Abstract：
OBJECTIVE: Tinnitus, the perception of sound without any external sound source, is a prevalent hearing health concern. Mounting evidence suggests that a confluence of genetic, environmental, and lifestyle factors can influence the pathogenesis of tinnitus. We hypothesized that alteration in DNA methylation, an epigenetic modification that occurs at cytosines of cytosine-phosphate-guanine (CpG) dinucleotide sites, where a methyl group from S-adenyl methionine gets transferred to the fifth carbon of the cytosine, could contribute to tinnitus. DNA methylation patterns are tissue-specific, but the tissues involved in tinnitus are not easily accessible in humans. This pilot study used saliva as a surrogate tissue to identify differentially methylated CpG regions (DMRs) associated with tinnitus. The study was conducted on healthy young adults reporting bilateral continuous chronic tinnitus to limit the influence of age-related confounding factors and health-related comorbidities.
METHODS: The present study evaluated the genome-wide methylation levels from saliva-derived DNA samples from 24 healthy young adults with bilateral continuous chronic tinnitus (> 1 year) and 24 age, sex, and ethnicity-matched controls with no tinnitus. Genome-wide DNA methylation was evaluated for > 850,000 CpG sites using the Infinium Human Methylation EPIC BeadChip. The association analysis used the Bumphunter algorithm on 23 cases and 20 controls meeting the quality control standards. The methylation level was expressed as the area under the curve of CpG sites within DMRs.The FDR-adjusted p-value threshold of 0.05 was used to identify statistically significant DMRs associated with tinnitus.
RESULTS: We obtained 25 differentially methylated regions (DMRs) associated with tinnitus. Genes within or in the proximity of the hypermethylated DMRs related to tinnitus included LCLAT1, RUNX1, RUFY1, NUDT12, TTC23, SLC43A2, C4orf27 (STPG2), and EFCAB4B. Genes within or in the proximity of hypomethylated DMRs associated with tinnitus included HLA-DPB2, PM20D1, TMEM18, SNTG2, MUC4, MIR886, MIR596, TXNRD1, EID3, SDHAP3, HLA-DPB2, LASS3 (CERS3), C10orf11 (LRMDA), HLA-DQB1, NADK, SZRD1, MFAP2, NUP210L, TPM3, INTS9, and SLC2A14. The burden of genetic variation could explain the differences in the methylation levels for DMRs involving HLA-DPB2, HLA-DQB1, and MUC4, indicating the need for replication in large independent cohorts.
CONCLUSIONS: Consistent with the literature on comorbidities associated with tinnitus, we identified genes within or close to DMRs involved in auditory functions, chemical dependency, cardiovascular diseases, psychiatric conditions, immune disorders, and metabolic syndromes. These results indicate that epigenetic mechanisms could influence tinnitus, and saliva can be a good surrogate for identifying the epigenetic underpinnings of tinnitus in humans. Further research with a larger sample size is needed to identify epigenetic biomarkers and investigate their influence on the phenotypic expression of tinnitus.
METHODS: The present study evaluated the genome-wide methylation levels from saliva-derived DNA samples from 24 healthy young adults with bilateral continuous chronic tinnitus (> 1 year) and 24 age, sex, and ethnicity-matched controls with no tinnitus. Genome-wide DNA methylation was evaluated for > 850,000 CpG sites using the Infinium Human Methylation EPIC BeadChip. The association analysis used the Bumphunter algorithm on 23 cases and 20 controls meeting the quality control standards. The methylation level was expressed as the area under the curve of CpG sites within DMRs.The FDR-adjusted p-value threshold of 0.05 was used to identify statistically significant DMRs associated with tinnitus.
RESULTS: We obtained 25 differentially methylated regions (DMRs) associated with tinnitus. Genes within or in the proximity of the hypermethylated DMRs related to tinnitus included LCLAT1, RUNX1, RUFY1, NUDT12, TTC23, SLC43A2, C4orf27 (STPG2), and EFCAB4B. Genes within or in the proximity of hypomethylated DMRs associated with tinnitus included HLA-DPB2, PM20D1, TMEM18, SNTG2, MUC4, MIR886, MIR596, TXNRD1, EID3, SDHAP3, HLA-DPB2, LASS3 (CERS3), C10orf11 (LRMDA), HLA-DQB1, NADK, SZRD1, MFAP2, NUP210L, TPM3, INTS9, and SLC2A14. The burden of genetic variation could explain the differences in the methylation levels for DMRs involving HLA-DPB2, HLA-DQB1, and MUC4, indicating the need for replication in large independent cohorts.
CONCLUSIONS: Consistent with the literature on comorbidities associated with tinnitus, we identified genes within or close to DMRs involved in auditory functions, chemical dependency, cardiovascular diseases, psychiatric conditions, immune disorders, and metabolic syndromes. These results indicate that epigenetic mechanisms could influence tinnitus, and saliva can be a good surrogate for identifying the epigenetic underpinnings of tinnitus in humans. Further research with a larger sample size is needed to identify epigenetic biomarkers and investigate their influence on the phenotypic expression of tinnitus.
摘要：
目标：耳鸣，没有任何外部声源的声音感知，是一个普遍的听力健康问题。越来越多的证据表明，遗传的融合，环境,生活方式因素可影响耳鸣的发病机制。我们假设DNA甲基化的改变，发生在胞嘧啶-磷酸-鸟嘌呤（CpG）二核苷酸位点的胞嘧啶处的表观遗传修饰，S-腺苷甲硫氨酸的甲基被转移到胞嘧啶的第五个碳原子上，可能会导致耳鸣。DNA甲基化模式是组织特异性的，但是与耳鸣有关的组织在人类中不容易接近。这项初步研究使用唾液作为替代组织来鉴定与耳鸣相关的差异甲基化CpG区(DMRs)。这项研究是针对报告双侧持续慢性耳鸣的健康年轻人进行的，以限制与年龄相关的混杂因素和与健康相关的合并症的影响。
方法：本研究评估了来自24名健康年轻成年人的唾液DNA样本的全基因组甲基化水平，这些成年人患有双侧持续慢性耳鸣（>1岁）和24岁，性别,和没有耳鸣的种族匹配的对照。使用Infinium人甲基化EPICBeadChip评估了>850,000个CpG位点的全基因组DNA甲基化。关联分析使用Bumphunter算法对23例和20例符合质量控制标准的对照进行分析。甲基化水平表示为DMRs内CpG位点的曲线下面积。使用0.05的FDR调整的P值阈值来鉴定与耳鸣相关的统计学显著的DMRs。
结果：我们获得了25个与耳鸣相关的差异甲基化区域（DMRs）。与耳鸣相关的高甲基化DMRs内或附近的基因包括LCLAT1，RUNX1，RUFY1，NUDT12，TTC23，SLC43A2，C4orf27（STPG2），EFCAB4B与耳鸣相关的低甲基化DMRs内或附近的基因包括HLA-DPB2，PM20D1，TMEM18，SNTG2，MUC4，MIR886，MIR596，TXNRD1，EID3，SDHAP3，HLA-DPB2，LASS3（CERS3），C10orf11(LRMDA),HLA-DQB1,NADK,SZRD1,MFAP2,NUP210L,TPM3、INTS9和SLC2A14。遗传变异的负担可以解释涉及HLA-DPB2，HLA-DQB1和MUC4的DMRs的甲基化水平的差异，表明在大型独立队列中需要复制。
结论：与有关耳鸣的合并症的文献一致，我们确定了与听觉功能有关的DMRs内或附近的基因，化学依赖性,心血管疾病,精神病,免疫疾病，和代谢综合征。这些结果表明，表观遗传机制可能影响耳鸣，唾液可以很好地替代人类耳鸣的表观遗传基础。需要更大样本量的进一步研究来鉴定表观遗传生物标志物并研究它们对耳鸣表型表达的影响。
方法：本研究评估了来自24名健康年轻成年人的唾液DNA样本的全基因组甲基化水平，这些成年人患有双侧持续慢性耳鸣（>1岁）和24岁，性别,和没有耳鸣的种族匹配的对照。使用Infinium人甲基化EPICBeadChip评估了>850,000个CpG位点的全基因组DNA甲基化。关联分析使用Bumphunter算法对23例和20例符合质量控制标准的对照进行分析。甲基化水平表示为DMRs内CpG位点的曲线下面积。使用0.05的FDR调整的P值阈值来鉴定与耳鸣相关的统计学显著的DMRs。
结果：我们获得了25个与耳鸣相关的差异甲基化区域（DMRs）。与耳鸣相关的高甲基化DMRs内或附近的基因包括LCLAT1，RUNX1，RUFY1，NUDT12，TTC23，SLC43A2，C4orf27（STPG2），EFCAB4B与耳鸣相关的低甲基化DMRs内或附近的基因包括HLA-DPB2，PM20D1，TMEM18，SNTG2，MUC4，MIR886，MIR596，TXNRD1，EID3，SDHAP3，HLA-DPB2，LASS3（CERS3），C10orf11(LRMDA),HLA-DQB1,NADK,SZRD1,MFAP2,NUP210L,TPM3、INTS9和SLC2A14。遗传变异的负担可以解释涉及HLA-DPB2，HLA-DQB1和MUC4的DMRs的甲基化水平的差异，表明在大型独立队列中需要复制。
结论：与有关耳鸣的合并症的文献一致，我们确定了与听觉功能有关的DMRs内或附近的基因，化学依赖性,心血管疾病,精神病,免疫疾病，和代谢综合征。这些结果表明，表观遗传机制可能影响耳鸣，唾液可以很好地替代人类耳鸣的表观遗传基础。需要更大样本量的进一步研究来鉴定表观遗传生物标志物并研究它们对耳鸣表型表达的影响。
