Correlation Of MtDNA^CD4977 In Hair Shaft And Hearing Loss In Presbycusis

Objective:This study was to evaluate the correlation of mitochondrial DNA4977bp common deletion (mtDNACD4977) in hair shaft and the severity of hearing loss in individuals with presbycusis and to clarify the meaning of obtaining mitochondrial DNA damage of hair shaft in the pathogenesis of presbycusis.Methods:1. Case Selection: From June2013to November2013, in Otolaryngology Head and Neck Surgery Clinic of Provincial Hospital affiliated to Shandong University, we collected samples in patients of presbycusis who are in search of treatment through questionnaires and rigorous hearing screening programs (pure tone audiometry and tympanogram). Inclusion criteria: Subjects must be bilateral, sensorineural hearing loss; subjects with bilateral hearing at the same frequency differences within10dB; pure tone audiometry thresholds increased by26dB or more of each subject; tympanometry test showed no other significant diseases which can cause hearing threshold increased.Detailed drug history, family history, past and current medical history are included in our survey. There is a clear exclusion of other causes of hearing loss disease attenders.2. The extraction of mitochondrial DNA in hair shaft:we collected4-6hair shafts of each subject included in the study and used Chelex-100method to extract mitochondrial DNA in hair shaft. 3.Ordinary PCR: Ordinary PCR validated mitochondrial DNA of hair shaft extracted successfully.4. Nested-PCR: The state of mitochondrial DNA and the ratios of mitochondrial DNA4977bp deletion in hair shaft is identified by using nested PCR5. Sequencing of the nested PCR products: Mitochondrial DNA deletion fragments amplified by Nested-PCR were sequenced to further verify the deletion fragments of mitochondrial DNA4977bp by ABI3730automatic sequencer.6. Real-time PCR Assay:The degree of mitochondrial DNA4977bp deletion and the relationship between mitochondrial DNA4977bp deletion mutation rate and the degree of hearing impairment were observed by Real-time PCR Assay.Results:1.87individuals with presbycusis (from60to83years of age) and95additional normal hearing controls (from63to81years of age) were selected based on strict audiometric criteria. We collect their4-6hair shafts in all cases.2. Ordinary PCR:135-bp fragment of the mitochondrial DNA could be amplified in all samples. Ordinary PCR validated mitochondrial DNA of hair shaft extracted successfully.3. Nested-PCR: The618bp fragment of mitochondrial DNA4977bp deletion could be amplified by the first-PCR in positive samples. DNA samples without deletions could not be amplified restriction fragment of about5kb due to time limits and Taq enzyme function restriction. The first-PCR positive samples were confirmed by second-PCR;396-bp amplified fragments were observed. In the presbycusis group, a total of59/87cases (67.82%) were detected to have the positive mitochondrial DNA4977bp deletion on hair shaft. Of those87cases, the mitochondrial DNA4977bp deletion was found in22/43cases (51.16%) with mild-to-moderate hearing loss.25/31cases (80.65%) with moderate-to-severe, severe hearing loss were indicated to have the mitochondrial DNA4977bp deletion.12/13cases (92.31%) with profound deafness were detected to have the mtDNACD4977(Fig.1B). In age-matched individuals with normal hearing,8/95cases (8.42%) were found to have the mitochondrial DNA4977bp deletion using nested PCR. In total,67.82%of the hair shafts was positive in mitochondrial DNA4977bp deletion. Using Pearson correlation coefficients analysis, a statistically significant difference was identified between the presence of mitochondrial DNA4977bp deletion and hearing loss (r=0.858; P<0.050). By the use of Chi-square, statistically significant differences of mitochondrial DNA4977bp deletion were indicated among all groups,(F=47.145; P<0.050).4. Sequencing of the nested PCR products:The presence of the mitochondrial DNA4977bp common deletion was identified by sequencing the nested PCR products generated.5. Real Time PCR Assay: The common deletion mean level in part of the specimens and auditory thresholds of mild-to-moderate, moderate-to-severe and severe, profound deafness hearing loss, age-matched individuals with normal hearing are11.97±4.12,19.75±5.29,33.68±10.30,4.91±4.16respectively. This difference in CD levels reached statistical significance in all groups (P<0.050). A trend toward increasing levels of the CD with more severe hearing loss was also observed (P<0.050). Furthermore, there was evidence for a significant association between the CD level and hearing loss based on audiometric thresholds at8kHz (r=0.778, P<0.050) and all ranges of frequency (r=0.858, P<0.050).Conclusions：1. Mitochondrial DNA4977bp deletion are present in hair shaft of Presbyacusis and age-matched individuals with normal hearing.2. The rate of mitochondrial DNA4977bp deletion in Presbyacusis is significantly higher than the normal hearing elderly population.3. The percentage of mitochondrial DNA4977bp deletion in hair shaft increases along with severity of hearing loss.