Identification of a genetic variant underlying familial cases of recurrent benign paroxysmal positional vertigo.

Benign paroxysmal positional vertigo (BPPV) is the most common cause of vertigo in humans, yet the molecular etiology is currently unknown. Evidence suggests that genetic factors may play an important role in some cases of idiopathic BPPV, particularly in familial cases, but the responsible genetic variants have not been identified. In this study, we performed whole exome sequencing [including untranslated regions (UTRs)] of 12 families and Sanger sequencing of additional 30 families with recurrent BPPV in Caucasians from the United States (US) Midwest region, to identify the genetic variants responsible for heightened susceptibility to BPPV. Fifty non-BPPV families were included as controls. In silico and experimental analyses of candidate variants show that an insertion variant rs113784532 (frameshift causing truncation) in the neural cadherin gene PCDHGA10 (protocadherin-gamma A10) is an exceedingly strong candidate (p = 1.80x10-4 vs. sample controls; p = 5.85x10-19 vs. ExAC data; p = 4.9x10-3 vs. NHLBI exome data). The mutant protein forms large aggregates in BPPV samples even at young ages, and affected subjects carrying this variant have an earlier onset of the condition than those without [average 44.0±14.0 (n = 16) versus 54.4±16.1 (n = 36) years old, p = 0.054]. In both human and mouse inner ear tissues, PCDHGA10 is expressed in ganglia, hair cells and vestibular transitional epithelia. Fluorescent RNA in situ hybridization using mouse inner ear tissues shows that expression increases with age. In summary, our data show that a variant in the PCDHGA10 gene may be involved in causing or aggravating some familial cases of recurrent idiopathic BPPV.

Functional cooperation between two otoconial proteins Oc90 and Nox3.

BACKGROUND: Otoconia-related vertigo and balance deficits are common in humans, but the molecular etiology is unknown at present. OBJECTIVE: In order to study mechanisms of otoconia formation and maintenance, we have investigated whether otoconin-90 (Oc90), the predominant otoconial constituent protein, and the NADPH oxidase Nox3, an essential regulatory protein for otoconia formation, are functionally interlinked. METHODS: We performed balance behavioral, electrophysiological, morphological and molecular cellular analyses. RESULTS: Double heterozygous mutant mice for Oc90 and Nox3 show severe imbalance, albeit less profound than double null mutants. In contrast, single heterozygous mutant mice have normal balance. Double heterozygous mice have otoconia defects and double null mice have no otoconia. In addition, some hair bundles in the latter mice go through accelerated degeneration. In vitro calcification analysis in cells stably expressing these proteins singly and doubly shows much more intense calcification in the double transfectants. CONCLUSIONS: Oc90 and Nox3 augment each other's function, which is not only critical for otoconia formation but also for hair bundle maintenance.

The Price of Immune Responses and the Role of Vitamin D in the Inner Ear.

OBJECTIVE: In this review the authors discuss evidence from the literature concerning vitamin D and temporal bone diseases (benign paroxysmal positional vertigo [BPPV], Menière's disease [MD], vestibular neuritis, idiopathic facial paralysis, idiopathic acute hearing loss). Common features shared by Menière's disease, glaucoma, and the possible influence by vitamin D are briefly discussed. DATA SOURCES, STUDY SELECTION: Publications from 1970 until recent times have been reviewed according to a keyword search (see above) in PubMed. CONCLUSIONS: MD, BPPV, vestibular neuritis, idiopathic facial paralysis, idiopathic acute hearing loss may all have several etiological factors, but a common feature of the current theories is that an initial viral infection and a subsequent autoimmune/autoinflammatory reaction might be involved. Additionally, in some of these entities varying degrees of demyelination have been documented. Given the immunomodulatory effect of vitamin D, we postulate that it may play a role in suppressing an eventual postviral autoimmune reaction. This beneficial effect may be enhanced by the antioxidative activity of vitamin D and its potential in stabilizing endothelial cells. The association of vitamin D deficiency with demyelination has already been established in other entities such as multiple sclerosis and experimental autoimmune encephalitis. Mice without vitamin D receptor show degenerative features in inner ear ganglia, hair cells, as well as otoconia. The authors suggest further studies concerning the role of vitamin D deficiency in diseases of the temporal bone. Additionally, the possible presence and degree of demyelination in these entities will have to be elucidated more systematically in the future.

Vitamin D supplementation may improve symptoms in Meniere's disease.

In the last 4 years the authors observed a trend that correcting vitamin D deficiency in newly diagnosed cases of Meniere's disease decreased the necessity of the ablative therapy with intratympanic gentamicin. According to their hypothesis, vitamin D supplementation may indeed have a beneficial effect in Meniere's disease if the symptoms are caused by a local postviral autoimmune reaction. Vitamin D has a strong immunomodulatory role, one of which is the regulation of the expression of pro-inflammatory mediators. The authors suggest further epidemiological studies to decide if there is a connection between vitamin D deficiency and Meniere's disease.

Spatiotemporal differences in otoconial gene expression.

Otoconia are minute biocrystals composed of glycoproteins, proteoglycans, and CaCO3 , and are indispensable for sensory processing in the utricle and saccule. Otoconia abnormalities and degeneration can cause or facilitate crystal dislocation to the ampulla, leading to vertigo and imbalance in humans. In order to better understand the molecular mechanism controlling otoconia formation and maintenance, we have examined the spatial and temporal expression differences of otoconial genes in the mouse inner ear at developmental, mature and aging stages using whole transcriptome sequencing (RNA-Seq) and quantitative RT-PCR. We show that the expression levels of most otoconial genes are much higher in the utricle and saccule compared with other inner ear tissues before postnatal stages in C57Bl/6J mice, and the expression of a few of these genes is restricted to the embryonic utricle and saccule. After the early postnatal stages, expression of all otoconial genes in the utricle and saccule is drastically reduced, while a few genes gain expression dominance in the aging ampulla, indicating a potential for ectopic debris formation in the latter tissue at old ages. The data suggest that the expression of otoconial genes is tightly regulated spatially and temporally during developmental stages and can become unregulated at aging stages. Birth Defects Research (Part A) 106:613-625, 2016. © 2016 Wiley Periodicals, Inc.

Vestibular dysfunction, altered macular structure and trait localization in A/J inbred mice.

A/J mice develop progressive hearing loss that begins before 1 month of age and is attributed to cochlear hair cell degeneration. Screening tests indicated that this strain also develops early onset vestibular dysfunction and has otoconial deficits. The purpose of this study was to characterize the vestibular dysfunction and macular structural pathology over the lifespan of A/J mice. Vestibular function was measured using linear vestibular evoked potentials (VsEPs). Macular structural pathology was evaluated using light microscopy, scanning electron microscopy, transmission electron microscopy, confocal microscopy and Western blotting. Individually, vestibular functional deficits in mice ranged from mild to profound. On average, A/J mice had significantly reduced vestibular sensitivity (elevated VsEP response thresholds and smaller amplitudes), whereas VsEP onset latency was prolonged compared to age-matched controls (C57BL/6). A limited age-related vestibular functional loss was also present. Structural analysis identified marked age-independent otoconial abnormalities in concert with some stereociliary bundle defects. Macular epithelia were incompletely covered by otoconial membranes with significantly reduced opacity and often contained abnormally large or giant otoconia as well as normal-appearing otoconia. Elevated expression of key otoconins (i.e., otoconin 90, otolin and keratin sulfate proteoglycan) ruled out the possibility of reduced levels contributing to otoconial dysgenesis. The phenotype of A/J was partially replicated in a consomic mouse strain (C57BL/6J-Chr 17(A/J)/NaJ), thus indicating that Chr 17(A/J) contained a trait locus for a new gene variant responsible to some extent for the A/J vestibular phenotype. Quantitative trait locus analysis identified additional epistatic influences associated with chromosomes 1, 4, 9 and X. Results indicate that the A/J phenotype represents a complex trait, and the A/J mouse strain presents a new model for the study of mechanisms underlying otoconial formation and maintenance.

Mechanisms of otoconia and otolith development.

BACKGROUND: Otoconia are bio-crystals that couple mechanic forces to the sensory hair cells in the utricle and saccule, a process essential for us to sense linear acceleration and gravity for the purpose of maintaining bodily balance. In fish, structurally similar bio-crystals called otoliths mediate both balance and hearing. Otoconia abnormalities are common and can cause vertigo and imbalance in humans. However, the molecular etiology of these illnesses is unknown, as investigators have only begun to identify genes important for otoconia formation in recent years. RESULTS: To date, in-depth studies of selected mouse otoconial proteins have been performed, and about 75 zebrafish genes have been identified to be important for otolith development. CONCLUSIONS: This review will summarize recent findings as well as compare otoconia and otolith development. It will provide an updated brief review of otoconial proteins along with an overview of the cells and cellular processes involved. While continued efforts are needed to thoroughly understand the molecular mechanisms underlying otoconia and otolith development, it is clear that the process involves a series of temporally and spatially specific events that are tightly coordinated by numerous proteins. Such knowledge will serve as the foundation to uncover the molecular causes of human otoconia-related disorders.

Gender-based comorbidity in benign paroxysmal positional vertigo.

It has been noted that benign paroxysmal positional vertigo (BPPV) may be associated with certain disorders and medical procedures. However, most studies to date were done in Europe, and epidemiological data on the United States (US) population are scarce. Gender-based information is even rarer. Furthermore, it is difficult to assess the relative prevalence of each type of association based solely on literature data, because different comorbidities were reported by various groups from different countries using different patient populations and possibly different inclusion/exclusion criteria. In this study, we surveyed and analyzed a large adult BPPV population (n = 1,360 surveyed, 227 completed, most of which were recurrent BPPV cases) from Omaha, NE, US, and its vicinity, all diagnosed at Boys Town National Research Hospital (BTNRH) over the past decade using established and consistent diagnostic criteria. In addition, we performed a retrospective analysis of patients' diagnostic records (n = 1,377, with 1,360 adults and 17 children). The following comorbidities were found to be significantly more prevalent in the BPPV population when compared to the age- and gender-matched general population: ear/hearing problems, head injury, thyroid problems, allergies, high cholesterol, headaches, and numbness/paralysis. There were gender differences in the comorbidities. In addition, familial predisposition was fairly common among the participants. Thus, the data confirm some previously reported comorbidities, identify new ones (hearing loss, thyroid problems, high cholesterol, and numbness/paralysis), and suggest possible predisposing and triggering factors and events for BPPV.

Menopause and benign paroxysmal positional vertigo.

OBJECTIVE: This study was designed to examine the age and sex distribution and the effects of menopause in a large cohort of participants diagnosed with benign paroxysmal positional vertigo (BPPV). METHODS: We analyzed 1,377 BPPV patients and surveyed 935 women from this group-all diagnosed at the Boys Town National Research Hospital in the last decade. RESULTS: A detailed age and sex distribution analysis of BPPV onset showed that aging had a profound impact on BPPV occurrence in both sexes, and that perimenopausal women were especially susceptible to BPPV (3.2:1 female-to-male ratio). The latter is a novel finding and was confirmed by a direct survey of female BPPV patients (168 participated). In addition, there was a pronounced female preponderance (6.8:1 female-to-male ratio) in BPPV in the teenage group despite its low prevalence in this age group. CONCLUSIONS: Data suggest that hormonal fluctuations (especially during menopause) may increase the tendency to develop BPPV.

Vitamin D deficiency and benign paroxysmal positioning vertigo.

Benign paroxysmal positional vertigo is a common cause of disabling vertigo with a high rate of recurrence. Although connections between vitamin D deficiency and osteoporosis, as well as between osteoporosis and benign paroxysmal positional vertigo have been suggested respectively in the literature, we are not aware of any publication linking vitamin D and benign paroxysmal positional vertigo. As a hypothesis, we suggest that there is a relation between insufficient vitamin D level and benign paroxysmal positional vertigo. In order to test this hypothesis, in a small retrospective pilot study, 25-hydroxyvitamin D levels in serum of patients with benign paroxysmal positional vertigo and frequency of recurrence after correction of serum level were assessed retrospectively. Patients with idiopathic positional vertigo had a low average serum level of 25-hydroxyvitamin D (23ng/mL) similar to that of the general Austrian population, which has a high prevalence of hypovitaminosis D. In 4 cases with chronically recurrent severe vertigo episodes, average levels of serum 25-hydroxyvitamin D were even significantly lower than in the other vertigo patients, who had their first episode. Vertigo attacks did not recur after supplementation with vitamin D. We raise the possibility that patients with benign paroxysmal positional vertigo who have low vitamin D levels may benefit from supplementation and suggest further epidemiological investigations to determine the effect of correcting vitamin D deficiency on the recurrence of vertigo. Given the many known benefits of vitamin D, the authors recommend the measurement of vitamin D in patients with benign paroxysmal positional vertigo and supplementation if necessary.

Peripheral blood monocyte-expressed ANXA2 gene is involved in pathogenesis of osteoporosis in humans.

Low bone mineral density (BMD) is a risk factor of osteoporosis and has strong genetic determination. Genes influencing BMD and fundamental mechanisms leading to osteoporosis have yet to be fully determined. Peripheral blood monocytes (PBM) are potential osteoclast precursors, which could access to bone resorption surfaces and differentiate into osteoclasts to resorb bone. Herein, we attempted to identify osteoporosis susceptibility gene(s) and characterize their function(s), through an initial proteomics discovery study on PBM in vivo, and multiscale validation studies in vivo and in vitro. Utilizing the quantitative proteomics methodology LC-nano-ESI-MS(E), we discovered that a novel protein, i.e. ANXA2, was up-regulated twofold in PBM in vivo in Caucasians with extremely low BMD (cases) versus those with extremely high BMD (controls) (n = 28, p < 0.05). ANXA2 gene up-regulation in low BMD subjects was replicated at the mRNA level in PBM in vivo in a second and independent case-control sample (n = 80, p < 0.05). At the DNA level, we found that SNPs in the ANXA2 gene were associated with BMD variation in a 3(rd) and independent case-control sample (n = 44, p < 0.05), as well as in a random population sample (n = 997, p < 0.05). The above integrative evidence strongly supports the concept that ANXA2 is involved in the pathogenesis of osteoporosis in humans. Through a follow-up cellular functional study, we found that ANXA2 protein significantly promoted monocyte migration across an endothelial barrier in vitro (p < 0.001). Thus, elevated ANXA2 protein expression level, as detected in low BMD subjects, probably stimulates more PBM migration through the blood vessel walls to bone resorption surfaces in vivo, where they differentiate into higher number of osteoclasts and resorb bone at higher rates, thereby decreasing BMD. In conclusion, this study identified a novel osteoporosis susceptibility gene ANXA2, and suggested a novel pathophysiological mechanism, mediated by ANXA2, for osteoporosis in humans.

Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the null mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

Osteopontin is not critical for otoconia formation or balance function.

Unlike the structural and mechanical role of bone crystals, the inertial mass of otoconia crystals provides a shearing force to stimulate the mechanoreceptors of the utricle and saccule (the gravity receptor organ) under the stimuli of linear motion. It is not clear whether otoconia, composed primarily of CaCO3 and glycoproteins, go through similar calcification processes as bone. We have recently shown that otoconin-90 (Oc90) regulates the growth of otoconia crystals as osteopontin does bone crystals. Here, we analyzed the role of this non-collagenous bone matrix protein, osteopontin, in otoconia formation and balance function utilizing its knockout mice, whose inner ear phenotype has not been examined. Despite the presence of the protein in wild-type otoconia and vestibular hair cells, morphological, ultrastructural, and protein and calcium composition analyses of osteopontin null otoconia show that the protein is not needed for crystal formation, and no evidence of compensatory protein deposition is found. Employment of a wide spectrum of balance behavioral tests demonstrates that the protein is not critical for balance function either, which is confirmed by the normal function of the gravity receptor organ directly measured with linear vestibular-evoked potentials (VsEPs). When compared with findings on other otoconins, the data manifest a hierarchy of importance of proteins in crystallization and indicate mechanistic similarities and differences between bone and otoconia calcification.

Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix.

A critical part of the functional development of our peripheral balance system is the embryonic formation of otoconia, composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the gravity receptor in the inner ear. To date neither the functions of otoconial proteins nor the processes of crystal formation are clearly defined. Using gene targeting and protein analysis strategies, we demonstrate that the predominant mammalian otoconin, otoconin-90/95 (Oc90), is essential for formation of the organic matrix of otoconia by specifically recruiting other matrix components, which includes otolin, a novel mammalian otoconin that we identified to be in wildtype murine otoconia. We show that this matrix controls otoconia growth and morphology by embedding the crystallites during seeding and growth. During otoconia development, the organic matrix forms prior to CaCO3 deposition and provides optimal calcification efficiency. Histological and ultrastructural examinations show normal inner ear epithelial morphology but reduced acellular matrices, including otoconial, cupular and tectorial membranes, in Oc90 null mice, likely due to an absence of Oc90 and a profound reduction of otolin. Our data demonstrate the critical roles of otoconins in otoconia seeding, growth and anchoring and suggest mechanistic similarities and differences between otoconia and bone calcification.

Assembly of the otoconia complex to the macular sensory epithelium of the vestibule.

In the inner ear, specificity of stimulus perception is achieved by associating the sensory epithelia of the three mechanoreceptor organs, the utricle/saccule, cristae, and cochlea, with distinct types of acellular matrices. Only the utricle and saccule have an extremely dense matrix, the otoconial complex, which overlies the sensory epithelium (macula) and provides inertial mass to generate shearing forces essential for the mechanoreceptors to sense gravity and linear acceleration. Such sensation is necessary for spatial orientation and balance. The importance of otoconia is clearly demonstrated by the impact of balance disorders upon the elderly population that involve otoconia degeneration, as well as by canalithiasis and cupulolithiasis, in which otoconia are dislocated. This underscores the need to understand how otoconia are formed and maintained and how to prevent their degeneration. To date, a number of otoconia-related proteins have been identified mostly in mice and bony fish. Although most of these proteins are also present in other structures of the inner ear, a distinct collection of proteins in the macula plus the unique ionic microenvironment of the endolymph near its epithelium likely contribute to the site-specific calcification of otoconia. Based on the current literature and ongoing research, this mini-review postulates a working model of how the otoconia complex is assembled specifically above the macular sensory epithelium of the vestibule. The central hypothesis of this model is that proteins are critical in sequestering calcium for crystallization in the calcium-poor endolymph. The review also sets forth some issues that need to be resolved in the future.

Mapping a chromosomal locus for valproic acid-induced exencephaly in mice.

Human neural tube defects (NTDs) are among the most common congenital defects. They have a highly heterogeneous etiology, and, in addition to those seen in association with genetic syndromes, there are also NTDs induced by pharmaceutical compounds in utero, such as the widely used anti-epileptic drug valproic acid (VPA). Although familial studies have suggested a genetic contribution to VPA-induced NTDs, this trait has not been adequately studied, nor have the responsible genetic factors been identified. We generated a series of mouse crosses and backcrosses using the highly inbred SWV/Fnn and C57BL/6J strains, in order to identify possible chromosomal loci contributing to VPA sensitivity. When exposed to a high dose of sodium VPA (600 mg/kg) via maternal intraperitoneal injection on gestational day E8.5, the fetuses manifested exencephaly in a strain-dependent manner. Our data show an autosomal recessive trait, plus a gender-related effect or an overall X-Chromosome (Chr) effect, as being primarily responsible for determining sensitivity to VPA-induced exencephaly. Genome scanning and further linkage analysis of 131 exencephalic backcross fetuses identified a major locus linked to D7Mit285 (p < 2 x 10(-6)), exceeding the threshold for significant linkage. These results suggest a major chromosomal locus associated with the sensitivity to VPA-induced exencephaly in mice.

Genetic dissection of hyperthermia-induced neural tube defects in mice.

BACKGROUND: Maternal hyperthermia has been shown to induce neural tube defects (NTD) in humans and in experimental animal systems. We report the first genetic dissection of maternal hyperthermia-induced NTD in mice. METHODS: After maternal exposure on E8.5 to 43 degrees C water bath for 10 min, we observed exencephaly frequencies among E15.5-17.5 fetuses from the following crosses and backcrosses, SWV/Fnn(SWV)xSWV, C57BL/6J(C57)xC57, SWVxC57 (F1), F1xSWV and SWVxF1. RESULTS: The fetuses with maternal hyperthermia exposure developed exencephaly in a strain-dependent manner and the exencephaly frequencies among the above crosses were 46.2, 14.3, 13.6, 11.3, and 27.0%, respectively, expressed over total live fetuses. The fetal death rates were 47.3, 24.6, 37.1, 4.3, and 35.5%, respectively, expressed over total implants. CONCLUSION: The data demonstrate that a single fetal genetic locus, plus a maternal effect, have likely caused the strain differences in the susceptibility to hyperthermia-induced exencephaly. A maternal effect alone may have caused the higher prenatal mortality rates in the SWVxF1 cross versus the reciprocal cross. Analysis of gender ratios among those affected from these crosses excludes an X- or Y-linked effect in causing the higher numbers of affected females.