A novel missense variant in MYO3A is associated with autosomal dominant high‐frequency hearing loss in a German family

Abstract Background MYO3A, encoding the myosin IIIA protein, is associated with autosomal recessive and autosomal dominant nonsyndromic hearing loss. To date, only two missense variants located in the motor‐head domain of MYO3A have been described in autosomal dominant families with progressive, mild‐to‐profound sensorineural hearing loss. These variants alter the ATPase activity of myosin IIIA. Methods Exome sequencing of a proband from a three‐generation German family with prelingual, moderate‐to‐profound, high‐frequency hearing loss was performed. Segregation analysis confirmed a dominant inheritance pattern. Regression analysis of mean hearing level thresholds per individual and ear was performed at high‐, mid‐, and low‐frequencies. Results A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in the kinase domain of MYO3A was identified that is predicted in silico as disease causing. High‐frequency, progressive hearing loss was identified. Conclusion Correlation analysis of pure‐tone hearing thresholds revealed progressive hearing loss, especially in the high‐frequencies. In the present study, we report the first dominant likely pathogenic variant in MYO3A in a European family and further support MYO3A as an autosomal dominant hearing loss gene.

showed bilateral, progressive, high-frequency HL beginning in the second decade of life caused by three different recessive loss-of-function variants in MYO3A (Walsh et al., 2002). Since then, several additional recessive loss-of-function and missense variants with a wide range of HL characteristics were identified in patients with nonsyndromic HL (Choi et al., 2013;Miyagawa, Naito, Nishio, Kamatani, & Usami, 2013;Qu et al., 2016;Sommen et al., 2016;Wu et al., 2015). In 2016, the inheritance pattern of HL due to variants in MYO3A expanded with the discovery of a missense variant in an African-American family with progressive, postlingual childhood onset HL (Grati et al., 2016). Later, in 2018, two Brazilian families with late-onset nonsyndromic HL were identified with the same segregating missense variant (Dantas et al., 2018). To date, only three autosomal dominant families with mild-to-profound, progressive HL have been identified with two different segregating missense variants in MYO3A (Dantas et al., 2018;Grati et al., 2016).
In vertebrates, two different isoforms have been identified; the longer Myo3A isoform (209 kDa) and a shorter Myo3B isoform (155 kDa), that are both expressed in the retina and testis (Dose et al., 2003). The encoded myosin IIIA protein consists of a N-terminal kinase domain, a highly conserved motor-head domain, followed by three calmodulin binding (IQ) motifs and a C-terminal actin-binding domain (3THD-II) (Dose & Burnside, 2000;Salles et al., 2009). Both previously described dominant variants in MYO3A are located in the motor-head domain and are thought to affect the ATPase activity of the gene (Dantas et al., 2018;Grati et al., 2016). Expression is present in the retina (Dose & Burnside, 2000) and the inner ear of mammals (Schneider et al., 2006;Walsh et al., 2002), specifically at the tips of both inner and outer hair cells in all stereocilia rows, as well as in vestibular hair cell stereocilia in mice (Walsh et al., 2011). Mutant mice homozygous for a nonsense allele, show progressive, high-frequency HL, advancing to all frequencies over time (Walsh et al., 2011).
We describe the first European family with dominant, moderate-to-profound, high-frequency sensorineural HL with a novel heterozygous missense variant c. 716T>C,p.(Leu239Pro) in MYO3A (NM_017433.4). This finding supports and consolidates the association of autosomal dominant HL due to variants in this gene. Due to a relative lack of studies on the genetic basis of autosomal dominant HL and the challenging occurrence of a typical later age of onset, characterization of large families is important for unraveling the distinction between autosomal recessive and dominant alleles in the personalized medicine era.

| Ethical compliance
Written informed consent was obtained from the family and all procedures were approved by the Ethics Commission of the University of Würzburg (46/15, approval date: 31 March 2015).

| Clinical evaluation
We recruited the genomic DNA from a three-generation German family with seven affected (I.2, II.2, II.3, III.1, III.2, III.4, III.5) and one unaffected individual (II.1). Audiological testing, including pure-tone audiometry, was done for all affected family members and the unaffected family member III.3 complying with guidelines described by Mazzoli et al. (2003).

| Genomic analysis and exome sequencing
Genomic DNA from participating affected and unaffected individuals was extracted from whole blood. We excluded pathogenic variants in the most common gene, GJB2, by diagnostic Sanger sequencing of the index patient. Exome sequencing of the index patient (III.1) was performed. Exome library preparation was performed with the Nextera Rapid Capture Exome kit (Illumina) according to manufacturer's instructions and paired-end sequenced (2 × 76 bp) with a v2 high-output reagent kit with the NextSeq500 sequencer (Illumina). The human reference genome GRCh37 (hg19) was used for data alignment.

| Protein modeling prediction of wild type MYO3A
The secondary protein structure at amino acid position 239 (Leu) of wild type MYO3A was predicted in silico with I-TASSER . C-scores ranging from −5 to 2 indicate the confidence of the predicted models, where a C-score of a higher value indicates a model with a higher confidence.

| Clinical presentation of the German family
We present a three-generation German family with prelingual, bilateral, sensorineural HL (Figure 1a). All affected individuals (I.2, II.2, II.3, III.1, III.2, III.4, III.5) showed high-frequency HL ranging from moderate to profound in severity (Figure 1d). Individual III.3 underwent audiometry and revealed normal hearing (data not shown). Our data show a clear correlation between the average thresholds at high-(4-8 kHz) and mid-(1-3 kHz) frequencies and the age at audiometric examination for both ears, indicating a progressive HL (Pearson's r: 0.62 (right ear), 0.76 (left ear) for high-frequencies; 0.71 (right ear), 0.83 (left ear) for mid-frequencies) ( Figure S1). All affected individuals use hearing aids and show a good hearing outcome. After clinical examination, additional symptoms and risk factors for hearing loss such as infections and trauma were excluded. Tinnitus was not reported for all affected individuals.

| Identification and analysis of a novel missense variant in MYO3A
The index patient (III.1) underwent exome sequencing and bioinformatics analysis that included 174 deafness-associated genes (Table S1) as an initial approach to screen variants in clinically relevant hearing loss-associated genes that was followed by an exome-wide analysis. A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in exon 8 of the gene MYO3A (NCBI Reference Sequence: NM_017433.4) was identified that was predicted in silico as disease causing and resulted in a putative pathogenic amino acid exchange according to several in silico tools (PolyPhen-2, MutationTaster, and SIFT). The variant is classified as "likely pathogenic" according to the ClinGen hearing loss working group expert specification (Oza et al., 2018). The variant affects a highly conserved nucleotide and amino acid (Figure 1c) that is part of the catalytic kinase domain of the encoded MYO3A protein. Protein modeling prediction of wild type MYO3A indicated an alpha-helix at amino acid position 239 (Leu), based on the two predicted models with the highest C-score (−1.82, −2.00). Segregation testing of the c.716T>C variant followed a dominant inheritance pattern (Figure 1a,b). Bioinformatics analysis in 174 Hl genes excluded additional potentially disease-causing variants and copy number variations (CNVs) that could resolve the phenotype of the family. The c.716T>C variant has been submitted to the Leiden Open Variation Database version 3 (LOVD v.3.0) under variant ID 0000660455.

| DISCUSSION
MYO3A belongs to the unconventional myosins (class III) of the large myosin superfamily (Dose & Burnside, 2000). They mediate crucial cellular functions such as signal transduction, cell movement, and vesicle trafficking (Mermall, Post, & Mooseker, 1998). Grati  resulting in an amino acid substitution in the motor-head domain that modifies the ATPase activity of MYO3A at the hair cell stereocilia tips. These authors also showed that MYO3A interacts with PCDH15 (protocadherin 15), a crucial component of the mechanoelectrical transduction (MET) complex (Grati et al., 2016). The two previously described dominant variants in an African-American (c.1463G>A, p.(Gly488Glu)) and two Brazilian families (c.2090T>G, p.(Leu697Trp)) are both located in the motor-head domain of the myosin IIIa protein (Figure 2). The affected individuals showed nonsyndromic, bilateral, progressive HL, ranging from mild to profound in severity. The age of onset varied between early childhood (postlingual) and an average age of onset of 30 to 32 years (Table S2) (Dantas et al., 2018;Grati et al., 2016). Furthermore, one congenital case was reported in one of the Brazilian families (Dantas et al., 2018). Interestingly, there is a great variability regarding the age of onset and HL severity in families with previously described recessive variants in MYO3A, ranging from congenital to late-onset HL and a moderate-to-profound degree of severity. Additionally, there is no apparent clustering of recessive and dominant variants in a certain protein domain, such as the kinase or motor-head domain (Table S2).
The affected individuals in the German family we describe, all showed nonsyndromic, prelingual, progressive HL (Figure 1b,c), especially impacting high-(4-8 kHz) and mid-(1-3 kHz) frequencies ( Figure S1). Progressive HL is observed in other autosomal dominant MYO3A families. As previously described, MYO3A variants that are implicated in an autosomal dominant inheritance confer a dominant-negative effect, reducing actin protrusion initiation, and elongation activity of the encoded protein in inner ear hair cell stereocilia (Dantas et al., 2018). Dominant-negative effects are characterized by the adverse assembly of wild type and mutant protein subunits, preventing correct protein functionality and are frequently involved in various human diseases (Bergendahl et al., 2019;Herskowitz, 1987;Marziano, Casalotti, Portelli, Becker, & Forge, 2003). The c.716T>C, p.(Leu239Pro) missense variant in the German family is the first dominant variant that is located in the kinase domain of the gene (Figure 2). In vitro analysis suggests that a functional kinase domain of the MYO3A protein is important for proper regulation of actin dynamics and stability of actin bundles at filopodial tips (Quintero et al., 2010). The heterozygous variant results in an amino acid exchange from leucine to proline that is predicted to be disease causing in silico. The exchange from a branch-chain leucine to a cyclic proline possibly mediates the disruption of the present alpha-helix at amino acid position 239 (Roy, Kucukural, & Zhang, 2010;Yang et al., 2015;Zhang, 2008) and results in a structural protein change (Bajaj et al., 2007;Cordes, Bright, & Sansom, 2002). Although it was not directly tested, it is suspected that the potentially defective MYO3A protein also interacts with the existing wild type protein via a dominant-negative mechanism and is responsible for the HL phenotype in the German family.
Compared to its recessive counterpart, many dominant forms of HL lack in-depth clinical characterization. Clinical data from large families are essential to discriminate progression, which is of high interest to directing current and future treatment modalities. Several genes, including MYO3A, lack substantial clinical validity through lack of replication evidence. Here, we describe the first European family with a novel dominant variant in MYO3A, thus, providing further evidence for the association of this gene with an autosomal dominant HL phenotype.