Neuropatia auditiva: avaliação clínica e abordagem diagnóstica

Auditory neuropathy is a condition in which there is a change in the neuronal transmission of the auditory stimuli. Our objective was to describe the patients’ series within the clinical spectrum of auditory neuropathy. We designed a transversal, retrospective study, with a description of a consecutive case series. Auditory neuropathy was defined by the presence of acoustic otoemissions plus absent/abnormal auditory brainstem responses with cochlear microphonism. 34 patients with bilateral hearing loss, 23 males and 11 females, were included in the study. Eighty percent of the cases had congenital onset of hearing loss. Acoustic otoemissions were absent in 67% of them. Cochlear microfonism was present in 79% of all cases. Prenatal, perinatal or ambiental factors were present in 35.2% of the cases. Medical literature shows great variability in findings related to auditory neuropathy, both in its etiology and epidemiological data. Auditory neuropathy presents a broad spectrum of changes that may result from mild to severe changes in the functioning of the auditory pathway, and in our sample we observed that 80% of Auditory neuropathy have congenital onset of hearing loss and/or with cochlear microphonism identified. 91% of patients experience significant hearing impairment and 53% suffer from severe or profound deafness6353359CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESnão temnão temA neuropatia auditiva é uma condição na qual há alteração na condução neuronal do estímulo sonoro. Este trabalho pretende descrever e caracterizar a casuística de doentes com neuropatia auditiva. Realizámos um estudo transversal, retrospetivo, com descrição de uma série de casos consecutivos. O diagnóstico da neuropatia auditiva foi definido nas seguintes situações: Presença de otoemissões acústicas com potenciais auditivos de tronco encefálico ausente ou anormal e presença do microfonismo coclear independentemente da presença de otoemissões acústicas. Foram avaliados 34 doentes com perda auditiva bilateral, 67% deles do sexo masculino. O aparecimento dos sintomas foi congênito em 80% dos casos. Na pesquisa das otoemissões acústicas, a resposta foi ausente em 67% dos doentes. O microfonismo coclear foi detetado em 79% dos doentes. Antecedentes gestacionais, perinatais ou ambientais relevantes estiveram presentes em 35,3% dos casos. A literatura médica ainda apresenta grande variabilidade nos achados relacionados com a neuropatia auditiva, tanto na sua etiologia quanto nos dados epidemiológicos. A neuropatia auditiva apresenta um amplo espectro de alterações que podem resultar em disfunções leves a severas no funcionamento da via auditiva. Na nossa amostra, observámos que 80% das neuropatias auditivas terão tido origem congênita e/ou apresenta microfonismo coclear, 91% dos doentes apresenta défice auditivo significativo e 53% sofrem de surdez severa ou profunda

Screening Of Genetic Alterations Related To Non-syndromic Hearing Loss Using Massarray Iplex® Technology.

Recent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss. The technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR. An initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes. The MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.168

SGC-CAMKK2-1: A Chemical Probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2

Genetic study of hereditary hearing loss with new technologies based on array-CGH and next-generation sequencing

Orientador: Edi Lúcia SartoratoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A perda auditiva é uma das desordens sensoriais mais comuns em humanos. A identificação de alterações genéticas associadas à surdez pode contribuir para uma melhor compreensão das bases moleculares e dos mecanismos fisiopatológicos envolvidos nos diferentes fenótipos das perdas auditivas hereditárias. Além disso, pode proporcionar um diagnóstico preciso, desenvolvimento de tratamentos mais específicos e aconselhamento genético para pacientes e famílias. No entanto, a elevada heterogeneidade clínica e genética da perda auditiva dificulta o diagnóstico molecular. Até o momento, já foram descritos mais de 90 genes e 150 loci envolvidos com perdas auditivas não-sindrômicas e pelo menos 40 genes associados aos principais tipos de surdez sindrômica. Com a aplicação de tecnologias de nova geração é possível otimizar o diagnóstico genético e realizar uma investigação mais ampla das perdas auditivas hereditárias. Dessa forma, o objetivo do presente estudo foi esclarecer a etiologia genética em uma amostra de indivíduos brasileiros com perdas auditivas associadas a distintos padrões de herança, mediante aplicação de tecnologias de nova geração (array-CGH e next-generation sequencing). Foram analisados casos esporádicos ou familiares com perda auditiva neurossensorial bilateral, para os quais foram excluídos fatores ambientais e as principais mutações relacionadas à perda auditiva de origem genética. Foi realizado o estudo molecular de 19 pacientes mediante a aplicação do OTO-NGS-Panel, um painel de captura de sequências dirigido a 71 genes envolvidos com perdas auditivas hereditárias para sequenciamento massivo, desenvolvido no Departamento de Genética do Hospital Ramón y Cajal, Madri/Espanha. Foram identificadas alterações patogênicas em nove dos 19 pacientes (47,4%). Doze mutações em sete genes distintos foram detectadas, com destaque para sete mutações que estão sendo descritas pela primeira vez neste trabalho. Dentre os genes envolvidos estavam: CDH23 e OTOF, relacionados à surdez de herança autossômica recessiva; DFNA5, ACTG1, COCH e CRYM, associados à perda auditiva de herança autossômica dominante; e PRPS1 envolvido com a perda auditiva de herança ligada ao cromossomo X. Foram pesquisadas deleções e inserções em seis pacientes com surdez de herança autossômica recessiva, mediante hibridação genômica comparativa baseada em array (array-CGH). Foi utilizado o OTO-CGH array, desenvolvido no Departamento de Genética do Hospital Ramón y Cajal, Madri/Espanha, que permite a análise de loci e genes associados à perda auditiva de herança autossômica recessiva. Porém, não foram encontradas alterações patogênicas que pudessem ser relacionadas à surdez nesses casos. A pesquisa de alterações moleculares em uma amostra de pacientes brasileiros com perdas auditivas hereditárias, mediante a aplicação de tecnologias de nova geração, possibilitou a identificação de mutações em 47,4% dos casos. O OTO-NGS-Panel é eficiente para detectar a etiologia genética em um grupo heterogêneo de pacientes. Além de permitir a identificação de mutações novas, essa abordagem nos possibilitou reportar o envolvimento de genes nunca antes investigados na população brasileira. Já o OTO-CGH array não contribuiu para a elucidação da causa da surdez nos casos analisados. Este trabalho foi importante para introduzir informações a respeito da epidemiologia genética da perda auditiva no Brasil e também para adicionar novos dados referentes à diversidade de mutações que ocorrem nos genes relacionados à surdezAbstract: Hearing loss is one of the most common sensory disorders in humans. The identification of genetic variants associated with deafness can contribute to a better understanding of the molecular basis and the pathophysiological mechanisms involved in the different phenotypes of hereditary hearing loss. Moreover, it can provide an accurate diagnosis, development of specific treatments and genetic counseling for patients and families. However, molecular diagnosis has been a challenge, mainly due to clinical and genetic heterogeneity of hearing loss. To date, more than 90 genes and 150 loci have been described for nonsyndromic hearing loss and at least 40 genes have been linked to the main types of syndromic hearing loss. The application of new high-throughput technologies allows an optimization of genetic diagnosis and a comprehensive investigation of hereditary hearing loss. Therefore, the main goal of the present study was to elucidate the genetic etiology in a cohort of Brazilian subjects with hearing loss associated with different inheritance patterns, using next-generation technologies (array-CGH and next-generation sequencing). We investigated unrelated sporadic or familial patients with bilateral sensorineural hearing loss, for whom environmental factors and the most frequent mutations associated with genetic hearing loss have been excluded. Nineteen patients were screened using the OTO-NGS-Panel, a targeted next-generation sequencing panel containing 71 genes already known to be involved in hereditary hearing loss, designed by the Genetic Department at the Hospital Ramón y Cajal, Madrid-Spain. Pathogenic variations were identified in nine out of 19 patients (47.4%). Twelve mutations in seven different genes were detected, highlighting seven mutations that are being described for the first time in the present work. The following genes were involved: CDH23 and OTOF, related to autosomal recessive hearing loss; DFNA5, ACTG1, COCH and CRYM, associated with autosomal dominant hearing loss; and PRPS1, responsible for X-linked hearing loss. Deletions and insertions were investigated in six patients with autosomal recessive deafness using array-based comparative genomic hybridization (array-CGH) technology. The OTO-CGH array, designed by the Genetic Department at the Hospital Ramón y Cajal, Madrid-Spain, which allows the analysis of loci and genes associated with autosomal recessive hearing loss, was employed. However, no pathogenic variations that could explain the etiology of hearing loss for these cases were found. Molecular investigation in a cohort of Brazilian patients with hereditary hearing loss using next-generation technologies allowed the identification of mutations in 47.4% of cases. Our study demonstrates the usefulness of the OTO-NGS-Panel for detecting the genetic etiology of hearing loss in a heterogeneous group of patients. In addition to the identification of novel mutations, this approach enabled us to report the involvement of genes that had never been investigated before in the Brazilian population. On the other hand, the OTO-CGH array has not been successful in elucidating the cause of deafness in the cases analyzed. This work was important to introduce information to the genetic epidemiology of hearing loss in Brazil and also to add new data to the diversity of mutations occurring in genes related to deafnessDoutoradoGenetica Animal e EvoluçãoDoutora em Genética e Biologia Molecular229880/2013-4CAPESCNP

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-

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections

An open source plant kinase chemogenomics set

One hundred twenty-nine protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for expression in Escherichia coli. Forty of these rice kinases were purified and screened using differential scanning fluorimetry (DSF) against 627 diverse kinase inhibitors, with a range of structures and activities targeting diverse human kinases. Thirty-seven active compounds were then tested for their ability to modify primary root development in Arabidopsis. Of these, 14 compounds caused a significant reduction of primary root length compared with control plants. Two of these inhibitory compounds bind to the predicted orthologue of Arabidopsis PSKR1, one of two receptors for PSK, a small sulfated peptide that positively controls root development. The reduced root length phenotype could not be rescued by the exogenous addition of the PSK peptide, suggesting that chemical treatment may inhibit both PSKR1 and its closely related receptor PSKR2. Six of the compounds acting as root growth inhibitors in Arabidopsis conferred the same effect in rice. Compound RAF265 (CHIR-265), previously shown to bind the human kinase BRAF (B-Raf proto-oncogene, serine/threonine kinase), also binds to nine highly conserved rice kinases tested. The binding of human and rice kinases to the same compound suggests that human kinase inhibitor sets will be useful for dissecting the function of plant kinases

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934 (CAMKK2 IC50 = 3 nM), a total of 32 compounds, composed of single ring, 5,6-, and 6,6-fused heteroaromatic cores were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. These compounds were evaluated in vitro in biochemical and cellular assays for CAMKK2 inhibition. Compared to GSK650394 and STO-609, thirteen of our compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had greatly improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs aimed at the identification of CAMKK2 chemical probes and clinical candidates</div