Between January 2009 and April 2014, 190 patients with SSHL, referred to the Departments of Otorhinolaryngology Head and Neck Surgery at Kaihua People's Hospital (Quzhou, China) and Hangzhou First People's Hospital (Hangzhou, China), were selected, and included 108 males and 82 females with an average age of 38.5±4.8 years. A total of 210 age- and gender-matched healthy subjects were selected, including 115 males and 95 females with an average age of 38.9±5.3 years. Pure-tone audiometry was performed and tobacco smoking and alcohol consumption status were recorded in all participants. The selection criteria were based on the Sudden Sensorineural Hearing Loss Diagnosis and Treatment Guidelines published by the Chinese Medical Association Archives of Otolaryngology Head and Neck Surgery Branch (21). The diagnostic standards were as follows: SSHL occurring in a few minutes, hours or within 3 days; non fluctuating sensorineural hearing loss (categorized into mild, moderate severe or even complete deafness), sensorineural hearing loss of 20 dB or more over three contiguous audiometric frequencies; unknown cause due to systemic or local factors; accompanying with tinnitus, ear blockage sensation, and non-recurrent dizziness, nausea and vomiting; no other cranial nerve injury with the exception of eighth cranial nerve injury (http://d.wanfangdata.com.cn/Periodical_zhebyhk200608003.aspx). The study protocol was approved by the Institutional Review Board of the Kaihua People's Hospital (Quzhou, China). Written informed consent was signed by each subject.

A commercially available blood DNA purification kit (Beijing CWBio Co., Ltd., Beijing, China) was used to extract genomic DNA from blood clots. The eluted DNA solution was collected and stored at −20°C.

A UV 260 spectrophotometer (Shimadzu Corp., Kyoto, Japan) was used to measure the absorbance at wavelengths of 260 and 280 nm. The Lambert-Beer law was used to calculate sample concentration based on the c = A260/(ε × b) equation, where ε is the molar absorption coefficient, b is the optical path length and c is the molar concentration. The A260/A280 ratio was used to determine the sample purity. DNA concentration was adjusted at >15 ng/µl and the purity of DNA was between 1.6 and 1.9.

SNPs of the human GRHL2 gene on chromosome 8 (8q22.3) were retrieved and the data packet was downloaded from NCBI-dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and HapMap (http://SNP.cshl.org/cgi-Perl/gbrowse/haPmaP27_B36/)databases. Haploview 4.2 (http://www.broad.mit.edu/mpg/haploview/) statistical software was used to select the tag SNPs of GRHL2 and the parameters were set for Han Chinese in Beijing with a minor allele frequency (MAF) >10%, r2>0.8, D'=1. The base sequences of the selected site were suitable for primers designed for PCR amplification. Three tag SNPs termed rs611419, rs10955255 and rs6898650, were selected to stand for 100% SNP sites of the GRHL2 gene (Fig. 1). The loci of the three SNPs is present in Fig. 2.

The PCR amplification primers for the three SNPs were designed using Assay Designer 3.1 software (Sequenom, San Diego, CA, USA) and their feasibility was based on the following criteria: i) Primers and the templates should be complementary; ii) stable dimers or hairpins between primers should be avoided; iii) DNA mismatch with the template at non-target sites should be avoided. Upstream and downstream primers for PCR reaction should strictly comply with the design principles of primers. Three primers are shown in Table I.

PCR-RFLP was used to determine the SNP genotype. PCR reaction conditions were as follows: 5 min initial denaturation at 94°C, 36 cycles of 30 sec denaturation at 94°C, 40 sec annealing at 60°C and 45 sec extension at 72°C, followed by 7 min extension at 72°C. The amplification products were isolated and identified by agarose gel electrophoresis at a voltage of 120 V for 30 min. PCR products (15 µl) were digested with shrimp alkaline phosphatase and the restriction enzyme ExoI at 60°C for 37 min and at 75°C for 15 min, respectively. The resulting restriction fragments were electrophoresed in 2% agarose gels and visualized under UV light. An ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used to compare the resulting restriction fragments and PCR products to identify the genotype.

SPSS 17.0 statistical software package (SPSS, Inc., Chicago, IL, USA) was applied to analyze the data. Hardy-Weinberg equilibrium was used to confirm the sample representation in the population. Data are expressed as the mean ± standard deviation and χ² test was used to compare group differences. Logistic regression analysis was used to calculate the odds ratios and 95% confidence interval of each genotype was used to represent the relative risk. P<0.05 was considered to indicate a statistically significant difference.

Table II shows the profiles of the SSHL group and the control group. The auditory threshold in the SSHL group (36.6±10.5 dB) was significantly higher than the control group (13.2±3.0 dB) (P<0.01). No significant differences in age, gender, smoking status and drinking status were found between the SSHL group and the control group.

Table III describes the basic information of rs611419, rs10955255 and rs6898650. rs611419 is located in the 5′ region with the A allele and T allele. rs10955255 is located in intron 8 with the G allele and A allele. rs6898650 is located in the 3′UTR with the C allele and T allele. χ² goodness-of-fit test was used to detect the genotype frequency distribution of rs611419, rs10955255 and rs6898650 in the SSHL group and the control group, and the results demonstrated that the three sites in the two groups conformed to the Hardy-Weinberg equilibrium (all P>0.05).

Table IV shows the results from logistic regression regarding the risk of SSHL. rs611419 of the GRHL2 gene may be a protective factor for SSHL (AT+TT vs. AA: OR=0.63, 95% CI=0.41–0.98, P=0.038). Similarly, rs10955255 site polymorphisms may reduce the risk of SSHL (AA vs. GG: OR=0.54, 95% CI=0.31–0.95, P=0.32; GA+AA vs. GG: OR=0.58, 95% CI=0.38–0.89, P=0.012). However, genotype and allele frequencies of rs6989650 demonstrated no statistical differences in the SSHL group and the control group (P>0.05).

In order to examine the interaction between these polymorphisms, the three polymorphisms were combined for the analysis. There was a marked association between the combined genotypes and SSHL risk (P=0.035). Subjects carrying 3–8 variant alleles demonstrated a lower SSHL risk compared with subjects carrying 0–2 variant alleles (OR=0.59, 95% CI=0.36–0.96, P=0.034; Table V).

Stratification analysis based on age, gender, smoking status and drinking status was conducted to analyze the effect of the combined genotypes of the three SNPs on SSHL risk. In individuals who consumed alcohol, subjects carrying 3–8 variant alleles were more resistant to SSHL compared with subjects carrying 0–2 variant alleles (OR=0.40, 95% CI=0.21–0.76, P=0.004). However, no significant differences were found between the frequency of combined genotypes and age, gender and smoking status (all P>0.05; Table VI).