Is Mitochondrial Mutation a Predisposing Factor for Tinnitus? A Single Case Study

Tinnitus is a highly prevalent and impactful condition with a multifactorial etiology.1 Significant epidemiological research indicates that nearly half of tinnitus cases cannot be attributed to identifiable causes.2 These unaccounted causes for tinnitus could suggest a potential genetic factor influencing the condition.3 Among the genetic factors, mutations in nuclear and mitochondrial DNA can result in alterations within the cell’s DNA, potentially leading to tinnitus.4Table 1: Audiometric test results. (Abbreviations: PTA-Pure Tone Audiometry; SRT-Speech recognition Threshold; SIS-Speech Identification cores; SPIN-Speech Perception in Noise; UCL-Uncomfortable loudness levels).Figure 1: Peak amplitude and latencies of LLR.Figure 2: mtDNA mutation at m.3394T>C.Studies that demonstrate pathogenic point mutations in mitochondrial DNA provide strong evidence of a genetic link to the occurrence of tinnitus. This study aims to establish a potential connection between the m.3394T>C mutation and chronic tinnitus. This mutation has been linked to a range of clinical symptoms, which encompass Leber’s Hereditary Optic Neuropathy (LHON), auditory and vestibular dysfunction, as well as various metabolic disorders.5 The study’s primary focus is to investigate the relationship between auditory aspects of this mutation, particularly in a case presenting with chronic tinnitus. CASE PRESENTATION A 24-year-old female patient reported to the audiology outpatient department of our institution complaining of reduced hearing sensitivity and a ringing sensation accompanied by a sensation of blocking in the right ear. The sudden and progressive onset of the symptom occurred one year prior to reporting to the department. There was no history of other relevant otological complaints, and no medications/systemic as reported. The patient was recommended for detailed audiological and tinnitus evaluations by the otorhinolaryngologist. Written consent was obtained from the participant who enrolled in the research funded by the institute in which they underwent a genetic evaluation. Ethical approval was obtained from the Institutional Ethical Committee. AUDIOLOGICAL ASSESSMENT The pure tone audiometry thresholds were assessed using the Inventis Piano Diagnostic Audiometer at octave frequencies ranging from 250Hz to 8kHz. The extended high-frequency (EHF) audiometry thresholds at 8kHz, 10kHz, 12.5kHz, and 14kHz were also obtained. Middle ear function was confirmed through immittance audiometry using the GSI Tympstar Pro. Acoustic ipsilateral and contralateral reflexes, including those for the tone decay test, were also evaluated using the GSI Tympstar Pro. Transient evoked otoacoustic emissions (TEOAE) were measured using 85dB SPL click stimuli, with a pass criterion set at a 6dB signal-to-noise ratio at any three consecutive test frequencies. To investigate neural synchrony, site of lesion testing was conducted through auditory brainstem response (ABR) audiometry. Responses were elicited using a 90dBnHL click stimulus at two different rates (11.1/second and 90.1/second), delivered and controlled by the Intelligent Hearing System. Additionally, higher auditory system functioning was assessed by recording late latency responses (LLR) using a click stimulus at 70dBnHL. In the tinnitus evaluation, psychoacoustical assessment was performed using the audiometer. The subjective evaluation involved the use of the Tinnitus Handicap Inventory (THI).6 GENETIC EVALUATION DNA Isolation and Sequencing A blood sample was collected from the patient and DNA was isolated by the phenol-chloroform method.7 PCR amplification of the complete mtDNA genome was carried out using primers for a total volume of 10µl reaction mixture, having 1µl genomic DNA; 0.5µl dNTP; 1µl PCR buffer; 0.1µl Taq polymerase; 0.4µl primers (both forward and reverse); and 7.04µl deionized water with cycling conditions of 94°C for two minutes, 95°C for one minute, 58°C for 45 seconds, 72°C for two and a half minutes, and final extension at 72°C for seven minutes for 35 cycles.8 Fluorescent sequencing was done using the BigDye Terminator v3.1 Cycle Sequencing Kit and fluorescent amplimers were detected through the ABI 3500 Genetic Analyzer. Sequence data analysis was done with SeqScape v3 software and mutations were scored using the Revised Cambridge Reference Sequence (NC_012920). OUTCOME The pure tone audiometry thresholds revealed bilateral hearing sensitivity within normal limits (thresholds within 15dBHL) at octave frequencies ranging from 250Hz to 8kHz. The EHF audiometry thresholds at 8kHz, 10kHz, 12.5kHz, 14kHz, and 16kHz also found that the obtained thresholds were less than or equal to 20dBHL, suggesting no significant hearing loss at high-frequency regions. The audiometric findings such as pure tone average, speech recognition thresholds, speech identification scores, speech perception in noise scores, and uncomfortable loudness levels have been demonstrated in Table 1. The normal middle ear functioning was confirmed using the immittance audiometry with bilateral “A” type tympanogram and the presence of ipsilateral and contralateral acoustic reflexes within normal sensation levels, which was further suggestive of no significant cochlear or retro-cochlear pathology up to the level of the lower brainstem on both sides. The reflex decay and tone decay test showed negative results suggestive of no significant adaptation. The TEOAE results indicated normal and healthy cochlear outer hair cells functioning in both ears. ABR exhibited the absolute latencies, inter-peak and interaural latencies, and absolute amplitudes of I, III, and Vth peaks were within normal limits. It was apparent that absolute latencies and amplitudes of P1, N1, P2, and N2 were within normal limits and no abnormalities were observed in the LLR (Figure 1). THI total score obtained was 80, which fell into “catastrophic” severity according to the THI scoring chart. The psychoacoustical evaluation results revealed tinnitus was closely matched at 125Hz narrow band noise at 20dB SL with a negative residual inhibition. The genetic evaluation results revealed a mutation in m.3394 T > C (Figure 2). DISCUSSION In this study, we conducted a clinical and genetic evaluation of an individual presenting with chronic tinnitus. Specific mitochondrial DNA mutations are associated with various neurological and audiological dysfunctions, with hearing loss being a prominent characteristic of these mtDNA auditory abnormalities.9 Additionally, certain mitochondrial point mutations have been reported to be associated with tinnitus.4,10 The findings revealed a mutation in the ND1 gene of mitochondria, specifically a m.3394T>C mutation. Earlier research has linked this mutation to a genetic condition called LHON with various clinical symptoms.11 These symptoms encompass optic-related issues and auditory dysfunctions including tinnitus and vestibular issues.5,12 Pre-existing genetic findings postulated that a decrease in translation efficiency due to mitochondrial mutation can result in cellular damage.13 Analogously, there is evidence that suggests metabolic damage caused by cell dysfunction induced by mutant DNA can result in physiological changes in the inner ear and the auditory pathways within the central auditory system.14 Hence, the mitochondrial mutation m.3394T>C may serve as a significant indicator for identifying individuals who are at risk of experiencing heightened neural damage due to various factors, not limited to optic-related issues but also encompassing auditory factors. This hypothesis may provide a plausible explanation for the physiological manifestation of tinnitus and could indicate a predisposition to hearing loss and other auditory-related dysfunctions.15 This data currently lacks the evidence to establish a direct association with a specific gene and to delve into its role as a causative or predisposing factor for tinnitus within the context of mitochondrial inheritance. Further research is essential to comprehensively investigate this gene across clinical, genetic, biochemical, and molecular dimensions, providing a more comprehensive understanding of this relationship. Exploring the pathways that link mitochondrial mutations to hearing impairments and uncovering the factors that contribute to the variability in symptom expression could facilitate the identification of novel therapeutic targets in the future. CONCLUSION The client underwent a detailed audiological evaluation including EHF audiometry and LLR apart from routine audiometric tests like PTA, immittance audiometry, OAE, and ABR. All the test findings were within normative limits, suggestive of no auditory pathology/organic hearing loss, apart from chronic tinnitus perception. The findings of this case study highlight the possibility of mitochondrial mutation resulting in tinnitus, and the need for further efforts to explore the causes of tinnitus and its potential risk factors.