Combined Effects Of IL-6 C-572G And ICAM-1 469K/E Polymorphisms On Patients With Sudden Sensorineural Hearing Loss

BackgroundSudden sensorineural hearing loss is a medical emergency defined as sudden onset with no identified cause,and the loss of hearing of>20 decibels (dB) in at least 2 contiguous frequencies. The pathogenesis of SSNHL is not fully understood. Possible etiologies include viral infection, vascular occlusion, autoimmune diseases, inner ear pathology, lipid metabolic disorder, central nervous system anomalies. However, vascular impairment have been suggested as the final common pathway. Blood circulation in the inner ear is special. As the internal auditory artery (IAA) is a functional end artery with minimal collaterals, which supplies the cochlea and vestibular labyrinth, the labyrinth is particulary vulnerable to ischemia. The onset of SSNHL is similar to that of myocardial infarction and cerebral stroke,which are majorly caused by atherosclerosis. Accumulating evidences indicate that atherosclerosis is a chronic vascular inflammatory disorder and inflammatory reaction plays an essential role in the onset and progression of cardiovascular disease. Previous studies provided imaging findings of the involvement of inflammation in the inner ear in patients affected by SSNHL.Genetic alterations may influence the concentration and functional activity of corresponding products. Single Neocleotide Polymorphisms (SNPs) of inflammatory cytokines and adhesion molecules have been found in SSNHL. SSNHL is a multifactorial disorder with multiple risk factors contributing to its initiation and progression. Although the relationship between SNPs of some cytokines and SSNHL has been suggested, these genes might explain only a small part of the genetic component. It is necessary to give a clue to the right gene by analyzing other genes involved in inflammatory pathways.ICAM-1 is a member of the immunoglobin family and one of the adhesion molecules. Adhesion molecules are important in establishing the complex network of celluar adhesion events between platelets, leucocytes and endothelia cells, which link the processes of hemostasis, thrombosis and inflammation. ICAM-1 has been proved to be a biomarker of inflammation and endothelial dysfunction. Although previous study have investigated the plasma levels of ICAM-1 in patients with SSNHL, this is the first study to investigate the distribution of ICAM-1-469K/E polymorphism in patients with SSNHL.IL-6 is charaterized by pleiotropy, functions in inflammation and the maturation of B cells. IL-6 promotes atherosclerosis by the impairment of vascular endothelial cells, and the contribution of endothelial activation. Elevated serum concentration of circulation IL-6 have been reported in cardiovascular events and SSNHL. There is polymorphism in promoter region at position-572 of the IL-6 gene,which is C/G A significant association of SSNHL with the IL-6-572C/G polymorphism was found in Japanese patients. In addition, there is a reciprocal interaction between ICAM-1 and IL-6. Through the research on experimental animals, Romano et al. found that the IL-6 system induces the augmentation of ICAM-1 in leukocyte recruitment. Exposure of IL-6 induced endothellial cell activation. Consequently, the expression of ICAM-1 on the surface of activated endothellial cell was increased. After adding the IL-6 antibody or IL-6 receptor antibody into conditional medium, the role of IL-6 was blocked, and this resulted in the reduced expression of ICAM-1. Likewise, ICAM-1 can induce the expression of IL-6 by signal-regulated kinase and p38 mitogen-activated protein kinase pathways. The the interaction of IL-6 and ICAM-1 amplify inflammation, which could generate negative effect on lipid metabolism and microvascular perfusion failsure with the result of ischemia. The I/R (ischemia/reperfusion) injury, in turn, increases the expression of ILs(pro-and anti-inflammatory factors), generates oxygen free radicals and promotes neutrophil infiltration, which can significantly contribute to microvascular perfusion failure. Control animals revealed that ischemia of 30 min or longer induced irreversible damage to cochlea. The synergistic effect of IL-6 C-572G and ICAM-1469K/E Polymorphisms may have influence on regulating the reciprocal interaction between ICAM-1 and IL-6 that affect the circulation.ObjectiveWe hypothesized that the polymorphism in genes involved in inflammatory cytokines and adhesion molecules are associated with SSNHL. The aim of our study was to investigate the distribution and possible synergistic effect of ICAM-1 K469E and IL-6 C-572G polymorphism on SSNHL.MethodsPatients and controlsA total of 75 patients (39 male and 36 female patients, aged between 21 and 80 years old) were enrolled in this prospective study from June 2014 to June 2015. According to the clinical practice of SSNHL, patients with idopathic hearing loss of at least 20dB in 2 consecutive frequencies using a pure tone audiogram with the sudden onset within 3 day were included. Data on ototoxic mediation exposure, circulatory disease, clinical and family history were obtained via medical interview or self-reporting. Audiological examinations including ear microscopy, pure tone audiometry, tympanometry, auditory brainstem-evoked responses, stapedial reflexes were performed in all patients. Retrocochlear disease was ruled out by MRI. Patients with a history of familiar deafness, chronic otological history, and other diseases which are the causes of SSNHL were excluded. We calculated the hearing levels by pure tone audiometry at frequencies of 0.25Hz,1 kHz,2 kHz,4 kHz, and 8 kHz. The healthy subjects (86 male and 79 female, aged between 22 and 79 years old) without a history of SSNHL were derived from the same region. Informed consent was obtained from all patients.Blood samples and DNA extractionTwo milliliters of venous blood obtained from each participant was collected into EDTA coated tubes. Genomic DNA was extracted from leukocytes separated from venous blood, using DNA extraction kit (Sangon Biotech), and quantified by spectrophotometer.GenotypingDetection of SNPs rs5498 and rs1800796 were performed by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism (RFLP) analysis, then the restriction enzyme-digested PCR product were analyzed by 2% agarose gel electrophoresis, with visualization under UV light.Statistical AnalysesStatistical analysis was performed using SPSS software version 19.0. The analysis of continuous variables between two groups applied Student’s t test or Mann-Whitney test. Chi-square test or Fisher’s exact test was used to evaluate the frequency of allele and genotype between case and control group. Logistic regression for odds ratio (OR) were assessed to test the risk of SSNHL in subjects with polymorphism. The p-value was considered statistically significant when it equals or is less than 0.05.Results1.This case-control study consisted of 165 controls and 75 patients. Details including three lifestyle-related disease in all subjects shown that there were no significant differences between SSNHL cases and controls in sex, dyslipidemia and diabetes. The prevalence of hypertension and age were significantly higher in patients than in controls.2. The Chi square test revealed that the distribution of the genotype in case and control groups was consistent with Hardy-Weinberg equilibrium.3. It shows the distribution of genotypes and alleles between patients and controls. The E-allele frequency was higher in patients than in controls. However, no significant difference in ICAM-1469K/E genotype and allele frequencies were observed between the 2 groups. The prevalence of the E-allele in ICAM-1469K/E was significant higher in patients with pantonal hearing loss.4. For IL-6 C-572G, the frequency of the G allele was significant higher in subjects with SSNHL than in controls, the G-allele containing genotypes(CG and GG) were higher in cases than in controls, but failed to reach significant difference.5. We used a multiple logistic regression to obtain ORs and 95%CI in three genetic models. For the ICAM-1 rs5498 polymorphism, there was no significant difference in three genetic models. For the IL-6 rs1800796 polymorphism, the ORs was increased with an increase in the frequency of the minor allele, but it failed to reach significant difference. The ORs was 1.828 (95%CI,1.033-3.233)in dominant model after moderating variables.6. We then assessed combination effect of IL-6 C-572G and ICAM-1469K/E on SSNHL. The combined G+/E+alleles and G-/E+alleles associated with higher prevalence in patients than controls. There was no significant difference in distribution of other combined alleles. Multiple logistic regression was used in the analysis to obtain odds ratios with 95% CI, with adjustment for age, gender, three lifestyle-related disease hypertension, diabetes, and dyslipidemia in all 4 models. The genotypes carrying G allele of IL-6 and the combined alleles were considered as independent variable in these logistic regression models. Multivariable analysis showed that the p value under model 4 was 0.038. However, the G-/E-should not be considered as an independent risk factor but as a protective factor for SSNHL(OR= 0.432,95% CI:0.195-0.956). Instead, The genotypes carrying G allele of IL-6(CG and GG genotypes) were significantly associated with an increased risk for SSNHL. Interestingly,the IL-6-572G and ICAM-1469E alleles carriers showed a 1.7 fold increased risk for SSNHL(OR=3.122,95% CI:1.662-5.866) than subjects with the genotypes carrying G allele.Conclusions1. The Chi square test revealed that the distribution of the genotype in case and control groups was consistent with Hardy-Weinberg equilibrium.2. The results of this study revealed a positive association between IL-6 rs 1800796 polymorphism and SSNHL. In this study, the CG and GG genotypes showed higher level of SSNHL risk compared with those of the CC genotypes. Thus, the genotype carrying G allele might confer susceptibility to the development of SSNHL.3. The Chi square test revealed that the distribution of the genotype in case and control groups was consistent with Hardy-Weinberg equilibrium.4. No differences were observed in the ICAM-1-469K/E genotype and allele frequencies between cases and controls. Although the E-allele presented an increased frequency in patients, it was not significantly associated.5. Furthermore, we evaluated the combined effect of IL-6 C-572G and ICAM-1-469K/E in the development of SSNHL. Our results suggested that there is a reciprocal interaction between ICAM-1469K/E and IL-6 C-572G in patient with SSNHL:the IL-6 and ICAM-1 gene polymorphism may act synergistically to raise the risk of SSNHL. The greatest effect was observed among patients carrying IL-6 G and ICAM-1 E alleles.