Synaptopathy in the noise-exposed and aging cochlea: Primary neural degeneration in acquired sensorineural hearing loss

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.

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From glutamate co-release to vesicular synergy: vesicular glutamate transporters

The extreme genetic heterogeneity of nonsyndromic hearing loss (NSHL) makes genetic diagnosis expensive and time consuming using available methods. To assess the feasibility of target-enrichment and massively parallel sequencing technologies to interrogate all exons of all genes implicated in NSHL, we tested nine patients diagnosed with hearing loss. Solid-phase (NimbleGen) or solution-based (SureSelect) sequence capture, followed by 454 or Illumina sequencing, respectively, were compared. Sequencing reads were mapped using GSMAPPER, BFAST, and BOWTIE, and pathogenic variants were identified using a custom-variant calling and annotation pipeline (ASAP) that incorporates publicly available in silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2, and Align-GVGD). Samples included one negative control, three positive controls (one biological replicate), and six unknowns (10 samples total), in which we genotyped 605 single nucleotide polymorphisms (SNPs) by Sanger sequencing to measure sensitivity and specificity for SureSelect-Illumina and NimbleGen-454 methods at saturating sequence coverage. Causative mutations were identified in the positive controls but not in the negative control. In five of six idiopathic hearing loss patients we identified the pathogenic mutation. Massively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss.

https://www.pnas.org/content/pnas/107/49/21104.full.pdf

Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.

Sensory neuron diversity in the inner ear is shaped by activity

Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C→T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca2+-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.

Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice.

To the Editor:

The prevalence of significant hearing loss (≥ 25 db HL) is 15-20% in young adults and rises to approximately 50% in individuals 80 years of age or older [Morton 1991]. Autosomal dominant nonsyndromic hearing loss (ADSNHL) accounts for approximately 15% of inherited hearing loss. To date, 22 genes have been implicated as causative for ADNSHL, and a further 30 loci mapped to autosomal chromosomal regions [Van Camp and Smith 2007].

ADSNHL at the DFNA9 locus is unusual amongst ADNSHL forms of deafness in that it is also associated with vestibular dysfunction. This type of hearing loss is caused by mutations in the COCH gene (chromosome 14q12), which encodes cochlin, a protein that consists of a signal peptide, an LCCL module, and two von Willebrand factor A (vWFA) domains (Table I) [de Kok et al., 1999]. Cochlin is the most highly expressed protein in the human and mouse inner ear, but its precise function remains unclear [Dessens et al., 2004; Robertson et al., 2006]. A possible role in either structural integrity or antimicrobial activity is predicted by its protein structure [Liepinsh et al., 2001] and immunohistochemistry of a temporal bone from a person with DFNA9-related deafness showing abundant aggregation of homogeneous acellular eosinophilic deposits with loss of cellularity in the cochlea and the vestibular labyrinth [Robertson et al., 2006].



Table I

DFNA9-causing mutations in the COCH gene



Most DFNA9-causing mutations are located within the LCCL domain, which is predicted to be involved in protein folding or a host-defense function (Table I) [Trexler et al., 2000]. The p.P51S variant that affects the LCCL module is the most frequently observed mutation in DFNA9 families [Bischoff et al., 2005; de Kok et al., 1999; Fransen et al., 2001; Fransen et al., 1999; Lemaire et al., 2003]. The identification of a large number of DFNA9 families carrying this variant has facilitated a detailed longitudinal analysis of the audiometric and vestibular impairment in 74 mutation carriers, which has shown that vestibular failure precedes and progresses more rapidly than hearing impairment [Bischoff et al., 2005].

In this study (approved by the University of Iowa Institutional Review Board), we recruited American subjects with ADNSHL and obtained DNA samples and audiograms. Audiograms were formatted for audioprofile analysis, which was conducted using the AudioGene v2.0 system [Hildebrand et al., 2008]. This system predicts the likely underlying genetic cause of hearing loss based on a number of different phenotypic parameters, including auditory thresholds, and rate of decrease in hearing relative to age and frequency. AudioGene v2.0 was trained with audiometric data from families with known deafness-causing mutations in KCNQ4 (DFNA2), DFNA5 (DFNA5), WFS1 (DFNA6/14/38), TECTA (DFNA8/12), COCH (DFNA9) and COL11A2 (DFNA13).

To validate AudioGene v2.0 as a clinical and research tool, we studied a cohort of 77 families segregating presumed ADSNHL represented by audiograms from 160 individuals. Individuals from seven families were identified by AudioGene v2.0 as having a DFNA9 profile, and in six families, mutation screening of the COCH gene was completed (genomic DNA was unavailable for screening in one family). While no mutations were identified in five families, one family was found to segregate the p.P51S mutation (positive predictive value, ∼16.7%) (Figs ​(Figs1,1, ​,2).2). To our knowledge, this family is the first outside Western Europe to be reported with this mutation. However by comparing highly heterozygous short tandem repeat polymorphisms (STRPs) tightly linked to the COCH gene, we could show that this family is related to five previously reported Belgian p.P51S families [Fransen et al., 2001] (data not shown), providing further evidence of a founder effect in the Benelux region of Western Europe.



Figure 1

Pedigree of the five-generation American family with nonsyndromic autosomal dominant HFSNHL, vestibular dysfunction and SSCD. Genotypes for nuceotide c.151 in the COCH gene are shown for those individuals included in the genetic analysis. Individuals ...





Figure 2

The causative mutation in the COCH gene in the American family. The mutation is a heterozygous C-to-T base change in exon 3 (c.151C→T) that results in substitution of a proline residue for a serine residue at position 51 (p.P51S).



The clinical presentation of all p.P51S families is very similar (Table I) [de Kok et al., 1999; Fransen et al., 2001; Fransen et al., 1999; Lemaire et al., 2003]. The hearing loss initially preferentially affects high frequencies (down-sloping) and progresses to become profound across all frequencies at a rate of ∼1.8 dB annually [Bom et al., 1999]. In a few exceptional cases, however, hearing at low frequencies declines much more rapidly, at greater than 24 dB annually [Bom et al., 1999]. In the American family, the self-reported age-of-onset of hearing loss ranged from 20 to 60 years. The hearing loss was progressive, with a general trend of initial moderate-to-severe high frequency (> 2000 Hz) hearing loss (HFSNHL) that became profound (Fig.3). In younger persons, the audioprofile was down-sloping, preserving hearing in the low and mid frequencies; with advancing age, low and mid frequency loss developed and the audioprofile flattened. Individuals IV:5 and IV:6 represent phenocopies.



Figure 3

Audiograms of representative affected family members at various ages. Affected individuals initially exhibit high frequency (> 2000 Hz) mild-to-moderate SNHL (HFSNHL) that progresses to become severe-to-profound before flattening out later in ...



All hearing impaired persons in this family excluding IV:5 and IV:6 also had vestibular impairment characterized by intermittent unsteadiness, particularly in the dark, and sporadic periods of dizziness. In addition, one person was diagnosed with Meniere’s-like symptoms and another with autoimmune inner ear disease requiring treatment with a diuretic and a low-salt diet. This variety of complaints is similar to that reported in other p.P51S families [Bischoff et al., 2005]. In general, p.P51S mutation carriers over 40 years of age usually have prominent vestibular symptoms however these complaints only emerge from the medical history if a targeted vestibular history is obtained.

Computed tomography (CT) scanning on a number of family members revealed intact ossicles, cochleae and semicircular canals with the exception of individual V:1. This person presented at age 31 in October, 2006, for an audiogram because of the family history of hearing loss. He has no complaints and by audiometry had 10 dB thresholds bilaterally through 2 kHz, a drop to 25 dB between 3-6 kHz, and normal hearing again at 8 kHz (Fig.4A). In February, 2008, he returned with a complaint of left aural fullness and persistent otalgia (6-9 months) and underwent temporal bone CT, which revealed bilateral superior semicircular canal dehiscence (SSCD) (Fig.4B).



Figure 4

Audiometry and temporal bone CT of individual V:1. A Audiogram recorded at 31 years of age showing slightly elevated hearing thresholds at high frequencies. B Coronial high-resolution CT scan of the temporal bones showing bilateral superior semicircular ...



SSCD is a rare disorder. Originally described by Minor and colleagues, it is characterized by the absence of bone overlying the superior semicircular canal, which creates a third labyrinthine window (with the oval and round windows) [Minor 2000; Minor 2005]. The consequence is the loss of acoustic energy and abnormal stimulation of the vestibular system; the clinical manifestations include Valsalva- and exercise-induced vertigo, sound-induced vertigo (Tullio phenomenon), and variable conductive hearing loss [Cox et al., 2003; Mikulec et al., 2004; Minor et al., 2003]. Diagnosis requires high resolution temporal bone CT [Belden et al., 2003; Hirvonen et al., 2003].

The genetics of SSCD have not been studied, and our finding of a COCH mutation in a single patient with SSCD is noteworthy since both DFNA9-related deafness and SSCD are rare. Although it seems unlikely that SSCD is directly related to the well-documented type of vestibular impairment that is part of the DFNA9 phenotype, SSCD may be present in other DFNA9 patients. We therefore recommend high resolution temporal bone CT in patients with DFNA9-related deafness and screening of COCH in sporadic or familial cases of SSCD. This diagnostic algorithm is important because SSCD and its associated symptoms can be partially or fully corrected with surgery [Carey et al., 2007; Limb et al., 2006; Mikulec et al., 2005; Wilkinson et al., 2008].

A remarkable finding in the process of identifying DFNA9/COCH as the underlying cause of deafness in this American family was not only that a dedicated computer program, AudioGene v2.0, hinted at this possibility, but that this occurred in the course of a screening procedure for DFNA2 families [Hildebrand et al., 2008]. It is reassuring to find that AudioGene v2.0 can distinguish between the audioprofiles of genetically different types of ADNSHL, especially when both DFNA2 and DFNA9 show remarkably similar high frequency hearing loss with steeply downsloping thresholds.

Admittedly, in the case of p.P51S mutation carriers genetic screening should be preferentially directed to DFNA9 and DFNA11, on the basis of the vestibular impairment findings. However, it is realistic to acknowledge the possibility that vestibular symptoms can go unnoticed. Furthermore, with other DFNA9/COCH mutations vestibular failure can be less prominent. It is not invariably penetrant and/or occurs at a more advanced age [Kemperman et al., 2005; Pauw et al., 2007a].

Jana M. Van Rybroek

Papers

Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice.

Mutation in the COCH gene is associated with superior semicircular canal dehiscence