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

Optimization simultaeous screening of the main mutations involved in non-syndromic deafness using TaqMan 'TRADEMARK' OpenArray 'TRADE MARK' Genotyping

Orientador: Edi Lúcia SartoratoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A perda auditiva é a deficiência sensorial mais frequente em humanos, atingindo aproximadamente 10% de toda a população mundial. A restrição da comunicação pela expressão oral resulta em alterações no desenvolvimento cognitivo e psicológico do indivíduo afetado. Em países desenvolvidos, um a cada 500 indivíduos apresenta perda auditiva neurossensorial bilateral profunda/severa. Já nos casos de indivíduos com até 5 anos, a porcentagem é maior, atingindo 0,27% de 1000 indivíduos, número que se torna maior ainda nos casos em jovens, chegando a 0,35%. Dentre as causas da perda auditiva, mais de 60% dos casos de perda auditiva congênita são genéticos. Até o momento já se tem conhecimento de 150 loci e 103 genes envolvidos com a perda auditiva, sendo que a maioria deles apresenta, no mínimo, 20 alterações (mutações de ponto, deleções, inserções, etc.) que podem causar a perda. O gene que apresenta maior número de alterações é o GJB2, codificador da conexina 26, uma proteína relacionada a trocas iônicas intercelulares, mantendo a homeostase de potássio do sistema auditivo, essencial para a audição. Apenas este gene apresenta mais de 302 alterações confirmadas até o presente momento, sendo o principal gene relacionado aos casos de perda auditiva de origem genética. Tendo em vista a grande heterogeneidade clínica e genética da perda auditiva e a importância do diagnóstico molecular correto dos indivíduos que apresentam perda auditiva hereditária, o presente trabalho propôs padronizar um layout para diagnóstico através de genotipagens utilizando uma tecnologia 'high-throughput' baseada em PCR (Polymerase Chain Reaction) em tempo real denominada TaqMan® OpenArrayTM Genotyping. Com esta, foi desenvolvido um layout das placas de genotipagem de OpenArrayTM, sendo possível analisar 32 alterações de 96 indivíduos simultâneamente por placa. Ao todo, foram analisados 376 indivíduos, sendo 94 deles controles ouvintes, totalizando 4 placas em duplicata. Todas as 31 alterações analisadas estavam presentes nos genes nucleares GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, OTOF e nos genes mitocondriais 12S rRNA e MT-TR1. As reações foram validadas posteriormente por técnicas previamente estabelecidas (sequenciamento direto, PCR Multiplex e RFLP-PCR) nos testes utilizados para o diagnótisco molecular da perda auditiva do Laboratório de Genética Humana do Centro de Biologia Molecular e Engenharia Genética (CBMEG) da Universidade Estadual de Campinas (UNICAMP). Ao total, foram realizadas 11.656 reações de genotipagem. Apenas 353 reações falharam, representando, aproximadamente, 3,03% das reações. Dentre as reações que falharam, estavam as amostras de nove indivíduos que não obedeciam aos requisitos mínimos de concentração, pureza e integridade do DNA para a realização dos experimentos. Com isso, calculou-se o rendimento médio das placas de genotipagem utilizando as placas de OpenArrayTM, que apresentou acurácia de, aproximadamente, 96,97%. Tais resultados comprovam a ótima acurácia, o baixo custo e a fácil reprodutibilidade da técnica, tornando este layout customizado para a plataforma TaqMan® OpenArrayTM Genotyping uma ferramenta ótima e confiável a ser empregada nos teste de diagnóstico molecular da perda auditiva no nosso paísAbstract: Hearing loss is the most common sensory deficit in humans, affecting approximately 10% of the entire world population. The restriction of communication by the oral expression results in changes in cognitive and psychological development of the affected individual. In developed countries, one in every 500 individuals has severe/profound bilateral sensorineural hearing loss. In cases of individuals with up to 5 years, the percentage is higher, reaching 0.27% of 1000 individuals, that number becomes even greater in cases where young people, reaching 0.35%. Among all the causes of hearing loss, more than 60% of congenital hearing loss is genetics. So far already aware of 150 loci and 103 genes involved in hearing loss, and most of them have at least 20 changes (point mutations, deletions, insertions, etc.) which may cause the loss. The gene that has the higher number of changes is the GJB2, encoding connexin 26, a protein related to ion exchange intercellular maintaining homeostasis potassium auditory system, essential for hearing. Only this gene has over 302 changes confirmed so far, being the main gene related to cases of hearing loss with genetic origin. Due to the great clinical and genetic heterogeneity of hearing loss and the importance of correct molecular diagnosis of individuals with hereditary hearing loss, this work proposes standardize a layout to the diagnosis by a genotyping technology using a high-throughput technique based on real-time PCR called TaqMan® OpenArrayTM Genotyping. With this, we customized a layout to the OpenArrayTM genotyping plates, being possible to analyze 32 changes of 96 individuals per plate simultaneously. Were analyzed 376 individuals, being 94 of them controls listeners, totaling 4 plates in duplicate. All 31 changes analyzed were present in the nuclear genes GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, OTOF and in the mitochondrial genes 12S rRNA and MT-TR1. Reactions were subsequently validated by previously established techniques (direct sequencing, multiplex PCR and RFLP-PCR), tests used for the molecular diagnostic of the hearing loss at Human Genetics Laboratory of the Center for Molecular Biology and Genetic Engineering (CBMEG), located at State University Campinas (UNICAMP). In total, 11.656 reactions of genotyping were performed using this platform. Only 353 reactions failed, representing approximately 3.03% of the reactions. Among the reactions that failed, were samples of nine individuals who did not meet the minimum concentration, purity and integrity of the DNA for the experiments. With this, was calculated the average income of the OpenArrayTM genotyping plates, which showed an accuracy of approximately 96.97%. These results and the comparative analysis of the costs among OpenArrayTM platform and the others molecular techniques demonstrated the great accuracy, low cost and easy reproducibility of the technique, making this layout customized for the platform TaqMan® OpenArrayTM Genotyping a good and reliable tool to be used in the molecular diagnostic of hearing loss in our countryMestradoGenetica Animal e EvoluçãoMestre em Genética e Biologia Molecula

Massive parallel sequencing for identification and characterization of genes related to hearing loss

Orientadores: Edi Lúcia Sartorato, Karen Bouskela AvrahamTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A perda auditiva é a deficiência sensorial mais frequente em humanos, afetando aproximadamente 5% da população mundial. Em países desenvolvidos, a cada mil indivíduos, um apresenta perda auditiva neurossensorial bilateral profunda para severa. Dentre as causas, 50-60% dos casos congênitos estão associados à fatores genéticos, distribuídos em mais de 100 genes envolvidos tanto com a perda auditiva sindrômica quanto não-sindrômica. Porém, estima-se que este número seja superior a 300 genes, o equivalente a 1% de todos os genes do genoma humano. Tendo conhecimento da heterogeneidade da perda auditiva, genética e clinicamente, assim como das limitações das tecnologias convencionais de diagnóstico molecular, novas estratégias tiveram de ser desenvolvidas para o melhor entendimento dos mecanismos relacionados ao processo da audição. Dentre tais tecnologias, o sequenciamento paralelo massivo mostrou-se uma ótima ferramenta, permitindo identificar e associar mais de 40 genes à perda auditiva em pouco menos de sete anos. Assim sendo, para este estudo envolvendo indivíduos afetados com perda auditiva, foram utilizadas tecnologias baseadas no sequenciamento paralelo massivo para a identificação e caracterização de variantes genéticas que pudessem esclarecer o fenótipo observado. Das 25 famílias estudadas, 20 eram provenientes do Oriente Médio e foram analisadas pelo painel de captura HEar-Seq v3, contendo 284 genes associados à perda auditiva em humanos e camundongos. As cinco famílias restantes, provenientes do Brasil, foram analisadas por meio do sequenciamento completo do exoma. A identificação de novos genes e variantes candidatas, relacionadas à perda auditiva nas famílias, foram analisadas funcionalmente, tendo seu efeito patogênico caracterizado por estudos in silico e in vitro. O uso do painel de captura e sequenciamento completo do exoma permitiu elucidar a causa molecular da perda auditiva em 12 dos 25 casos estudados, correspondendo a 48% dos casos. Variantes novas e conhecidas foram identificadas nos genes SLC26A4, USH2A, GATA3, OTOF, MYO6, STRC, CEACAM16, COL11A2 e MYH9, esclarecendo a etiologia da perda auditiva nas famílias estudadas. Além disso, este trabalho permitiu a associação de um novo gene, SLC12A6, com a perda auditiva em humanos. Tais resultados demonstraram o poder do sequenciamento paralelo massivo no estudo de desordens genéticas, principalmente em casos complexos e heterogêneos. Adicionalmente, as análises contribuíram para o diagnóstico conclusivo da perda auditiva, permitindo o aconselhamento genético e prognóstico adequado para estas famíliasAbstract: Hearing loss is the most common sensory deficit in humans, affecting approximately 5 % of the world population. In developed countries, one in every thousand individuals presents severe to profound bilateral sensorineural hearing loss. Of the congenital cases, 50-60 % are associated with genetic factors, which affect more than 100 genes involved in both syndromic and non-syndromic hearing loss. However, this number is estimated to be over 300 genes, i.e. 1 % of all genes in the human genome. Due to the genetic and clinical heterogeneity of hearing loss, as well as the limitations of conventional molecular diagnostic technologies, new strategies are constantly being developed for better understanding of the mechanisms of hearing. Among these, massive parallel sequencing has proven to be efficient, enabling the identification and association of more than 40 genes to hearing loss in seven years. In this study, massive parallel sequencing based technologies were used to identify and characterize genetic variants that could clarify the observed phenotype in individuals with hearing loss. Of the 25 families studied, 20 were from the Middle East. They were analyzed by the HEar-Seq v3 capture panel, containing 284 genes associated with hearing loss in humans and mice. The five remaining families were from Brazil and analyzed by complete exome sequencing. Novel candidate variants and their related genes causing the familial hearing loss were functionally analyzed and their pathogenic effect was characterized in silico and in vitro. With the use of the capture panel and complete exome sequencing, the molecular cause of the hearing loss in 12 of the 25 studied cases (48 % of the cases) was elucidated. Novel, as well as known variants, were identified in the SLC26A4, USH2A, GATA3, OTOF, MYO6, STRC, CEACAM16, COL11A2 and MYH9 genes, thereby clarifying the etiology of the hearing loss in the studied families. Moreover, this work identified a new gene, SLC12A6, as related to hearing loss in humans. These results demonstrate the power of massive parallel sequencing for studying genetic disorders, especially in complex and heterogeneous cases. Additionally, the analyzes aided a conclusive diagnosis of the hearing loss, which enabled adequate genetic counseling and prognosis for the affected familiesDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecular2013/05823-5, 2013/22538-2FAPES

A rare case of deafness and renal abnormalities in HDR syndrome caused by a de novo mutation in the GATA3 gene

Abstract HDR syndrome is a rare autosomal dominant disorder caused by mutations in the GATA3 gene and characterized by hypoparathyroidism, sensorineural deafness and renal abnormalities. Here we report a Brazilian family, from which the proband, his mother and his grandfather were diagnosed with bilateral sensorineural hearing loss. Molecular screening of the GJB2, GJB6 and MTRNR1 genes in the proband showed no alterations; however, whole exome sequencing detected a heterozygous mutation, c.1099C > T (p.Arg367*), in the GATA3 gene. Segregation analyses showed that the mother also had the mutation, but not the grandparents, hence indicating a different hearing impairment type for the grandfather. Paternity test of the mother of the proband confirmed that she has a de novo mutation. Furthermore, HDR syndrome was confirmed with new clinical evaluations showing right kidney agenesis in the proband. This is the first study reporting only deafness and renal abnormalities as symptoms of the p.Arg367* mutation in the GATA3 gene, and also the sixth HDR syndrome case in the world, and the first on the American continent. Together with other reported cases, this study highlights the variability of HDR syndrome symptoms in individuals with the p.Arg367* mutation, emphasizing the importance of molecular analyses for correct diagnosis