint, the results of the present study suggest that the BDNF Val66Met polymorphism is unlikely to have a large effect on OAB. While a small effect cannot be entirely ruled out, before completing any future studies, the directions of which have been mentioned above, the BDNF polymorphism should not be considered as a diagnostic or response marker in OAB.

In conclusion, the results of our present study do not support a link between the BDNF: rs6265 polymorphism and the susceptibility to overactive bladder in Polish women, or with efficacy in the reduction of OAB severity, with scores of ICIQ-OAB and ICIQ-LUTS-QoL questionnaires or with urodynamic response to intradetrusor injection of BoNT/A in these patients.

Author contributions

Conceptualization, S.C., D.K., J.K. and A.C.; methodology, S.C., A.B. and A.C.; validation, A.C. and J.S.C.; formal analysis, J.S.C.; investigation, S.C., K.L. and A.B.; resources, K.L. and K.G.; data cu-ration, S.C. and K.G.; writing—original draft preparation, S.C., K.L., A.B., D.K., J.K., K.G. and A.C.; writing—review and editing, J.S.C. and A.C.; supervision, S.C.; funding acquisition, K.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially (analysis of the control group) supported by the Program of the Polish Minister of Science and Higher Education “Regional Initiative of Excellence” in 2019–2022 (project no. 002/RID/2018/19).

Data availability

The data that support the findigns of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The study was conducted in accordance with the Declaration of Helsinki and approved by the bioethics committee of the Pomeranian Medical University in Szczecin (decision no. KB-0012/125/17 on November 16, 2017, and decision no. KB-0012/16/19 on January 14, 2019).

Consent to participate

Written informed consent was obtained from all OAB patients and parents of all children involved in the study.