⁴Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea

⁵Department of Ophthalmology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea

Corresponding author: Byung Yoon Choi Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Bundang Hospital, Seoul National University College of Medicine, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620, Korea Tel: +82-31-787-7406, Fax: +82-31-787-4057, Email: choiby@snubh.org

Received 2024 July 4; Revised 2024 August 5; Accepted 2024 August 6.

Abstract

Objectives.

FDXR encodes mitochondrial ferredoxin reductase, which is associated with auditory neuropathy spectrum disorder (ANSD) and optic atrophy. To date, only two studies have described FDXR-related hearing loss. The auditory rehabilitation outcomes of this disease entity have not been investigated, and the pathophysiological mechanisms remain incompletely understood. Here we report a hearing-impaired individual with co-segregation of the FDXR variant and post-synaptic type ANSD, who underwent cochlear implantation (CI) with favorable outcomes. We suggest a possible pathophysiological mechanism of adult-onset ANSD involving mitochondrial dysfunction.

Methods.

A 35-year-old woman was ascertained to have ANSD. Exome sequencing identified the genetic cause of hearing loss, and a functional study measuring mitochondrial activity was performed to provide molecular evidence of pathophysiology. Expression of FDXR in the mouse cochlea was evaluated by immunohistochemistry. Intraoperatively, electrically evoked compound action potential (ECAP) responses were measured, and the mapping parameters were adjusted accordingly. Audiological outcomes were monitored for over 1 year.

Results.

In lymphoblastoid cell lines (LCLs) carrying a novel FDXR variant, decreased ATP levels, reduced mitochondrial membrane potential, and increased reactive oxygen species levels were observed compared to control LCLs. These dysfunctions were restored by administering mitochondria isolated from umbilical cord mesenchymal stem cells, confirming the pathogenic potential of this variant via mitochondrial dysfunction. Partial ECAP responses during CI and FDXR expression in the mouse cochlea indicate that FDXR-related ANSD is post-synaptic. As a result of increasing the pulse width during mapping, the patient’s CI outcomes showed significant improvement over 1-year post-CI.

Conclusion.

A novel FDXR variant associated with mitochondrial dysfunction and post-synaptic ANSD was first identified in a Korean individual. Additionally, 1-year post-CI outcomes were reported for the first time in the literature. Excellent audiologic results were obtained, and our results reiterate the correlation between genotype and CI outcomes in ANSD.

Keywords: FDXR; Postlingual; Post-Synaptic; Auditory Neuropathy; Optic Atrophy; Mitochondria; Cochlear Implantation; Auditory Rehabilitation

INTRODUCTION

Hearing and visual impairment rarely co-occur due to a shared genetic etiology [1-3]. However, the ferredoxin reductase (FDXR) gene is a common etiology of these conditions, and alterations in FDXR have been associated with auditory neuropathy spectrum disorder (ANSD) and optic atrophy (OMIM #617717) [4]. Other common etiologies include mutations in the OPA1 and TMEM126A genes [1,2]. FDXR is the most recently reported of these genes; the first study implicating FDXR in hearing loss was published in 2017, and only one other article regarding FDXR-related hearing loss has been published since [5]. Thus, the phenotypic manifestations of this condition, including auditory rehabilitation outcomes, have not been well described. Moreover, there has been no description of the outcomes of cochlear implantation (CI) in patients with FDXR-related hearing loss.

FDXR encodes a mitochondrial ferredoxin reductase involved in the biosynthesis of iron-sulfur (Fe-S) clusters and heme formation [6]. A previous study reporting the first case of FDXR-related hearing loss confirmed the critical role of Fe-S biogenesis in the function of auditory and optic neurons. It also presented indirect evidence of mitochondrial iron overload in ARH1-null mutation (arh1∆) yeast cells expressing the human mutant protein, where Arh1 is the ortholog of human ferredoxin reductase [4]. Furthermore, increased mitochondrial reactive oxygen species (ROS) production was observed in fibroblasts from individuals affected by FDXR-related hearing loss. However, direct assessments of mitochondrial functions other than ROS production have not been performed for this disease entity.

ANSD, which is associated with FDXR mutation, is a distinct type of hearing loss characterized by absent auditory brainstem response (ABR) and normal otoacoustic emissions, with poor speech discrimination given the pure tone audiometry threshold [7]. There exist several etiologies for ANSD, among which alterations in OTOF are the most common [8]. CI has been a mainstay treatment for successful auditory rehabilitation in several, if not all, types of ANSD [9,10]. Recently, OTOF-related auditory neuropathy (DFNB9) has become the first target for successful inner ear gene therapy [11]. However, given the paucity of literature regarding FDXR-related ANSD, auditory rehabilitation data are limited for this disease entity, and CI outcomes have never been reported.

In the study, we report the first Korean pedigree with FDXR-related ANSD and present the audiologic outcomes of CI in this disease entity for the first time in the literature. We also provide evidence for mitochondria-related pathogenesis based on a direct assessment of mitochondrial function in control- and mutant-derived lymphoblastoid cell lines (LCLs) and further suggest mitochondrial administration as a potential treatment for this disease.

MATERIALS AND METHODS

The human subjects research in this study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (No. IRB-B-1007-105-402), and written informed consent was obtained from all subjects.

Subject and clinical features

A 35-year-old woman (SB1113-1825) presented with progressive hearing loss on the left side since childhood and on the right side, which had started 2 years ago. She particularly complained of difficulty hearing in noisy environments. History-taking revealed that visual impairment, more severe on the left side, had been present since childhood. Audiologic tests showed no response on both sides in the ABR and normal responses on both sides in the distortion product otoacoustic emissions (DPOAE) and transient evoked otoacoustic emissions (TEOAE) tests. These findings were consistent with a diagnosis of ANSD (Fig. 1). Intraoperative electrically evoked compound action potential (ECAP) thresholds were measured in every channel for the subject under an automated measurement mode of neural response telemetry, as previously described [9,10,12,13]. Preoperative and postoperative evaluations of auditory rehabilitation were performed using the speech recognition test and CAP score, as previously described [14,15]. An ophthalmologic consultation was conducted to investigate the involvement of the eye.

Fig. 1.

Pedigree, audiologic tests, and Sanger chromatograms. (A) The pedigree demonstrated a sporadic or autosomal recessive inheritance pattern. (B) A pure-tone audiogram (PTA) showed asymmetric sensorineural hearing loss, and auditory brainstem response (ABR) testing resulted in no response at 90 dB in both ears. (C) DPOAE and TEOAE showed normal responses in both ears. (D) The Sanger chromatogram identified a homozygous variant only in the proband. R, right; L, left; DPOAE, distortion product otoacoustic emissions; TEOAE, transient evoked otoacoustic emissions.

Genetic diagnosis and generation of LCLs

Genomic DNA samples from the blood of the proband and her siblings were collected and analyzed for genetic diagnosis. Initially, 11 recurrent variants of five deafness genes were screened as described elsewhere [16-19]. However, these did not identify potential candidate variants to explain the hearing loss in the pedigree. Subsequently, we proceeded with exome sequencing (ES) and narrowed down the candidate variants using bioinformatics filtering steps [20]. The potential causative variant identified through the above steps was confirmed by Sanger sequencing and was checked for co-segregation with the phenotype in the pedigree. Whole blood in the EDTA (ethylenediaminetetraacetic acid) bottle from the proband was sent to Seoul Clinical Laboratories in Gyeonggi-do, South Korea, for the generation of LCLs.

Cell culture

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were obtained through the primary culture of the umbilical cord from a healthy pregnant woman who provided informed consent. The study was approved by the Public IRB designated by the Ministry of Health and Welfare, Korea (IRB No. P01-202002-31-008). UC-MSCs were cultured in Minimum Essential Medium Eagle Alpha Modification (α-MEM; HyClone) supplemented with 10% fetal bovine serum and 10 ng/mL basic fibroblast growth factor (FGF-2; CHA Meditech Co.).

Isolation of mitochondria from UC-MSC

UC-MSCs were used at passage 7 for mitochondria preparation. Cells were harvested from culture flasks, depressurized in SHE buffer (0.25 M sucrose, 20 mM HEPES [pH 7.4], 2 mM EGTA, 0.1% defatted bovine serum albumin [BSA]) using nitrogen cavitation (Parr Instrument) [21], and then centrifuged at 2,000 ×g for 10 minutes at 4 °C to remove cellular debris and nuclei. The supernatant was subsequently centrifuged at 12,000 ×g for 15 minutes at 4 °C to pellet the mitochondria. The pellet was washed twice by resuspension in 500 μL SHE buffer followed by centrifugation at 20,000 ×g for 10 minutes at 4 °C. The final pellet was resuspended in 100 μL of suspending buffer and kept on ice until use. Isolated mitochondria were quantified by determining protein concentrations using a bicinchoninic acid assay. The treatment of mitochondria was as follows: 1.0×10⁵ LCLs were seeded in 24-well plates and cultured overnight, followed by the administration of 10 mg of the isolated mitochondria.

Measurement of intracellular ATP

Intracellular ATP concentration was measured using a luciferin-luciferase reaction with CellTiter-Glo Luminescent reagent (Promega), following the manufacturer’s recommendations. A total of 5×10³ LCLs were seeded into a 96-well white tissue culture plate and incubated with PN-101 for 24 hours. After washing with DPBS, 100 μL of CellTiter-Glo reagent (Promega) was added to each well. The LCLs were then incubated for 10 minutes at room temperature on an orbital shaker to induce cell lysis. The luminescence signal was measured using a Synergy HTX multi-mode reader (BioTek), and the data were analyzed using Gen5 3.09 software. The study was performed in triplicate to ensure consistent results. All data are presented as the percentage of control.

Intracellular ROS measurements

ROS production in cell lysates was evaluated using CM-H2DCFDA (Invitrogen) according to the supplier’s instructions. Briefly, 50 µL of cell lysate was treated with 1 µM CM-H2DCFDA at 37 °C for 60 minutes. After staining with CM-H2DCFDA, ROS production was measured using a Synergy HTX multi-mode reader (BioTek), and the data were analyzed using Gen5 3.09 software. Relative CM-H2DCFDA fluorescence intensities were normalized to Hoechst 33342. The study was performed in triplicate. All data are presented as the percentage of control.

Measurement of mitochondrial membrane potential

The mitochondrial membrane potential (MtMP) was measured by the fluorescence of the potential-dependent TMRE (tetramethylrhodamine ethyl ester). Briefly, cells were incubated with 500 nM TMRE at 37 °C for 30 minutes, protected from light. After staining with TMRE, fluorescence was measured using flow cytometry. Cells were collected, suspended in phosphate-buffered saline (PBS), and analyzed with a CytoFLEX LX flow cytometer. Data analysis was performed using CytExpert software.

Antibody validation

The GFP-tagged FDXR (NM_001258012), Human Tagged ORF Clone (RG23436, OriGene), was transfected into HEK293 cells using Lipofectamine 3000 (Invitrogen). For controls, HEK293 cells were transfected with either distilled water (negative control) or pCMV6-AC plasmid DNA (empty vector; mock). Twenty-four hours post-transfection, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by three additional washes with PBS. They were then treated with blocking buffer (2% BSA, 0.1% Triton X-100, 5% normal goat serum) for 1 hour at room temperature. For the primary antibody reaction, the cells were incubated with rabbit anti-FDXR antibody (ab204310, Abcam) diluted 1:200 for 2 hours at room temperature and washed three times with PBS. The secondary antibody, Alexa Fluor 555 goat anti-rabbit (A21428, Invitrogen), diluted 1:500 was used, and phalloidin-647 (A22287, Invitrogen) was added for 1 hour at room temperature. After three washes with PBS, the cells were counterstained with mounting medium containing DAPI (H-1200, Vector Laboratories) to prepare slides for imaging with the LSM800 (Zeiss) confocal microscope.

Immunohistochemistry

C57BL/6 mice, aged 4 weeks, were sacrificed, and their whole mount cochleae were fixed in 4% paraformaldehyde at room temperature for 1 hour, followed by three washes with PBS. The organ of Corti was then isolated from each fixed cochlea. The samples were treated with a blocking buffer containing 2% BSA, 0.1% Triton X-100, and 5% normal goat serum for 1 hour at room temperature. The samples were incubated with anti-FDXR antibody diluted 1:100 at 4 °C for 2 days and subsequently washed three times with PBS. The secondary antibody, Alexa Fluor 488 goat anti-rabbit (ab150077, Abcam), was diluted 1:400 and mixed with Rhodamine phalloidin (Invitrogen, R415), then incubated overnight at 4 °C. After three washes with PBS, the samples were mounted with a mounting medium containing DAPI to prepare slides for imaging with the THUNDER Imager microscope (Leica).

RESULTS

Audiologic characteristics of hearing loss from SNUBH1113

Sporadic asymmetric hearing loss was observed in a 35-year-old patient (SNUBH1113-1825) from family SNUBH1113. The diagnosis of ANSD was confirmed by absent ABR responses and normal DPOAE/TEOAE. This was further supported by discrepancies between pure tone audiometry thresholds and word recognition scores, which are characteristic of the audiologic phenotype of SNUBH1113-1825 as postlingual ANSD (Fig. 1A-C). During the ophthalmologic examination, her best-corrected visual acuity was 20/30 in the right eye and 20/40 in the left eye. Fundus photography revealed temporal pallor of the optic disc in both eyes and mild thinning of the retinal nerve fiber layer. More severe involvement of the papillomacular bundle was observed on optical coherence tomography of both eyes, which is characteristic of mitochondrial optic neuropathy (Supplementary Fig. 1).

Genetic diagnosis of postlingual ANSD in SNUBH1113

ES identified a potentially causative, novel variant of FDXR (c.940G>T: p.Val314Leu) in a homozygous state, and the variant status was confirmed with Sanger sequencing. The patient’s phenotypes of ANSD and optic atrophy matched perfectly with previous reports in the literature related to FDXR [4]. Additionally, the variant co-segregated well with the hearing loss phenotype in the pedigree, as expected for autosomal recessive inheritance (Fig. 1A and D). Detailed genetic information related to the pathogenic potential of the variant is summarized in Table 1.

Table 1.

Genetic information of the potential causative variant associated with deafness in the pedigree

Mitochondrial dysfunction of LCLs derived from patients harboring FDXR variants

To investigate whether the FDXR variant (c.940G>T: p.Val314Leu) is indeed pathogenic and thus affects mitochondrial function, intracellular ATP, ROS, and MtMP were evaluated in LCLs derived from both a control and a patient with the FDXR variant (SNUBH 1113-1825). As shown in Fig. 2A-C, intracellular ATP and MtMP were significantly reduced in FDXR-mutant LCLs compared to the control, whereas intracellular ROS levels were remarkably increased (Fig. 2D).

Fig. 2.

Intracellular mitochondrial function in the FDXR mutant-derived lymphoblastoid cell lines (LCLs). (A) Schematic diagram of the treatment of PN-101 into LCLs. Intracellular ATP, reactive oxygen species (ROS), and mitochondrial membrane potential (MtMP) were measured in LCLs derived from both the control and the FDXR mutant. Compared to the control, the LCLs derived from the FDXR mutant exhibited mitochondrial dysfunction, including decreased intracellular ATP and MtMP (B, C), and increased intracellular ROS (D). Treatment with PN-101 restored the levels of ATP, ROS, and MtMP in the FDXR mutant-derived LCLs to those of normal LCLs (B-D). The introduction of PN-101 into FDXR mutant LCLs was confirmed using fluorescence-labeled PN-101 (E). The fluorescence of control cells was set to 100%. Values are presented as mean±standard error of the mean and the Student t-test was used for comparison. NS, not significant; MT, mitochondria. ^a)vs. normal. ^b)vs. non-treatment of PN-101. *P<0.05, ***P<0.001.

Next, we attempted to rule out the possibility that the alterations in intracellular ATP, ROS, and MtMP were secondarily induced by factors other than mitochondrial dysfunction itself. To accomplish this, we investigated whether the impairments in intracellular ATP, ROS, and MtMP observed in FDXR-mutant LCLs could be reversed through direct mitochondrial administration. For this purpose, functionally intact mitochondria were isolated from human UC-MSCs and designated as PN-101. We confirmed that PN-101 could be successfully introduced into FDXR-mutant LCLs by simple co-incubation using fluorescence-labeled PN-101 (Fig. 2E). Interestingly, treatment with PN-101 restored the intracellular ATP and MtMP levels in FDXR-mutant LCLs to those of the control. Although not statistically significant, PN-101 tended to decrease intracellular ROS levels in the FDXR-mutant LCLs (Fig. 2B-D). Collectively, these observations confirmed that this FDXR variant (c.940G>T: p.Val314Leu) is clearly pathogenic and significantly affects mitochondrial function.

Expression pattern of FDXR in the mouse cochlea

The specificity of the anti-FDXR antibody was validated in HEK 293 cells transfected with GFP-tagged FDXR (FDXR-GFP), distilled water (negative control), and an empty vector (mock) (Supplementary Fig. 2). Subsequently, the expression of FDXR was examined in a whole mount of the mouse cochlea, which revealed prominent expression in the spiral ganglion neuron area and moderate expression in the inner hair cell area (Fig. 3). The localization of FDXR in the cochlea suggests that FDXR may play a role in the synaptic region and the spiral ganglion, thus prompting the hypothesis that ANSD due to alterations in FDXR would be primarily post-synaptic.

Fig. 3.

Expression of FDXR in the mouse cochlea. The organ of Corti was stained with phalloidin (red), FDXR antibody (green), and DAPI (blue). The upper images were captured at ×25 magnification, while the lower images are captured at ×200 magnification. The white arrow indicates the inner hair cells (IHCs), and the location of the spiral ganglion neuron (SGN) is marked on the image.

Intraoperative electrophysiological findings and CI outcomes

According to the internal auditory canal magnetic resonance imaging oblique sagittal view, the cochlear nerve exhibited significant atrophy. Therefore, the prognosis for CI did not appear favorable (Fig. 4A). Indeed, during CI, after the electrode insertion, when measuring the ECAP response in the default mode with a pulse width of 25, ECAP responses were only obtained in the nine apical channels (channels 14–22). This type of ECAP pattern—with meaningful responses, but only in some consecutive electrodes—was previously proposed to be associated with post-synaptic pathologies [9]. Therefore, the intraoperative electrophysiological findings (Fig. 4A) correspond well to the expression pattern of FDXR in the mouse cochlea (Fig. 3). However, after increasing the pulse width to 50, ECAP responses were obtained in all channels (Fig. 4B). Therefore, this parameter was maintained during the switch-on and subsequent mapping sessions.

Fig. 4.

Preoperative (Preop) imaging, intraoperative electrically evoked compound action potential (ECAP) response, and postoperative (Postop) cochlear implantation (CI) outcomes. (A) The atrophic cochlear nerve is shown (red arrow), and a poor initial ECAP response was recorded in only nine channels. (B) Increased pulse width (PW) enabled the recording of ECAP responses in all channels. (C) Both the speech recognition test and CAP score showed gradual improvement over time after. IHC, inner hair cell; SGN, spiral ganglion neuron.

Given these mapping parameters, post-CI audiologic outcomes demonstrated gradual improvements, almost plateauing at 6 months postoperative follow-up, with a sentence recognition score of 98% and a CAP score of 6 (Fig. 4C). The speech discrimination score on the left side also increased from 4% with a hearing aid to 56% with the CI at the 1-year post-CI follow-up.

DISCUSSION

Since the first report of the FDXR gene as a deafness-related gene, only one paper has presented support for this gene as a cause of ANSD, with five FDXR-related hearing loss pedigrees [4,5]. In this study, FDXR was reconfirmed as a gene related to ANSD, in a Korean pedigree for the first time, suggesting its relevance in various ethnicities, including previous reports from Tunisian, Algerian, French, Azerbaijani, Russian, and Chinese families. Furthermore, we provided evidence that FDXR-related ANSD is post-synaptic, through expression studies using immunohistochemistry and intraoperative ECAP response measurements. Our study is also the first report in the literature on post-CI auditory rehabilitation outcomes for this type of post-synaptic ANSD due to FDXR mutation, demonstrating successful outcomes for at least 1 year. Regarding the underlying pathophysiology of this disease entity, significant mitochondrial dysfunction was identified through functional studies using a patient-derived cell line, consistent with a previous report [4]. An interesting finding is that the alterations in intracellular ATP, ROS, and MtMP observed in the patient-derived cells were significantly ameliorated by the administration of healthy mitochondria. This finding not only provides stronger evidence that the FDXR variant causes mitochondrial dysfunction but also suggests mitochondrial administration as a potential therapeutic approach for FDXR alterations in the future.

So far in the literature, only nine subjects from five pedigrees have been reported to have FDXR-related hearing loss and visual impairment [4,5]. Previous studies have reported that patients exhibited a diverse onset of hearing loss, ranging from congenital to 20 years old. However, all reported patients shared characteristics of both ANSD and visual impairment, as seen in our patient. Our patient exhibited a relatively late onset of hearing loss and asymmetric hearing loss, which is distinct from other reported cases. To date, eight missense variants, including those identified in our study, and one nonsense variant have been reported to be associated with FDXR-related hearing loss and visual impairment. Although the American College of Medical Genetics and Genomics/Association for Molecular Pathology variant interpretation guidelines for genetic hearing loss classified the c.1069G>T variant in our study as of uncertain significance (Table 1) [22,23], the characteristic phenotype of ANSD and optic atrophy was highly suggestive of a causative variant in the pedigree, and PP4 can be added through the curation of ClinGen. Additionally, a functional study on mitochondrial function supported the pathogenic potential of the variant.

The cochlea is an important organ of the peripheral auditory system with a high energy demand. Because mitochondria are the powerhouses that produce intracellular energy in the form of ATP through oxidative phosphorylation, mitochondrial function in the cochlea is critical for maintaining auditory capacity. In this context, mitochondrial abnormalities have frequently been implicated in hearing loss caused by mutations in either mitochondrial DNA or nuclear DNA-encoding proteins, which are closely linked to mitochondrial function [24-26]. For example, the mitochondrial 12SrRNA gene A1555G mutation results in non-syndromic hereditary hearing loss, and mutations in genes coding for nuclear DNA encoding proteins (e.g., OPA1, MFN2, and MSRB3) contribute to mitochondrial dysfunction, leading to sensorineural deafness [2,27-29]. Therefore, the best option for treating hearing loss would be one that directly repairs or replaces damaged mitochondria. It has recently been reported that mitochondria isolated from different cell sources can be taken up by cells and tissues via local or systemic injections [30,31]. Moreover, intravenously administered mitochondria preferentially migrate to tissues and cells with damaged mitochondria [10]. In this context, administering healthy mitochondria to replace damaged ones has received attention as an attractive therapeutic strategy for treating diseases caused by mitochondrial dysfunction. The current study demonstrated that the FDXR variant induced mitochondrial dysfunction, including decreases in ATP production and mitochondrial membrane potential, and an increase in ROS. Interestingly, UC-MSC-derived mitochondria (PN-101) were able to restore the mitochondrial damage induced by the FDXR variant, suggesting that mitochondrial replacement can be developed as a novel therapeutic approach for hearing loss.

CI is a definitive solution for many patients with severe-to-profound hearing loss, including ANSD. The postoperative audiologic improvements in this study provide additional evidence for the important role of CI in the auditory rehabilitation of FDXR-related adult-onset ANSD, which is post-synaptic. Generally, a hearing aid, which could play a crucial role in bridging hearing ability to CI, can be beneficial for most types of hearing loss. However, as observed in our pedigree, where the word recognition score was very poor (4%) despite relatively preserved pure tone audiometry thresholds (45 dBHL), the role of hearing aids in ANSD is less efficacious in bridging to CI. Thus, CI instead of a hearing aid should cover a significant portion of the gap between no assistive device and CI. This may support early CI implementation (Fig. 5). The clinical decision to advance the timing of CI drastically, while accepting the risk of some damage to residual pure-tone hearing, should be based on an accurate etiological assessment, including a molecular genetic diagnosis. Another consideration for the auditory rehabilitation of ANSD, which differs from that of general sensorineural hearing loss (SNHL), is that cochlear nerve dystrophy can progress relatively rapidly. This progression could be supported by our in-house database (unpublished) and a similar tendency in optic nerve dystrophy, which shares a common etiology with FDXR-related ANSD [32]. Therefore, in cases of ANSD, a more proactive approach to CI is necessary compared to the standard indications for CI in general SNHL. The proband in our study experienced significantly worse hearing on the left side, and even on the right side, which was better, she had poor speech discrimination scores (50% for one-syllable words, 75% for two-syllable words, and 80% for sentences). These scores could not be adequately compensated using a hearing aid. As a working professional, it was difficult for her to maintain a normal daily life, which led to her desire for CI. Furthermore, magnetic resonance imaging of the temporal bone in the patient revealed severe atrophy of the cochlear nerve on the left side (red arrow) and progressive atrophy on the right side, despite an almost normal pure tone threshold (Supplementary Fig. 3). Overall, a genetic diagnosis in post-synaptic, postlingual ANSD might have significance in providing a basis for confidently advancing CI. Previously, Kim et al. [9] demonstrated in 2023 that even in post-synaptic ANSD involving the spiral ganglion neuron or cochlear nerve, there is potential for better synchronous stimulation, indicating that the post-synaptic nature of ANSD does not preclude the benefits of CI. In fact, in their study, performing early CI and adjusting mapping parameters enabled significant improvements in speech discrimination after CI in these post-synaptic ANSD cases. In our present study, we confirmed these findings once again in FDXR-related ANSD.

Fig. 5.

Difference in proposed auditory rehabilitation protocols between ordinary sensorineural hearing loss (SNHL) and auditory neuropathy spectrum disorder (ANSD). ANSD needs early cochlear implantation (CI), unlike ordinary SNHL, which can benefit from hearing aid use bridging to CI.

In this study, we reconfirmed FDXR as a gene related to ANSD and optic neuropathy, and reported the first Korean pedigree with this disease entity. Post-CI audiologic outcomes showed that FDXR-related post-synaptic ANSD could be a good candidate for CI, although long-term follow-up is needed. Functional studies also provided a possible link between mitochondrial dysfunction and ANSD, suggesting the potential role of mitochondrial replacement in the treatment of hearing loss.

HIGHLIGHTS

▪ A novel missense variant of the FDXR gene (p.Val314Leu) was identified as a causative variant for adult-onset auditory neuropathy spectrum disorder (ANSD) with optic atrophy in a Korean pedigree for the first time.

▪ Mitochondrial dysfunction is suggested as a possible pathogenesis of FDXR-related hearing loss, based on decreased mitochondrial function in patient-derived cell lines and restoration of function after administration of normal mitochondria to the cell lines.

▪ Immunohistochemistry and intraoperative electrophysiological findings suggest that FDXR mutations manifest as post-synaptic ANSD, posing concerns about the outcomes of cochlear implantation (CI).

▪ For the first time in the literature, 1-year post-CI outcomes for FDXR-related ANSD were reported, with favorable results after increasing the pulse width during mapping.

▪ Our results could provide a scientific basis for advancing CI timing with clinical confidence for post-synaptic ANSD caused by alterations in genes with similar expression patterns to FDXR.

Notes

Young Cheol Kang, Yu Jin Kim, and Chun-Hyung Kim are employees of Paean Biotechnology, Inc.

The remaining authors declare no conflicts of interest concerning the research, authorship, and publication of this article.

Byung Yoon Choi and Chae-Seo Rhee are editorial board members of the journal but were not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

AUTHOR CONTRIBUTIONS

Conceptualization: BJK, CHK, BYC. Data curation: YK, JHH, MYK. Formal analysis: JHH, YCK. Funding acquisition: BJK, YCK, BYC. Methodology: MYK, YK. Project administration: BJK. Visualization: BJK, YK, JAK, HKY, BYC. Writing–original draft: BJK, YK, JAK, CHK, BYC. Writing–review and editing: BJK, JAK, HKY, CSR, CHK, BYC. All authors read and agreed to the published version of the manuscript.

Acknowledgements

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (Grant 2021R1A2C2092038 to BYC), Bio Core Facility center program through the NRF-2022 M3A9G1014007 to BYC and also by the Basic Research Laboratory program through the NRF, funded by the Ministry of Education (Grant RS-2023-0021971031482092640001 to BYC) and the Technology Innovation Program (K_G012002572001 to BYC) funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea). This study is also funded by SNUBH intramural research fund (18-2023-0004, 16-2022-0005, 13-2024-0004, 13-2023-0002, and 16-2023-0002 to BYC). This work was supported by an NRF grant funded by the Korean government (MSIT) (2021R1C1C1007980 and RS-2024-00355990 to BJK), Chungnam National University Sejong Hospital Research Fund, 2022 and Chungnam National University (BJK). This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2023-00283926 to YCK).

SUPPLEMENTARY MATERIALS

Supplementary materials can be found online at https://doi.org/10.21053/ceo.2024.00184.

Supplementary Fig. 1.

Optic disc atrophy. (A) Fundus photography reveals temporal pallor of the optic discs in both eyes (indicated by white arrows). (B) Optical coherence tomography reveals mild thinning of the retinal nerve fiber layer (RNFL) and more severe involvement of the papillomacular bundle (indicated by black arrows) in both eyes. NAS, nasal; INF, inferior; TMP, temporal; SUP, superior; OD, right; OS, left.

ceo-2024-00184-Supplementary-Fig-1.pdf

Supplementary Fig. 2.

Validation of FDXR antibody specificity. To confirm the specificity of the FDXR antibody, HEK293 cells were transfected with GFP-tagged FDXR (FDXR-GFP), distilled water (negative), or an empty vector (mock). The localization of the FDXR-GFP signal in HEK293 cells demonstrates the antibody’s specificity (upper panel).

ceo-2024-00184-Supplementary-Fig-2.pdf

Supplementary Fig. 3.

Cochlear nerve status on both sides on magnetic resonance imaging. Cochlear nerve dystrophy is more prominent on the left side, and even on the right side, which is the better ear with an almost normal pure tone threshold, progressive dystrophy of the cochlear nerve is noted.

ceo-2024-00184-Supplementary-Fig-3.pdf

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Subject ID	Phenotype	Causative gene	Genomic position: change (GRCh37/hg19)	HGVS		Type of mutation	Zygosity/inheritance pattern	In silico predictions			Alternative allele frequency		ACMG/AMP 2018 guideline
Subject ID	Phenotype	Causative gene	Genomic position: change (GRCh37/hg19)	Nucleotide change	Amino acid change	Type of mutation	Zygosity/inheritance pattern	CADD Phred	REVEL	GERP++	KOVA (5,305 individuals)	GMAF (dbSNP)	Criteria	Classification
SB1113-1825	Postlingual progressive ANSD, ANOA	FDXR	Chr17:72860332 C>A	c.940G>T	p.Val314Leu	Missense	Homozygote/autosomal recessive	21.90	0.130	4.58	Absent	A=0.000043 (6/140268, gnomAD)	PM2, PM3_Supporting, PS3_Supporting, BP4	Uncertain significance
												A=0.00005 (1/18520, ALFA)
												A=0.0002 (1/4480, Estonian)
												A=0.0007 (2/2922, KOREAN)