Detailed Clinical Features of Deafness Caused by a Claudin-14 Variant

Tight junctions are cellular junctions that play a major role in the epithelial barrier function. In the inner ear, claudins, occludin, tricellulin, and angulins form the bicellular or tricellular binding of membrane proteins. In these, one type of claudin gene, CLDN14, was reported to be responsible for human hereditary hearing loss, DFNB29. Until now, nine pathogenic variants have been reported, and most phenotypic features remain unclear. In the present study, genetic screening for 68 previously reported deafness causative genes was carried out to identify CLDN14 variants in a large series of Japanese hearing loss patients, and to clarify the prevalence and clinical characteristics of DFNB29 in the Japanese population. One patient had a homozygous novel variant (c.241C>T: p.Arg81Cys) (0.04%: 1/2549). The patient showed progressive bilateral hearing loss, with post-lingual onset. Pure-tone audiograms indicated a high-frequency hearing loss type, and the deterioration gradually spread to other frequencies. The patient showed normal vestibular function. Cochlear implantation improved the patient’s sound field threshold levels, but not speech discrimination scores. This report indicated that claudin-14 is essential for maintaining the inner ear environment and suggested the possible phenotypic expansion of DFNB29. This is the first report of a patient with a tight junction variant receiving a cochlear implantation.


Hearing Loss
Hearing loss (HL) is the most common sensory impairment and is diagnosed in approximately two in every 1000 children [1]. At least 60% of all childhood nonsyndromic sensorineural hearing loss is caused by genetic factors [2]. The inheritance patterns of this form of HL include autosomal recessive, autosomal dominant, X-linked, and mitochondrial. Autosomal recessive nonsyndromic hereditary HL (ARNSHL) is typically prelingual, and accounts for approximately 70% of nonsyndromic hereditary HL patients [3]. Thus far, 75 causative genes for ARNSHL have been identified [4]. One form of ARNSHL is DFNB29 (OMIM #: 614035), which is caused by variants in the CLDN14 gene.

Tight Junctions in the Inner Ear
For our sound receiving process, it is necessary to convert sound vibration to nerve action potential (mechano-transduction) [5]. The center for this mechano-transduction is the cochlea, located in the inner ear. The cochlea is filled with two types of lymph fluid, the endolymph and perilymph. These

CLDN14 Variants in Deafness
To date, nine different variants in CLDN14 [13,[26][27][28][29] have been reported to cause HL in the Pakistani, Greek, and Canadian populations. Seven of the nine variants were reported from Pakistani consanguineous families. The CLDN14 variants are a relatively common cause of recessive hearing loss, which is responsible for 2.25% of HL patients in a Pakistani study cohort [28], whereas no pathogenic variants were reported from east Asian populations [30,31]. Although previously reported papers have shown some clinical characteristics of patients with CLDN14 variants, the audiovestibular findings, such as progression and details of vestibular function, remain unclear. In addition, there are no reports on patients with cochlear implantation (CI), thus the outcome of CI is also unknown. In the present study, we used massively parallel DNA sequencing (MPS) to detect pathogenic variants in CLDN14 among a large series of Japanese HL patients.
Here, we present a patient with a novel variant in the CLDN14 gene identified from a group of non-consanguineous HL patients and discuss additional clinical features including CI outcomes. This is the first report of a diagnosis of hearing caused by CLDN14 in an east Asian population and in such a patient receiving a cochlear implant as intervention.

Detected Variant
One possible disease-causing variant, CLDN14: NM_144492: c.241C>T, leading to p.Arg81Cys, was homozygously detected in one of 1577 probands with autosomal recessive HL (Table 1, Figure 1), whereas no pathogenic variants were found in the other 972 probands with autosomal dominant or inheritance pattern of unknown HL. At the same amino acid residue, which is within the first extracellular loop, a different missense change c.242G>A, leading to p.Arg81His, has been reported ( Figure 2) [27]. No candidate variants in the other 67 deafness genes were identified in the proband. In detail, the variants in two other genes, PTPRQ and EYA4, were also detected from the proband but were not segregated. As shown in Figure 1, family segregation was confirmed by using Sanger sequencing. The variant was not identified in ExAC, gnomAD, 3.5KJPN or the 1208 Japanese exome variants, in addition to the 333 in-house Japanese normal hearing control databases.

Detected Variant
One possible disease-causing variant, CLDN14: NM_144492: c.241C>T, leading to p.Arg81Cys, was homozygously detected in one of 1577 probands with autosomal recessive HL (Table 1, Figure  1), whereas no pathogenic variants were found in the other 972 probands with autosomal dominant or inheritance pattern of unknown HL. At the same amino acid residue, which is within the first extracellular loop, a different missense change c.242G>A, leading to p.Arg81His, has been reported ( Figure 2) [27]. No candidate variants in the other 67 deafness genes were identified in the proband. In detail, the variants in two other genes, PTPRQ and EYA4, were also detected from the proband but were not segregated. As shown in Figure 1, family segregation was confirmed by using Sanger sequencing. The variant was not identified in ExAC, gnomAD, 3.5KJPN or the 1208 Japanese exome variants, in addition to the 333 in-house Japanese normal hearing control databases.  We employed in silico software (SIFT, polyphen-2, LRT, Mutation Taster, etc.), and almost all scores indicated "damaging" ( Table 1). The corresponding amino acid was well conserved across species (170/170 (100%) in vertebrates). Taken together, according to the American College of Medical Genetics (ACMG) guideline, the variant was classified as "likely pathogenic" (PS4, PM2, and PM5). We employed in silico software (SIFT, polyphen-2, LRT, Mutation Taster, etc.), and almost all scores indicated "damaging" ( Table 1). The corresponding amino acid was well conserved across species (170/170 (100%) in vertebrates). Taken together, according to the American College of Medical Genetics (ACMG) guideline, the variant was classified as "likely pathogenic" (PS4, PM2, and PM5).

Clinical Findings
The proband was a 37-year-old female. The newborn hearing screening program had not yet started at birth and there were no particular complications in the perinatal period. The female proband also passed an elementary school health checkup at six years of age, but was suspected of hearing loss at age nine and was referred to an otolaryngology clinic at a local general hospital. Pure-tone audiograms showed steep high-frequency sensorineural HL (SNHL) with an average of 77 dBHL in both ears ( Figure 3A). It appeared to progress slowly, and the proband began to wear hearing aids bilaterally at 11 years of age. Over a period of 20 years, their residual hearing in the lower frequencies gradually deteriorated and hearing aids became ineffective. At the age of 34, the proband consulted our hospital for further examination. Otoscopic examination revealed a normal tympanic membrane. Computed tomography (CT) and magnetic resonance imaging (MRI) of the temporal bones showed no malformations, hearing level was approximately 100 dBHL in both ears, but the left ear showed moderate residual hearing in the lower frequencies. We performed vestibular assessment (caloric test and cervical-ocular vestibular evoked myogenic potentials, i.e., c/oVEMPs) as preoperative examinations prior to CI. The caloric test, cVEMPs, and oVEMPs represent the function of the semicircular canal, the saccule and inferior vestibular nerve, and the utricle and superior vestibular nerve, respectively. As shown in Figure 3B, all vestibular testing showed normal vestibular functions. The proband was a 37 year old female. The newborn hearing screening program had not yet started at birth and there were no particular complications in the perinatal period. The female proband also passed an elementary school health checkup at six years of age, but was suspected of hearing loss at age nine and was referred to an otolaryngology clinic at a local general hospital. Puretone audiograms showed steep high-frequency sensorineural HL (SNHL) with an average of 77 dBHL in both ears ( Figure 3A). It appeared to progress slowly, and the proband began to wear hearing aids bilaterally at 11 years of age. Over a period of 20 years, their residual hearing in the lower frequencies gradually deteriorated and hearing aids became ineffective. At the age of 34, the proband consulted our hospital for further examination. Otoscopic examination revealed a normal tympanic membrane. Computed tomography (CT) and magnetic resonance imaging (MRI) of the temporal bones showed no malformations, hearing level was approximately 100 dBHL in both ears, but the left ear showed moderate residual hearing in the lower frequencies. We performed vestibular assessment (caloric test and cervical-ocular vestibular evoked myogenic potentials, i.e., c/oVEMPs) as preoperative examinations prior to CI. The caloric test, cVEMPs, and oVEMPs represent the function of the semicircular canal, the saccule and inferior vestibular nerve, and the utricle and superior vestibular nerve, respectively. As shown in Figure 3B, all vestibular testing showed normal vestibular functions.   The proband underwent a CI (MED-EL FLEX28) in the right ear at the age 35. Sound field threshold levels were improved from 77.5 dBSPL (with hearing aids) to 37.5 dBSPL (with the cochlear implant) ( Figure 3A). To evaluate speech perception outcomes, the proband underwent speech discrimination testing (using the 67S Japanese monosyllable test, presenting in 65 dBSPL). Contrary to the good improvement in sound field threshold levels, the proband's speech perception outcomes improved from 12% to only 32% two years after receiving a CI.

Frequency of CLDN14-Associated HL in the Japanese Population
We discovered a novel causative variant in the CLDN14 gene as a cause of progressive SNHL in a Japanese patient. To date, a total of nine pathogenic variants in CLDN14 have been reported (Table 2, Figure 2). Among them, seven were reported from Pakistan, and one each from Greece and Canada. Otherwise, according to the previous reports, no pathogenic variants of CLDN14 have been detected in the Korean and Chinese populations [30,31]. Hence, this is the first report of a causative CLDN14 variant in an east Asian population.
The incidence of CLDN14 variants was 0.04% (1/2549) among the Japanese HL patients, and 0.06% (1/1577) among the families with autosomal recessive HL in the Japanese population. In clear contrast to this, variants of CLDN14 account for 2.25% of autosomal recessive HL in the Pakistani population. In this study, the patient was identified from a non-consanguineous family and the parents of the proband were from a geographically remote area, whereas almost all of the previous reports were from consanguineous families [13,[27][28][29]. As no pathogenic CLDN14 variants were found in our cohort, but one HL patient with a homozygous CLDN14 variant was identified, it is necessary to consider such rare HL cases. The proband underwent a CI (MED-EL FLEX28) in the right ear at the age 35. Sound field threshold levels were improved from 77.5 dBSPL (with hearing aids) to 37.5 dBSPL (with the cochlear implant) ( Figure 3A). To evaluate speech perception outcomes, the proband underwent speech discrimination testing (using the 67S Japanese monosyllable test, presenting in 65 dBSPL). Contrary to the good improvement in sound field threshold levels, the proband's speech perception outcomes improved from 12% to only 32% two years after receiving a CI.

Frequency of CLDN14-Associated HL in the Japanese Population
We discovered a novel causative variant in the CLDN14 gene as a cause of progressive SNHL in a Japanese patient. To date, a total of nine pathogenic variants in CLDN14 have been reported (Table 2, Figure 2). Among them, seven were reported from Pakistan, and one each from Greece and Canada. Otherwise, according to the previous reports, no pathogenic variants of CLDN14 have been detected in the Korean and Chinese populations [30,31]. Hence, this is the first report of a causative CLDN14 variant in an east Asian population.
The incidence of CLDN14 variants was 0.04% (1/2549) among the Japanese HL patients, and 0.06% (1/1577) among the families with autosomal recessive HL in the Japanese population. In clear contrast to this, variants of CLDN14 account for 2.25% of autosomal recessive HL in the Pakistani population. In this study, the patient was identified from a non-consanguineous family and the parents of the proband were from a geographically remote area, whereas almost all of the previous reports were from consanguineous families [13,[27][28][29]. As no pathogenic CLDN14 variants were found in our cohort, but one HL patient with a homozygous CLDN14 variant was identified, it is necessary to consider such rare HL cases. * The prediction scores of each algorithm included on the ANNOVAR software were converted from the original scoring system. Scores closer to 1.0 indicated the mutation was more damaging, and those closer to 0 indicated they were more tolerant. ** Conserved in 170 vertebrates from the HGMD professional.

Progression and Onset of DFNB29
Until Pater et al. reported the progression of HL [29], the phenotype of DFNB29 was associated with non-progressive, congenital or prelingual HL with a variable degree of severity [13,[26][27][28]. In this report, serial audiograms showed hearing deterioration especially at low frequencies (125 to 500 Hz). The average threshold progression rate of HL in the low frequencies was about 1.6 dB/year. Moreover, in this case, HL in the high frequencies was already impaired from the first decade, but there may have been no problem at birth as there was no problem in articulation. According to Pater's report, the phenotype of DFNB29 showed no hearing impairment until three years of age, and its onset occurred after age four especially in the higher frequencies. After that it progressed to almost deaf at above 2 kHz by nine years of age, therefore, it might be that our patient had a similar course. Indeed, in our case, no hearing impairment was detected at the elementary school health checkup at six years of age and it was only suspected at nine years of age. Lee et al. reported HL patients with another amino acid change, p.Arg81His [27], at the same amino acid residue. In that report, the phenotype was described as non-progressive prelingual HL. The audiogram indicated bilateral severe to profound HL with a gentle slope at 20 years of age. From our study and the previous reports, the hearing deterioration in patients with CLDN14 remains unclear, and further study including serial audiograms is required to clarify this.

Vestibular Examination
We could not identify any dysfunction on the vestibular examinations. According to past reports, claudin-14 is also expressed in the vestibule of mice (such as in the sensory epithelia) [19]. Vestibular function in Cldn14 knockout model mice has not been reported and the details remain unknown. This is the first report to mention in detail the vestibular function, including caloric testing, cVEMPs, and oVEMPs. Endolymph in cochlear is characterized by extremely high resting potential called EP, in addition to high K + concentration. In contrast, in the vestibular endolymph, the K + concentration is high, but no EP can be detected. Claudin-14 may be necessary for maintaining a barrier to resist EP, but not K + .

Outcome of Cochlear Implantation
This is the first report showing the outcome for a CI for a CLDN14-associated HL patient. A CI represents the most successful neural prosthesis in clinical cases [32]. For patients with severe to profound SNHL, CI has been established as the standard therapy [33]. The implant is surgically implanted and works by transducing acoustic energy into an electrical signal, with an electrode array in the cochlea used to stimulate the surviving spiral ganglion cells of the auditory nerve [34].
As mentioned above, the patient showed relatively poor improvement in speech discrimination scores, whereas good improvement in sound field threshold levels was observed. Since the cochlear implant directly stimulates the cochlear nerve, the influence of the inner ear should be excluded. Thus, it is likely that the unsatisfactory speech discrimination was due to deficiencies in the spiral ganglion or central auditory pathway. In our previous study, CLDN14 was expressed at the same level in the organ of Corti and spiral ganglions [35]. Including this report, there have been no cases with intellectual disability indicating central involvement; therefore, it is possible that the spiral ganglion may be involved in claudin-14 associated HL.

Subjects
All procedures were approved by the Shinshu University Ethical Committee (No. 387-4 September 2012 and No. 576-2 May 2017) as well as the respective Ethical Committees of the other participating institutions described elsewhere [36] and were carried out after obtaining written informed consent from all subjects (or from their next of kin, caretaker, or guardian in the case of minors or children). A total of 2549 probands from unrelated Japanese HL families were enrolled from the 67 otolaryngology departments across Japan participating in the present study from May 2012 to September 2016. The age of the probands ranged from 0 to 79 years (mean ± SD: 22.1 ± 19.7). The hereditary patterns of the HL in the families of the probands were autosomal dominant in 602, autosomal recessive in 1577, and unknown inheritance mode in 370.

Variant Analysis
For the genetic analysis for this proband, we performed target genome enrichment for 68 previously reported genetic causes for deafness and MPS analysis described elsewhere [37]. In brief, amplicon libraries were prepared using an Ion AmpliSeq™ Custom Panel (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer's instructions, for 68 genes reported to cause nonsyndromic hereditary HL [37]. Emulsion PCR and sequencing was performed according to the manufacturer's instructions. The detailed protocol has been described elsewhere [38,39]. MPS was performed with an Ion Proton™ system using the Ion PI™ Hi-Q™ Sequencing 200 Kit and Ion PI™ Chip (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturers' instructions. The sequence data were mapped against the human genome sequence (build GRCh37/hg19) with a Torrent Mapping Alignment Program. The mean depth of coverage of 68 target genes was 363.0. The percentage of each region with more than 20 times coverage (indicating the percentage of each region sequenced 20 times or more by MPS) was 95.31%. After sequence mapping, the DNA variant regions were piled up with Torrent Variant Caller plug-in software. After variant detection, their effects were analyzed using ANNOVAR software [40,41].

Clinical Evaluations
The age of onset of HL, the incidence of progressive HL, and episodes of vertigo and dizziness were analyzed based on the medical charts of the probands with the CLDN14 variant.
Pure-tone audiometry was performed to evaluate HL. The pure-tone average (PTA) was calculated from the audiometric thresholds at four frequencies (0.5, 1, 2, and 4 kHz). If an individual did not respond to the maximum hearing level at a frequency, 5 dB was added to the maximum hearing level. The severity of HL was classified into mild (PTA: 20-40 dBHL), moderate (41-70 dBHL), severe (71-95 dBHL), and profound (>95 dBHL). The audiometric configurations were categorized as low-frequency, mid-frequency, high-frequency (gently or steeply sloping), flat, and deaf [56].
The vestibular examination findings, including caloric testing and the measurement of cervical and ocular vestibular evoked myogenic potentials (c/oVEMPs), were analyzed. Caloric testing involved the measurement of the maximum slow phase velocity (SPV) by cold water irrigation (20 • C, 5 mL, 20 s). We defined a maximum SPV value below 10 deg/s as representing areflexia and a value between 10 and 20 deg/s as representing hyporeflexia. For cVEMPs testing, electromyography (EMG) was performed using a pair of surface electrodes mounted on the upper half and sternal head of the sternocleidomastoid muscle, respectively. The electrographic signal was recorded using a Neuropack evoked potential recorder (Nihon Kohden Co Ltd., Tokyo, Japan). Clicks lasting for 0.1 ms at 105 dBnHL were presented through a headphone. The stimulation rate was 5 Hz, the bandpass filter intensity was 20 to 2000 Hz, and the analysis time was 50 ms. The responses to 100 stimuli were averaged twice. The oVEMPs testing was measured by bone-conductive vibration (BCV). The BCV was delivered in 4 ms tone bursts of 500 Hz vibration (rise and fall time = 1 ms and plateau time = 2 ms) by using a hand-held 4810 mini-shaker (Bruel and Kjaer, Naerum, Denmark), which was placed on the midline (Fz) of the forehead. The active electrode was located over the inferior orbital margin and a reference electrode was placed 2 cm below the active electrode. The ground electrode was placed on the chin. The patients laid in a supine position on a bed and looked up at an angle of approximately 30 degrees above straight ahead during the recording. The signals were amplified and bandpass filtered between 20 and 2000 Hz. The stimulus intensity was 115 dB force level, 500 Hz with an analysis time of 40 ms, and 50 responses were averaged for each run. The VEMPs asymmetry was calculated as follows: asymmetry ratio (AR) = (larger amplitude -smaller amplitude) × 100/(larger amplitude + smaller amplitude). In this study, an asymmetry ratio of >30% was defined as a decreased reaction and no reaction in amplitude VEMPs as absent.
Intervention for HL, including the use of hearing aids or cochlear implants, was investigated. To evaluate speech perception outcomes, speech discrimination scores (using the 67S Japanese monosyllable test) were used. The subjects sat 1 m away from the sound source facing zero-degree azimuth, and recorded monosyllable words in quiet were presented in the sound field at 65 dBSPL.

Conclusions
We present a patient with a novel variant in the CLDN14 gene identified from a non-consanguineous family. This is the first report of CLDN14-associated HL in an east Asian population. Serial audiograms indicated high-frequency hearing loss type, and the deterioration gradually spread to other frequencies, finally resulting in deafness. The patient showed normal vestibular function for caloric testing, cVEMPs, and oVEMPs. This is also the first report of an HL patient with a tight junction variant receiving a CI. The CI improved the proband's sound field threshold levels, but not their speech discrimination scores. This information contributes to our understanding, diagnosis, and treatment of HL caused by TJ disorders.