Eur Arch Otorhinolaryngol DOI 10.1007/s00405-015-3804-5

OTOLOGY

Phosphodiesterase 4D gene polymorphisms in sudden sensorineural hearing loss Chen-Yu Chien1,2,5,8 • Shu-Yu Tai1,3,6,10 • Ling-Feng Wang2,5,9 • Edward Hsi4,7 Ning-Chia Chang2,5,8 • Hsun-Mo Wang5,9 • Ming-Tsang Wu1,3,6,11 • Kuen-Yao Ho1,2,5

•

Received: 19 March 2015 / Accepted: 15 October 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract The phosphodiesterase 4D (PDE4D) gene has been reported as a risk gene for ischemic stroke. The vascular factors are between the hypothesized etiologies of sudden sensorineural hearing loss (SSNHL), and this genetic effect might be attributed for its role in SSNHL. We hypothesized that genetic variants of the PDE4D gene are associated with susceptibility to SSNHL. We conducted a case–control study with 362 SSNHL cases and 209 controls. Three single nucleotide polymorphisms (SNPs) were selected. The genotypes were determined using TaqMan technology. Hardy–Weinberg equilibrium (HWE) was tested for each SNP, and genetic effects were evaluated according to three inheritance modes. We carried out sex-specific analysis to analyze the overall data. All three

SNPs were in HWE. When subjects were stratified by sex, the genetic effect was only evident in females but not in males. The TT genotype of rs702553 exhibited an adjusted odds ratio (OR) of 3.83 (95 % confidence interval = 1.46–11.18) (p = 0.006) in female SSNHL. The TT genotype of SNP rs702553 was associated with female SSNHL under the recessive model (p = 0.004, OR 3.70). In multivariate logistic regression analysis, TT genotype of rs702553 was significantly associated with female SSNHL (p = 0.0043, OR 3.70). These results suggest that PDE4D gene polymorphisms influence the susceptibility for the development of SSNHL in the southern Taiwanese female population. Keywords Sudden sensorineural hearing loss
Phosphodiesterase 4D
Polymorphism

L.-F. Wang and K.-Y. Ho have equal contributions to this study. & Ling-Feng Wang [email protected] & Kuen-Yao Ho [email protected]

6

Department of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

7

Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

1

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

8

2

Department of Otorhinolaryngology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

Department of Otorhinolaryngology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

9

3

Department of Family Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

Department of Otorhinolaryngology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

10

4

Department of Medical Genetics, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

Department of Family Medicine, Kaohsiung Municipal TaTung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

11

5

Department of Otorhinolaryngology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, No. 100, Tzyou 1st Road, Kaohsiung 807, Taiwan

Department of Public Health, College of Health Sciences, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan

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Eur Arch Otorhinolaryngol

Introduction Sudden sensorineural hearing loss (SSNHL) is defined as a loss of at least 30 dB in 3 contiguous frequencies over a period of 3 days or less [1]. The incidence rates per 100,000 people in the Taiwan population are 8.85 for men and 7.79 for women [1]. The etiology and pathogenesis of SSNHL are not known, but the primary proposed causes include viral infection, vascular disease, and autoimmunity [1]. SSNHL is a complex multifactor disease that may include genetic factors, as it has been associated with heat shock protein 70 (HSP 70), factor V Leiden G1691A, prothrombin G20210A, and methylene-tetrahydrofolate reductase (MTHFR) C677T [2–4]. SSNHL, in patients with factor V Leiden, has been associated with life-threatening medical conditions such as leg and pulmonary venous thromboembolism and cerebral sinus venous thrombosis as reported by Lovato et al. [5]. Acquired and inherited cardiovascular risk factors have been associated with an increased risk of developing SSNHL [6]. A personal or familial history of cardiovascular events, including coronary insufficiency, myocardial infarction, stroke, and transient ischemic attack, have also been found to be more prevalent in patients with SSNHL than the control group [7, 8]. Phosphodiesterase 4D (PDE4D) (OMIM 600129) is a large gene, which spans [1.5 Mb region on chromosomal region 5q12 and has 22 exons [9]. Previous studies revealed that the PDE4D [9], factor V Leiden [10], and MTHFR gene [11] were associated with stroke [12]. Previous studies also revealed a significant association of rs702553 (SNP56) of PDE4D with ischemic stroke [13– 15]. A meta-analysis by Yoon et al. also found the most significant association of rs705223 (SNP56) with ischemic stroke [16]. PDE4D gene encodes phosphodiesterase 4D, which is a member of a large superfamily of cyclic nucleotide phosphodiesterases. PDE4D regulates the second messenger cyclic adenosine monophosphate (cAMP) signal transduction in various cell types including inflammatory, endothelial, and smooth muscle cells [17]. Decreased cAMP level enhances vascular smooth muscle cell proliferation and migration, the events representing atherosclerosis [9]. This dreadful complication of atherosclerosis can result in stroke and/or myocardial infarction [18]. PDE4D protein has been hypothesized to be involved in stroke risk through this atherosclerotic pathway [9, 19]. Manipulation of the PDE4D pathway has been exploited therapeutically in an experimental model of stroke [20]. Therefore, PDE4D gene polymorphisms may play a role in the pathogenesis of SSNHL. No data have been published on the relationship between PDE4D genetic polymorphisms

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and the risk of SSNHL. This study investigated whether PDE4D gene polymorphisms were risk factors for SSNHL in southern Taiwanese subjects. We conducted a case– control study to investigate the role of PDE4D single nucleotide polymorphisms (SNPs) in the development of SSNHL in a southern Taiwanese population.

Materials and methods Subjects We recruited 362 patients with the SSNHL and 209 controls at the Kaohsiung Medical University Hospital between October 2010 and July 2013. The diagnostic criteria of SSNHL included sensorineural hearing loss of at least 30 dB in three contiguous frequencies using a pure tone audiogram with an onset within 3 days [1]. Healthy volunteers without a history of hearing loss or any ear disorders were enrolled as controls. Information on demographic characteristics was collected. Audiometric tests, including pure tone audiometry, auditory brainstem-evoked responses, and computer tomography or magnetic resonance imaging (to exclude acoustic neuroma) were performed in SSNHL patients. The Institutional Review Board (IRB) of the Kaohsiung Medical University Hospital approved this study, and informed consent was signed by each subject. SNP selection and Genotyping Genomic DNA was extracted from peripheral blood using a standard method. Three SNPs were selected according to previous studies associated with stroke in southern Taiwan [13, 21]. The three SNPs included the SNP rs702553 (SNP56), rs966221 (SNP83) and rs2910829 (SNP87) of the PDE4D gene. Genotyping was performed using TaqMan technology (7500 Real Time PCR System, Applied Biosystems, Foster City, CA, USA), and reactions were performed in 96-well microplates in ABI 9700 thermal cyclers (Applied Biosystems, Foster City, CA, USA). Fluorescence was measured using an ABI 7500 Real-Time PCR System and analyzed with System SDS software version 1.2.3. Each subject was typed for all SNPs. Statistical analysis Continuous variables were analyzed using independent t tests, and the results were presented as means ± SD. The allele frequency was obtained using direct gene counting. The Hardy–Weinberg equilibrium (HWE) was examined in

Eur Arch Otorhinolaryngol

controls using the V2 test. Multivariate logistic regression analysis was performed to adjust for the effects of age, sex, smoking, diabetes mellitus, hypertension, and hyperlipidemia while assessing the genetic effects. We examined the effect of the minor allele of each SNP in three genetic models: dominant, additive, and recessive. We analyzed the sex-specific genetic effect by stratifying the subjects into male and female. Multivariate logistic regression analysis was used to determine the factors associated with SSNHL. The JMP software version 9.0 Stat View version 5.0 (SAS institute Inc.) for Windows was used for statistical analysis. Two-tailed p values \0.05 were considered significant. Ethical concerns The Institutional Review Board of the Kaohsiung Medical University Hospital approved this study (approval No. KMUH-IRB-990243), and informed consent was obtained from all subjects.

Results Study participants The demographic features of the study population are shown in Table 1. The mean age of the 362 cases (187 male and 175 female) was 51.1 ± 14.6 years, and the mean age of the 209 control volunteers (120 male and 89 female) was 34.8 ± 7.3 years. The age of SSNHL patients was older than that of the control group (p \ 0.001). The sex distribution was 1.07:1 (male–female) in SSNHL patients. The sex characteristics were not significantly different between the two groups. The prevalences of diabetes mellitus and hypertension were higher in the SSNHL patients than in the control group (p \ 0.001), although hyperlipidemia was not significant between the two groups. The prevalence of personal history of cardiovascular events was similar in SSNHL patients (10.8 %) Table 1 Baseline demographics of subjects Age (mean ± SD, year old)

and controls (6.7 %, p = 0.106). The familial history of cardiovascular events was significantly higher in SSNHL patients (33.4 %) than controls (18.2 %, p \ 0.0001, OR 2.26, 95 % CI 1.49–3.42). Single SNP results The distribution of PDE4D genotypes was in HWE among control subjects using p = 0.05 as the cutoff point. The distributions of genotypes and allele frequencies of the PDE4D SNPs are listed in Table 2. The adjusted odds ratios (ORs) were computed for the association analyses between PDE4D genotypes and SSNHL. The genotypic and allelic distributions were not significantly different between the SSNHL patients and control group for SNPs rs702553, rs966221, and rs2910829 (Table 2). Stratified by sex The SNP rs702553 rare allele T was significantly higher in the SSNHL patients compared to the control group in females (41 vs. 29 %) (p = 0.011) (Table 3). For rs702553 in females, after adjusting for age, smoking, diabetes mellitus, hypertension, and hyperlipidemia, the results indicated that genotype TT [adjusted odds ratio (OR) = 3.83, p = 0.006] and AT (adjusted OR 1.08, p = 0.821) carried a higher risk than the reference AA genotype (Table 3). Neither rs966221 nor rs2910829 was related to female SSNHL. Three genetic models (assuming the rare allele having a dominant, recessive, or additive effect) were used to evaluate the associations between PDE4D gene and SSNHL. A significant result was observed under the recessive model, which yielded an adjusted OR of 3.70 [95 % confidence interval (CI) 1.48–10.37, p = 0.004] for TT vs. AA ? AT of rs702553 after adjustment for age, smoking, diabetes mellitus, hypertension, and hyperlipidemia in females (Table 4). Compared with the A allele carriers (AA ? AT genotypes), the TT genotype conferred SSNHL Case (n = 362)

Control (n = 209)

p value

51.1 ± 14.6

34.8 ± 7.3

\0.001a

187 (51.7)

120 (57.4)

0.184b

Sex (n, %) Male

175 (48.3)

89 (42.6)

Smoking (n, %)

Female

63 (17.4)

52 (24.9)

Diabetes mellitus (n, %)

64 (17.7)

6 (2.9)

\0.001b

Hypertension (n, %)

103 (28.5)

20 (9.6)

\0.001b

Hyperlipidemia (n, %)

88 (24.3)

49 (23.4)

a

Independent t test

b

v2 test

0.035b

0.816b

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Eur Arch Otorhinolaryngol Table 2 Association of three single nucleotide polymorphisms (SNPs) of PDE4D gene and sudden sensorineural hearing loss (SSNHL) in overall study subjects

SNP

Overall, n (%) SSNHL Case

Control

p valuea

Adjusted OR(95 % CI)b, p valuea

0.284

1.00c

rs702553 Genotypes AA

133 (36.7)

90 (43.1)

AT

161 (44.5)

87 (41.6)

OR 1.07 (0.68–1.70), P = 0.761

TT

68 (18.8)

32 (15.3)

OR 1.55 (0.85–2.83), P = 0.151

Alleles A

427 (59)

267 (64)

T

297 (41)

151 (36)

TT TC

250 (69.1) 96 (26.5)

145 (69.4) 51 (24.4)

CC

16 (4.4)

13 (6.2)

T

596 (82)

341 (82)

C

128 (18)

77 (18)

0.103

rs966221 Genotypes 0.583

1.00c OR 1.27 (0.78–2.06), P = 0.336 OR 1.15 (0.45–2.92), P = 0.772

Alleles 0.753

rs2910829 Genotypes 0.627

1.00c

CC

254 (70.2)

149 (71.3)

CT

100 (27.6)

53 (25.4)

OR 1.09 (0.68–1.75), p = 0.731

TT

8 (2.2)

7 (3.3)

OR 0.78 (0.21–2.77), p = 0.694

C

608 (84)

351 (84)

T

116 (16)

67 (16)

Alleles

a

v2 test

b

Adjusted for age, sex (male; female), smoking, diabetes mellitus, hypertension, and hyperlipidemia

c

Reference group

an increased risk for SSNHL in females (OR 3.70, p = 0.004) but not in males (OR 0.84, p = 0.647) (Table 4). The multivariate logistic regression model yielded an adjusted OR of 3.70 (95 % CI 1.48–10.37, p = 0.0043) for the genotype TT vs. AA ? AT of rs702553 after adjustment for age, smoking, diabetes mellitus, hypertension, and hyperlipidemia in females (Table 5). No significant results for SNPs rs702553, rs966221 and rs2910829 were observed in any of the three genetic models in male SSNHL patients. The final multivariate regression model suggested that age and TT genotype of rs702553 were the major risk factors for female SSNHL (Table 5).

Discussion We investigated the relationship between three SNPs in the PDE4D gene and SSNHL in a southern Taiwanese population. Our results revealed a sex-differential effect of the PDE4D gene on SSNHL. When subjects were stratified by

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0.998

sex, the genetic effect was only evident in females but not in males. The significant result stemmed from the rs702553 (SNP56) polymorphism of the PDE4D gene, which indicates that the rare T allele significantly increases the risk for females SSNHL in southern Taiwan. The multivariate logistic regression model yielded an adjusted OR of 3.70 (95 % CI 1.48–10.37, p = 0.0043) for the genotype TT vs. AA ? AT of rs702553 after adjustment for age smoking, diabetes mellitus, hypertension, and hyperlipidemia in females. No significant differences existed between the SSNHL and control groups at SNP rs966221 and SNP rs2910829. However, SNP rs702553 has not been associated with hearing impairment before. Previous study revealed an association between PDE4D polymorphism and ischemic stroke among normotensive patients [13] in the SNP rs702553, but not in rs966221 or rs2910829 of PDE4D. Furthermore, significant results were observed under recessive models in this study, which yielded an adjusted OR of 3.70 (p = 0.004) for TT vs. AA ? AT of rs702553 after adjustment for age, smoking, diabetes mellitus,

Eur Arch Otorhinolaryngol Table 3 Association of three SNPs of PDE4D gene and SSNHL in sex-differential subjects (male and female) SNP

Male, n (%)

Female, n (%) b

SSNHL Case

Control

p valuea

Adjusted OR(95 % CI) , p valuea

SSNHL Case

Control

p valuea

Adjusted OR(95 % CI)b, p valuea

rs702553 Genotypes AA AT

66 (35.3) 87 (46.5)

46 (38.4) 49 (40.8)

TT

34 (18.2)

25 (20.8)

0.611

1.00c OR 1.08 (0.55–2.09), p = 0.827

67 (38.3) 74 (42.3)

44 (49.4) 38 (42.7)

OR 0.88 (0.39–1.99), p = 0.752

34 (19.4)

7 (7.9)

0.033*

1.00c OR 1.08 (0.56–2.08), p = 0.821 OR 3.83 (1.46–11.18), p = 0.006*

Alleles A

219 (59)

141 (59)

T

155 (41)

99 (41)

0.962

208 (59)

126 (71)

142 (41)

52 (29)

126 (72.0)

59 (66.3)

0.011*

rs966221 Genotypes TT 124 (66.3)

86 (71.7)

0.251

1.00c

0.632

1.00c

TC

55 (29.4)

26 (21.7)

OR 1.93 (0.97–3.95), p = 0.059

41 (23.4)

25 (28.1)

OR 0.75 (0.37–1.51), p = 0.416

CC

8 (4.3)

8 (6.6)

OR 0.71 (0.17–2.82), p = 0.634

8 (4.6)

5 (5.6)

OR 1.91 (0.49–7.83), p = 0.348

T

303 (81)

198 (82)

293 (84)

143 (80)

C

71 (19)

42 (18)

57 (16)

35 (20)

Alleles 0.643

0.334

rs2910829 Genotypes CC

127 (67.9)

87 (72.5)

CT

55 (29.4)

28 (23.3)

TT

5 (2.7)

5 (4.2)

C

309 (83)

202 (84)

T

65 (17)

38 (16)

0.423

1.00c

0.867d

1.00c

127 (72.6)

62 (69.7)

OR 1.51 (0.76–3.04), p = 0.235

45 (25.7)

25 (28.1)

OR 0.83 (0.42–1.62), p = 0.577

OR 0.61 (0.14–3.07), p = 0.608

3 (1.7)

2 (2.2)

OR 0.84 (0.08–8.78), p = 0.877

299 (85)

149 (84)

51 (15)

29 (16)

Alleles 0.617

0.602

* p \ 0.05 a

v2 test

b

Adjusted for age, smoking, diabetes mellitus, hypertension, and hyperlipidemia

c

Reference group

d

Fisher’s exact test

hypertension, and hyperlipidemia. This result revealed the tendency for the A-carrier subjects (AA ? AT) to exhibit a lower incidence of SSNHL than subjects with the TT genotype in females. This result indicated that the A-carrier is a protective factor for SSNHL in females. The T allele of rs702553 was found to be a risk factor for female SSNHL in our study. The sex-differential effect of the PDE4D gene had been reported in previous study. The SNP rs702553 was significant in young stroke males (OR 1.8, p = 0.025) but not in females (p = 0.27) [21]. Zee et al. also revealed the sex-

dependent effect of the PDE4D gene in ischemic stroke in the United States [15]. Sex-specific effect has been demonstrated in other genes. For example, the endothelial NO synthase gene (NOS3) affects vascular stiffness in females but not in males [22]. The solute carrier family 2 member 9 (SLC2A9) gene accounts for a larger proportion of uric acid variation in females than in males [23]. The SSNHL is a complex disease, and is affected by various etiologies and genetic risk factors. The gender discrepancy of association with SSNHL may be attributed to the complex etiology and the multifactorial

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Eur Arch Otorhinolaryngol Table 4 The association between PDE4D SNP rs702553 and SSNHL in overall study subjects and sex-differential subjects under recessive model SNP

Overall, n (%) SSNHL

Male, n (%)

Female, n (%)

Adjusted OR (95 % CI)b, p valuea

SSNHL Case

Control

Adjusted OR (95 % CI)c, p valuea

SSNHL Case

Control

Adjusted OR (95 % CI)c, p valuea

Case

Control

AA ? AT

294 (36.7)

177 (84.7)

1.00d

153 (81.8)

95 (79.2)

1.00d

141 (80.6)

82 (92.1)

1.00d

TT

68 (18.8)

32 (15.3)

OR 1.49 (0.86–2.61), p = 0.152

34 (18.2)

25 (20.8)

OR 0.84 (0.40–1.76), p = 0.647

34 (19.4)

7 (7.9)

OR 3.70 (1.48–10.37), p = 0.004*

rs702553 Genotypes

* p \ 0.05 a

v2 test

b

Adjusted for age, sex (male; female), smoking, diabetes mellitus, hypertension, and hyperlipidemia

c

Adjusted for age, smoking, diabetes mellitus, hypertension, and hyperlipidemia

d

Reference group

Table 5 Factors associated with SSNHL in female cases and controls Female SSNHL

Univariate

Multivariatec

Case (n = 175)

Control (n = 89)

p value

OR (95 % CI)

p value

Age (mean ± SD, year old)

50.9 ± 15.4

35.0 ± 6.9

\0.0001*,a

1.10 (1.07–1.14)

\0.0001*

Smoking (n, %)

9 (5.1)

6 (6.4)

0.5958b

1.24 (0.37–4.34)

0.7263

Diabetes mellitus (n, %) Hypertension (n, %)

28 (16.0) 42 (24.0)

3 (3.4) 5 (5.6)

0.0026*,b 0.0002*,b

1.18 (0.27–6.81) 1.07 (0.32–4.09)

0.8354 0.9072

Hyperlipidemia (n, %)

33 (18.9)

9 (10.1)

0.0663b

1.11 (0.39–3.25)

0.8465

PDE4D rs702553 [TT, n (%)]

34 (19.4)

7 (7.9)

0.0142*,b

3.70 (1.48–10.37)

0.0043*

* p \ 0.05 a

Independent t test

b

v2 test

c

Multivariate analyses was conducted by stepwise logistic regression adjusting for correlated covariates (age, smoking, diabetes mellitus, hypertension, hyperlipidemia, and PDE4D rs702553(TT))

background of the pathogenesis of SSNHL. A possible biological explanation is that sex hormones may regulate the expression of these candidate genes. The major limitation of this study is that it was only performed in subjects in Eastern Asia. Further studies among different ethnic groups are needed to further generalize our results. In addition, further functional analyses of SNP rs702553 in the PDE4D gene in SSNHL are warranted.

the SNP rs702553 may be a risk genotype for female SSNHL. Acknowledgments This work was supported by a Grant from the Kaohsiung Municipal Hsiao-Kang Hospital (Kmhk-102-016). Compliance with ethical standards Conflict of interest The authors have no conflict of interest to declare.

Conclusions The results of this study support the influence of genetic polymorphisms of PDE4D gene for the risk of SSNHL in a southern Taiwanese population. The TT genotype of

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