Rare coding variants involving MYO7A and other genes encoding stereocilia link proteins in familial meniere disease

: The MYO7A gene encodes a motor protein with a key role in the organization of stereocilia in auditory and vestibular hair cells. Rare variants in the MYO7A (myosin VIIA) gene may cause autosomal dominant (AD) or autosomal recessive (AR) sensorineural hearing loss (SNHL) accompanied by vestibular dysfunction or retinitis pigmentosa (Usher syndrome type 1B). Familial Meniere’s disease (MD) is a rare inner ear syndrome mainly characterized by low-frequency sensorineural hearing loss and episodic vertigo associated with tinnitus. Familial aggregation has been found in 6–8% of sporadic cases, and most of the reported genes were involved in single families. Thus, this study aimed to search for relevant genes not previously linked to familial MD. Through exome sequencing and segregation analysis in 62 MD families, we have found a total of 1 novel and 8 rare heterozygous variants in the MYO7A gene in 9 non-related families. Carriers of rare variants in MYO7A showed autosomal dominant or autosomal recessive SNHL in familial MD. Additionally, some novel and rare variants in other genes involved in the organization of the stereocilia links such as CDH23, PCDH15 or ADGRV1 co-segregated in the same patients. Our findings reveal a co-segregation of rare variants in the MYO7A gene and other structural myosin VIIA binding proteins involved in the tip and ankle links of the hair cell stereocilia. We suggest that recessive digenic inheritance involving these genes could affect the ultrastructure of The MYO7A gene encodes a motor protein with a key role in the organization of stereocilia in auditory and vestibular hair cells. Rare variants in the MYO7A (myosin VIIA) gene may cause autosomal dominant (AD) or autosomal recessive (AR) sensorineural hearing loss (SNHL) accompanied by vestibular dysfunc- tion or retinitis pigmentosa (Usher syndrome type 1B). Familial Meniere’s disease (MD) is a rare inner ear syndrome mainly characterized by low-frequency sensorineural hearing loss and episodic vertigo as- sociated with tinnitus. Familial aggregation has been found in 6–8% of sporadic cases, and most of the reported genes were involved in single families. Thus, this study aimed to search for relevant genes not previously linked to familial MD. Through exome sequencing and segregation analysis in 62 MD families, we have found a total of 1 novel and 8 rare heterozygous variants in the MYO7A gene in 9 non-related families. Carriers of rare variants in MYO7A showed autosomal dominant or autosomal recessive SNHL in familial MD. Additionally, some novel and rare variants in other genes involved in the organization of the stereocilia links such as CDH23, PCDH15 or ADGRV1 co-segregated in the same patients. Our ﬁnd-ings reveal a co-segregation of rare variants in the MYO7A gene and other structural myosin VIIA binding proteins involved in the tip and ankle links of the hair cell stereocilia. We suggest that recessive digenic inheritance involving these genes could affect the ultrastructure of the stereocilia links in familial MD. ( )


Introduction
Monogenic sensorineural hearing loss (SNHL) is sometimes associated with vestibular dysfunction, leading to a complex clinical syndrome which may range from episodic vertigo to a progressive vestibular dysfunction ( Roman-Naranjo et al., 2017 ). Mutations in the MYO7A gene are involved in several types of SNHL with variable vestibular dysfunction, including autosomal recessive (DFNB2), autosomal dominant (DFNA11) SNHL, and Usher syndrome type 1B (USH1B) ( Friedman et al., 2020 ). DFNB2 is caused by homozygous or compound heterozygous variants in MYO7A , and it was firstly described in a large consanguineous family in which 22 individuals presented profound bilateral deafness affecting all frequencies.
Additionally, 4 of these individuals presented vestibular symptoms ( Weil et al., 1997 ). Later, this family was revaluated, reporting mild retinal degeneration in 5 patients and an abnormal vestibular function in 7 patients. On the other hand, DFNA11 has been reported in different families with progressive mild-to-profound hearing loss and an age of onset ranging from 1 to 47 years. No evidence of retinal degeneration has been described for DFNA11 ( Yamamoto et al., 2020 ). USH1B disease is an autosomal recessive disorder characterized by profound deafness affecting all frequencies, variable vestibular dysfunction, and early onset retinitis pigmentosa (RP) ( Friedman et al., 2020 ). The MYO7A gene encodes an unconventional myosin whose expression in the inner ear is restricted to the stereocilia of the hair cells of the vestibular and cochlear organs. The functional effect of rare variants in this gene has been demonstrated in several animal models such as mice or zebrafish. Homozygous mutations in MYO7A in mice led to a phenotype known as shaker-1, characterized by deafness and balance impairment ( Gibson et al., 1995 ), showing hair bundles anomalies ( Batiste Boëda et al., 2002 ). Zebrafish carrying mutations in myo7a , known as mariner mutants, showed a phenotype very similar to shaker-1 mice, resulting in hair bundle disorganization in the inner ear and lateral line, leading to hearing loss and balance dysfunction (S Ernest et al.,20 0 0 ). To date, a total of 683 rare variants affecting the MYO7A gene have been reported in humans and classified as likely pathogenic or pathogenic ( http://deafnessvariationdatabase.org/ ; Release: 4 Jan 2021; Accessed: 24 Jun 2021), causing mostly USH1B.
Meniere disease's (MD [OMIM 156,0 0 0]) is a rare inner ear disorder with an estimated prevalence 75/10 0.0 0 0 in Southern European population ( Morales Angulo et al., 2003 ). It is characterized by SNHL affecting initially low and medium frequencies, episodes of vertigo, tinnitus, and aural fullness ( Lopez-Escamez et al., 2015 ). Familial aggregation has been described in 9% of European descendant patients with MD ( Requena et al., 2014 ). Most families with MD show an autosomal dominant inheritance pattern and rare missense variants in genes such as DTNA, PRKCB, DPT or SEMA3D have been described in four unrelated MD families ( Escalera-Balsera et al., 2020 ;Gallego-Martinez et al., 2020 ). However, none of these variants have been reported in other families with MD, supporting genetic heterogeneity in familial MD. More recently, applying multiallelic inheritance models in SNHL genes in 46 unrelated families, we found an enrichment of rare missense variants in the OTOG gene in 15 families with MD . Homozygous and compound heterozygous mutations in OTOG cause DFNB18, an autosomal recessive monogenic SNHL with vestibular dysfunction ( Schraders et al., 2012 ).
Here, we have performed exome sequencing in a total of 62 MD families and 9 of them showed rare heterozygous variants in the MYO7A gene associated with the MD phenotype.

Diagnosis of patients & clinical evaluation
Patients included in this study were diagnosed according to the diagnostic criteria defined by the International Classification Committee for Vestibular Disorders of the Barany Society in 2015 ( Lopez-Escamez et al., 2015 ). Vestibular and hearing functions were assessed in all cases, discarding other vestibular diseases that could explain the phenotype. Pure-tone audiograms were retrieved to assess hearing loss since the onset of the disease. A total of 94 Spanish and Swiss patients with familial MD over 18 years old from 62 different families were selected for exome sequencing.

Whole exome sequencing
Saliva or blood samples were collected to obtain DNA as previously described ( Szczepek et al., 2019 ). DNA samples were extracted with prepIT-L2P (DNA Genotek, Ottawa, Canada) and QI-Aamp DNA Mini Kit (Qiagen, Venlo, The Netherlands) following manufacturer's protocols, according to the quality controls required for exome sequencing. Coding regions were selected using SureSelect Human All Exon V6 capture kit (Agilent Technologies, Santa Clara, CA, USA) and paired-end sequenced on the Illumina HiSeq 40 0 0 platform with a mean coverage of 100X.

Processing and dataset generation
The raw unmapped paired-end reads of each sample, contained in FASTQ files, were aligned to the GRCh37/hg19 reference genome using the Burrows-Wheeler Aligner with maximal exact matches algorithm. After a post-alignment processing, where duplicated reads were located and removed and the quality of the alignment itself was assessed ( McKenna et al., 2010 ), genetic variants were called using the Haplotypecaller function from GATK. Thus, a Variant Call Format (VCF) file for each individual was generated, retrieving single nucleotide variants (SNVs), insertion and deletions (indels) from the sequenced exomes. Annovar was used for prediction and annotation purposes ( Wang et al., 2010 ).

Data analysis and prioritization strategy
Based on the data from a multi-ethnic study that assessed the pathogenicity of reported non-syndromic hearing loss variants ( Shearer et al., 2014 ), we selected MAF thresholds of 0.005 and 0.0 0 05 to identify, autosomal-recessive and autosomal-dominant rare variants in the familial MD cohort, respectively. Allelic frequencies were retrieved for non-Finish European (NFE) population from the gnomAD ( N = 33,365) and ExAC (32,299) databases, and for the Spanish population ( N = 1942) from the Collaborative Spanish Variant Server (CSVS) database ( Peña-Chilet et al., 2020 ).
Candidate variants were classified according to the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) guidelines ( Richards et al., 2015 ), considering the specific guidelines for variant interpretation in genetic hearing loss ( Oza et al., 2018 ). Furthermore, in silico tools, such as SIFT or CADD were used to prioritize and classify each variant according to its predicted pathogenicity.
As a method of variant prioritization, candidate genes harbouring rare variants were associated to mammalian phenotypes using the Mouse Genome Database (MGD; http://www.informatics. jax.org ). Likewise, the Human Phenotype Ontology Project (HPO; https://hpo.jax.org/app/ ) and the Online Mendelian Inheritance in Man (OMIM; https://omim.org/ ) databases were used to determine associations between candidate genes and phenotypes in humans.

Statistical analysis
To assess the statistical significance of the candidate variants, European and Spanish databases (i.e., ExAC, gnomAD and CSVS) were used as reference to compare the observed MAF in FMD. Thus, odds ratios (OR) with 95% confidence interval (CI) were calculated for each single or set of variants. One-sided p-values were corrected for multiple testing following the Bonferroni approach. The same approach was followed to assess whether a single variant or a combination of variants drive the association.

Protein modeling
The effect of candidate variants on the myosin VIIA protein structure was evaluated by protein modeling. When it was possible, wild-type and mutated functional domains were modelled using SWISS-MODEL, using as templates crystal structures already published. Thus, crystal structures of the human myosin VIIA MyTH4-FERM domains in complex with harmonin-a (PDB ID: 5MV9) and the mouse myosin VIIA IQ5 domain in complex with apocalmodulin (apo-CaM, PDB ID: 5WSU) were used to assess the effects of R836H and A2083T variants.

Hearing assessment and analysis
Standard audiometric evaluations for air and bone conduction elicited by pure tones from 125 to 80 0 0 Hz were retrieved from the clinical records to analyze the time course of the hearing profile in FMD cases with candidate variants. Regression analysis was performed to estimate the outcome of hearing loss for each frequency.

Rare missense variants in MYO7A and other genes in the stereocilia links suggest digenic inheritance in familial MD
A total of 18,998 and 12,164 nonsynonymous or splice site variants were considered for FMD after applying quality controls for recessive and dominant inheritance models, by using MAF thresholds of 0.005 and 0.0005, respectively. Using the most restrictive MAF threshold, we found variants in a total of 7389 genes, whereas variants in 9612 genes were retained for MAF threshold ≤ 0.005. From them, 355 genes (393 for MAF threshold 0.005) were already related to hearing/vestibular/ear phenotype (MP:0,005,377) in different mouse models (data not shown).
The gene with the majority of rare variants in our dataset for both MAF thresholds was the MYO7A gene. Nine variants in 9 individuals were identified fulfilling frequencies criteria in at least one of the reference populations used in this study ( Table 1 ).
The 9 variants found were scattered across the gene sequence, affecting different functional domains in Myosin VIIA protein ( Fig. 1 ). Variants were found in heterozygous state in 9 over 62 families, accounting for the 14.5% of the studied families. Of note, we found two Loss of Function (LoF) variants: a start loss variant p.Met1Ile found in F1 segregating the MD in the three affected individuals of this family (Supplemental Table 2), and a novel stop gain variant p.Trp1545Ter found in F6. There were no mutations in the 5 ′ untranslated region (UTR) that generated a novel transcription start site to compensate the start loss variant identified in F1. The predicted pathogenicity of these two LoF variants was high, being classified as likely pathogenic variants (Supplemental Table 3). Frameshift indels in the MYO7A gene were analyzed to search for additional LoF variants, however only a common deletion (rs111033223) found in 11,084/21,494 Non-Finish European from gnomAD and classified as benign was identified in F1, F2, F3, F7 and F9.
Our segregation analyses in familial MD revealed several rare variants in genes encoding proteins involved in the formation of the mechanotransduction complex in the stereocilia of hair cells. We assessed digenic inheritance in families carrying rare variants in the MYO7A gene and genes codifying for proteins involved in the stereocilia links. Interestingly, two families (F4 and F5) carried rare missense variants in MYO7A and ADGRV1 genes, other two families (F2 and F6) carried rare variants in MYO7A and CDH23 genes, and three families carried variants in SHROOM2 (F1), USH1C (F7) and PCDH15 (F8) genes (Supplemental Table 4). Thus, 7 of the 9 families carrying rare variants in the MYO7A gene also carried rare variants in genes with a structural function in the hair cell stereocilia.
Statistically, some of these variants in the MYO7A gene could explain the phenotype of certain families by themselves (Supplemental Table 5), such as M1I (F1), R836 (F4), W1545X (F6), A2083T (F7) and R2209Q (F8). On the other hand, a second variant in a gene involved in the stereocilia of hair cells is needed in some families to show statistical significance (Supplemental Table 6). In families such as F2 and F5 the phenotype could be explained by the combination of rare variants in MYO7A and CDH23 genes, and MYO7A and ADGRV1 genes, respectively. The co-segregation of these variants was confirmed by Sanger sequencing in F2 (II-1 and II-6) and F5 (I-1 and II-1).

Carriers of rare variants in MYO7A show autosomal dominant or autosomal recessive inheritance in familial MD
The segregation of rare variants in the MYO7A gene was assessed in each family. Among the 9 families, 3 followed an autosomal dominant inheritance pattern (F1, F5 and F7), whereas in the other 6 families was found an autosomal recessive inheritance pattern ( Fig. 2 ). Digenic or compound heterozygous inheritance patterns could likewise explain these latter families. In total, 29 individuals with MD were diagnosed in these 9 families, and in 3 families (F1, F7 and F8) were observed partial syndromes in 3 individuals (i.e., only hearing loss or vertigo), suggesting a variable expressivity. The LoF variant p.Met1? found in F1 causes a start loss in the MYO7A gene, codon 12 being the next in-frame start codon. Although this variant could explain the MD phenotype in F1 by itself, it was found in conjunction with a variant in the SHROOM2 gene (p.Gly211Ser). Two variants in genes involved in the architecture of the mechanotransduction complex in the hair cell stereocilia ( MYO7A and CDH23 ) were found in F2, which seems to follow a recessive digenic inheritance pattern. Despite its frequency (0.22% of NFE population), the variant p.Arg336His in the MYO7A gene was classified as variant of unknown significance (VUS), since it is predicted to impact the protein (CADD = 24.1; M-CAP = 0.139). The third family (F3) appears to be following a recessive inheritance pattern, likely caused by digenic inheritance. Only a heterozygous variant in the MYO7A gene, affecting a highly conserved arginine (p.Arg686His), classified as VUS was found in this family. MYO7A rare variant in the functional domain IQ together with a rare variant in the ADGRV1 gene were found in F4 (p.Arg836His & p.Pro2528Ser) and F5 (p.Arg873Trp & p.Arg5547His). The pedigree of F4 suggests a recessive/digenic inheritance pattern, whereas F5 is suggestive of a dominant/digenic inheritance pattern. In the last four families (F6; F7; F8 and F9) we found variants affecting the FERM functional domain of the MYO7A gene following a recessive/digenic inheritance pattern. The variant found in F6 cause a novel stop codon (p.Trp1545Ter) in exon 35 of the canonical and longest isoform of the MYO7A gene (NM_0 0 0260.4). In addition, a rare variant classified as a VUS in the CDH23 gene (p.Arg2171His), which is part of the tip link complexes, was found in this family. Variants in genes of these complexes were also found in F7 and F8, who presented variants in the USH1C gene (p.Pro608Arg) and the PCDH15 gene (p.Pro1748Thr), respectively. Supplemental Table 4 shows the most relevant associations in these families.

Table 1
Rare variants in the MYO7A gene ( N = 9) found in the studied familial MD cohort ( N = 62

Hearing assessment in MYO7A patients
Standard audiometric evaluations were obtained and hearing profiles for the patients (2 males, 7 females) with rare variants in the MYO7A gene were studied. Six patients showed bilateral hearing loss, whereas the other three individuals presented unilateral hearing loss (2 left-sided and 1 right-sided SNHL). So, 3 ears had a normal pure-tone audiogram (16.6%), 6 ears from 5 patients showed a flat shaped audiogram (33%), 4 ears from 3 patients showed a ski-slope shaped audiogram (22.2%), 3 ears from 2 patients showed a reverse ski-sloped audiogram (16.6%) and only an ear from a patient showed a tent shaped audiogram (5.5%). The age of onset of the first symptom considered to be caused by MD was 38.3 ± 11. 5 (Supplemental Table 7). The estimated hearing threshold at onset was 72.1 ± 1.2 dB for low frequencies (125-250-500 Hz) and 58.9 ± 2.1 dB for high frequencies (10 0 0-20 0 0-40 0 0 Hz) (Supplemental Figure 1).

Protein modeling
The identified p.Arg836His and p.Arg873Trp variants were in (or near of) IQ5 domain of myosin VIIA, and the p.Arg2083Thr was in the FERM domain ( Fig. 3 ). Complexes of apo-CaM with p.Arg836His mutant IQ5 domains were modelled ( Fig. 3 A). In the IQ5 wild-type (IQ5-WT) model, no bonds were observed between the highly conserved arginine 836 (R836) of MYO7A and the phenylalanine 92 (F92) of apo-CaM. On the other hand, the IQ5 histidine 836 mutant (H836-Mutant) model shows the formation of a new NH/ π interaction between this histidine and the F92 of apo-CaM. The variant affecting ar ginine 873 (R873) was also assessed with the IQ5-WT model ( Fig. 3 B), observing three hydrogen bonds between R873 and the glutamic acid 877 (E877) of MYO7A. In the tryptophan 873 mutant (W873-mutant) model, these three hydrogen bonds were lost. This loss is predicted to affect the stability/folding of the protein. The opposite occurs with the variant involving the alanine 2083 (A2083) in the FERM domain (FERM-WT) model of MYO7A ( Fig. 3 C). In the threonine 2083 mutant (T2083-mutant) model, a hydrogen bond not showed between T2083 and serine 2080 (R2079) in FERM-WT appears between T2083 and R2079.

Discussion
Rare coding variants in the MYO7A gene are known to underlie different cochleovestibular syndromes, such as DFNB2, DFNA11 and USH1B ( Friedman et al., 2020 ). Hearing loss is a common trait for these three syndromes, however the onset and affected thresholds vary widely between them. Vestibular dysfunction and RP are other manifestations that can appear in the carriers of rare variants in MYO7A . In this study, 9 rare variants in the MYO7A gene were identified in 9 unrelated families with MD by exome sequencing. Interestingly, three of these variants have been reported before in Usher syndrome patients. The start loss variant (MYO7A:c.3G > A ;p.Met1?) that segregates the MD phenotype in F1 has been previously found along with the p.Leu1839Pro MYO7A variant in a patient causing Usher Syndrome type 2 ( Fuster-García et al., 2018 ). Moreover, the p.Arg873Trp variant in the MYO7A gene found in F5 has been previously observed in two patients with Usher Syndrome type 2 ( Västinsalo et al., 2013 ); and the MYO7A variant found in F2 (p.Arg336His) has been already observed in patients with Usher Syndrome type 1, being classified as a disease causing variant ( Jaijo et al., 2006 ). We suggest that rare missense variations in this gene could also affect the stability of myosin VIIA in the hair cell stereocilia in familial MD.
Familial MD typically shows an autosomal dominant inheritance pattern with incomplete penetrance ( Escalera-Balsera et al., 2020 ;Requena et al., 2014 ), however compound recessive inheritance has been recently reported in several families with MD, revealing OTOG as a key gene in familial MD . The OTOG gene encodes otogelin, an extracellular protein that participates in, among others, the otolith tethering in the otolithic membrane, the tectorial membrane attachment crowns, and the horizontal top connectors between stereocilia ( Avan et al., 2019 ). Stereocilia are connected to each other through different links involving a large number of proteins, one of them being myosin VIIA ( Goodyear et al., 2005 ).
Myosin VIIA, encoded by the MYO7A gene, is a motor protein highly expressed in the cochlea involved in the formation of tip links and ankle links in the stereocilia of hair cells ( Morgan et al., 2016 ). Its canonical isoform encodes for a 2215 amino acid large protein (ENST0 0 0 0 0409709) containing, from N-terminal to Cterminal, several functional domains: a motor domain, five IQ motifs and two repeated regions consisting of a MyTH4 and a FERM domain separated by a SH3 domain. Through these domains, myosin VIIA interacts with other proteins involved in the tip and ankle links of stereocilia, encoded by genes such as USH1C, CDH23 (Batiste Boëda et al., 2002 ), ADGRV1 ( Michalski et al., 2007 ) or USH1G ( Weil et al., 2003 ) ( Fig. 4 ).
Because of its location and function in the transduction complex of hair cells, Myosin VIIA has been proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex in the tip links ( Li et al., 2020 ). We suggest that rare variations in the MYO7A gene, alone or combined with rare variants in genes encoding myosin VIIA binding proteins, will lead to fragile tip and ankle links, loss of cohesiveness among stereocilia, abnormal opening of the MET complex with sustained depolarization of hair cells, and ultimately hearing loss and/or vestibular dysfunction. Thus, we found rare variants in the MYO7A gene in 9 MD families, associated with missense variants in genes encoding myosin VIIA binding proteins, including ADGRV1, CDH23, PCDH15, USH1C and SHROOM2 genes. Seven of these 9 families showed, at least, a rare variant in MYO7A and a variant in one of the genes mentioned above, suggesting a potential digenic inheritance in MD.
The contribution of the MYO7A gene to digenic inheritance in hearing loss has been studied in the Myo7a sh1-8J mouse model, characterized by carrying a complex deletion affecting exons 38-40 and 42-46 ( Zheng et al., 2012 ). The combination of recessive mutations was assessed making use of double heterozygotes of Myo7a and other Usher genes involved in the organization of the hair cell stereocilia. Thus, hearing loss was assessed in double heterozygotes of Myo7a and Ush1g, Cdh23 or Pcdh15 genes. Myo7a, Ush1g, Cdh23 and Pcdh15 single heterozygotes mice showed normal phenotype, whereas double heterozygotes had progressive hearing loss. This study reinforces the key role of the MYO7A gene in the architecture of hair bundles and raises the hypothesis of recessive digenic inheritance in familial MD by the combination of rare variants in MYO7A with variants in genes involved in the cohesiveness of the stereocilia.
In this study, two rare missense heterozygous variants were found in the ADGRV1 gene in two families (F4 & F5) carrying MYO7A gene variants. The ADGRV1 gene, an ankle-link component which encodes for the adhesion G-coupled receptor V1, has been associated in different studies with Usher syndrome type 2C (USH2C) ( Weston et al., 2004 ). USH2C is an autosomal recessive disorder mainly characterized by prelingual sensorineural hear-6 ing loss and progressive RP. To date, two different mouse models have been generated, revealing defects in hair bundle formation at P2, progressively decreasing their cohesion and becoming disorganized. This is likely caused because of the role of the Adhesion G-protein coupled receptor V1 protein in the ankle link complex ( Michalski et al., 2007 ). Although it has not been replicated, digenic inheritance was reported in USH2C patients with variations in ADGRV1 and PDZD7 genes ( Bonnet et al., 2016 ;Ebermann et al., 2010 ), however there are no reports of any phenotype caused by the combination of variants in MYO7A and ADGRV1 genes in humans. Nevertheless, some studies suggest a direct MYO7A-ADGRV1 interaction and a key role of functional MYO7A in the localization of ADGRV1 in the ankle link region ( Michalski et al., 2007 ;Morgan et al., 2016 ). The additive effect of rare missense variants in MYO7A and ADGRV1 could affect the stability of ankle link proteins leading to loss of cohesiveness among stereocilia.
Similarly, two rare variants were found in the CDH23 gene in two unrelated MD families (F2 & F6) with rare variants in the MYO7A gene. The CDH23 gene encodes cadherin-related 23, a structural protein of the tip links associated to autosomal recessive deafness 12 (DFNB12) and Usher syndrome type 1D (USH1D) ( Bork et al., 2001 ). DFNB12, mainly diagnosed in patients carrying homozygous or compound heterozygous variants in CDH23 gene, is characterized by severe to profound SNHL affecting high or all frequencies and normal vestibular function. On the other hand, USH1D, characterized by SNHL, RP and abnormal vestibular function, is assumed to be caused by variants disrupting more severely the function of the cadherin-related 23 protein. At least four mouse models have been generated to study the function of Cdh23 gene (i.e., waltzer v2J , salsa, Cdh23 12J , Cdh23 Ahl and Cdh23 CBA/CaJ ). In short, these mice were characterized by flat progressive hearing loss, mainly caused by a reduction of outer hair cells and progressive loss of tip links ( Bolz et al., 2001 ;Schwander et al., 2009 ). The variant found in II-1 and II-6 of F2 in the CDH23 gene, which is chr10:73491873A > G (NM_022124.6:c.3845A > G ; p.Asn1282Ser), was considered likely benign since it is a neutral amino acid change. However, the variant chr10:73553197G > A (NM_022124.6:c.6512G > C ; p.Arg2171His), found in F6 in the same gene, is affecting a highly conserved calcium-binding element (DXNDNR). These motifs are thought to be essential in the function of cadherins ( Nagar et al., 1996 ), and variants affecting them have been described causing hearing loss ( De Brouwer et al., 2003 ). Digenic inheritance has not been postulated in humans for MYO7A and CDH23 genes. However, although myosin VIIA does not bind to cadherin-related 23, they participate and cooperate together with the adaptor protein harmonin (encoded by the USH1C gene), providing the scaffold by which the Factin-bound motor connects to the upper end of the tip link in the sensory hair cell bundles (Batiste Boëda et al., 2002 ).
A variant in USH1C gene, that encodes harmonin, was found in our familial MD cohort. Variants in this gene are phenotypically related to Usher syndrome type 1C and autosomal recessive deafness 18A (DFNB18A) ( Ahmed et al., 2002 ). We found a variant in F7, chr11:17531093G > C (NM_153,676.4:c.1823C > G ; p.Pro608Arg), previously described in a Chinese family causing Usher syndrome 1C, when present in homozygous state ( Ouyang et al., 2002 ). Although it was found as a heterozygous variant in our cohort, the presence of a variant in the MYO7A gene in F7 raises the possibility of digenic inheritance.
In addition, variants in PCDH15 and SHROOM2 genes were found in F8 and F1, respectively. The PCDH15 gene encodes protocadherin related 15, a component of the tip links, and it is associated to autosomal recessive deafness 23 (DFNB23) and Usher syndrome 1F ( Ahmed et al., 2003 ). Notably, Yoshimura et al. (2014) reported the first case of MYO7A / PCDH15 digenic inheritance ( Yoshimura et al., 2014 ), two genes that encode proteins that can directly inter-act with each other in the tip links in hair cell stereocilia ( Senften et al., 2006 ). The co-segregation of MYO7A :p.Arg2209Gln and PCDH15 :p.Pro1706Thr variants was confirmed in both relatives from F8 (II-1 and II-IV). On the other hand, although the SHROOM2 gene is expressed in the hair cells, and an interaction with the tail of myosin VIIA has been suggested, its function is not well-known ( Etournay et al., 2007 ;Hickox et al., 2017 ).
The audiograms of FMD patients carrying rare variants in the MYO7A gene showed moderate-to-severe hearing loss (73.2 ± 1.9 dB) at low frequencies and moderate hearing loss (58.8 ± 1.9 dB) at high frequencies since the first years of the disease.
The phenotypes and traits associated with the variants in MYO7A and related genes showed in this study are expected to be milder compared to phenotypes showed in the different knockout mouse models mentioned before. We can speculate that the variants in myosin VIIA, or in other proteins of its network, may lead to hearing loss because of a disorganization in the stereocilia, suggesting a key role in MD of the proteins involved in the tip links and ankle links that connect the stereocilia among them.
The results of this study suggest that some of these families may follow a composite digenism inheritance pattern, where a variant in a main gene (the MYO7A gene) is enough to produce the phenotype, but an additional variant in a second candidate gene (e.g., CDH23, PCDH15, ADRV1 …) can modify the phenotype (e.g., modifying the age of onset). Composite digenism could explain the variable expressivity typically found in MD. Examples of this type of inheritance could explain the phenotype in F1 ( MYO7A/SHROOM2 ), F4 ( MYO7A/ADGRV1 ), F6 ( MYO7A/CDH23 ) or F8 ( MYO7A/PCDH15 ). However, we cannot discard other inheritance patterns involving MYO7A variants in MD, such as true digenism (two variants needed to show the phenotype) or dominant inheritance.

Conclusions
We have found co-segregation in several novel and rare variants in the MYO7A gene with other genes such as CDH23, PCDH15 or ADGRV1 involved in the mechanotransduction complex and the intercilliary links of the hair cells in families with MD. These findings suggest that digenic inheritance could be a novel mechanism in familial MD.

Declarations of Competing Interest
None.

Data availability
The datasets during and/or analyses during the current study are available from the corresponding author upon reasonable request.
National Science Foundation (SNSF). AHE is supported by a career development grant ("Filling the Gap") from the University of Zurich, Switzerland. Funding for open access charge: Universidad de Granada/CBUA. The authors declare no conflict of interest.

Ethics declaration
This study protocol was approved by the local ethics committees (MS/2014/02, Institutional Review Board for Clinical Research, Universidad de Granada, Spain; KEK-ZH-Nr. 2019-01006, Kantonale Ethikkommission, Zurich, Switzerland) and a written informed consent to donate biological samples was obtained from all subjects.

Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.heares.2021.108329 .