Types of Inheritance and Genes Associated with Familial Meniere Disease

Meniere disease (MD) is a rare disorder of the inner ear defined by sensorineural hearing loss (SNHL) associated with episodes of vertigo and tinnitus. The phenotype is variable, and it may be associated with other comorbidities such as migraine, respiratory allergies, and several autoimmune disorders. The condition has a significant heritability according to epidemiological and familial segregation studies. Familial MD is found in 10% of cases, the most frequently found genes being OTOG, MYO7A, and TECTA, previously associated with autosomal dominant and recessive non-syndromic SNHL. These findings suggest a new hypothesis where proteins involved in the extracellular structures in the apical surface of sensory epithelia (otolithic and tectorial membranes) and proteins in the stereocilia links would be key elements in the pathophysiology of MD. The ionic homeostasis of the otolithic and tectorial membranes could be critical to suppress the innate motility of individual hair cell bundles. Initially, focal detachment of these extracellular membranes may cause random depolarization of hair cells and will explain changes in tinnitus loudness or trigger vertigo attacks in early stages of MD. With the progression of the disease, a larger detachment will lead to an otolithic membrane herniation into the horizontal semicircular canal with dissociation in caloric and head impulse responses. Familial MD shows different types of inheritance, including autosomal dominant and compound recessive patterns and implementation of genetic testing will improve our understanding of the genetic structure of MD.


Introduction
Meniere disease (MD) is a term used to describe patients with an audio-vestibular phenotype that includes episodes of vertigo associated with variable aural symptoms (hearing loss, tinnitus, and aural pressure) [1]. The phenotype is not limited to the inner ear, and it may be associated with other comorbidities such as migraine, allergic rhinitis, asthma, and several autoimmune or autoinflammatory disorders [1][2][3][4]. Most of the episodes are reported as spontaneous, but some patients report that a loud noise may trigger vestibular symptoms [5]. There is a great variability in the onset of the symptoms [6], and many patients initially show a partial syndrome [7]. This clinical heterogeneity makes the diagnosis a challenge in the first few years, since no biological marker is available to define MD [8].
The diagnostic criteria were initially proposed by the Japanese Ménière's disease study group in 1974. The American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) developed guidelines for diagnosis and therapy evaluation of MD in 1972 and revised them in 1985 and 1995 [9]. In this century, the criteria were redefined by the Classification Committee for Vestibular Disorders of the Bárány Society, the Japan Society for Equilibrium Research, the European Academy of Otology and Neurotology (EAONO), the Equilibrium Committee of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS), and the Korean Balance Society in 2015 [10]. These criteria improved the clinical diagnosis by excluding patients with conductive hearing loss or isolated high-frequency sensorineural hearing loss (SNHL) and may include several comorbidities such as allergy, migraine, or autoimmune diseases in the condition [8].
Histopathological studies in human temporal bones have consistently shown an accumulation of endolymph, termed endolymphatic hydrops, in the vestibular end organs (saccule and utricle) and the cochlear duct in most patients with MD [11,12]. This finding probably reflects a histopathological damage in the inner ear, since it has also been found in other patients with SNHL > 50 dB without episodes of vertigo [13] or patients with vestibular migraine [14].
Two major hypotheses are currently accepted to explain the pathophysiology of MD: a chronic autoinflammatory process defined by a immune dysfunction with high levels of several cytokines and chemokines [15,16], and rare allelic variants in coding regions reported in several genes, many of them previously associated with non-syndromic SNHL [17]. Although there have been considerable advances in the last 10 years, the contribution of genetic factors to the occurrence of MD symptoms is not yet fully understood.
The purpose of this review is to summarize the evidence that supports the genetic contribution to MD, including familial aggregation and exome sequencing studies. Furthermore, we describe the main genes reported in multiplex familial MD and elaborate a hypothesis regarding the potential role of stereocilia, otolithic membrane (OM), and tectorial membrane (TM) proteins in MD. For this, we conducted a Pubmed search with the following keywords: (

Familial MD
Madeleine Ray Brown was the first that reported two families with MD in 1941. The first family consisted of two sisters and a brother in one French Canadian consanguineous family with paroxysmal vertigo associated with sensorineural hearing loss. All started with tinnitus before the vertigo attacks and the age of onset were 46, 32, and 35 respectively [18]. In the second family, two identical twins showed audio-vestibular symptoms; one reported a sudden increase of deafness, tinnitus, and paroxysmal attacks of vertigo since he was 31 years old; the second only showed a non-progressive hearing loss [18].
Bernstein described in 1965 seven families in which more than one member had episodic vertigo or hearing loss. Two families had histories of allergy and members of three other families were suffering from migraine headaches [19]. Although some of these patients were partial syndromes and cannot be defined as MD, these early studies started to define clinical subgroups of MD patients that have been confirmed in large MD cohorts [8].
Morrison et al. reported a series of 46 British families with MD. Most of these families showed an autosomal dominant inheritance with reduced penetrance [20]. Anticipation was also observed, although this could be a bias of the search strategy. In this set of British families, maternal transmission was more frequent than paternal inheritance.
Familial clustering has been reported in about 9% of cases in Spanish population [21], and in 6% of cases in South Korea [22]. The sibling recurrence risk ratio for MD that estimates the odds to develop MD if the proband has a first degree relative with MD compared to the prevalence in the general population was 16-48 [21].
Most MD patients do not report relatives with the same clinical picture; however, it is not uncommon to find relatives with SNHL or episodic vertigo that have not been investigated in detail and could be partial syndromes [23]. For this reason, most cases are considered sporadic, but familial MD (FMD) has been repeatedly described in European descendent population in 5-20% of cases [24].
Several types of inheritance have been reported in FMD, including autosomal dominant (AD) and autosomal recessive (AR) inheritance [18,25]. Moreover, digenic and multiallelic inheritance have also been found in FMD [26]. These findings start to define a complex inheritance that combined with some environmental triggers may result in a familial disorder with variable expressivity [23] that it is observed even in the same family (i.e., uni/bilateral hearing loss, early/ late age of onset).

Exome Sequencing Studies in Familial MD
The application of exome sequencing technology to the diagnosis of MD has contributed to decipher the genetics underpinnings of familial MD [24,25]. Several rare mutations and target genes have been reported in different families with MD in Spain, South Korea, Finland, Sweden, and Iran [27]. The first Spanish family was reported by Requena et al. in 2015 [28]. The family consisted of three women with the complete MD phenotype over three generations which segregated two heterozygous rare variants in FAM136A and DTNA genes which were classified as pathogenic [28].
The first variant was a nonsense novel variant in the FAM136A gene leading to a stop codon (GRCh38 chr2:70300842G > A; NM_032822.3); the second variant was an ultrarare missense heterozygous variant found in the DTNA gene (GRCh38 chr18: 34882130G > T; NM_001390.5) that results in a p.Val715Phe substitution and generates a novel splice-site sequence predicted as a constitutive acceptor [28].
Thirteen genes have been associated with AD or AR familial MD. Intriguingly, each of these genes has a different function within the inner ear ranging from playing a role in the cytoskeleton structure of cochlear hair cells, the stress oxidative to axonal guidance pathways [29]. The main criticism on these familial studies is that most of the reported variants were only found in one family, and it cannot be ruled out that these mutations were private mutations restricted to each one of these families. For this reason, additional families with pathogenic or likely pathogenic variants in the same gene segregating the phenotype are needed to support the association between these candidate genes and FMD, according to the criteria for variant prioritization of the American College of Medical Genetics modified for hearing loss genes [30].
However, this issue was solved by Roman-Naranjo et al. in 2020, with the report of 6 Spanish families segregating 2 missense variants in the OTOG gene [25]. Two heterozygous variants of unknown significance (VUS), chr11:17557227G > A and chr11:17611374C > T (GRCh38; NM_001292063.2), were found in four unrelated patients from four different families with MD. Moreover, another heterozygous variant (chr11:17553211G > A), classified as pathogenic, was observed in two MD cases from another two unrelated families; both families also shared a novel variant chr11:17573200G > A, and one of them also had a third variant, chr11:17606001G > A. Although a double Otog mutant mouse will be needed to confirm the functional effect of these VUS, these findings support that OTOG gene is associated with heterozygous compound recessive inheritance in the 6 families [25].
Multiple MD families carrying rare variants in genes encoding proteins involved in the architecture of the hair cells stereocilia and their attachment to the TM have been found [31]. Roman-Naranjo et al. found co-segregation in several novels and rare variants in the MYO7A gene with other genes including CDH23, PCDH15, and ADGRV1 involved in the mechanoelectric transduction (MET) complex and the interciliary links of the hair cells in several MD families, suggesting a digenic inheritance model [31] (Fig. 1).
CDH23 and PCDH15 genes encode for cadherin-23 and protocadherin-15, two calcium-dependent cell adhesion proteins that show a direct interaction in the apical surface of hair cells to form the tip links between stereocilia.
These links are required for maintaining the proper organization of the stereocilia bundle of hair cells in the sensory epithelia of the organ of Corti and the vestibular organs during embryonic and early postnatal development [32]. Furthermore, cadherin-23 and protocadherin-15 mediate SNHL and Usher syndrome type 1 by digenic recessive inheritance [32]. They are part of the functional gene network formed by USH1C (harmonin b), USH1G (SANS), CDH23, and MYO7A (myosin-VIIa) that regulate MET in cochlear hair cells [33,34].
Moreover, by using a gene burden analysis and applying multiallelic inheritance models in SNHL genes, enrichment of rare missense variants in the OTOG gene were found in 15 families with MD suggesting multiallelic inheritance, including the 6 families previously mentioned with compound recessive inheritance [25]. Finally, the presence of rare missense variants and frameshift deletions in the TECTA gene within 6 MD families suggests a role of this gene in the pathophysiology of the disease [35].
Although familial MD has been associated to several genes, the incomplete penetrance and variable expressivity within families remain unexplained and the role of regulatory elements (promoters, enhancers, non-coding RNA species) in the MD phenotype deserves further research. Several microRNAs and other non-coding elements are known to be associated with SNHL and they are candidate targets for therapy [36]. An epigenetic study was performed using whole-genome bisulfite sequencing (WGBS) suggesting that the DNA methylation signature could allow distinguishing between MD patients and controls [37]. In this study, a great number of differentially methylated CpGs were found when comparing MD patients to controls. Of note, few of these CpGs involved several hearing loss genes, including CDH23, PCDH15, or ADGRV1, that encode for stereocilia link proteins [37]; however, the study was performed in a small group of sporadic patients with MD and further studies in familial cases are needed to clarify the role of methylated CpGs in these genes.
All these studies point to a complex inheritance model, including digenic and multiallelic inheritance. Genes encoding proteins linking the hair cells stereocilia in the sensory epithelia and proteins in the TM and OM should be considered potential molecular targets associated with the onset of FMD.
In this study, Roman-Naranjo et al. have reported 6 families with rare missense and frameshift variants [35]. The variant p.Val1494Ala was found in two families and one sporadic case and with a minor allelic frequency 8.8 × 10 -5 in Non-Finish European was classified as VUS. Further studies are needed to clarify the functional effect of this variant.
Of note, two of the 6 families showed two heterozygous frameshift deletions that were classified as likely pathogenic (p.Asn1474LysfsTer91 in exon 14 and p.Gly2118ProfsTer22 in exon 23, respectively). Both deletions generate a shorter α-tectorin with a modified C-terminal region that involves the glycosylphosphatidylinositol (GPI) anchorage signal. This signal peptide is essential to prevent diffusion of secreted TM proteins, and these deletions lead to a random aggregation of collagen fibrils and thinner TM with low tolerance to changes in endolymphatic pressure [38].
COCH is the causal gene for DFNA9, characterized by a progressive high-frequency SNHL with variable progressive vestibular impairment [39]; however, a family was reported in South Korea with episodic vertigo and bilateral SNHL with the mutation p.Cys162Tyr was considered a MD-like phenotype [40]. In the adult mouse cochlea, Cochlin is also a protein expressed in the fibrocytes of spiral ligament and spiral limbus, but not in the organ of Corti or the stria vascularis. In the vestibular, cristae show intense staining in the Fig. 1 Schematic representation of the mammalian cochlear hair cell stereocilia. Proteins and their coding genes are listed for the stereocilia links (tip links, horizontal top connectors, shaft connectors, and ankle links) and between the stereocilia and the tectorial membrane. In addition, proteins that are part of the mechanoelectrical transduction (MET) complex [61], the tectorial membrane [62], and some of the most relevant proteins that constitute the stereocilium are indicated [63,64]. fibrocytes and stroma underlying the sensorineural epithelium and the ampullary wall [41].
DTNA encodes α-dystrobrevin, a structural component of the dystrophin-glycoprotein complex, leading to progressive brain oedema in the knockout mouse [42]. Evidence to involve α-dystrobrevin in familial MD have been from a Drosophila model [43]. Requena et al. (2022) have investigated two Drosophila homologues, Dystrobrevin (Dyb) and Dystrophin (Dys), in Johnston's Organ function, showing that Dyb mutant flies exhibit defects in proprioception and early onset hearing loss caused by a progressive loss MET associated with a reduction of sensitive transducers [43].
However, for the seven remaining genes, pathogenicity is based on bioinformatic predictors and additional evidence from new families segregating rare variants in these genes are required to support the association. The most relevant gene in familial MD is OTOG which encodes for otogelin Spanish families have compound recessive inheritance in 6% of the cases, but rare VUS or likely pathogenic variants are found in 15% of the families [25].

Autosomal Recessive MD
Otogelin is a secreted protein related to epithelial mucins required for the anchoring of the OM to the hair cell stereocilia in the sensory epithelia in the vestibule and the organ of Corti [44]. It is involved in the organization and stabilization of the structure of the TM in the organ of Corti, and it may play a role in MET [45]. In the adult mouse, otogelin is still produced by the vestibular supporting cells, which suggests a continuous process of otogelin renewal in the OM. In contrast, in the TM, otogelin should be a long-lasting protein since OTOG gene has a low expression in the adult cochlear supporting cells [46].
A novel heterozygous missense variant p.Tyr273Asn was found in the HMX2 gene in a Finnish family with MD affecting a child and his paternal grandfather [47]. The HMX2 gene encodes a highly conserved protein involved in the inner ear development in mice [48] and zebrafish [49].
The gene encoding the limbic system associated membrane protein (LSAMP) was described in two sisters from a consanguineous Iranian Lur family [50]. The homozygous variant p.Tyr273Asn was classified as likely pathogenic and segregated the MD phenotype. The gene LSAMP is a neuronal surface adhesion glycoprotein in cortical and subcortical regions of the limbic system [51], but its function in the inner ear is not known.
STRC encodes stereocilin, a protein that interacts with otogelin and otogelin-like to form crowns in the TM attachment to stereocilia tip and horizontal top connectors in mouse cochlear hair cells [45]. A non-consanguineous Swedish-Norwegian family consisting of two brothers and their first cousin with moderate SNHL and a history of episodic vertigo starting before 6 years old was reported [52]. This child onset MD-like phenotype segregated the homozygous nonsense variant p.Gln1343 in the STRC gene [52].
Variants in STRC gene cause DFNB16B representing at least 10% of cases with AR, non-syndromic SNHL [53]; however, vestibular symptoms are usually missing.
Taken together, OTOG is the most common gene found in familial MD, but cellular or animal models are required to demonstrate the pathogenic effect, particularly in variants of unknown significance with compound recessive inheritance.

Digenic Inheritance in MD
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 gene may cause AD or AR SNHL accompanied by vestibular dysfunction or retinitis pigmentosa (Usher syndrome type 1B) [54]. Nine rare coding variants in MYO7A gene have been reported in familial MD [31]. Two of them (p.Met1? and p.Trp1545) were loss of function variants, leading to a start loss and stop codon in the sequence, respectively, and classified as likely pathogenic (Table 3); however, the rest of the variants were classified as VUS or likely benign. Of note, some of these families showed a second missense variant in the genes ADGRV1, CDH23, PCDH15, USH1C, or SHROOM2 which also segregated the phenotype, leading to the hypothesis of digenic/polygenic inheritance in familial MD associated with protein in the stereocilia links.

Molecular Hypothesis to Explain Episodic Symptoms in Meniere Disease
The TM may contribute to regulating Ca 2+ levels around the hair cell stereocilia and MET channel adaptation [55]. Apparently, behind this function are the VFWD domains of α-tectorin and otogelin, which can bind Ca 2+ ions acting as a reservoir for Ca 2+ cations [56,57]. Several constitutive proteins of the OM and TM, including otogelin and α-tectorin, show rare variations that may result in new electrostatic interactions affecting the 3D structure [25. 35]. These changes may affect the formation of the OM or TM or the attachment of these extracellular structures to the hair cells stereocilia [3]. Since α-tectorin functions as a structural organizer on the surface of the supporting cells to establish the layers of the TM, mutations involving the glycosylphosphatidylinositol anchorage sequence will produce a release of α-tectorin into the luminal space and impair the TM self-assembly process [38].
Moreover, proteins involved in the stereocilia links seems to be also involved in the pathophysiology of MD, Table 2 Genes and variants reported in autosomal recessive familial MD   including myosin VIIa, cadherin-23, protochadherin-15, or adhesion G-protein coupled receptor V1 (ADGRV1) [31]. The stereocilia in the mature outer hair cells have 2 types of links: the tip links, a filamentous protein formation that connect adjacent stereocilia formed by two cadherin-related proteins, cadherin-23, and protochadherin-15, which is linked to the MET channel [33], and the crownshaped structures located at the tips of the tall stereocilia that form the TM-attachment crown that involve otogelin, otogelin-like, stereocilin [43], tubby protein homolog, and microtubule-associated protein 1 A [58].
The network of proteins that connects stereocilia, OM, or TM is essential to preserve not only the OM or TM architecture but also the ionic microenvironment in hair cell bundles. The ionic homeostasis of the otolithic and TM could be critical to suppress the innate motility of individual hair cell bundles and focal detachment of these membranes may cause random depolarization of hair cells and explaining the changes in tinnitus loudness or triggering vertigo attacks [59].
With the progression of the disease, a large detachment will lead to an otolithic membrane herniation into the horizontal semicircular canal with dissociation in caloric and head impulse responses [60]. However, further studies in cellular and animal models are needed to confirm this hypothesis.

Ackowledgements
We would like to thank the patients with MD and their families for their enthusiastic participation.
Funding Open Access funding enabled and organized by CAUL and its Member Institutions. Alberto M Parra-Perez is a PhD student in the Biomedicine Program at Universidad de Granada and his salary was supported by Andalusian University, Research and Innovation Department (Grant# PREDOC2021/00343). Jose A Lopez-Escamez has received funds to support research on genetics in MD by Instituto de Salud Carlos III (Grant# PI20-1126), CIBERER (Grant# PIT21_GCV21), Andalusian University, Research and Innovation Department (PY20-00303, EPIMEN), Andalusian Health Department (Grant# PI027-2020), Asociacion Sindrome de Meniere España (ASMES) and Meniere's Society, UK.
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