Molecular analysis of genes related to Pendred syndrome in individuals with deafness and functional study of pendrin protein

Orientador: Edi Lúcia SartoratoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: 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 SLC26A4Abstract: 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 geneDoutoradoGenetica Animal e EvoluçãoDoutora em Genética e Biologia Molecula

Interaction between audiology and genetics in the study of a family: the complexity of molecular diagnosis and genetic counseling

Hearing loss is a multifaceted condition with many etiologies, among which genetic mutation is. Therefore, it is important to connect audiological investigation to etiological diagnosis. AIM: this study aims to establish the audiological and genetic profiles of three non-syndromic children with sensorineural hearing loss. MATERIALS AND METHOD: three brothers aged 3, 5 and 16 were enrolled in this study. They were submitted to behavioral and electrophysiological hearing tests and molecular studies. RESULTS: the hearing tests showed moderate to moderately severe bilateral symmetric sensorineural hearing loss and an accentuated descending slope. Transient and Distortion Product Otoacoustic emissions were absent in the two younger children. ABR showed a bilateral moderately severe to severe sensorineural hearing loss. P300 showed bilateral normal latencies in the older brother. Molecular tests showed that the two younger children were heterozygote for mutation 35delG on gene GJB2. CONCLUSION: The combination of speech and hearing tests and genetic analysis allows for the etiologic diagnosis of seemingly similar hearing loss cases, which however display different genetic backgrounds. Molecular studies must be comprehensive enough to avoid precipitated diagnosis which may impair genetic counseling.A deficiência auditiva como déficit sensorial mais comum tem dentre suas diferentes etiologias as alterações genéticas. Assim, é importante que a investigação audiológica se associe à busca do diagnóstico etiológico. OBJETIVO: Relatar o perfil audiológico e genético de três irmãos portadores de deficiência auditiva neurossensorial não-sindrômica. MATERIAL E MÉTODO: Estudo de caso de três irmãos, do sexo masculino, com 3, 5 e 16 anos, respectivamente, submetidos à avaliação audiológica comportamental e eletrofisiológica, e estudo molecular. RESULTADOS: Os achados audiológicos mostraram: audiometria do tipo neurossensorial, bilateral, simétrica, de grau moderado a moderadamente severo e configuração descendente acentuada. EOAT e EOAPD ausentes nos dois irmãos mais novos. PEATE compatível com perda auditiva moderadamente severa a severa. Presença do P300 com latências dentro da normalidade bilateralmente no irmão mais velho. Os achados do exame molecular mostraram que as duas crianças mais novas eram heterozigotos para a mutação 35delG no gene GJB2 e o mais velho não apresentava essa mutação. CONCLUSÃO: A associação das avaliações fonoaudiológicas e genéticas permite o diagnóstico etiológico de perdas auditivas que à primeira vista são semelhantes, mas que não obedecem à mesma estrutura genética. Os estudos moleculares devem ser abrangentes, evitando diagnósticos precipitados que prejudiquem o aconselhamento genético.Instituto de Estudos Avançados da AudiçãoUniversidade Federal de São Paulo (UNIFESP)Instituto de Estudos da AudiçãoUniversidade Estadual de CampinasUniversidade Estadual de Campinas Departamento de Genética MédicaUSP Faculdade de Medicina Departamento de Fisioterapia, Fonoaudiologia e Terapia OcupacionalFAPESPUNIFESPSciEL

Interaction between audiology and genetics in the study of a family: the complexity of molecular diagnosis and genetic counseling

Hearing loss is a multifaceted condition with many etiologies, among which genetic mutation is. Therefore, it is important to connect audiological investigation to etiological diagnosis. AIM: this study aims to establish the audiological and genetic profiles of three non-syndromic children with sensorineural hearing loss. MATERIALS AND METHOD: three brothers aged 3, 5 and 16 were enrolled in this study. They were submitted to behavioral and electrophysiological hearing tests and molecular studies. RESULTS: the hearing tests showed moderate to moderately severe bilateral symmetric sensorineural hearing loss and an accentuated descending slope. Transient and Distortion Product Otoacoustic emissions were absent in the two younger children. ABR showed a bilateral moderately severe to severe sensorineural hearing loss. P300 showed bilateral normal latencies in the older brother. Molecular tests showed that the two younger children were heterozygote for mutation 35delG on gene GJB2. CONCLUSION: The combination of speech and hearing tests and genetic analysis allows for the etiologic diagnosis of seemingly similar hearing loss cases, which however display different genetic backgrounds. Molecular studies must be comprehensive enough to avoid precipitated diagnosis which may impair genetic counseling.A deficiência auditiva como déficit sensorial mais comum tem dentre suas diferentes etiologias as alterações genéticas. Assim, é importante que a investigação audiológica se associe à busca do diagnóstico etiológico. OBJETIVO: Relatar o perfil audiológico e genético de três irmãos portadores de deficiência auditiva neurossensorial não-sindrômica. MATERIAL E MÉTODO: Estudo de caso de três irmãos, do sexo masculino, com 3, 5 e 16 anos, respectivamente, submetidos à avaliação audiológica comportamental e eletrofisiológica, e estudo molecular. RESULTADOS: Os achados audiológicos mostraram: audiometria do tipo neurossensorial, bilateral, simétrica, de grau moderado a moderadamente severo e configuração descendente acentuada. EOAT e EOAPD ausentes nos dois irmãos mais novos. PEATE compatível com perda auditiva moderadamente severa a severa. Presença do P300 com latências dentro da normalidade bilateralmente no irmão mais velho. Os achados do exame molecular mostraram que as duas crianças mais novas eram heterozigotos para a mutação 35delG no gene GJB2 e o mais velho não apresentava essa mutação. CONCLUSÃO: A associação das avaliações fonoaudiológicas e genéticas permite o diagnóstico etiológico de perdas auditivas que à primeira vista são semelhantes, mas que não obedecem à mesma estrutura genética. Os estudos moleculares devem ser abrangentes, evitando diagnósticos precipitados que prejudiquem o aconselhamento genético.69870

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Study of modulators genes associated to mutations in mitochondrial genes in individuals with non-syndromic deafness

Orientador: Edi Lucia SartoratoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A perda auditiva é a mais comum das deficiências sensoriais da população em geral. A surdez congênita ocorre em cerca de 1 em cada 1000 nascidos vivos, dos quais aproximadamente 50% têm origem hereditária nos países desenvolvidos. A perda auditiva não-sindrômica pode ser causada por mutações em genes nucleares e genes mitocondriais (mtDNA). Mutações no mtDNA foram associadas ao uso de aminoglicosídicos e à surdez não-sindrômica em muitas famílias no mundo todo. No entanto, o background genético do indivíduo influencia a expressão fenotípica destas mutações patogênicas. Dessa forma, foi proposto que os genes nucleares modificadores modulam a manifestação fenotípica da mutação mitocondrial A1555G no gene MTRNR1. Ambos os genes nucleares modificadores TRMU e MTO1 codificam uma proteína mitocondrial altamente conservada, a qual acredita-se estar envolvida na modificação do tRNA. Estudos propuseram que o TRMU humano, assim como o gene nuclear MTO1 poderiam modular a manifestação fenotípica da surdez associada a mutações mitocondriais. O objetivo deste trabalho foi elucidar a contribuição de mutações mitocondriais, de mutações em genes nucleares modificadores e a exposição aos aminoglicosídeos no fenótipo da perda auditiva. Nossos achados sugerem que o background genético dos indivíduos pode desempenhar um papel importante na patogênese da surdez associada à mutação mitocondrial e ao uso de aminoglicosídeos.Abstract: Hearing loss is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non- yndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafnessassociated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.MestradoGenetica Animal e EvoluçãoMestre em Genética e Biologia Molecula

Etiologic And Diagnostic Evaluation: Algorithm For Severe To Profound Sensorineural Hearing Loss In Brazil.

Evaluation of the effectiveness of imaging and genetic testing, and establishment of a cost-effective diagnostic protocol for the etiologic diagnosis of sensorineural hearing loss (SNHL) in Brazil. Prospective cohort study. Analysis of 100 unrelated Brazilian patients with severe to profound bilateral SNHL submitted to cochlear implant (CI) between 2002 and 2010 at the University of Campinas hospital. The study was based upon three groups: individuals with congenital, progressive, and sudden SNHL. After the diagnostic investigation, the number of cases with unknown etiology was reduced from 72 to 42 (a 42% reduction); 25% of cases were due to environmental factors, 19% to genetic causes, and 14% to inner-ear abnormalities or other clinical features. The genetic and imaging findings contributed to the diagnosis of SNHL in 19% and 20% of the cases analysed, respectively. Molecular testing mainly contributed to the diagnosis of patients with congenital SNHL, while the contribution of radiologic examination was higher for individuals with progressive or sudden SNHL. A sequential diagnostic protocol was proposed based on these data. The proposed diagnostic workup algorithm could provide better optimization of etiologic diagnosis, as well as reduced costs, compared to a simultaneous testing approach.52746-5

Molecular Analysis Of Slc26a4 Gene In Patients With Nonsyndromic Hearing Loss And Eva: Identification Of Two Novel Mutations In Brazilian Patients.

The SLC26A4 gene has been described as the second gene involved in most cases of sensorineural non-syndromic hearing loss, since the first is the GJB2 gene. Recessive mutations in the SLC26A4 gene encoding pendrin, an anion transporter, are responsible for non-syndromic hearing loss associated with an enlarged vestibular aqueduct (EVA) and Pendred syndrome, which causes early hearing loss and affects the thyroid gland. Typically, the hearing loss is profound and prelingual. However, in some individuals, hearing impairment may develop later in childhood and then progress. Over 200 different SLC26A4 mutations have been reported, with each ethnic population having its own distinctive mutant allele series including a few prevalent founder mutations. Perform the screening of the 20 coding exons of SLC26A4 gene in Brazilian deaf individuals with EVA. Among the 23 unrelated non-syndromic hearing loss Brazilian patients with EVA, in whom no deafness-causing mutations of the GJB2 gene, the direct sequencing was performed to screen the 20 exons and their flanking regions of the SLC26A4 gene. The sequencing results revealed 9 cases (39%) carrying 13 different SLC26A4 mutations, including 11 known mutations (279delT, V138F, T193I, IVS8+1G>A, T410M, Q413R, R409H, L445W, IVS15+5G>A, V609G, and R776C) and 2 novel mutation (G149R and P142L). The SLC26A4 mutations have a high carrying rate in non-syndromic hearing loss Brazilian patients. The identification of a disease-causing mutation can be used to establish a genotypic diagnosis and provide important information to the patients and their families.77410-