Doctor of Philosophy / Biochemistry Interdepartmental Program / Antje Philine Wangemann / Mutations of SLC26A4 are the most common cause of the hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in the cochlea, the vestibular labyrinth, and the endolymphatic sac of the inner ear. Slc26a4Δ/Δ mice, devoid of pendrin expression, develop an enlarged membranous labyrinth which leads to the failure to develop hearing, thereby recapitulating the human disease. Identifying the ionic composition of the endolymph and evaluating the importance of pendrin expression at various sites are initial steps towards developing strategies for preventing enlargement of the endolymph volume and subsequently restoring the inner ear functions. The major aims of the present study are 1) To determine the ionic composition of inner ear fluids during the developmental phase in which the enlargement of the endolymph volume occurs; 2) To test the hypothesis that pendrin expression in the endolymphatic sac is more important than its expression in the cochlea and the vestibular labyrinth. Here, we determined the Na+ and K⁺ concentrations in the cochlea and the endolymphatic sac with double-barreled ion-selective electrodes and generated a mouse model that restores pendrin expression in the endolymphatic sac while lacking expression in the cochlea and the vestibular labyrinth. High Na⁺ and low K⁺ concentrations were found in the cochlear endolymph during the embryonic stage. A rise of the K⁺ concentration along with a decline of the Na⁺ concentration occurred shortly before birth. The site-specific restoration of pendrin to the endolymphatic sac prevented enlargement and rescued hearing and balance. In conclusion, these data demonstrate that endolymph, in the phase of luminal enlargement during the embryonic development, is a Na⁺-rich fluid that is modified into a K⁺-rich fluid just before birth; restoration of pendrin in the endolymphatic sac is sufficient for developing normal inner ear function. Furthermore, these data suggest enlargement of endolymph volume caused by the loss of Slc26a4 is a consequence of disrupted Na⁺ absorption. Moreover, pharmacological strategies that correct fluid transport, as well as spatially and temporally limited restorations of pendrin, might restore normal inner ear functions in humans carrying mutations of SLC26A4.

Master of Science / Department of Anatomy and Physiology / A. Philine Wangemann / SLC26A4 and its murine ortholog Slc26a4 code for pendrin, an anion-exchanger that is expressed in the inner ear. Patients with mutations in SLC26A4 have syndromic or nonsyndromic hearing loss that is associated with a prenatal enlargement of the membranous labyrinth. The mouse model Slc26a4[superscript]Δ/Δ recapitulates the enlargement, develops an enlargement of the inner ear and fails to acquire hearing. The vestibular labyrinth secretes fluid, accounting for enlargement of the membranous labyrinth. The objective of the current study was to measure K⁺ concentrations in the utricular endolymph of Slc26a4[superscript]Δ/+ and Slc26a4[superscript]Δ/Δ mice as a first step toward a mechanistic understanding of fluid secretion during perinatal development. Doublebarreled K⁺-selective electrodes were used to measure K⁺ concentrations of the utricular endolymph in vitro. Potassium concentrations were ~10 mM in both genotypes at embryonic (E) day 16.5. The K⁺ concentrations started to rise at E17.5 in Slc26a4[superscript]Δ/+ mice. There was a 1-day delay in Slc26a4[superscript]Δ/Δ mice. This delay may be the consequence of a larger fluid volume. K⁺ concentrations rose to 150 mM and 132 mM in Slc26a4[superscript]Δ/+ and Slc26a4[superscript]Δ/Δ adult mice, respectively. Consistently, expression of KCNQ1 and the Na⁺/2Cl⁻/K⁺ cotransporter SLC12A2 was found in the utricle at E19.5 in Slc26a4[superscript]Δ/+ and Slc26a4[superscript]Δ/Δ mice. In conclusion, the data suggest that K⁺ secretion is not the major driving force of fluid secretion in the utricle of the developing mouse inner ear.

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Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.