Study of dyslexia candidates genes in brazilian individuals

Orientador: Edi Lúcia SartoratoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A dislexia é definida como um distúrbio, ou transtorno de aprendizagem na área da leitura, escrita e soletração, que ocorre apesar de uma adequada inteligência e oportunidade sociocultural. A etiologia desse distúrbio se deve, em parte, a um importante componente genético. Ao todo, nove loci no genoma foram identificados por meio de estudos de ligação, e sete genes proeminentes foram propostos como candidatos à dislexia: DYX1C1, KIAA0319, DCDC2, ROBO1, MRPL19, C2ORF3 e KIAA0319L, mas nenhuma mutação funcional nesses genes foi efetivamente relacionada com a causa do distúrbio até o momento. O objetivo deste estudo foi verificar a relação de dados moleculares com a manifestação do distúrbio em 49 famílias de escolares brasileiros com diagnóstico de dislexia. Para isso, foi investigada a presença de grandes deleções e duplicações em algumas regiões dos genes candidatos DCDC2, KIAA0319 e ROBO1 pela técnica de Multiplex Ligation-dependent Probe Amplification (MLPA), utilizando o kit SALSA MLPA P150 (MRC-Holland, Amsterdam, The Netherlands). E além disso, foi realizado um estudo de associação utilizando 4 SNPs (Single Nucleotyde polymorphisms) presentes no gene DYX1C1, que já haviam sido relacionados com o fenótipo na literatura. A técnica de MLPA foi aplicada pela primeira vez na pesquisa de mutações em genes candidatos para a dislexia, este método foi reprodutível e o padrão de variação total por sonda foi baixo, porém a análise não revelou nenhuma deleção ou duplicação nas regiões de ligação das sondas nos genes estudados. Algumas modificações no kit de dislexia P150 foram sugeridas ao fabricante visando o aprimoramento para estudos futuros. Na etapa seguinte, a genotipagem dos SNPs foi realizada por PCR em tempo real, e a estratégia utilizada no estudo de associação foi o Teste de Transmissão de Desequilíbrio de Ligação (TDT). Nenhuma associação foi obtida para os marcadores estudados. As aparentes discrepâncias de nossos resultados com estudos anteriores podem ser explicados pelas diferenças na definição do fenótipo, a ancestralidade da amostra, o desenho do estudo, e as interações com efeitos ambientais que diferem entre populações. Esse resultado não descarta a participação do gene DYX1C1 na etiologia da dislexia, o aumento do número da amostra e de marcadores para estudos posteriores é fundamental para que se possa fazer uma análise mais completa do envolvimento desse gene no fenótipo, o que poderá fornecer importantes informações para o entendimento da dislexia e para futuros protocolos de diagnósticos e de conduta para os indivíduos afetadosAbstract: Dyslexia or reading disability is a learning disorder associated with difficulty in learning to read, writing and spelling, despite adequate intelligence and educational opportunities, with a significant heritable trait. At least nine loci in the genome were related through linkage studies, and seven prominent genes were associated with dyslexia: DYX1C1, KIAA0319, DCDC2, ROBO1, MRPL19, C2ORF3 and KIAA0319L but no functional mutation in these genes was indeed related with the disorder cause so far. The intent of this study was access the contribution of these genes in the learning disorder molecular etiology, in a sample 49 families of dyslexic Brazilian individuals. Large deletions and duplications in the candidate genes DCDC2, KIAA0319 and ROBO1 were investigated by Multiplex Ligation-dependent Probe Amplification (MLPA) technique, using the SALSA MLPA P150 kit (MRC-Holland, Amsterdam, The Netherlands). In addition, an association study was performed using 4 SNPs (Single Nucleotyde polymorphisms) in DYX1C1 gene, which had already related to the phenotype in the literature. The MLPA technique was applied for the first time in the search for candidate genes mutations in dyslexia, this method was reproducible and the overall standard variation per probe was low, but the analysis revealed no deletion or duplication in probes binding regions in the studied genes. Some modifications in the SALSA MLPA P150 kit have been suggested to the manufacturer attempting to improve it for future studies. In the next stage, SNPs genotyping was performed by real time PCR, and the strategy used in the association study was the Transmission Disequilibrium Test (TDT). No association was obtained for the markers. The apparent discrepancies of our results with previous studies can be explained by phenotype definition differences, the sample ancestry, study design, and interactions with environmental effects that differ between populations. This result does not rule out the involvement of DYX1C1 gene in the dyslexia etiology, the increase of the sample and markers numbers for future studies is essential to make a more complete analysis of this gene involvement in phenotype, which may provide important information to the dyslexia understanding and future diagnostic protocols and conduct for affected individualsMestradoGenetica Animal e EvoluçãoMestre em Genética e Biologia Molecula

Association between phenotype, performance with hearing aids, and genotype of childhood hearing loss in children with and without genetic alteration

PURPOSE: To establish the frequency of genetic mutations related to sensorineural hearing loss (SNHL); to verify if there is association between the degree of SNHL and the presence of genetic alteration; and to verify if the Minimal Response Levels (MRL) with hearing aids vary according to the genetic alteration. METHODS: Thirty hearing aids users with ages between 8 and 111 months were evaluated. The evaluation procedures used were: pure-tone audiometry; the auditory steady state response (ASSR) on sound field, with and without hearing aids; and genetic study of the hearing loss. RESULTS: Three genetic mutations were diagnosed: 35delG, A1555G and A827G, and the children with these mutations showed higher degree of SNHL. There was no difference between the genetic patterns regarding the degree of SNHL, except for patients with A827G mitochondrial mutation, because all subjects with this mutation had profound SNHL. The difference between the MRL obtained with and without amplification, considering the presence of mutation and the degree of SNHL, was higher in children with moderate SNHL without genetic alterations, both in behavioral and electrophysiological evaluations. CONCLUSION: Genetic mutations were found in 36.7% of the sample, justifying the importance of genetic tracking in the hearing habilitation process. Children with genetic mutations showed higher degrees of hearing loss. The different mutation patterns do not directly determine the degree of hearing loss. The best thresholds with amplification were found in children with moderate hearing loss without genetic alterations.OBJETIVO: Estabelecer a frequência de mutações genéticas relacionadas à deficiência auditiva neurossensorial (DANS); verificar se há associação entre grau da DANS e presença de alteração genética e verificar se os Níveis Mínimos de Resposta (NMR) com próteses auditivas variam em função da alteração genética. MÉTODOS: Foram avaliadas 30 crianças, com idades entre 8 e 111 meses, usuárias de próteses auditivas. Os procedimentos de avaliação utilizados foram: audiometria tonal e resposta auditiva de estado estável (RAEE) em campo livre, com e sem as próteses auditivas e estudo genético da DANS. RESULTADOS: Foram diagnosticadas três mutações genéticas: 35delG, A1555G e A827G, sendo que as crianças com tais mutações apresentaram maior grau de DANS. Não houve diferença entre os padrões genéticos em relação ao grau de DANS, com exceção dos pacientes com mutação mitocondrial A827G, pois todos com essa mutação eram portadores de DANS de grau profundo. A diferença entre os NMR obtidos sem e com o uso da amplificação, considerando a presença de mutação e grau de DANS, foi maior nas crianças portadoras de DANS de grau moderado sem alteração genética, tanto na avaliação comportamental quanto na eletrofisiológica. CONCLUSÃO: As mutações genéticas foram encontradas em 36,7% da amostra, o que justifica a importância do rastreamento genético no processo de habilitação auditiva. Crianças com mutações genéticas apresentam o maior grau de DANS. Os diferentes padrões de mutações não determinam diretamente o grau da DANS. Os melhores limiares com o uso da amplificação foram encontrados nas crianças com DANS moderada, sem alteração genética.Universidade Federal de Santa Maria Departamento de FonoaudiologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de FonoaudiologiaUniversidade Estadual de Campinas Laboratório de Genética HumanaUNIFESP, Depto. de FonoaudiologiaSciEL

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

Development of a diagnostic panel for simultaneous screening of the main mutations related to hearing loss

Orientador: Edi Lúcia SartoratoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A perda da audição é uma doença sensorial comum, que pode ser causada por fatores genéticos ou ambientais. Os recentes avanços na biologia molecular têm permitido a determinação das causas genéticas da perda auditiva, e mais de 100 genes já foram relacionados com este fenótipo. Devido à elevada heterogeneidade genética envolvida, um grande número de pacientes permanece sem diagnóstico molecular, pois apenas um pequeno conjunto de genes é geralmente analisado. Isso indica a necessidade de novas estratégias metodológicas para o rastreamento simultâneo de mutações em múltiplos genes. Neste trabalho, um painel para genotipagem utilizando o sistema de espectrometria de massa iPLEX MassARRAY® (Sequenom Inc.), contendo 86 alterações em 17 genes, foi otimizado e testado, visando sua aplicação no diagnóstico molecular de indivíduos com perda auditiva. Inicialmente, um teste com indivíduos controles mostrou falha em 20% das alterações. Na tentativa de se obter uma maior taxa de genotipagem, os primers das alterações que falharam foram redesenhados e ressintetizados. Novos testes com 25 indivíduos controles mostraram um significativo aumento na taxa de genotipagem, e a especificidade e sensibilidade da técnica foram estimadas em mais de 95%. Após a otimização dos ensaios, um grupo de 180 indivíduos, com perda auditiva não-sindrômica com causa presumidamente genética, foi rastreado utilizando o painel desenvolvido, e a validação das mutações identificadas foi realizada por sequenciamento de DNA ou PCR seguido de análise de restrição enzimática. Alterações genéticas foram confirmadas em 31% dos indivíduos, sendo que em 20,6% dos casos analisados foi possível esclarecer a etiologia da perda auditiva. Alterações no gene GJB2 foram as mais prevalentes, porém outras mutações também foram identificadas nos genes SLC26A4, CDH23, MT-RNR1, MT-TS1, MYO15A e OTOF. A genotipagem de mutações usando o sistema iPLEX MassARRAY® mostrou melhor custo-benefício em comparação com as técnicas convencionais, e permitiu rastrear um conjunto mais abrangente de genes do que aqueles atualmente analisados. Portanto, o painel desenvolvido é viável para o rastreamento inicial de mutações envolvidas com a perda auditiva não-sindrômica e poderá auxiliar no diagnóstico dos pacientes afetadosAbstract: Hearing loss (HL) is a common sensorial disorder, which can be caused by genetic or environmental factors. Recent advances in molecular biology have allowed the determination of the genetic causes of HL, and more than 100 genes have been linked to the phenotype. Due to the extraordinary genetic heterogeneity involved in HL a large percentage of patients remain without any molecular diagnosis. This indicates the need for new methodological strategies for simultaneously screening for mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes, screened using the mass spectrometry system, iPLEX MassARRAY® (Sequenom Inc.), in order to determine the molecular etiology of hearing loss. An initial genotyping of control subjects, showed failures in 20% of the selected alterations. In an attempt to achieve greater genotyping rates, the tests that failed were redesigned and new primers were synthesized. Further tests with 25 control subjects were then performed and showed significant increase in genotyping rate. The sensitivity and specificity of the technique showed average values above 95%. Additionally, a group of 180 individuals, with nonsyndromic hearing loss with presumably genetic causes, was tested using the developed panel, and the validation of the identified mutations was performed by DNA sequencing or PCR followed by restriction enzyme analysis. Genetic alterations were confirmed in 31% of patients, and in 20.6% of the individuals was possible to elucidate the etiology of HL. Mutations in the GJB2 gene were the most prevalent, but other mutations in the SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes were also identified. Genotyping of mutations using iPLEX MassARRAY® Spectrometry System proved to be faster and more cost-effective, compared to conventional techniques, and enabled the screening of a broader set of genes and mutations than those currently analyzed to establish the molecular etiology of hearing loss. Hence, the developed panel is feasible for the initial screening mutations involved in non-syndromic hearing loss and may aid in the diagnosis of affected patientsDoutoradoGenetica Animal e EvoluçãoDoutora 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-

Karyotypic variability in Iheringichthys labrosus (Teleostei, Pimelodidae) from the Tibagi River basin (Paraná State, Brazil)

Cytogenetic analyses were carried out in a populational sample of Iheringichthys labrosus from the Guaraúna River (Upper Tibagi River; Paraná State, Brazil) in order to provide a karyotypic comparison with another previously studied population from the Lower Tibagi River, characterized by the presence of 32m + 8sm + 6st + 10a (2n = 56, FN = 102) and occurrence of supernumerary chromosomes (80% of individuals). The 17 specimens of I. labrosus (6 females, 10 males and 1 of unknown sex) from the Upper Tibagi River showed 2n = 56 chromosomes, a karyotype formula of 14m + 32sm + 4st + 6a (FN = 106), without evidence of sex chromosome heteromorphism or supernumerary chromosomes. The heterochromatin was detected at telomeric and centromeric positions in several chromosomal pairs. The Ag-nucleolar organizer regions were heteromorphic and located at terminal position on short arms of the 16th chromosomal pair, suggesting a positive association with heterochromatic regions. The inter-populational karyotypic differentiation reported indicates distinct evolutionary pathways within I. labrosus in the Tibagi River basin. ©FUNPEC-RP

Novel mutations associated with pyruvate kinase deficiency in Brazil

Background: Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype–phenotype correlations. Method: Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A). Results: Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. Conclusion: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants. Keywords: Red cell disorder, Pyruvate kinase, Mutation, Hemolytic anemia, PKLR gen