Auditory neuropathy (AN) is a kind of hearing disorder involving different lesion sites beyond the outer hair cells (OHCs), ranging from the inner hair cells (IHCs) and synapses to auditory nerve and higher auditory centers (Rance and Starr, 2015; Moser and Starr, 2016). AN features impaired or absent response in auditory brainstem responses (ABR) and the presence of cochlear microphonics and/or detectable otoacoustic emission (OAE), showing evidence of intact outer hair cell function. Varying degrees of hearing loss are present in subjects with AN; however, their speech recognition rates are generally poor, disproportionate to the degree of hearing loss (Harrison et al., 2015; Rance and Starr, 2015; Moser and Starr, 2016). A variety of etiologies can trigger AN, generally considered to be hypoxia, infection, kernicterus, cytotoxic oncologic drugs, and genetic factors (Harrison et al., 2015).

Considering postlingual-onset AN, many syndromic forms that cause sensory and motor neuropathy have been documented, including Charcot-Marie-Tooth disease, Friedreich’s ataxia, deafness-dystonia-optic neuropathy (DDON) syndrome, autosomal dominant optic atrophy (ADOA), and AUNX1 due to AIFM1 gene mutations in apoptosis-inducing factor (Han et al., 2017). Han et al. (2017) explored the molecular etiology of three unrelated Korean subjects manifesting AN with postlingual onset, identified a mutation in c.2452 G > A (p.E818K) of ATP1A3 gene, and demonstrated ATP1A3 gene to be an important and prevalent causative gene for progressive AN with postlingual onset. ATP1A3 gene has thus been said to occur frequently de novo in both Caucasians and Koreans (Han et al., 2017). However, p.E818K in the ATP1A3 gene has rarely been documented in the Chinese population.

In this study, we reported four Chinese AN patients carrying a de novo variant, p.E818K mutation of ATP1A3 gene, identified through next generation sequencing (NGS). ATP1A3 p.E818K has been generally recognized as a genetic cause of cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome. A genotype–phenotype correlation study of ATP1A3 was performed, and one AN patient underwent cochlear implantation (CI) to reconstruct hearing.

Hearing loss is the most common sensorineural disorder. It is estimated that more than half of hearing loss cases are attributable to genetic factors (He Y. et al., 2017; Wang et al., 2017; Zhu et al., 2018; Fang et al., 2019; He Z. et al., 2019; Fu et al., 2021), while the other half of hearing loss could be caused by aging, chronic infections, infectious diseases, ototoxic drugs, and noise exposure (Liu L. et al., 2016; He Z. et al., 2017; Cheng et al., 2019; Gao et al., 2019; Tan et al., 2019; Zhang et al., 2019; Zhong et al., 2020). The functions of these hearing loss genes play an essential role in the development and function of hair cells and synaptic transmission of spiral ganglion neurons (Qi et al., 2020; Qian et al., 2020; Zhang et al., 2020, 2021; Cheng et al., 2021; Lv et al., 2021). Thus, hearing loss is often induced by loss of sensory hair cells (Liu Y. et al., 2019; Qi et al., 2019; Han et al., 2020; He et al., 2020, 2021; Chen et al., 2021) and spiral ganglion neurons (Guo et al., 2016, 2019, 2020, 2021; Liu W. et al., 2019; Hu et al., 2021; Liu W. et al., 2021) in the inner ear cochlea. AN is a hearing disorder with functional outer hair cells and disrupted function of inner hair cells and/or the auditory nerve itself. Han et al. (2017) first proposed that p.E818K in ATP1A3 gene, which encodes the NKAα3 subunit of Na, K-ATPases, can cause AN in Caucasians and Koreans, implying that ATP1A3 serves as a global contributor to AN (Supplementary Table 1). In our study, four AN cases were tested with p.E818K in the ATP1A3 gene. The incidence of p.E818K accounting for AN in Chinese individuals is still under investigation. Nevertheless, we found that the p.E818K mutation accounts for a high proportion of AN with postlingual onset, not only in Koreans and Caucasians but also in Chinese individuals.

ATP1A3 p.E818K has been regarded as a causal mutation of CAPOS syndrome since identified in 2014 (Carecchio et al., 2018), long after the initial description in 1996 (Nicolaides et al., 1996). CAPOS syndrome is a rare disorder, with fewer than 100 CAPOS patients worldwide, and is sporadically reported (Demos et al., 2014; Rosewich et al., 2014; Heimer et al., 2015; Potic et al., 2015; Maas et al., 2016; Duat Rodriguez et al., 2017). Growing evidence shows that sensorineural hearing loss in CAPOS syndrome is AN, which may progress slowly over time. The typical CAPOS phenotype is characterized by acute neurological deterioration manifesting in infancy and triggered by stressful episodes, such as a febrile illness (Paquay et al., 2018). Meticulous history as well as comprehensive neurologic examination should be performed on postlingual-onset AN subjects, to detect any episode of visual disturbance and ataxia associated with CAPOS syndrome triggered by a fever that might otherwise go unnoticed. CAPOS syndrome patients initially have cochlear apical turns, manifested in a low-frequency loss in PTA. However, neuronal distribution of NKAα3 is also observed in middle and basal turns of the cochlea (Paquay et al., 2018), indicating that mid and high frequencies may also be involved. Through 15 years follow-up, we detected progressing hearing defects involving all frequencies, predominantly the low frequencies in patient 1. It is worth continuing the follow-up periods for patients with p.E818K to detect changes in PTA over time, to clarify the significance of the p.E818K mutation in AN.

In previous study, Demos et al. (2014) reported follow-up of three patients with ATP1A3 p.E818K. All the patients showed slow progression of all symptoms with no further acute episodes. Demos et al. (2014) further reported two Caucasian families with ATP1A3 p.E818K. In the first family, all affected suffered from acute episodic ataxic encephalopathy and/or weakness triggered by a febrile illness onset in infancy or early childhood. Recovery was not as desirable after these episodes, when variable neurologic abnormalities was lingering. All affected individuals eventually had optic atrophy and sensorineural hearing loss. One patient exhibited mild cognitive dysfunction at age 10 years. In the second family, the affected mother and her two children presented with recurrent acute episodes of neurologic disorders associated with febrile illnesses beginning in the first years of life. Features included weakness, ataxia, and a progressive decline in hearing and vision. In our study, patient 1 exhibited dystonia and ataxia at a very early age after having a fever. He exhibited optic atrophy and auditory neuropathy at an early age, which were slowly progressive over time. Follow-up period was needed for every patient to further clarify whether there was a slow progression of all symptoms, be it neurological disorders or visual and hearing impairment.

The ATP1A3 gene has also been reported in two other clinical entities, alternating hemiplegia of childhood (AHC, AHC2, OMIM #614820) and rapid-onset dystonia Parkinsonism (RDP, DYT12, OMIM #128235) (Blanco and Mercer, 1998; Starr et al., 2005; Rodacker et al., 2006; Blanco-Arias et al., 2009). Mutation sites are distributed across the entire coding region of ATP1A3 in RDP and AHC (Supplementary Table 1; Clausen et al., 2017). Interestingly, all CAPOS patients reported without exception share a common mutation, p.E818K in the ATP1A3 gene. Also, p.E818K is seemingly tightly coupled with CAPOS syndrome and has never been reported in other two disorders (Han et al., 2017; Figure 3). There is no report of hearing or visual impairment in AHC and RDP, which is vastly different from CAPOS syndrome (Figure 4; Tranebjærg et al., 2018a). However, those three entities present with an expanding phenotypic spectrum and are more often reported to share somewhat overlapping neurological phenotypes. In addition, atypical phenotypes due to ATP1A3 gene mutations have recently been identified (Gropman et al., 2020; Guo et al., 2020). It becomes gradually clear that these overlapping phenotypes may be a series of ATP1A3-related symptoms, rather than allelic disorders (Carecchio et al., 2018). Moreover, the expression timing of neurological deficits of the mutation varies and has still not been fully characterized until now (Han et al., 2017). In our study, it was not very hard to observe the latency of occurrence of numerous symptoms in patients 1 and 2. Notably, patients 3 and 4 denied all neurological episodes. Continuation of the follow-up periods for these four patients is therefore necessary for the discovery of other delayed neurological symptoms. Nevertheless, whether AN is simply a symptom of CAPOS or a completely new phenotype of the ATP1A3 gene remains to be seen.

Early diagnosis of hearing loss and timely prevention of sensory deprivation are of vital importance and confer lifelong benefits to CAPOS patients, especially for those with visual interference. The 818 position is previously confirmed located at the cytoplasmic end of transmembrane helix 6 of NKAα3, forming a salt bridge with the backbone carbonyl of Arg930, which is a residue known to stabilize the C-terminus. Tranebjærg et al. (2018b) have evaluated functional impact of Glu818Lys to the C-terminal structure and demonstrated that Glu818Lys leads to low affinity for extracellular sodium and a high rate of sodium release from the third ion-binding portion; therefore, it disrupts the function of the NKAα3 subunit of Na, K-ATPases, which is a key regulator of regaining the resting potential of the membrane after excitatory activity in the auditory pathway. In the auditory pathway, NKAα3 is expressed in the membranes of type I afferent terminals contacting the inner hair cells, spiral ganglion, and medial efferent terminals that contact the outer hair cells (Erichsen et al., 1996; McLean et al., 2009; Bottger et al., 2011), providing evidence that CI in those patients could generate likely preferable outcomes. One example of CI having an ideal outcome is CI in AN together with ADOA (OMIM #165500) owing to OPA1 variations (Santarelli et al., 2008; Huang et al., 2009; Moser and Starr, 2016). The expression site of OPA1 significantly overlaps with that of NKAα3, being it in the hair cells as well as in the neuronal fibers innervating the inner hair cells, ganglion cells of the cochlea, and vestibular organs (Bette et al., 2007).

Auditory neuropathy with ATP1A3 mutation are generally considered to have ideal CI results. However, CI produces dubious outcomes in some reported cases. Tranebjaerg et al. (Fang et al., 2019) stated that four CAPOS patients received CI, two of whom gained significant benefits and the other two was relatively poor. In contrast, Atilgan et al. (2019) documented a much more beneficial effect of CI in a patient with CAPOS syndrome as compared to patients with SNHL. These findings could be explained by the fact that the ATP1A3 gene is expressed not only in synapse but also in postsynaptic spiral ganglion cells (SGNs). If the lesion is mainly at the synapse, good CI results can be expected, similar to those in OPA1 variants (Han et al., 2017), whereas CI is less useful with lesions dominantly at the SGNs. In fact, in most postsynaptic AN, CI has a dubious outcome (Supplementary Table 2). An example is CI in the SGNs (Fasquelle et al., 2011; Eppsteiner et al., 2012; Atilgan et al., 2019). CI performed earlier in TMPRSS3 generated a preferable outcome, while the outcomes in later implantees were not as favorable (Chung et al., 2014). Patients who benefit most from CI were reported to be relatively younger (Han et al., 2017; Tranebjærg et al., 2018b), yet an accurate CI efficacy estimation with age and other possible impact factors taken into consideration is still awaiting discussion. Chaudhry et al. (2020) summarized 25 postsynaptic CI results, with 22 displaying modest to significant benefit, and demonstrated that CI behaves variably but generally good in postsynaptic AN. Moreover, the best functional outcome of CI observed at the short-term follow-up may decrease over time. Although earlier electrical stimulation of SGNs may attenuate the degeneration of the neurons, progressive degeneration of SGNs could possibly occur due to a disrupted function of NKAα3 (Han et al., 2017). In our study, patient 2 underwent CI on his left ear, but the result was hardly desirable. One possible explanation is that his age by the time he received surgery and the duration of his hearing loss are too long to ensure SGN activities. Moreover, the result of CI may be in relation with the characteristic of the PTA of the patients. Therefore, a continuous follow-up period for CI performance in those patients is warranted, to better determine if the current benefits from CI decreases over time.

The identification of p.E818K in ATP1A3 has implications for future risk calculation and genetic counseling. In CAPOS families with an autosomal dominant inheritance pattern, the chance of offspring carrying pathogenic mutations is 50%, with varying degrees of penetrance. In addition, all reported CAPOS patients had unaffected siblings without exception, revealing that all pedigrees of CAPOS syndrome are sporadic cases. It is still unclear why de novo variants frequently occur in ATP1A3. The age of the father has been reported to be a strong impactor of de novo mutations (Niederberger, 2013). However, that study failed to explain all de novo cases. A genetic context in ATP1A3 that favors the occurrence of de novo variants may account for this phenomenon, awaiting further clarification.

Here, four AN patients were identified with p.E818K mutation in the gene ATP1A3, causing progressive AN with postlingual onset and varying degrees of syndromic features. Our study confirms that ATP1A3 in Chinese individuals is undoubtedly an important genetic cause of progressive AN with postlingual onset. Genetic testing for this specific mutation site allows for identification of a special subtype of AN with somewhat preferable CI results, as well as accurate genetic counseling.

The data presented in the study are deposited in the ClinVar repository, accession number VCV000156238.

The studies involving human participants were reviewed and approved by Committee of Medical Ethics of Chinese PLA General Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

HW and WW conceived and designed the experiments and wrote the manuscript. HW, WW, JL, LX, LL, and FX performed the experiments. HW analyzed the data. JL, LL, LX, and FX contributed to the reagents, materials, and analysis tools. HW, JG, and QW critically read and discussed the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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