Orientador: Edi Lúcia Sartorato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T05:36:29Z (GMT). No. of bitstreams: 1 Martins_FabioTadeuArrojo_M.pdf: 6195877 bytes, checksum: 88704a1b2448dcb2851e78b62c37e6b9 (MD5) Previous issue date: 2013 / Resumo: 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 país / Abstract: 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 country / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), characterized by lesions formed due to demyelination. MS is a complex disease thought to be triggered by environmental factors in genetically predisposed individuals. The strongest associated susceptibility allele is HLA-DRB1*1501. Environmental factors include smoking, latitude and previous infection of Epstein-Barr virus (EBV), a common herpes virus. There is no cure for MS, but several inhibitor and symptomatic drugs. Tysabri® (natalizumab) is the most effective drug, but it may lead to progressive multifocal leukoencephalopathy (PML), a rare but often fatal disease caused by reactivation of JC virus. The aim of this thesis was to replicate previous findings from a genome-wide association study and to find host genetic factors influencing JCV seropositivity and EBNA1 IgG titers in Swedish MS patients and healthy controls. Samples from the EIMS and IMSE studies were genotyped by TaqMan® OpenArray™ PCR, an end-point SNP genotyping analysis. 1143 cases and 556 healthy controls were genotyped. Due to poor call rates, genotype data from an Immunochip study was added. A total of 3408 samples (1664 cases and 1744 controls) were analyzed. EBNA1 IgG antibodies were previously measured as a detection of EBV infection and increased MS risk, and JCV IgG antibodies were measured to find patients potentially at risk for PML. One significant result was found, gene 105 (p = 0.01674, OR 0.68, CI 95% 0.49-0.93), with a protective effect in MS. More significant results might have been found with better loading of the plate, or with a different genotyping method.

The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.