Mutations in GJB2 have been reported as the most frequent cause of hereditary HL [14, 15]. For this reason, we first analyzed the GJB2 gene by Sanger sequencing in all 851 probands. Using this method, we determined the genetic etiology in 245 (29%) of the probands, who were then excluded from further analyses.
In the next step, the remaining probands were selected based on the clinical phenotype. Although we focused on a relatively uniform group of patients from the audiological point of view (mid-frequency HL), the number of potentially involved genes was large (> 226). Moreover, the spectrum of the genes responsible for this particular form of HL is mostly unknown. It is not practical to analyze each gene separately in such a case. Therefore, next generation sequencing (NGS) allowing the analysis of many genes simultaneously seemed to be the best solution [16, 17].
Using ES, we identified the genetic aetiology in six of the 30 probands (20%) with mid-frequency HL. Additionally, novel candidate variants (VUS) were found in eight probands. A compound heterozygous pathogenic and a VUS variant were observed in one proband. The detected variants were found in nine genes (COL4A5, DIAPH1, MYO7A, MYO15A, OTOGL, TECTA, TMC1, TMPRSS3, TSPEAR) harbouring four pathogenic variants, three likely pathogenic variants and nine VUS variants associated with hearing impairment. Seven of these variants have not yet been described in the literature or have not yet been submitted to deafness databases (Table 2). Five of the identified genes (MYO7A, MYO15A, TMC1, TMPRSS3 and TSPEAR) have not yet been reported as a cause of mid-frequency SNHL. Other genes which are known to cause non-syndromic mid-frequency HL but were not detected in our cohort include: CCDC50 [18], COL11A1 [19], COL11A2 [20, 21], EYA4 [22], KCNQ4 [23], OTOA [24] and POU4F3 [25]. Among these genes, we were able to identify three distinct groups based on the site of their expression and their function [18, 26–29].
Glycoproteins expressed in the tectorial membrane. The tectorial membrane (TM) of the cochlea is a ribbon-like strip of extracellular matrix that contacts the tips of the sensory hair bundles of the outer hair cells. Sound induces movement of these hair cells relative to the TM, deflects the stereocilia, and leads to fluctuations in hair-cell membrane potential, transducing sound into electrical signals [30, 31].
The TECTA gene encodes a protein called alpha-tectorin. Mutations in TECTA account for 2–4% of all autosomal dominant non-syndromic SNHL characterized by mild prelingual HL from a clinical point of view [15, 32–34]. TECTA contains several extracellular matrix (ECM)-interacting domains, including a nidogen-like domain and von Willebrand factor type D domains (vWFDs), which may allow binding to collagens and other glycoproteins, and a zona pellucida (ZP) domain important for the multimerization and stability of the protein. Missense mutations in different Tecta domains are known to cause different phenotypes with distinct changes in the structure of the tectorial membrane. Mutations in the zona pellucida domain cause mid-frequency HL with a typical U-shaped or shallow U-shaped audiogram, whereas mutations in the zonadhesin-like domain comprising vWFD1, vWFD2 and trypsin inhibitor-like (TIL) repeats lead to high-frequency HL [35, 37]. Our results are in agreement with the findings of previous studies: four of our variants (p.Gly1858Glu, p.Thr1866Met, p.Arg1890Cys and p.Tyr1942Cys) located in the ZP domain were identified in probands with a U-shaped audiogram. However, the p.Cys650Arg variant identified in the cysteine-rich TIL1 domain is responsible for mild HL, which became clinically manifest at the age of 16 years. The audiometric curve has a shallow U-shape profile. Similarly, Yasukawa et al. [37] identified the likely pathogenic p.Cys606Gly variant in the TIL1 domain in a proband with mid-frequency HL. Variant p.Lys2150Asnfs*9 is located in the last exon and causes replacement of the last six amino acids and extension of the glycosylphosphatidylinositol (GPI) anchorage of TECTA protein by two amino acids. It has been shown that GPI-dependent release of TECTA is required for elongation and bundled organization of collagen fibrils on the apical surface of inner supporting cells during formation of the TM [38]. Therefore, any change in the conformation or length of the GPI anchorage may impair this process and lead to a clinical phenotype. However, further study of these mechanisms is required. In our study, 10% of all probands with a U-shaped audiogram carried pathogenic variants in TECTA, and when we also take into account all the identified VUS variants, the theoretical contribution of TECTA to hearing impairment in our cohort may be as high as 47%. This is more than the published prevalence of TECTA in patients with U-shaped audiograms, for example in Japan, where 6% of patients with U-shaped audiograms could be attributed to TECTA [36]. The difference may be explained by high variability in the prevalence of causal deafness variants and genes among different ethnic populations as a well-known phenomenon in hereditary HL [39].
The OTOGL gene encodes an otogelin-like protein, which has structural similarities to the epithelial-secreted mucin protein family [40]. Its mutations lead to non-syndromic prelingual HL, which is mild to moderate and stable, with autosomal recessive inheritance [41]. The 2,343-amino acid protein contains an N-terminal signal peptide, four paired von Willebrand factor (VWF) and cysteine-rich (C8) domains, four trypsin inhibitor-like (TIL) domains, and a C-terminal cystine knot motif [40]. We identified two heterozygous variants in proband D1315. The first is the known pathogenic nonsense variant p.Gln520* in vWFD2 [42]. The second likely pathogenic variant, c.4032_4054 + 30del, has not yet been published. It is located at the interface of the 34th exon and 34th intron and, according to HSF and MaxEnt, it can cause an alteration of splicing due to activation of several cryptic acceptor and donor sites. However, we cannot rule out exon skipping, which may result in a more significant shortening of the protein sequence located between domains vWFD3 and vWFD4. The exon skipping does not lead to a frameshift, and therefore, according to Abou Tayoun et al. [43], only the PVS1_strong classification criterion can be applied. On the other hand, vWFD domains containing the multimerization consensus site CGLC are essential for the multimer assembly of proteins expressed in the tectorial membrane (like α-tectorin, otogelin or otogelin-like protein) to form a filament and higher order structures. Although little is known about these domains in OTOGL, mutations in the vWFDs of TECTA have been clearly connected with hearing impairment [37, 44]. Moreover, the shape of the audiometric curve observed in our proband is almost identical to the audiogram obtained from the compound heterozygous patient carrying mutations p.Gln520* and p.Arg925* reported by Bonnet et al. [42]. The vestibular hypofunction observed in patients by Oonk et al. [45] was not diagnosed in our proband.
Collagens expressed in the cochlea. The collagen superfamily comprises 28 types (I–XXVIII) in vertebrates [46]. Collagens are fibrous structural proteins involved in the construction of skin, cartilage, bone, eye, and other tissues [47]. All collagen molecules are comprised of one, two, or three different types of α-chain subunits tightly wrapped into a triple helix. A single missense mutation of a glycine to another residue in the triple helix can result in almost 40 genetic diseases [46]. Collagens encoded by genes COL2A1, COL4A3, COL4A4, COL4A5, COL4A6, COL9A1, COL9A3, COL11A1 and COL11A2 are essential for proper auditory function. Mutations in these genes are linked to non-syndromic or syndromic HL (Marshall syndrome, fibrochondrogenesis, Alport syndrome, Stickler syndrome) [19, 48]. In this study, we present a novel variant in the COL4A5 gene. Variants in this gene are associated with X-linked Alport syndrome [49]. In 80% of all cases, Alport syndrome is caused by mutations in the COL4A5 gene [50]. The disorder shows considerable heterogeneity in the age of onset of end-stage renal disease and the occurrence of deafness among particular families [51]. Most patients have mid-frequency HL, although high frequency and flat SNHL curves may be present, as well. SNHL in males may first manifest around the age of 10 years and usually gets progressively worse [49, 52]. In family FAM618 we identified the variant p.Gly472Glu, which interrupts the Gly-Val-Lys triplet in the α-chain and results in disruption of the triple helix’s function, changing the scaffolding and flexibility of collagen IV [46]. This results in an Alport syndrome phenotype in both siblings (D815, D817).
Genes expressed in hair cells. Myosins play an important role in the cellular organization of the cochlea. In general, myosins are motor proteins, and based on their variable C-terminal binding domains, they are included in conventional myosins (class II) and unconventional myosins (classes I and III to XV) [53]. Unconventional myosins IA, IIIA, VI, VIIA and XVA occur in the cochlea. MYO7A and MYO15A, encoding unconventional myosins, are expressed in the outer and inner hair cells of the Organ of Corti [54, 55].
MYO15A is known to be the third-most mutated gene causing severe to profound ARNSHL, after GJB2 and SLC26A4 mutations. Mutations in this gene cause both pre- and post-lingual forms of progressive HL [41, 56]. Our patient D1321 clinically manifests profound non-syndromic HL. Interestingly, she passed the neonatal hearing screening, and her HL became apparent at the age of 1.5 years. We identified two variants, c.5693G > A and c.8050T > C, the latter of which was previously described in trans with other pathogenic variants [57, 58], whereas c.5693G > A variant was submitted only to the ClinVar archive and at the Deafness Variation Database (https://deafnessvariationdatabase.org/) [59]. It is located in the myosin motor domain of the MYO15A protein. This domain consists of ATP- and actin-binding sites, which can generate force and move actin filaments; therefore, motor domain dysfunction results in shorter stereocilia with an ectopic staircase structure of stereocilia associated with a severe deafness phenotype [60].
The MYO7A tail contains a pair of myosintail homology 4–protein 4.1, ezrin, radixin, moesin (MyTH4-FERM) tandems separated by an SH3 domain. Mutations in the head and tail regions of MYO7A lead to defects in mechanotransduction and result in Usher syndrome 1B (USH1B), an autosomal recessive disorder with sensory impairment [61]. In addition to syndromic conditions, mutations in MYO7A can cause dominant (DFNA11) [62] and recessive (DFNB2) [63] non-syndromic HL. The p.Arg.1338His variant detected in FAM411 is located in the FERM 1 domain of the tail of the myosin VIIA protein. The nonsense variants p.Arg1338AlafsTer61, p.Gln1336Ter or p.Glu1337SerfsTer62, linked to Usher syndrome, were identified close to our identified p.Arg1338His variant. VUS missense variants p.Arg1338Ser or p.Arg1338Cys with unclear clinical phenotype were also identified (https://deafnessvariationdatabase.org/). Our proband had onset at the age of 22 years with mild progressive hearing impairment and a U-shape audiogram. Moreover, no retinal abnormalities were recorded, indicating the dominant non-syndromic HL.
Transmembrane channel-like protein 1 is a protein encoded by the TMC1 gene. The predicted structure of TMC1 shows a dimeric channel with 10 transmembrane domains (S1–S10) and intracellular amino- and carboxyl-termini in each monomer. Transmembrane helices S4–S7 form a groove that lines the pore of sensory transduction channels [64]. The transmembrane channels are proteins necessary for hair cell mechanotransduction [65]. Dominant mutations in TMC1 cause the late-onset progressive HL phenotype, whereas ARSNHL cases are linked to congenital severe-to-profound HL [66, 67]. We identified a missense variant, c.1949C > T, which is predicted to substitute a conserved proline at position 650 for leucine, which may influence the correct folding or assembly of TMC1 into multimers. It is located in the helical S9 domain. Two pathogenic autosomal recessive variants were previously identified in close proximity to our variant: p.Met654Val [68] and p.Ser647Pro [69]. However, according to the phenotype observed in FAM416, we cannot rule out the dominant character of inheritance of the p.Pro650Leu variant or a yet undetected causal variant in a different gene. One young family member, D1337, showed still normal hearing, although she carried the same variant as her father, thus supporting the possibility of late-onset HL.
The TMPRSS3 gene encodes a member of the serine protease family. Transmembrane Serine Protease 3 is linked to hair cell sterocilia mechanics and to actin network formation, which supports cell motility and integrity. Apart from hair cells, the TMPRSS3 gene is also expressed in the inner and outer pillar cells and the Deiters cells, stria vascularis or ganglion spirale [70, 71]. This gene was identified by its association with childhood onset of an autosomal recessive deafness. The hearing impairment in families with mutations in the TMPRSS3 gene has a prelingual to postlingual onset. A common feature of patients with TMPRSS3 mutations is progressive HL beginning in high frequencies and often leading to partial or complete deafness. However, Sasamori et al. [72] reported a case of progressive mid- to low-frequency sensorineural HL associated with mutation of the TMPRSS3 gene. We identified a likely pathogenic variant, p.Arg106Cys, which is located within exon 4 and causes the negatively charged residue arginine to change to a neutral hydrophobic cysteine at position 106. This change can result in the loss of hydrogen bonds and/or disturb the correct folding of TMPRSS3. It has been observed in the compound heterozygote state with p.(Phe13Serfs∗12) and p.Ala306Thr, where it cosegregated with prelingual profound hearing impairment or postlingual milder hearing impairment, respectively [70]. The homozygous form observed in proband D298 has not previously been observed. Although Gao et al. [70] predicted a very subtle or no effect on auditory function of homozygous p.Arg106Cys, we have shown that it is associated with moderate to severe perilingual progressive HL with a typical U-shape audiogram.
The DIAPH1 gene encodes human protein DIA1, a formin that elongates unbranched actin [73, 74]. The correct polymerization of actin is critical for the formation and elongation of stereocilia on the apical surface of cochlear hair cells [75]. Expression of DIAPH1 in the Organ of Corti has been proven in the inner pillar hair cells as well as at the base of the outer hair cells and in the outer pillar cells [76]. DIA1 consists of the GTPase-binding domain (GBD), a partially overlapping N-terminal diaphanous inhibitory domain (DID), formin homology (FH) domains FH1 and FH2, and the C-terminal DAD. It is held inactive in the resting state through an autoinhibitory intramolecular interaction between the DID and the DAD, which is regulated by Rho family GTPases [74]. In this study, we identified the VUS variant p.Gly1197Asp which, according to the Human Splicing Finder, has no significant impact on splicing signals. It is located in close proximity to the MDxLLxxL recognition site (1199-1206aa), which presents the central consensus motif of the DAD domain and is essential for binding to the DID domain. We hypothesize that our missense c.3590G > A variant results in a change from a small, uncharged amino acid glycine to a larger, charged residue aspartate. This may have a profound effect on the conformation of the amphipathic helix of the DAD domain and subsequently impair interaction with the acidic groove of the DID. Disruption of the autoinhibitory intramolecular DID-DAD interaction and consequent activation of DIA1 may explain the DFNA1 pathology [74]. DIAPH1 mutations have been associated with mild- and low-frequency SNHL, characterized by progressive deafness starting in childhood [73]. In contrast, some described variants showed hearing impairment beginning in the high-frequency range and progressing to deafness involving all frequencies [74]. Audiograms of our proband and family members showed a typical U-shape. Autosomal dominant deafness caused by mutations in the DIAPH1 gene can be associated with thrombocytopenia [76, 77]. However, our family was lost in follow-up, and therefore we cannot rule out this phenotype.
The TSPEAR gene encodes the thrombospondin-type laminin G domain and EAR repeats protein (TSPEAR), which was detected at the basal region of the stereocilia of hair cells. Mutations in TSPEAR cause non-syndromic SNHL with prelingual onset [78]. We identified the nonsense variant p.Glu624* in exone 12 resulting in a loss of the EAR7 repeat. Seven epilepsy-associated repeats (EARs) of TSPEAR are predicted to form a beta-propeller structure, function as part of a ligand-binding domain and mediate protein-protein interactions [79]. Delmaghani et al. [78] identified in 3 affected brothers from a consanguineous Iranian family segregating autosomal recessive nonsyndromic sensorineural deafness a homozygous frame-shift mutation (c.1726G > T + c.1728delC) that was predicted to result in termination of translation in the EAR6 repeat, connected with defective secretion of the mutant protein and congenital profound sensorineural deafness. As both affected siblings in our family FAM174 segregated a similar HL phenotype, but only one of them carried the homozygous variant in TSPEAR, we may assume that in addition to the identified p.Glu624* variant in the TSPEAR gene a second, yet unknown gene may contribute to HL in this family.
In general, autosomal recessive variants are mainly responsible for prelingual forms of severe to profound hearing impairment, whereas autosomal dominant variants cause HL that usually appears later and is often mild or moderate. Exceptions comprising postlingual HL include recessive variants in the TMPRSS3 gene; prelingual include dominant variants in the TECTA gene [32].