Background
Autosomal dominant sensorineural hearing loss (ADSNHL) accounts for about 20% of all hereditary nonsyndromic SNHL cases. Currently, at least 38 genes are known to be associated with nonsyndromic ADSNHL [
1]. In contrast to the autosomal recessive SNHL, which is typically congenital or prelingual, the onset of ADSNHL is often delayed and escapes the neonatal hearing screening. It may develop as late as in adulthood and it may even overlap with presbycusis [
2,
3]. Therefore, genetic etiology may not raise sufficient awareness in the diagnostic workup. The course of hearing loss in ADSNHL is generally progressive, although the speed of hearing deterioration and its severity may vary among different genes or even among different affected individuals. More importantly, progressive bilateral SNHL is often the first symptom in a number of syndromic forms of hearing loss [
4].
One of these genes is
EYA4 (
Eyes
Absent Homolog
4) which encodes a 640-amino-acid protein that serves as a transcription coactivator. In higher animals
EYA4 is a component of network composed of genes belonging to the Pax, Six, Eya, and Dach families which play a key regulatory role in the development of multiple organs including the eye, muscle, ears, heart, lungs, endocrine glands, placodes, pharyngeal pouches, craniofacial skeleton, and parathyroid [
5]. This protein contains a highly conserved 271 amino acid Eya domain (eya homologous region, eya-HR) at the C-terminus and a poorly conserved proline-serine-threonine (PST)-rich transactivation domain called variable region (eya-VR) at the N-terminus [
6]. It has been proposed that truncations of the C-terminal Eya domain cause nonsyndromic ADSNHL (DFNA10) whereas upstream truncations deleting the N-terminal variable region cause hearing loss with dilation cardiomyopathy [
7]. To date, there are only a few reported families in who ADSNHL segregated with
EYA4 mutations.
Here we present a Slovak pedigree with late-onset progressive SNHL analyzed by whole exome sequencing (WES), which allowed us to identify a novel pathogenic variant in the EYA4 gene.
Discussion & Conclusions
In the present study, we report a four-generation Slovak family in which postlingual nonsyndromic autosomal dominant SNHL segregates with the c.804G > C mutation in EYA4 discovered using whole exome sequencing approach. The mutation is located at the last base position of the exon 10. To the best of our knowledge, this is the first point mutation localized in exon 10 and the first known case of EYA4 gene associated with SNHL in the Slavic Caucasian population.
Nucleotide substitutions affecting the last base pair of exon should be regarded as a rare event of which the effect is difficult to predict. At least 10 similar nucleotide changes have been shown to cause abnormal mRNA splicing in mammalian genes [
28].
In-silico analysis using state-of-the-art prediction algorithms suggests that the mutation weakens splice donor site of intron 10. This may lead to complete or partial skipping of the exon 10, or to the activation of a cryptic splice donor site. As it is known that
EYA4 is not expressed in blood [
29], we tried to amplify
EYA4 by RT-PCR from a urine sample from proband as well as from healthy control to determine the exact impact of the mutation of the last exonic nucleotide on mRNA splicing. However, we did not detect any
EYA4 transcripts in the urine sample. Therefore, we applied the mini-gene assay to test the functionality of the exon 10 mutated splice site. Our findings confirmed the
in-silico predictions and showed that the c.804G > C mutation compromises donor site functionality and causes exon 10 skipping in vitro. Exon 10 skipping in the
EYA4 mRNA splicing would result in frame-shift p.(Ser243Leufs*29) that introduces premature termination codon (PTC) and would thus lead to nonsense-mediated mRNA decay (NMD). However, we cannot exclude the formation of other mutant transcripts, as disruption of one splice site can influence also splicing of other adjacent exons and this we have not tested.
To date, 19 pathogenic or probably pathogenic
EYA4 variants associated with non-syndromic autosomal dominant SNHL (DFNA10) have been reported in about 20 families. Moreover, a large deletion c.581_804del was associated with SNHL and dilation cardiomyopathy resulting in risk of premature death [
30,
31]. Genetic and clinical characteristics of known
EYA4 mutations are summarized in Table
4. Similar syndromic phenotype (hearing loss and cardiac malformation) plus microcephaly and mental retardation were observed in 9 Mb deletion 6q23.2–24.1 which also disrupts
EYA4 gene [
32].
Table 4
List of pathogenic variants in EYA4 identified to date and their hearing loss phenotypes
8 | c.464delC | p.(Pro155Glnfs*43) | frameshift | Dutch | childhood | moderate | mid- or high-frequency | c.464del | |
8 | c.511 G > C | p.(Gly171Arg) | missense | Chinese | 26–33 years | moderate to severe | gently sloping | c.511 G > C | |
8 | c.544_545insA | p.(Ser182Tyrfs*63) | nonsense | Chinese | 20–40 | moderate to profound | high-frequency to flat | c.544_545insA | |
8 | c.579_580insTACC | p.(Asp194Tyrfs*52) | frameshift | Swedish | 4–40 years | mild to profound | variable | c.579_580insTACC | |
9–10 | c.581_804del | p.(Asp194Glyfs*30) | frameshift | N/A | from schoolage | moderate to profound | mid-frequency | 4846-bp deletion | |
10 | c.804G > C | p.(Gln268His) | missense | Slovak | 10–40 years | mild to severe | gently sloping | c.804G > C | this paper |
11 | c.863 C > A | p.(Ser288*) | nonsense | Korean | N/A | moderate | reverse U-shaped | c.863 C > A | |
11 | c.978C > G | p.(Phe326Leu) | missense | Korean | N/A | moderate | down-sloping | c.909 C > G | |
12 | c1026_1027dupAA | p.(Thr343Lysfs*62) | frameshift | North American Caucasian | 1st-3rd decade | moderate to profound | gently sloping to flat | c.1468insAA | |
12 | c.1048_1049dupAA | p.(Arg352Profs*53) | frameshift | North American Caucasian | 2nd -4th decade | moderate to severe | mid- to high-frequency | c.1490insAA | |
13 | c1115_1118dupTTTG | p.(Trp374Cysfs*6) | frameshift | Hungarian | postlingual | up to profound | variable | c.1558insTTTG | |
13 | c.1154C > T | p.(Ser385Leu) | missense | Italian | postlingual | mild to profound | mid-frequency | c.1154C > T | |
13 | c.1177C > T | p.(Gln393*) | nonsense | Korean | N/A | moderately severe | mid- and high-frequency | c.1177C > T | |
14 | c.1194delT | p.(Met401Trpfs*3) | frameshift | Korean | from 1st decade | moderate | down-sloping | c.1194delT | |
intron 14 | c.1282-12 T > A | splicing effect | splice site | Australian Caucasian | 6 years to 4th decade | mild to profound | mid-frequency to flat | c.1282-12 T > A | |
15 | c. 1301 T > A | p.(Ile434Lys) | missense | Chinese | 8–38 years | mild to severe | mid-frequency to flat | c. 1301 T > A | |
18 | c.1643C > G | p.(Thr548Arg) | missense | Chinese | 17–40 years | mild to profound | variable | c.1643C > G | |
20 | c.1759C > T | p.(Agr587*) | nonsense | Belgian | 6–40 years | mild to moderate | mid-frequency | c.2200C > T | |
The most frequent type of mutations observed so far in
EYA4 were frameshifts and nonsense mutations, both leading to the introduction of a premature termination codon (PTC) into mRNA sequence. Presence of the PTC in the mRNA molecule makes the molecule a potential subject of NMD pathway that selectively degrades transcripts carrying PTCs which are 50–55 nucleotides upstream from the last exon-exon junction [
33]. In result, mRNA molecule transcribed from the mutated allele would be completely degraded. In the remaining families, nonsynonymous mutations leading to substitutions of highly conserved amino acid residues were identified (Table
4). The prevalence of
EYA4/DFNA10 related deafness is not known and it was only detected in Caucasian and East Asian population to date. The only relevant data on epidemiology is from Korea, where the prevalence of
EYA4 among nonsyndromic ADSNHL is estimated at 7.4% [
34]. In our series,
EYA4 mutation was identified in 5.56% (1/18) of probands with nonsyndromic ADSNHL analyzed by next-generation sequencing.
The exact molecular pathomechanism of hearing impairment associated with mutations in
EYA4 has not been determined yet, but it may result from lowered gene dosage or reduced protein activity. The EYA proteins are transcriptional coactivators that interact with the transcription factors SIX and DACH but lack a DNA-binding domain [
35].
EYA4 acts as a histone phosphatase and promotes efficient DNA repair. Studies of expression in rodent inner ear suggest developmental role during maturation of the inner ear as well as survival role in the mature system [
36]. One of the possible pathomechanisms involved in hearing loss development due to
EYA4 mutations may be the impaired regulation of Na+/K + -ATPase by altered expression of its β2b subunit as demonstrated in zebrafish model [
37].
Hearing loss in the investigated family had postlingual onset from 10 to 40 years, which corresponds to the onset range (6–40 years) reported in the literature [
38]. This relatively broad interval was observed even among the same mutation carriers and members of the same families and is not yet explained. Environmental or intrinsic factors, such as chronic noise exposure or other genetic factors may play a role. However, self-reported hearing loss onset used in this and previous studies is also a matter of subjective disability evaluation, which may vary among affected subjects. Especially in mild degree cases with slow progression, the hearing loss may remain unnoticed for a longer period of time. Therefore, prospective audiological assessment of yet asymptomatic mutation carriers will be required in the future to obtain more precise data. The audiogram shape and degree of hearing loss in
EYA4 deafness are variable. Most often it is described as mid-frequency hearing loss (“cookie bite” audiogram) of a mild to moderate degree at the time of onset. Subsequent hearing deterioration also affects the high frequencies resulting in the flat or down-sloping audiometric curve of a moderate to profound degree (Table
4). In our investigated family, the shape of audiogram was relatively constant (gently downsloping) since its onset. The severity of hearing loss based on pure tone average (0.5–4 kHz) in the five subjects with deafness duration of ≥20 years ranged from 43.75 dB to 77.5 dB, not yet fulfilling the traditional criteria for cochlear implantation.
The effect of
EYA4 truncating mutations on cardiac functions is often discussed due to the cardiomyopathy found in patients with large deletions comprising the variable domain [
7,
30,
32]. However, despite the process of NMD is known now for almost four decades and was accepted to be the consequence of mutations introducing PTC [
39], only Pfister et al. [
40] suggested that the frameshift mutations in
EYA4 will cause NMD. In such a case it is irrelevant to discuss the localization of the so-called truncating mutations in different EYA4 domains. We agree with this view, as all the patients with PTC share the same phenotype without cardiac symptoms similarly to the individuals with a missense mutation, which is also the case of patients studied in this report. All of them had normal morphology and function of the heart, without any signs of dilation cardiomyopathy. This would argue for the haploinsufficiency as the mechanism of this dominantly inherited hearing loss. Thus, it is more probable, that the reported cardiomyopathy was caused by other genetic factors present in the large deleted regions or created by the event of deletion. The definitive answer could be attained by estimating the level of the
EYA4 mRNA transcripts in patient’s cells, but
EYA4 is not expressed in blood leukocytes making this test inaccessible without a biopsy.
In conclusion, a novel EYA4 mutation, c.804G > C (p.Gln268His), was identified in a Slovak nonsyndromic ADNSHL family using WES and Sanger sequencing. This variant represents a nucleotide change at the last base pair of exon 10. We demonstrate in vitro that c.804G > C leads to exon 10 skipping resulting in frame-shift that introduces premature termination codon. To the best of our knowledge, this is the first EYA4 mutation associated with ADSNHL in the Slavic Caucasian population. Regarding the auditory phenotype, most of the variability concerned the onset of hearing loss, whereas the shape of the audiometric curve and progression after clinical manifestation were relatively uniform.