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.

Mutações no gene SLC26A4 estão relacionadas a dois distúrbios de herança autossômica recessiva, a síndrome de Pendred (SP) e uma forma de surdez não sindrômica (DFNB4). Nesses distúrbios podem ocorrer anomalias da orelha interna, como dilatação do aqueduto vestibular ou displasia de Mondini. O objetivo foi verificar se mutações no gene SLC26A4 são causa de surdez genética em 70 famílias e em 15 casos isolados de surdez com anomalias da orelha interna ou suspeita de SP. Por meio da análise de microssatélites e sequenciamento, foi encontrado um total de 7 mutações diferentes provavelmente patogênicas (4 novas) distribuídas em 5 indivíduos. Os casos com mutações em um único alelo ou resultados normais foram investigados por MLPA e nenhuma variação foi encontrada. Mutações nesse gene explicaram cerca de 3% dos casos familiais de surdez. Cerca de 20% dos casos de surdez com anomalias da orelha interna ou suspeita de SP apresentaram pelo menos uma mutação patogênica nesse gene. Esse estudo reforça a importância da triagem molecular desse gene na população brasileira. / Mutations in the SLC26A4 gene are related with two autosomal recessive disorders, Pendred syndrome (PS) and a form of non-syndromic deafness (DFNB4). In these disorders can occur inner ear anomalies, such as dilatation of the vestibular aqueduct or Mondini dysplasia. The aim was to verify if mutations in the SLC26A4 gene are cause of genetic deafness in 70 families and 15 isolated cases of deafness with inner ear anomalies or suspected SP. By means of the microsatellite analysis and sequencing, was found a total of 7 different mutations probably pathogenic (4 novel) distributed in 5 subjects. The cases with mutations in one allele or normal results were investigated by MLPA and no variation was found. Mutations in this gene explained about 3% of familial cases of deafness. About 20% of cases of deafness with inner ear anomalies or suspected SP presented at least one pathogenic mutation in this gene. This study reinforces the importance of molecular screening of this gene in Brazilian population.

Orientador: Edi Lúcia Sartorato / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:12:43Z (GMT). No. of bitstreams: 1 DeMoraes_VanessaCristineSousa_D.pdf: 4935241 bytes, checksum: ce8c385da6e3872d30f08426c629d34d (MD5) Previous issue date: 2013 / Resumo: O alargamento do aqueduto vestibular (EVA) é uma malformação da orelha interna que pode ser identificado por tomografia computadorizada ou ressonância magnética. O EVA é um dos principais sinais clínicos da Síndrome de Pendred (PDS), uma doença genética com padrão de herança autossômico recessivo causada na maioria dos casos por mutações no gene SLC26A4. Além de EVA, o bócio e defeito na organificação do iodeto na tireóide são achados clínicos típicos da PDS. Por sua vez, mutações no gene SLC26A4 têm também sido observadas em indivíduos com surdez não sindrômica associada ao EVA. Recentemente os genes FOXI1 e KCNJ10 também foram implicados na PDS. O gene FOXI1 é um fator de transcrição do gene SLC26A4. Medições electrofisiológicas mostraram que a alteração da pendrina, proteína codificada pelo gene SLC26A4, em modelos animais levava indivíduos à surdez pela falta do potencial endococlear devido à perda de expressão de canais potássio. Sendo atribuído ao gene KCNJ10 a função de manutenção do potencial endococlear. Desta maneira, o presente estudo teve como objetivo avaliar a ocorrência de mutações nos genes SLC26A4, FOXI1 e KCNJ10 em 60 indivíduos brasileiros portadores de perda auditiva sensorioneural, associada ou não a alterações no aqueduto vestibular. Foram encontradas 14 diferentes alterações no gene SLC26A4, das quais 3 ainda não haviam sido descritas na literatura (P142L, G149R e C282Y) e 4 já haviam sido descritas, porém ainda não haviam sido caracterizadas funcionalmente (T193I, Q413R, L445W e R776C). Dessa forma, foi realizada a análise funcional e a co-localização celular da proteína Pendrina com estas 7 variações alélicas. Não foi encontrada nenhuma evidência de contribuição digênica relacionada ao gene FOXI1 e/ou KCNJ10, uma vez que nenhum paciente desta casuística com alteração no gene SLC26A4 apresentou mutações nesses genes. Além disso, no grupo composto por 30 indivíduos surdos que não apresentam EVA, ficou evidente que o rastreamento do gene SLC26A4 não foi suficiente para explicar a perda auditiva nesses pacientes, uma vez que foram encontradas apenas alterações em um alelo do gene. Por outro lado, no grupo formado por 30 indivíduos surdos que apresentam EVA, o rastreamento do gene SLC26A4 possibilitou o esclarecimento do diagnóstico etiológico da perda auditiva em 5 pacientes que apresentaram mutações nos dois alelos do gene SLC26A4 / Abstract: Enlargement of the vestibular aqueduct (EVA) is a malformation of the inner ear that can be identified by computed tomography or magnetic resonance imaging. EVA is the main feature of Pendred syndrome (PDS), a genetic disease with autosomal recessive inheritance pattern, in most cases caused by mutations in the SLC26A4 gene. Besides EVA, goiter and defective organification of iodide in the thyroid are other typical clinical signs of PDS. In turn, SLC26A4 gene mutations have been also observed in patients with non-syndromic deafness associated with EVA. Recently the genes FOXI1 and KCNJ10 were also implicated in the PDS. The FOXI1 gene is a transcription factor of SLC26A4 gene. Electrophysiological measurements in animal models showed that the mutated pendrin, the protein encoded by the SLC26A4 gene, led individuals to deafness by the lack of endocochlear potential due to loss of expression of potassium channels. Being assigned to the KCNJ10 gene the maintenance of endocochlear potential. Thus, the present study aimed to evaluate the occurrence of mutations in SLC26A4, and KCNJ10 FOXI1 genes in 60 Brazilian patients with sensorineural hearing loss, with or without changes in the vestibular aqueduct. We found 14 different mutations in SLC26A4 gene, of which 3 had not yet been described in the literature (P142L, G149R and C282Y) and 4 had already been described, but had not been characterized functionally yet (T193I, Q413R, L445W and R776C). Thus, we performed the functional analysis and cellular co-localization of Pendrin protein with these 7 allelic variants. We found no evidence of digenic contribution related to FOXI1 and/or KCNJ10 genes, since no patient in with mutations in SLC26A4 gene showed mutations in these genes. In addition, the screening of SLC26A4 gene in 30 deaf individuals with no EVA was not sufficient to explain the hearing loss in these patients, since mutations were found only in one allele of the gene. On the other hand, the screening of SLC26A4 gene in 30 deaf individuals with EVA allowed the elucidation of the etiology of hearing loss in 5 patients with mutations in both alleles of this gene / Doutorado / Genetica Animal e Evolução / Doutora em Genética e Biologia Molecular

Doctor of Philosophy / Department of Anatomy and Physiology / A. Philine Wangemann / Pendred syndrome is characterized by sensorineural deafness and post-pubertal goiter. It is caused by mutations in the anion exchanger, pendrin (SLC26A4). The purpose of this study was to understand the etiology of Pendred syndrome using a mouse model. Different methods of amplification from nanogram amounts of starting RNA were evaluated for gene array application. Gene arrays were performed and free radical stress markers were compared between the stria vascularis and the thyroid of the Slc26a4+/- and Slc26a4-/- mice. Hearing loss in Slc26a4-/- mice is linked to the loss of Kcnj10 protein expression and consequently the loss of endocochlear potential. To understand the mechanism of hearing loss in Slc26a4-/- mice, progressive loss of Kcnj10 protein expression in stria vascularis of Slc26a4-/- mice was assessed, the modulation of Kcnj10 protein expression by free radical stress in cultured stria vascularis and in an heterologous expression system was evaluated. To characterize the thyroid pathology, pendrin expression in the thyroid of Slc26a4+/- mice was measured in a developmental study and correlated with serum thyroxine (T4) levels of Slc26a4+/- and Slc26a4-/- mice over a developmental time course. All tested methods of RNA amplification were suitable for gene array application and demonstrated high internal consistency. Intermethod comparisons revealed variations in data, suggesting that a single amplification method ought to be used within a given experiment. Markers of free radical stress were increased in the stria vascularis of Slc26a4-/- mice before the onset of hearing. Progressive loss of Kcnj10 expression was seen in Slc26a4-/- mice at the onset of hearing. Furthermore, free radical stress modulated the expression of Kcnj10 in cultured stria vascularis and in a heterologous expression system. The pendrin mRNA expression was marginal in the thyroid and did not correlate with serum T4 levels. Further, absence of pendrin did not affect free radical stress markers in the thyroid. These data suggest that free radical stress-mediated loss of Kcnj10 expression in stria vascularis contributes to deafness in the Pendred syndrome mouse model and that pendrin is not essential for the function of mouse thyroid gland.

Pendrin is an anion transporter that is expressed in several organs. In the thyroid gland, pendrin is localized at the apical pole of thyrocytes and it is responsible for the iodide efflux from thyrocytes into the colloid in the follicular lumen where iodide is organificated. The extrathyroidal expression was shown in the inner ear, kidney, placenta and mammary gland. Carriers of mutations in the pendrin gene (PDS, SLC26A4) display variable phenotypical features following the autosomal recessive manner of the inheritance: combined thyroid and hearing affection (Pendred syndrome - OMIM274600), nonsyndromic autosomal recessive neurosensory deafness (DFNB4 - OMIM600791) or isolated enlarged vestibular aqueduct (EVA - OMIM603545). The thyroid affection is usually manifested as euthyroid or hypothyroid goitre in the second decade of life. In a minority of patients, dyshormonogenesis is present at birth, and the disease is diagnosed in the frame of the nation-wide neonatal screening for congenital hypothyroidism.

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.