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Orientador: Solange Bento Farah Dissertação (mestrado) - Universidade Estadual de Campinas. Instituto de Biologia Mestrado Genética Humana Mestre em Ciências Biológicas

Orientador: Solange Bento Farah Kooke Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Doutorado Genética Doutor em Ciências Biológicas

Purpose Leber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m.G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan® OpenArray™ Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON. Methods The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan® OpenArray™ Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing. Results The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m.G11778A mutation, and seven had the m.T14484C mutation. Conclusions The TaqMan® OpenArray™ Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h and specific TaqMan probes are used.

Purpose Leber hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by bilateral vision loss. More than 95% of LHON cases are associated with one of the three main mtDNA mutations: G11778A, T14484C, and G3460A. The other 5% of cases are due to other rare mutations related to the disease. The aim of this study was to identify the prevalence and spectrum of LHON mtDNA mutations, including the haplogroup, in a cohort of Brazilian patients with optic neuropathy and to evaluate the usefulness of iPLEX Gold/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in detecting LHON mutations. Methods We analyzed a total of 101 patients; 67 had a clinical diagnosis of LHON and 34 had optic neuropathy of unknown etiology. Direct sequencing and iPLEX Gold/MALDI-TOF MS were used to screen for the most common pathogenic point mutations in LHON, together with the rare mutations G3733A, C4171A, T10663C, G14459A, C14482G, A14495G, C14568T, and C14482A. Results We identified mutations in 36 patients, of whom 83.3% carried the G11778A mutation and 16.7% carried the T14484C mutation. In individuals with mutations, the haplogroups found were L1/L2, L3, C, R, U, D, and H. Rare mutations were not detected in any of the patients analyzed. Conclusions The frequencies of the main LHON mutations were similar to those previously reported for Latin America. A different frequency was found only for the A3460G mutation. The most frequent haplogroups identified were of African origin. The iPLEX Gold/MALDI-TOF MS technology proved to be highly accurate and efficient for screening mutations and identifying the haplogroups related to LHON. The MassArray platform, combined with other techniques, enabled definitive diagnosis of LHON in 36% (36/101) of the cases studied.

Aim: To identify the occurrence of 35delG mutation and of environmental factors related to hearing loss in patients with cochlear implant, determining the functional gain post-implant. Methods: It was a cross-sectional study conducted with six volunteers, aged from five to twenty-one years old. The studied variables consisted on the identification of risk factors for hearing loss in prenatal, perinatal and postnatal periods. For genetic investigation, the occurrence of 35delG mutation in the connexin 26 gene was studied. The functional gain due to the implant was evaluated with “pure tone” stimulus in modulated frequencies of 250Hz, 500Hz, 1000Hz, 2000Hz, 3000Hz and 4000Hz. Results: In the study of environmental factors, we evidenced reports of TORCH infections (Toxoplasmosis, Rubella, Cytomegalovirus, Herpes and Syphilis), maternal hypertension, abortive usage, Meningitis, Measles, oxygen therapy and Jaundice. The genetic investigation revealed two cases of 35delG mutation, one in homozygosis and other in heterozygosis. The functional gain due to the cochlear implant ranged from 60 to 85dBHL. Conclusion: In the studied sample, both the occurrences of 35delG mutation and of environmental factors related to the hearing loss were evident. The values of functional gain were satisfactory, regardless from the etiology related to the loss. Objetivo: Identificar a ocorrência da mutação 35delG e de fatores ambientais relacionados à perda auditiva em pacientes usuários de implante coclear, determinando o ganho funcional auditivo após o implante. Métodos: Tratou-se de um estudo transversal realizado em seis voluntários, com idades variando de cinco a vinte e um anos. As variáveis estudadas consistiram da identificação de fatores de risco para a perda auditiva no período pré-natal, peri-natal e pós-natal. Para o estudo genético, pesquisou-se a ocorrência da mutação 35delG no gene da conexina 26. O ganho funcional devido ao implante foi avaliado com o estímulo “tom puro”, em frequências moduladas de 250Hz, 500Hz, 1000Hz, 2000Hz, 3000Hz e 4000Hz. Resultados: Na pesquisa de fatores ambientais, evidenciaram-se relatos de infecções do grupo TORCH (Toxoplamose, Rubéola, Citomegalovírus, Herpes e Sífilis), hipertensão materna, uso de abortivos, Meningite, Sarampo, oxigenoterapia e Icterícia. O estudo genético revelou dois casos de mutação 35delG, um em homozigose e outro em heterozigose. O ganho funcional do implante coclear variou de 60 a 85dBNA. Conclusão: Na amostra estudada, evidenciaram-se as ocorrências da mutação 35delG e de fatores ambientais relacionados à deficiência auditiva. As medidas de ganho funcional foram satisfatórias, independente da etiologia relacionada à perda.

Purpose There are many similarities in the clinical presentation of Leber hereditary optic neuropathy (LHON) and in patients who have optic neuropathy and a history of heavy tobacco and alcohol consumption. The main objective of this study is to investigate the frequency of primary and secondary mitochondrial DNA (mtDNA) mutations for LHON in patients diagnosed as having alcohol and tobacco optic neuropathy (ATON). Methods Twenty-six patients who had a history of heavy alcohol and tobacco consumption and who developed bilateral optic neuropathy were tested for primary mutations (G11778A, T14484C, and G3460A) by restriction analysis, and 14 secondary mutations in the genes mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), mitochondrially encoded NADH dehydrogenase 5 (MT-ND5), mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), and mitochondrially encoded cytochrome B (MT-CYB) by direct sequencing. Results Four (15.4%) of 26 patients tested positive for LHON primary mutations, two for the G11778A mutation, and two for the T14484C mutation. No patient tested positive for any of the 14 secondary mutations. Familial recurrence was present in four patients, and only three of these patients have presented the LHON mutation. Conclusions The diagnosis of LHON should be considered in all patients diagnosed as having optic neuropathy, particularly those with familial recurrence of vision loss.

OBJETIVOS: Calcular a incidência da perda auditiva sensorioneural (PASN) em recém-nascidos de alto risco, verificar se existe associação causal entre o uso de fármacos ototóxicos em neonatos de alto risco e a PASN e estabelecer a frequência das mutações genéticas relacionadas à PASN em neonatos de alto risco. MÉTODOS: A pesquisa foi uma coorte retrospectiva e prospectiva realizada em 250 crianças. Foi realizada coleta de dados em prontuários e com os responsáveis, triagem auditiva por emissões otoacústicas-produto de distorção, timpanometria, audiometria com reforço visual, potencial evocado auditivo de tronco encefálico e emissões otoacústicas transientes. A pesquisa das mutações genéticas 35delG e mitocondriais A1555G e A7445G, foi fundamental para avaliar a possibilidade da PASN ser de origem genética não-sindrômica. RESULTADOS: A incidência da PASN foi de 11,6%, as associações causais entre a PASN e os fármacos administrados foram: amicacina e cefotaxima (OR 5,35), cefotaxima e furosemida (OR 7,02), ceftazidima e vancomicina (OR 9,12). A frequência da mutação 35delG foi de 0,8% e para as mutações mitocondriais A1555G e A7445G foi 0%. CONCLUSÃO: A incidência de PASN em recém-nascidos de alto risco foi alta, apresentando importante relação causal com o uso de medicamentos e pequena relação com mutações genéticas. PURPOSE: To calculate the incidence of sensorineural hearing loss (SNHL), to verify if there is a causal association between the use of ototoxic drugs and SNHL, and to establish the frequency of genetic mutations related to SNHL in high risk newborns. METHODS: The study was a retrospective and prospective cohort research with 250 children. Data was gathered from subjects' charts and with their caregivers. Moreover, subjects were submitted to auditory evaluation with distortion product otoacoustic emissions, timpanometry, visual reinforcement audiometry, auditory brainstem response and transient otoacoustic emissions. The study of the genetic mutation 35delG, and the mitochondrial mutations A1555G and A7445G was essential to evaluate the possibility that SNHL had a non-syndromic genetic origin. The association between the medicine use and the occurrence of hearing loss had been analyzed. RESULTS: The incidence of SNHL in high risk newborns was 11.6%, and causal associations between SNHL and the drugs administered were: amikacin and cefotaxime (OR=5.35), cefotaxime and furosemide (OR=7.02), ceftazidime and vancomycin (OR=9.12). The frequencies of the mutation 35deIG and mitochondrial mutations A1555G and A7445G were, respectively, 0.8% and 0%. CONCLUSION: The incidence of SNHL in high risk newborns was high, showing an important causal relation with the use of ototoxic drugs and a small relation with genetic mutations.