ABSTRACT
AbstractNonsyndromic oral clefts are considered a problem of public health in Brazil, presenting a multifactorial etiology that involves genetic and environmental components, such as maternal alcohol consumption. Several candidate genes have been investigated to identify some association with nonsyndromic clefts risk. The epidermal growth factor (EGF) gene is implicated in the normal craniofacial development and its functional +61 A>G polymorphism has been related to cancer susceptibility. It has been suggested that cancer and oral clefts may share the same molecular pathways.Objective Our goal was to evaluate the association between the EGF+61 A>G polymorphism and nonsyndromic oral clefts susceptibility.Material and Methods The case-control study included 218 cleft cases and 253 controls from Brazil. The control group was comprised of individuals without congenital malformations, dental anomalies and family history of clefts. The cleft phenotypes and subphenotypes were determined based on clinical examination. Genomic DNA was extracted from oral mucosa cells obtained by mouthwash. The EGF+61 A>G polymorphism genotype was determined by polymerase chain reaction-restriction fragment length polymorphism.Results We noticed the association between maternal alcohol consumption during pregnancy and cleft occurrence. The A allele and AA genotype were over-represented in cleft cases compared with control group when we considered the bilateral cleft lip with or without cleft palate (CL±P) cases, cleft cases with tooth agenesis and cleft cases presenting family history of cleft, but the differences were not statistically significant. Contradictorily, the G allele was higher in cleft palate only (CP) cases than in control group, showing a borderline p value. Comparing the different cleft phenotypes, we observed statistical differences between CP and CL±P cases. Our data suggest the EGF+61 A>G polymorphism was not related with nonsyndromic oral clefts susceptibility in a Brazilian population, but supported the different genetic background between CL±P and CP. Moreover, we confirmed the potential effect of maternal alcohol intake on cleft risk in our population.
Subject(s)
Humans , Male , Female , Pregnancy , Child , Adolescent , Adult , Young Adult , Cleft Lip/genetics , Cleft Palate/genetics , Epidermal Growth Factor/genetics , Genetic Association Studies , Polymorphism, Restriction Fragment Length , Alcohol Drinking/adverse effects , Brazil , Case-Control Studies , Gene Frequency , Genotype , Polymerase Chain Reaction , Risk Factors , Sex Factors , Smoking/adverse effectsABSTRACT
Introduction: The tumor protein p53 gene (TP53) is a constant target of investigation in cancer pathogenesis. Analysis by immunohistochemistry provides limited data about p53 in oral carcinogenesis, and TP53 sequencing can contribute to this analysis. However, obtaining high-quality and contamination-free deoxyribonucleic acid (DNA) for a proper amplification can be a difficult task when using paraffin-embedded tissues. Objective: Standardize DNA extraction, polymerase chain reaction (PCR) amplification and DNA sequencing techniques for TP53 mutation analysis. Material and methods: Thirty-nine cases of oral squamous cell carcinoma (OSCC) were selected from the Pathology Division of Instituto Nacional de Câncer (Inca). The DNA extraction method used was the QIAamp® DNA minikit® system. After DNA quantification by spectrophotometry, 250 ng of genetic material obtained from TP53 gene were amplified by PCR for exon 2 and by nested PCR for exon 6. Out of the total sample, 11 cases were selected for exon 2 sequencing. Results: The DNA samples presented mean concentration of 119.74 ± 88.86 ng/µl (28.9-556.4) and purity of 1.69 ± 0.18 (1-1.9). Thirty-three (84.6%) samples were amplified for exon 2, and all samples for exon 6 (39/100%). Readable sequencing data were obtained in 10 (90.9%) cases. Conclusion: Optimization of conditions for TP53 sequencing was obtained, and this will facilitate the analysis of mutations in paraffin-embedded tissues, allowing molecular retrospective studies...
Introdução: O gene TP53 (proteína tumoral p53) é alvo constante de investigação na patogênese do câncer. A imuno-histoquímica fornece dados limitados na análise de p53 no processo da carcinogênese bucal e o sequenciamento de TP53 pode contribuir nessa investigação. Contudo, a obtenção de ácido desoxirribonucleico (DNA) com qualidade para amplificação e livre de contaminação pode constituir uma tarefa difícil na utilização de material parafinado. Objetivo: Padronizar as técnicas de extração de DNA, amplificação por reação em cadeia da polimerase (PCR) e sequenciamento para a análise de mutações em TP53. Material e métodos: Foram selecionados 39 casos de carcinomas de células escamosas bucal da Divisão de Patologia do Instituto Nacional de Câncer (Inca). O DNA foi extraído utilizando o sistema comercial QIAamp® DNA minikit®. Após quantificação do DNA por espectrofotometria, 250 ng de amostra foram amplificados pela técnica de PCR para o éxon 2 e por nested PCR para o éxon 6 do gene TP53. Da amostra total, 11 casos foram selecionados para a padronização da reação de sequenciamento do éxon 2. Resultados: As amostras de DNA apresentaram concentração média de 119,74 ng/µl ± 88,86 (28,9-556,4 ng/µl) e pureza de 1,69 ± 0,18 (1-1,9). Do total das amostras analisadas, 33 (84,6%) foram amplificadas para o éxon 2, e todas (39/100%), para o éxon 6. No sequenciamento do éxon 2 obtiveram-se sequências passíveis de leitura em 10 (90,9%) casos. Conclusão: A otimização das condições para o sequenciamento de TP53 foi obtida, o que facilitará a análise de mutações em tecidos parafinados, permitindo...
Subject(s)
Humans , Sequence Analysis, DNA/methods , Carcinoma, Squamous Cell/genetics , /genetics , Mutation/genetics , Paraffin Embedding , Mouth Neoplasms/genetics , Polymerase Chain ReactionABSTRACT
As tonsilites recorrentes têm sido objeto de muitos estudos. Eventos considerados na predisposição e causa incluem a utilização errônea de antibióticos em crises agudas, alterações da microflora, mudanças estruturais nas criptas epiteliais tonsilares e infecções virais. A infecção pelo vírus Epstein-Barr (EBV) ocorre freqüentemente na infância persistindo em linfócitos de tonsilas, podendo causar tonsilites recorrentes. Pouco se conhece sobre a persistência e reativação do EBV em pacientes imunocompetentes. Alguns métodos como a hibridização in situ, a reação em cadeia da polimerase (PCR) e a imuno-histoquímica têm sido utilizados no estudo da patogenia do vírus. OBJETIVO: Para caracterizar a associação do vírus Epstein-Barr com tonsilites recorrentes examinamos a presença do EBV pela PCR e por imuno-histoquímica usando como alvo a proteína viral LMP-1. FORMA DE ESTUDO: Estudo transversal com análise de prevalência amostral. MATERIAL E MÉTODOS: Foram selecionados 24 blocos parafinados de tonsilas, provenientes do Serviço de Anatomia Patológica, removidas de crianças de 2 a 12 anos com diagnóstico de tonsilite recorrente. Resultados: O genoma do EBV foi detectado em 13 (54,1%) e a LMP-1 em 9 (37,5%) dos casos. CONCLUSÃO: As tonsilas das crianças podem ser colonizadas pelo EBV e este pode estar associado à patogenia das tonsilites recorrentes.
Recurrent tonsillitis has been the subject of frequent investigation. Misuse of antibiotic therapy in acute tonsillitis, changes to the tonsillar microflora, structural changes to the tonsillar crypts, and viral infections have been listed as predisposing or causal factors for recurrent tonsillitis. Epstein-Barr virus (EBV) infection usually occurs in early childhood and may persist in tonsillar lymphocytes, thus leading to the onset of recurrent tonsillitis. Little is known about the persistence and reactivation of EBV strains in immunocompetent patients. Methods such as in situ hybridization, polymerase chain reaction (PCR), and immunochemistry have been used to study the pathogenesis of the EBV. AIM: this study aims to characterize the association between EBV and recurrent tonsillitis by investigating the presence of EBV through PCR and immunohistochemistry, using viral protein LMP-1 as a target. STUDY DESIGN: this is a cross-sectional study with analysis of sample prevalence. MATERIALS AND METHOD: twenty-four paraffin-embedded tonsil specimens from the Pathology Service were selected. The specimens were removed from children aged between 2 and 12 years diagnosed with recurrent tonsillitis. RESULTS: EBV genome was detected in 13 (54.1%) specimens, whereas viral protein LMP-1 was found in 9 (37.5%) specimens. CONCLUSION: children's tonsils can be colonized by EBV and such colonies may be associated with the pathogenesis of recurrent tonsillitis.