Based on WES data as well as segregation and genotype-phenotype correlational studies, mutations in MYO15A gene were identified as a main contributor of NSHL in the first and second surveyed families.
MYO15A, as a new branch of the myosin protein-coding gene superfamily, has a role in stereocilia formation.[11] This gene is considered as the third leading cause of ARNSHL in many populations [12, 13], including Iran, with a prevalence rate ranged from 4.8-9.6%[14-16] . Moreover, mutations in this gene have been associated with severe-to-profound HL. Screening of 66 coding exons through Sanger sequencing is expensive and more time-consuming. On the contrary, high-throughput techniques can save time and money[17].
MYO15A is a different form of myosins protein with long N-terminal extensions following by the conserved motor domain, IQ motifs (calmodulin/ myosin light chain binding), MyTh4 domains (Myosin-Tail like Homology region 4), FERM motifs (4.1 protein, Ezrin, Radixin, and Moesin), SH3 domain (Src Homology 3), and the PDZ ligand domain (Post synaptic density protein (PSD95), Dlg1 (Drosophila disc large tumor suppressor), and zo-1 (Zonula occludens-1 protein) [18]. The identified c.T6442A: p.W2148R variant in our study is located in the first (MyTH4) domain and is also conserved among different species (data not shown). The association of (MyTH4) domain mutations with hearing loss was firstly reported in 1998 [19]. Due to documents, variants in this domain have related severe to profound hearing loss, which is consistent with our pedigree phenotype [5, 15, 16, 20-22]. To the best of our knowledge, p.P2073S, p.R2124Q [22] p.P2073L, p.V2114G [16], R2146Q [23], p.R2146W [24] and c.6273+1G>A [25] variants have been detected in Iranian population until now [15, 16, 22]. The p.R2124Q and p.P2073S were the first reported mutations in the MyTH4 domain of MYO15A protein which was located in conserved fourth helix of MyTH4. Variations in the MyTH4 domains interfere in forming of transmembrane actin microfilament assembly complex at the stereocilia tips [22].
Mehregan et al, reported p.Arg2146Gln in the fourth helix of the first MyTH4 core, which results in severe to profound hearing loss. Structural analysis of this variation has revealed that this substitution alters binding properties at the domain surface [24].
The substitution of a highly conserved amino acid (Table 1), hydrophobic non-polar tryptophan, with arginine can lead to a loss of hydrophobic pocket. The counterparts of p.W2148 in the more studied family members, i.e. MYO7A is W1192. Sans CEN2 (a scaffold protein) interacted with MyTH4 domain by extensive hydrogen bonding, hydrophobic contacts, and charge-charge interactions. The hydrophobic pocket in MYO7A comprised the conserved A1189, W1192, I1193, P1220, and Y1223 residues. Substitution of Ala1189 by Glu, leads to a
~10-fold decrease of the binding affinity between MyTH4 domain and CEN. The complex of myosins and SANS linked cadherins to the actin cytoskeleton [26]. Woo et al, explained that p.R2146Q in myosin 15A and R1190 of MYO7A had similar structure in MyTH4- FERM domains and interfere to binding CEN2 to this domain [5].
In addition, Myosin interacts with other scaffolding proteins (whirlin and Eps8 (Epidermal Growth Factor Receptor Pathway Substrate 8)) and can transport them to the tip of stereocilia to form a stereocilia tip complex, which can facilitate maturation of stereocilia. These scaffold proteins are essential for normal hearing in humans [27, 28]. MyTH4 domain contains the actin-binding sites. The overall surface of the microtubule (MT) is negatively charged. The positively charged motifs with surface-exposed hydrophobic side chains found in the myosin MyTH4 domain can serve as an MT binding site [29].
We speculate the, c.T6442A:p.W2148R variant interferes with the formation of the Myosin 15A-whirlin-Eps8-CEN2 complexs and microtubule-binding.
In the second studied family, WES could successfully detect a novel homozygous insertion variant i.e. c.10504dupT:p.C3502Lfs*15 in MYO15A gene, co-segregated with the disease within the pedigree. This variant between the second FERM and PDZ domains could also lead to a reading frame shift at position 10504 and a stop codon (p.C3502Lfs*15) with truncation and translation of mRNA resulting in lack of its conserved amino acids at C-terminal (data not shown). Lezirovitz et al reported a frameshift mutation c.10573delA to cause profound
hearing loss. This mutation in the PDZ binding ligand of MYO15A altered interaction of this protein with whirlin. Thus, stereocilia elongation did not occurred [30].
Zhang et al identified p.Leu3501Glu variant was associated with profound hearing loss [17].
Today, the topic of oligogenic inheritance traits has made a big wave in diagnostic medicine; since, in many monogenic diseases, it represents that there is not just one gene affected phenotype which causes new challenges in diagnosing these diseases and it can be more complicated for diseases with heterogenic pathology in which many genes are involved [31].