The present large-cohort study revealed that the prevalence of CDH23-gene associated hearing loss was 2.38% (289/12,139) among SNHL probands and 3.73% (258/6,912) among ARSNHL/sporadic probands in this Japanese population. Some patients (n=32) were found in ADSNHL families, which is probably due to a pseudo-dominant inheritance pattern. These frequencies are slightly higher than those in our previous report (1.6% in total, 2.5% in ARSNHL) (Miyagawa et al. 2012), but this is due to differences in the methodology; this study used sequencing for the entire exons of the CDH23 gene in contrast with the previous screening based on TaqMan for a limited number of common variants. Based on the use of over 100,000 samples collected in a more unbiased manner, the present study indicated the prevalence of CDH23-gene associated hearing loss among non-syndromic SNHL patients. The CDH23-gene associated hearing loss, along with SLC26A4, is the second or third most frequent type of hearing loss after GJB2-gene associated hearing loss. (Nishio and Usami 2015; Usami and Nishio, submitted), and that CDH23 variants are an important cause of non-syndromic SNHL.
The present study demonstrated a total of 131 possible disease-causing CDH23 variants, including 39 previously reported and 92 novel variants (Table 1). As in our previous reports on DFNB12 (Miyagawa et al. 2012), a majority of variants was found in the EC domain with only few exceptions found in the cytoplasmic domain. Thirteen out of the 123 possible causative variants were found in the DRE, DXNDN, and DXD motifs, which are thought to be important for calcium binding. These highly conserved EC calcium-binding motifs are thought to be essential for linearization, rigidification, and dimerization of the cadherin molecules (Nagar et al. 1996; Angst et al. 2001). It should be noted that p.E956K is located in the DRE motif, which is in agreement with the comparatively severe DFNB12 phenotype. According to recent computer analysis for the prediction of the impact of amino acid changes to protein structures, some possible pathologic variants are predicted to cause severe damage to the protein function of CDH23 (Supplemental Figure 1).
This study revealed that there are several common variants in the Japanese hearing loss population; p.P240L accounts for 33% of all CDH23 variants in the Japanese population, followed by p.R1588W (14%), p.R2029W (10%), and p.E956K (5%). These 4 common variants account for 62% of all variants. For such recurrent variants, founder effects have been demonstrated in many deafness genes. For example, with regard to GJB2, it is reported that c.35delG, which was predominant throughout Europe, the Middle East, North Africa, North and South America, and Australia; and c.235delC, which is commonly found in East Asians, are due to founder effects (see review; Tsukada et al. 2015). The p.P240L variant in the CDH23 gene, the most frequent variant in the Japanese as well as the Korean population, has been proven to be due to a founder effect using the STR marker (Kim et al. 2015). In fact, the MAF in Japanese control is exceptionally high compared to those in other ethnic groups (Table 1), which is consistent with the fact that there are many patients with CDH23-related hearing loss due to the founder effect.
DFNB12 phenotype vs. Usher phenotype
The majority of the causative variants (either pathogenic, likely pathogenic, or VUS) identified in this study (Table 1) were missense mutations, which are supposed to have a residual function. With regard to genotype/phenotype correlations, the DFNB12 phenotype is reported to be associated with biallelic missense mutations, whereas the USH1D phenotype is associated with presumably functional null alleles, including nonsense, splice-site, frameshift, or some missense mutations (Bork et al. 2001; Wagatsuma et al. 2007; Ohshima et al. 2008; Miyagawa et al 2012). It has been reported that cases in which an Usher allele and DFNB12 allele are present in trans configuration, the DFNB12 allele is phenotypically dominant (Schultz et al. 2011).
In this study, the majority of patients with biallelic missense mutation or those with compound heterozygous of missense and truncating mutations showed the DFNB12 phenotype, which is generally in line with this rule. In our cohort, a limited number of patients were found to show the Usher phenotype (Table 3). Three out of five were associated with truncating mutations and had visual impairment (Usher phenotype), which also supports this rule. However, patient No.3330 with a biallelic homozygous missense mutation (p.[R2489C]; [R2489C]) and the patient No. 4177 with a nonsense mutation and missense mutation (p.[Y288X]; [G2017S]) also showed the Usher phenotype. Although the functional significance of these missense mutations is unknown, they are supposed to be functionally null alleles.
A wide range of hearing loss: clinical characteristics and genotype/phenotype correlations
Using more than 10,000 hearing loss patients, the present updated study clearly demonstrated that CDH23 variants cause a wide range of hearing loss from non-syndromic hearing loss (DFNB12) to syndromic hearing loss and Usher syndrome type ID (USH1D). Also, the present results showed that most cases of CDH23-gene associated hearing loss are congenital/early-onset, but nonetheless demonstrated that there is also a certain number of cases of late-onset (up to the 60s) progressive hearing loss. As shown in Fig. 2A, B, a wide range of onset ages (awareness of hearing loss) was found from congenital to 60+ years old, although the majority of cases were congenital or early-onset. Genotype (variant combination) and phenotype (onset age) were shown to be well correlated. The patients with p.[P240L];[P240L] and p.[P240L];[E956K] showed congenital and severe hearing loss, whereas the patients with the p.R2029W or p.R1588W variant showed late-onset high frequency-involved hearing loss (Fig. 2A,B, 3). We have previously reported the clinical characteristics of CDH23-related hearing loss to be high frequency-involved progressive hearing loss (Miyagawa et al. 2012). With regard to audiogram configurations, the majority of patients had some residual hearing in the lower frequencies, as reported previously (Wagatsuma et al. 2007; Miyagawa et al. 2012). Further, the progressive nature of hearing loss was demonstrated by serial audiograms (Miyagawa et al., 2012), and reconfirmed using audiograms with the average for each age plotted (Fig. 4). To date, several replication studies stating the same clinical features have been reported (Mizutari et al. 2015; Kim et al. 2016; Ramzan et al. 2020). Combined with these reports, the present large-cohort data confirmed that this clinical feature tends to be constant regardless of the type of variant.
In addition, we have previously reported some types of age-related hearing loss (ARHI) due to CDH23 variants, both of which had hearing loss due to a homozygous mutation in p.R2029W (Usami et al. 2012). Such a late-onset phenotype is not surprising because a series of animal studies have shown that CDH23 variants are involved in the C57BL/6 mouse strain, which is the most common mouse model for ARHI (Noben-Trauth et al. 2003). ARHI is believed to be a typical complex disorder associated with both genetic factors and environmental factors. Degeneration and age-related changes in the cochlea might be accelerated by accumulated external and internal factors. These environmental factors including exposure to noise, ear disease, ototoxic drugs, associated disease (circulatory disease and diabetes mellitus, etc.) play important causative roles in ARHI. Therefore, in addition to the monogenically inherited particular type of ARHI (late-onset hereditary hearing loss) shown in this study, various SNPs may be involved in susceptibility to ARHI. It should be noted that variants of CDH23 are reported to be associated with noise-induced hearing loss (Kolwalski et al., 2014). Taken together, these findings suggest that the residual function defined by the CDH23 variants can cause various types of hearing loss. (Fig. 5).
In this study, at least 100 out of the 103 patients for which clinical data were available, used HAs or CI/EAS, indicating that these are common therapeutic interventions used in Japan. In terms of CI/EAS, our series of papers demonstrated the CI is a good therapeutic option for patients with hearing loss over all frequencies, and EAS is a good option for patients with residual hearing (Usami et al. 2012b; Miyagawa et al. 2013b; Moteki et al. 2017, 2018; Yoshimura et al. 2020; Usami et al. 2020). As a significant portion of patients with CDH23 variants have residual hearing, it is extremely important to perform atraumatic CI surgery to preserve residual hearing for this particular category of patients. With regard to post-operative residual hearing after EAS, we have demonstrated that the hearing preservation rate among patients with mutations in stereocilia-related genes, such as CDH23, MYO7A, or MYO15A, was statistically better compared to the patients with other etiologies (Yoshimura et al. 2020). Genetic testing is also useful for estimating the presence of residual hearing for very young children whose residual hearing is difficult to measure by auditory brainstem response (ABR).