Prognostic impact of gene polymorphisms in patients with idiopathic sudden sensorineural hearing loss

Abstract

Objective: To investigate whether genetic polymorphisms (single-nucleotide polymorphism [SNPs]) have a prognostic influence on hearing recovery after standardized corticosteroid therapy.

Methods: A total of 192 gene samples from idiopathic sudden sensorineural hearing loss (SSNHL) patients registered in the Intractable Inner Ear Disease Gene Bank were enrolled and, as the candidate genes, 16 SNPs from 13 genes were selected for this study. Fischer’s exact test was used to compare allele frequencies in each SNP between the patients with good hearing recovery and patients with poor hearing recovery.

Results: The SNPs of the GSR gene (rs2251780 and rs3779647) and NOS3 gene (rs1799983) were associated with treatment outcome (p < .05). Furthermore, there was a marginal correlation between the SNP of the NR3C1 gene (rs4912910) and treatment outcome (p = .05).

Conclusions: The results of this study indicate that the analysis of genetic factors might make it possible to predict the treatment outcome, at least in part, in patients with idiopathic SSNHL.

Chinese abstract

目的: 探讨基因多态性 (单核苷酸多态性[SNPs]) 是否对标准化皮质类固醇治疗后的听力恢复有预后性影响。

方法: 难治性内耳疾病基因库登记的特发性突发感觉神经性听力损失 (SSNHL) 患者的192个基因样本被纳入研究, 并从13个基因中选择的16个SNP作为候选基因。 将Fischer精确检验用于比较听力恢复良好的患者和听力恢复差的患者之间的每个SNP中的等位基因频率。

结果: GSR基因 (rs2251780和rs3779647) 和NOS3基因 (rs1799983) 的SNPs与治疗结果具有相关性 (p <0.05) 。此外, 在NR3C1基因 (rs4912910) 的SNP和治疗结果 (p = 0.05) 之间存在边际相关性。

结论: 这项研究的结果表明, 遗传因素的分析, 至少是部分的, 对于特发性SSNHL患者, 会使预测治疗结果成为可能。

Keywords:

• Sudden sensorineural hearing loss
• genetic polymorphisms
• SNPs
• treatment outcome
• GSR
• NOS3
• NR3C1

Introduction

Idiopathic sudden sensorineural hearing loss (SSNHL) is one of the diseases specified by the Japanese government as an ‘intractable disease’ resulting from an unidentifiable cause and without a clearly established treatment, and entailing a considerably high risk of disability.

Many hypotheses have been put forward to explain the etiology of idiopathic SSNHL, including viral infection induced inflammation, ischemic events, and autoimmune responses. However, the pathological cause remains uncertain.

Recently, disease susceptibility genes have been identified for common diseases, such as diabetes mellitus, bronchial asthma, and cerebral infarction. We are now constructing a gene bank of intractable inner ear diseases including idiopathic SSNHL in collaboration with other institutions associated with the Sudden Deafness Research Committee of the Ministry of Health and Welfare, Japan. We examined gene associations with idiopathic SSNHL using samples collected for the above-mentioned gene bank, and our results suggested that SOD1 rs4998557 could be associated with susceptibility to idiopathic SSNHL in the Japanese population [1]. Furthermore, the relevance of some genes to idiopathic SSNHL, such as MTHFR [2,3], PRKCH [4], CFH [5], and LTA [6], has also been reported in the literature.

Not only the etiology of idiopathic SSNHL, but also the hearing prognosis in individual cases with idiopathic SSNHL remains uncertain. Among the various treatment regimens, systemic corticosteroids have been the most widely used since the randomized placebo-controlled trial by Wilson et al. [7]. Several clinical prognostic factors associated with idiopathic SSNHL have been reported, such as age, initial hearing level, time from onset to treatment start, and symptom of vertigo/dizziness; however, there have been no reports on genetic analysis as a prognostic factor for idiopathic SSNHL.

The aim of the present study was to investigate whether genetic polymorphisms have prognostic influence on hearing recovery after standardized corticosteroid therapy.

Material and methods

Cases

The gene samples (n = 192) from idiopathic SSNHL patients registered in the Intractable Inner Ear Disease Gene Bank Japan were enrolled. Idiopathic SSNHL was defined according to the criteria established by the Sudden Deafness Research Committee of the Ministry of Health and Welfare, Japan (1973). Details of the criteria are shown in Table 1.

Table 1. Criteria for the diagnosis of sudden deafness.

Main symptoms

1. Sudden onset of hearing loss; patient can say clearly when it appeared

2. Sensorineural hearing loss, usually severe

3. Unknown cause

Accessory symptoms

1. May be accompanied by tinnitus

2. May be accompanied by vertigo, nausea, and/or vomiting without recurrent episodes

3. No cranial nerve symptoms other than from the eighth nerve

Definite: all of the above criteria

Probable: main symptoms 1 and 2

We performed gene association analysis with the patient clinical data collected for the gene bank. Evaluation of hearing level was performed at 250, 500, 1000, 2000, and 4000 Hz. The clinical data for the 192 cases are shown in Table 2. In this study, we included only those patients who received initial treatment within 1 month from onset.

Table 2. Clinical data for the 192 cases.

Patient number	192
Age (average year)	56.4 ± 14.3
Gender (M:F)	89:92
Affected ear (R:L:Bil)	89:90:2
Symptom
Tinnitus (%)	87.8
Vertigo (%)	39.9
Initial pure tone average (PTA) (average dB)	73.9 ± 22.3
Duration from onset to treatment (day)	5.5 ± 6.4

Ethics statement

The study protocol for DNA sampling of the patients and controls was reviewed and approved by the Ethics Committee of each collaborative institution, and written informed consent was obtained from all subjects.

SNP selection and genotyping

As the candidate genes, 16 single-nucleotide polymorphisms (SNPs) from 13 genes were selected for this study (Table 3) as follows. (1) As oxidant stress is considered to be one of the mechanisms of inner ear injury, 13 SNPs from 10 genes related to the oxidative stress cascade and protection against oxidative stress were analyzed. (2) As an adrenal cortical steroid is used for medical treatment in general, 3 SNPs from 3 of the steroid hormone receptor genes were also included.

Table 3. Selected candidate 13 SNPs.

Gene	SNPs	Minor allele	MAF of control (%)	Gene function
(1) Oxidative stress-related genes
SOD1	rs4998557	A	45.6	Converts superoxide anion into H2O2
	rs1041740	T	39.4
GSTP1	rs1695	G	8.8	Conjugation of glutathione with xenobiotics
GCLM	rs7549683	T	21.2	Glutamate-cysteine ligase modifier subunit
CAT	rs769217	T	49.1	Catalyzes breakdown of H2O2 to H2O and O2
SOD2	rs4880	G	9.7	Converts superoxide anion into H2O2
CYBA	rs3794622	T	23.0	Superoxide production and phagocytosis
GSR	rs2251780	T	18.2	Reduces oxidized glutathione
	rs2253409	G	14.8
	rs3779647	T	37.2
MPO	rs7208693	A	14.3	Produced strong oxidant, hypochlorous acid
PON1	rs662	T	28.4	Hydrolyzes specific oxidized lipids
NOS3	rs1799983	T	6.8	Modulates flow-mediated vasodilation
GPX	rs1800668	T	10.2	Catalyzes breakdown of H2O2 to H2O and O2
(2) Steroid hormone receptors and inflammation-related genes
NR3C1	rs4912910	A	40.7	Glucocorticoid receptor
NR3C2	rs2070951	C	30.5	Mineralocorticoid receptor
ESR1	rs2234693	C	42.0	Estrogen receptor

The SNPs were selected mainly by reference to previous reports. Some of the SNPs were selected from the list of SNPs/genes available in the NCBI database and using the search program of the LD block of SNPs, using the SNP Browser Software (Applied Biosystems, Foster City, CA).

Real-time PCR using a TaqMan probe (Applied Biosystems) was used for the typing of gene polymorphisms, with reactions performed in 96-well microplates using the StepOnePlus Real-Time PCR System (Applied Biosystems). Fluorescence was measured and analyzed with System SDS software, which uses an advanced multicomponent algorithm to calculate the distinct signal contribution of each allele of a marker.

Statistical analysis

In this study, the cases were divided into two groups: those with good hearing recovery, including a complete recovery and remarkable improvement, and those with poor recovery, including a slight improvement and no change, based on the recovery criteria defined by the Ministry of Health and Welfare, Japan (Table 4). A Fischer’s exact test was then used to compare allele frequencies in each SNP between the two groups. Odds ratios were calculated with 95% confidence intervals. A p value of less than .05 was considered statistically significant.

Table 4. Hearing improvement criteria for SSHNL as defined by the Ministry of Health and Welfare, japan.

Hearing improvement status	Criteria
Complete recovery	All five frequencies on final audiograms are 20 dB or less, or improvement to the same degree of hearing in the unaffected side
Marked improvement	Pure tone average (PTA) improvement ≧30 dB
Slight improvement	10 dB ≦ PTA improvement <30 dB
No change	PTA improvement <10 dB

PTA: arithmetic mean of the five frequencies (250, 500, 1000, 2000, and 4000 Hz).

Results

The call rate in the SNP typing by this method was 99.1%. Hardy-Weinberg equilibrium (HWE) was tested using a χ² test and no SNPs showed any significant deviation from the HWE (p < .05). In this study, we used case data for the analysis that not only presented the results of polymorphism genotyping but also recorded treatment outcomes. Genotyping results for each SNP are summarized in Table 5. The SNPs of the GSR gene (rs2251780 and rs3779647) and NOS3 gene (rs1799983) were associated with treatment outcome (p < .05). Furthermore, there was a marginal correlation between the SNPs of the NR3C1 gene (rs4912910) and treatment outcome (p = .05) (Figure 1).

Figure 1. Details of the SNPs with statistically significant correlations between genotype and treatment outcome. The vertical axis shows the number of minor alleles, so that ‘0’ means homozygous of major allele, ‘1’ means heterozygous, and ‘2’ means homozygous of minor allele. (A) A significant difference was observed in the dominant model study of the GSR gene (rs2251780) (p = .02, OR = 2.56, 95% CI 1.16–5.66). (B) A significant difference was observed in the recessive model study of the GSR gene (rs3779647) (p = .03, OR = 0.39, 95% CI 0.17–0.91). (C) A significant difference was observed in the dominant model study of the NOS3 gene (rs1799983) (p = .02, OR = 2.13, 95% CI 1.11–4.10). D) An almost significant difference was observed in the recessive model study of the NR3C1 gene (rs4912910) (p = .05, OR = 0.38, 95% CI 0.15–0.97).

Table 5. Association results for genotyping analysis in SD patients with good recovery or poor recovery.

		Treatment outcome			Results (p value)
Gene (SNPs)	Genotype of SNPs (allele)	Good recovery (n)	Poor recovery (n)	Rate of poor outcome	Dominant	Recessive
SOD1 (rs4998557)	Major homozygous (GG)	14	21	0.60	0.69	0.86
	Heterozygous (GA)	28	49	0.64
	Minor homozygous (AA)	17	32	0.65
SOD1 (rs1041740)	Major homozygous (CC)	28	54	0.66	0.52	1.00
	Heterozygous (CT)	24	36	0.60
	Minor homozygous (TT)	7	12	0.63
GSTP1 (rs1695)	Major homozygous (AA)	46	75	0.62	0.45	1.00
	Heterozygous (AG)	11	25	0.69
	Minor homozygous (GG)	1	2	0.67
GCLM (rs7549683)	Major homozygous (GG)	40	56	0.58	0.12	0.09
	Heterozygous (GT)	17	37	0.69
	Minor homozygous (TT)	0	6	1.00
CAT (rs769217)	Major homozygous (AA)	20	33	0.62	0.86	0.11
	Heterozygous (AG)	31	44	0.59
	Minor homozygous (GG)	8	25	0.76
SOD2 (rs4880)	Major homozygous (TT)	48	78	0.62	0.55	0.55
	Heterozygous (TC)	10	23	0.70
	Minor homozygous (CC)	1	1	0.50
CYBA (rs3794622)	Major homozygous (CC)	38	61	0.62	0.50	0.33
	Heterozygous (CT)	18	33	0.65
	Minor homozygous (TT)	2	8	0.80
GSR (rs2251780)	Major homozygous (CC)	49	67	0.58	0.02	0.30
	Heterozygous (CT)	10	31	0.76
	Minor homozygous (TT)	0	4	1.00
GSR (rs2253409)	Major homozygous (GG)	44	73	0.62	0.57	0.55
	Heterozygous (GC)	13	25	0.66
	Minor homozygous (CC)	0	3	1.00
GSR (rs3779647)	Major homozygous (CC)	23	46	0.67	0.51	0.03
	Heterozygous (CT)	21	44	0.68
	Minor homozygous (TT)	15	12	0.44
MPO (rs7208693)	Major homozygous (CC)	24	43	0.64	0.87	1.00
	Heterozygous (CA)	14	23	0.62
	Minor homozygous (AA)	21	36	0.63
PON1 (rs662)	Major homozygous (CC)	25	51	0.67	0.52	0.64
	Heterozygous (CT)	25	40	0.62
	Minor homozygous (TT)	9	13	0.59
NOS3 (rs1799983)	Major homozygous (GG)	37	43	0.54	0.02	1.00
	Heterozygous (GT)	22	57	0.72
	Minor homozygous (TT)	0	1	1.00
GPX (rs1800668)	Major homozygous (CC)	19	46	0.71	0.14	0.33
	Heterozygous (CT)	7	8	0.53
	Minor homozygous (TT)	1	0	0.00
NR31C (rs4912910)	Major homozygous (GG)	23	41	0.64	1.00	0.05
	Heterozygous (GA)	23	50	0.68
	Minor homozygous (AA)	12	9	0.43
NR3C2 (rs2070951)	Major homozygous (GG)	47	94	0.67	0.27	0.16
	Heterozygous (GC)	0	1	1.00
	Minor homozygous (CC)	8	7	0.47
ESR1 (rs2234693)	Major homozygous (TT)	17	43	0.72	0.09	0.28
	Heterozygous (TC)	28	52	0.65
	Minor homozygous (CC)	13	15	0.54

Bold value represents p values under .05.

Discussion

In the present study, we analyzed the relationship between the genotype of the genes associated with oxidative stress or steroid hormone receptors and idiopathic SSNHL prognosis.

In recent years, several studies have reported on the relationship between oxidative stress and various disorders including some inner ear disorders such as noise-induced hearing loss [8], drug-induced ototoxicity [9], and age-related hearing loss [10]. We found that polymorphisms in the GSR and NOS3 genes were related to prognosis in patients with idiopathic SSNHL. Glutathione reductase (GR) is an enzyme that catalyzes the reduction of glutathione disulfide to the reduced form glutathione, which is a critical molecule in resisting oxidative stress and maintaining the reducing environment of the cell. Although no articles have been published on the correlation between GR and hearing disorders, GR deficiency was reported to be a rare cause of enzymopenic hemolytic anemia, and a partial GR deficiency was also reported to be a cause of an unstable hemoglobinopathy, whereas riboflavin supplementation improved glutathione metabolism and ameliorated hemolysis in these patients [11]. In our previous report, significant differences in allele frequency were observed in glutathione-S-transferase Pi 1 (GSTP1) between idiopathic SSNHL patients and control subjects as a result of the first-stage analysis. Glutathione-S-transferase enzymes catalyze the conjugation of glutathione to xenobiotic substrates and other compounds for the purpose of detoxification. In this way, the antioxidative reaction associated with the glutathione pathway was found to be correlated with the development and prognosis of idiopathic SSNHL.

Nitric oxide (NO) is a free radical, and is an important cellular signaling molecule involved in many physiological processes such as vasodilation, inhibition of platelet aggregation, immune response, and neurotransmission. Nitric oxide synthases (NOSs) are a family of enzymes catalyzing the production of NO from L-arginine. There are three distinct genes encoding NOS in mammals: neuronal NOS (nNOS or NOS1), cytokine-inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). NOS3 is primarily responsible for the generation of NO in the vascular endothelium, and NO produced by eNOS plays a number of crucial roles in the regulation of vascular tone. To date, a large number of studies have shown that polymorphisms in the NOS3 gene affect susceptibility to diseases such as hypertension [12], diabetes mellitus [13], and several autoimmune diseases including Behçet’s disease [14]. The G894T (rs1799983) polymorphism in the endothelial nitric oxide synthase (eNOS/NOS3) gene has been implicated in susceptibility to essential hypertension [12], and a decreased frequency of the NOS3 (rs1799983) GG genotype and an increased frequency of the GT genotype was found in patients with Behçet’s disease [14]. In addition, the same NOS3 polymorphism was significantly associated with a risk of idiopathic SSNHL; the OR for the NOS3 polymorphism and idiopathic SSNHL risk was 2.108 (CI: 1.343–3.309) with adjustment for age and sex [15]. In our present study, the NOS3 (rs1799983) GT and TT genotypes were related to a poor prognosis in idiopathic SSNHL patients, thus it seemed that genotyping of the NOS3 polymorphism would be meaningful as not only the cause of idiopathic SSNHL but also as a prognostic factor.

Our results suggested that there was a marginal correlation between the SNP of the NR3C1 gene (rs4912910) and treatment outcome (p = 0.05). NR3C1 (nuclear receptor subfamily 3, group C, member 1) is the receptor to which cortisol and other glucocorticoids bind. Corticosteroids had been used as a general treatment according to a past epidemiological survey in Japan [16], and 162 of the 173 patients recorded as having used corticosteroids in this study received intravenous or/and intratympanic corticosteroid. In recent years, there have been several reports on whether some disorders treated with corticosteroids generally showed a correlation between the genotype of NR3C1 gene polymorphisms and disease onset or response to corticosteroids treatment. Mohamed et al. reported that the frequencies of NR3C1 646 C > G genotypes and alleles differed significantly between asthmatic patients and controls [17]. Krupoves et al. reported that variations in the GR/NR3C1 gene are associated with corticosteroid resistance and dependency in pediatric-onset Crohn’s disease [18]. In addition, inter-individual variability was observed when the corticosteroids were administered to patients with inflammatory bowel disease and other chronic inflammatory diseases [19]. Treatment efficacy in the patients with idiopathic SSNHL also differs by patient, thus the results from this study might make it possible to predict the outcome of corticosteroid treatment.

Some limitations to the present study should be considered when interpreting its findings. Although we chose to analyze 16 gene polymorphisms, we considered a p value of less than 0.05 to be statistically significant without statistical correction; for example, a Bonferroni correction. Furthermore, we did not analyze other genetic polymorphisms such as macrophage migration inhibitory factor (MIF), which has previously been reported to be associated with response to glucocorticoid treatment in SSNHL patients [20]. We need to register more idiopathic SSNHL patients for this gene bank, and further studies are needed to investigate the association between prognostic factors and gene polymorphisms in idiopathic SSNHL.

Conclusions

In this study, we examined the association between polymorphisms in genes related to the oxidative stress cascade or steroid hormone receptors and treatment prognosis in the patients with idiopathic SSNHL. We found that gene polymorphisms in the GSR and NOS3 genes were related to prognosis in patients with idiopathic SSNHL. Furthermore, there was a marginal correlation between the gene polymorphism in the NR3C1 gene and treatment outcome. The results of this study indicate that the analysis of genetic factors might make it possible to predict, at least in part, treatment outcomes in patients with idiopathic SSNHL.

Acknowledgements

This study was supported by a Health and Labour Sciences Research Grant for Acute Profound Deafness Research Committee of the Ministry of Health, Labour and Welfare, Japan (http://www.mhlw.go.jp/english/) (K. O.), Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labour and Welfare, Japan (http://www.mhlw.go.jp/english/) (S. U.), and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (http://www.mext.go.jp/english/) (S. U.).

We thank the participants of the Intractable Inner Ear Disease Gene Bank Japan: Dr. Kaoru Ogawa (Keio University School of Medicine), Dr. Makito Okamoto (Kitasato University School of Medicine), Dr. Ken Kitamura (Tokyo Metropolitan Geriatric Hospital), Dr. Kiyofumi Gyo (Ehime University School of Medicine), Dr. Hiroaki Sato (Iwate Medical University), Dr. Tsutomu Nakashima (Nagoya University, Graduate School of Medicine), Dr. Satoshi Fukuda (Hokkaido University Graduate School of Medicine), Dr. Kunihiro Fukushima (Okayama University Graduate School of Medicine), Dr. Akira Hara (University of Tsukuba), and Dr. Tatsuya Yamasoba (University of Tokyo), for providing samples from their patients.

Disclosure statement

The authors alone are responsible for the content and writing of the paper. The Shinshu University Conflict of Interest Committee also approved the study.

Additional information

Funding

This study was supported by a Health and Labour Sciences Research Grant for Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labour and Welfare, Japan (S.U.). and Ministry of Education, Science and Culture of Japan (S.U.).