Association of a novel missense mutation in MYO15A with nonsyndromic hearing loss: a case report

loss is a common disease globally, and more than 50% of the cases are genetic. Autosomal recessive nonsyndromic hearing loss (ARNSHL) is one of the most common types of hereditary hearing loss. Here, a novel MYO15A mutation was identied in a Chinese family with ARNSHL, using targeted genetic sequencing and sequencing.


Conclusions
We found pathogenic compound heterozygous mutations in MYO15A, including a novel missense mutation, c.6353T > C (p.Leu2118Pro). It could provide help not only for genetic counseling but also for further understanding of the functional role of MYO15A mutations.

Background
Hearing loss (HL) is a common disease, with one to two in every 1,000 newborns suffering from severe congenital deafness [1]. The etiology of HL is diverse and includes genetic, environmental, or multifactorial factors. However, genetic factors predominate; more than 50% of HL is genetic [2].
Approximately 70% of deafness with no other symptoms can be categorized as nonsyndromic HL (NSHL). Autosomal dominant, autosomal recessive, mitochondrial, and x chromosome inherited are the four genetic modes of hereditary deafness. Autosomal recessive inherited nonsyndromic HL (ARNSHL) is the most common genetic type [3]. GJB2 was the rst gene shown to the responsible for hereditary HL, and to date, more than 80 genes and 100 genetic loci have been shown to cause ARNSHL (https://hereditaryhearingloss.org).
MYO15A (OMIM 602666), as a one of the most common pathogenic genes involved in nonsyndromic HL, is located on chromosome 17p11.2 (total length of 71 KB) and comprises 66 encoded exons [4]. The MYO15A gene encodes myosin XVA protein, which is comprised of 3530 amino acids and includes two isoforms. Both isoforms are expressed in the human inner ear [5].
In this study, we reported a novel MYO15A missense mutation using targeted region sequencing, Sanger sequencing, functional prediction, and three-dimensional protein structure modeling to identify and verify the responsible genes for deafness in this Chinese family with ARNSHL. The results identi ed compound heterozygous mutations in MYO15A, including the novel missense mutation c.6353T > C(p.Leu2118Pro) and the previously reported pathogenic mutation c.1185dupC(p.Glu396ArgfsTer36). To the best of our knowledge, this missense mutation has not been reported in any international database. Our ndings could provide help not only for genetic counseling for the family but also for further understanding of the functional role of MYO15A mutations.

Ethics statement
This study followed the guidelines of the Helsinki declaration and was approved by the Ethics Committee of the First A liated Hospital of Chongqing Medical University (Chongqing, China). All participants signed the relevant informed consent forms before the beginning of the study.

Participants and ndings
This study recruited a Chinese family with ARNSHL from the First A liated Hospital of Chongqing Medical University (Chongqing, China). Three family members, including the parents (normal hearing) and a deaf daughter, spanning two generations, participated in this study. Table 1 summarizes the clinical data of the ARNSHL family. The proband was a 6-year-old girl with deafness, whose parents had normal hearing (Fig. 1a). Pure tone audiometry (PTA), auditory brainstem responses (ABRs), tympanometry (TP), distortion product otoacoustic emission (DPOAE), and auditory steady state response (ASSR) were evaluated. Congenital inner ear malformation was excluded using temporal bone computed tomography (CT) and magnetic resonance imaging (MRI). PTA showed bilateral profound sensorineural HL with a threshold of more than 100 dBHL (Fig. 1b). Her ASSR suggested the same result (Fig. 1c). She failed the ABR and DPOAE. Type A tympanometric curves were obtained through acoustic immitance measurement, and the temporal bone CT and MRI of the patient showed no abnormalities in the inner ear.

Preparation of genomic DNA
Genomic DNA was extracted from the peripheral venous blood of the participants, using a blood DNA extraction kit (TIANGEN, Beijing, China), and was stored in a refrigerator at -20 ℃.

Target region sequencing
Targeted sequencing was used to analyze the known or suspected association of speci c mutations with phenotypes in a set of genes or regions. We could identify the candidate genes or mutations associated with the disease by using this approach. All exon, splicing site, and lateral intron sequences of ve HLrelated genes, including GJB2, MYO7A, MYO15A, MT-RNR1, and SLC26A4, were captured by multiplex polymerase chain reaction (PCR). Targeted deafness gene sequencing was conducted by Mingma Inc. (Shanghai, China), and data collection and bioinformatics analysis were conducted using an in-house pipeline.

Results of sequencing analyses
Targeted deafness gene capture sequencing Five HL-related genes were sequenced in the proband. We detected two mutations in the MYO15A gene, c.1185dupC (p.Glu396ArgfsTer36), and c.6353T > C (p.L2118P); and the co-segregation of mutations and deafness suggested that these mutations may be responsible for deafness in this ARNSHL family.

Con rmation of the variants
The compound heterozygous mutations in MYO15A, including c.1185dup (p.Glu396ArgfsTer36) and c.6353T > C (p.L2118P) were con rmed by direct sequencing in the proband. Parents with normal hearing carried only one heterozygous mutation (Fig. 2a). To the best of our knowledge, this missense mutation of c.6353T > C (p.L2118P) has not been reported in any international database. The mutation c.6353T > C in MYO15A, a missense mutation, leads to an alternation in a leucine with a proline at amino acid position 2118 (p.L2118P).
Amino acid sequences of MYO15A of different species: H. sapiens, R. norvegicus, G. gallus, Pongo abelii, Pan troglodytes, Macaca mulatta, B. taurus, and Macaca fascicularis were analyzed to look for conservation of amnio acid residues (Fig. 2b). It was revealed that this residue was important for the normal function of proteins, because p.L2118 was conserved in many species. Results of structural modeling and functional evaluation SWISS-MODEL (http://swissmodel.expasy.org) was used to construct the templates of the wild-type and the p.L2118P mutant MYO15A proteins (pdb ID: 3pvl.1.A). Compared with the template proteins, both the p.L2118P mutant MYO15A proteins and the wild-type proteins showed a consistency of more than 30% in the protein sequence; thus, these met the basic requirements of the homologous modeling program. The 3D protein structures simulated 1751-2380 residues of myosin XVA protein, and p.L2118P is in the rst MyTH4 domain of myosin XVA (Fig. 3a). The simulated structures of the wild type and mutant in the MYO15A MyTH4 region were visualized using PYMOL software (Fig. 3b). The results indicated that the mutant p.L2118P showed disruption of the amino acid side chain, which further affected the stability of the protein (Fig. 3c).

Discussion And Conclusions
MYO15A is one of the most common pathogenic genes for NSHL. Indeed, after GJB2 and MYO7A, MYO15A is the third most commonly identi ed gene associated with ARNSHL (https://hereditaryhearingloss.org). In this report, we found pathogenic compound heterozygous mutations in MYO15A, including a novel missense mutation c.6353T > C (p.Leu2118Pro) that has not been reported previously. PTA and ASSR of the proband showed profound deafness. In addition, the patient/s ABR and DPOAE were still negative under strong stimulation, suggesting that the patient was severely deaf. Moreover, Sanger sequencing con rmed that the new mutation p.L2118P co-segregated with the disease phenotypes of the family, and strong genetic evidence was obtained using sequence conservation analysis and prediction of the protein structure changes caused by mutations at those speci c points.
Myosin XVA is an unconventional myosin encoded by the human MYO15A gene and consists of 3530 amino acids. It is expressed at the tips of hair cell stereocilia in the inner cochlear region [7][8][9]. Three evolutionally conserved regions (head, neck, and tail) are present in this protein. The head region includes the N-terminal domain and the motor domain. The motor region plays a key role in ATP activity and contains two binding sites for adenosine triphosphate and actin. The neck contains two IQ motifs associated with calmodulin light chain binding. The longest tail region includes two MyTH4 domains, two band F/ezrin/radixin/moesin (FERM) domains, a Src-homology-3 (SH3) domain, and the C-terminal isoform I and PDZ-binding ligand domain [10][11][12][13]. The novel missense mutation, L2118P, found in this study was in exon 29 of the rst MyTh4 domain. In studies performed in different countries, several mutations, including c.V2114M, p.R2124Q, p.R2146Q, and p.A2153fs, have been reported in the rst MyTh4 domain [14][15][16][17] and these mutations may lead to deafness and play an important role in the functions of myosin XVA.
Mutations that cause HL were rst identi ed at the deafness, autosomal recessive 3 (DFNB3) site in the Bengkala kindred and two unrelated consanguineous Indian families [18]. Since then, many countries in South Asia, such as Pakistan, India, and Turkey, have reported various mutations [5,[19][20][21][22][23]. In East Asians, however, the rst mutations in MYO15A were not reported until 2013 [16]. A study had shown that the p.The R2146Q mutation broke the surface links between MYTH4 and FERM and caused deafness.
The p.The R2146Q protein contains a hydrophobic sac similar in structure to that of R1190 of MYO7A myth4-ferm reported previously. Other mutations reported in the MyTH4 region of myosin XVA have been shown to interfere with the interaction between myosin XVA and whirlin, thus inhibiting the formation of the complex needed for normal hearing. [15]. The MyTH4 region is closely associated with the microtubule binding and acting binding on the plasma membrane [24]. It has been shown that the MyTH4/FERM domains in myosin XVA are necessary for myosin XVA to locate to the tip of the stereocilia, which is the key to the formation of the transmembrane actin micro lament assembly complex [24][25][26].
Homozygous shaker-2 (sh2) mice show profound deafness similar to that observed in humans with DFNB3, but vestibular defects not present in human patients. The stereocilia of hair cells in sh2 mice are located correctly during development, but they are much shorter and lack the typical stepped structure compared with wild-type mice [27]. The other mutation, c.1185dupC, was rst reported in 2012 [28] and is a frameshift by the introduction of an eventual stop codon in the MYO15A open reading framework (p.E396fsX431).
With the widespread application of next-generation sequencing technology, reports on MYO15A mutations are no longer limited to the Middle East and other countries, where inbreeding is common. Reports of MYO15A mutation sites are increasing worldwide, particularly for complex heterozygous mutation sites, resulting in continuous enrichment of the MYO15A gene mutation database.
In summary, a novel mutation of the MYO15A gene was identi ed in this study. The mutation was in the rst MyTH4 domain of myosin XVA and affected its protein function. The results of this study indicated that second-generation target region sequencing was feasible for the identi cation of rare variants in HL. Our ndings expanded our knowledge of pathogenic variants in the MYO15A gene in patients with ARNSHL and could have implications in genetic counseling for families with HL.

Consent for publication
All participants or guardians provided written informed consent to publish the results of the study using their clinical case data and sequencing results.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
There is no con ict of interest.

Funding
This work was supported by the Chongqing Demonstration of Technological Innovation and Application Program (cstc2018jscx-msybX0006). The study was completed with funding from the project, including all clinical tests, DNA extraction, and the costs of second-generation sequencing and Sange sequencing.
Authors' contributions HK and SW initiated and designed the study. ZC and XO recruited suitable family members and completed relevant audiology and clinical examination. JD and SW collected blood samples and extracted DNA. The bioinformatics analysis was done by HK. LZ and CX assisted with follow-up. FY and SW studied molecular genetics and mutation ltration. HK and SW prepared the manuscript. All authors read and approved the nal manuscript.