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Genotipagem do gene KRAS em indivíduos tatuados
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Advisor(s)
Abstract(s)
Introdução: Mutações na família RAS de proto-oncogenes, particularmente no gene KRAS, foram identificadas como fator-chave no desenvolvimento de vários tipos de cancro de pele, incluindo melanomas e carcinomas de células escamosas. A mutação mais comum ocorre no codão 12, alterando a funcionalidade da proteína GTPase, que está envolvida na transdução de sinais celulares. Também foram relatados casos de neoplasias
cutâneas em indivíduos tatuados, levantando questões sobre se a tinta, o trauma mecânico da agulha ou o fotoenvelhecimento da pele poderiam ser fatores desencadeantes. No entanto, apesar dessas observações, ainda há uma falta de evidências científicas robustas que relacionem diretamente as tatuagens a mutações genéticas específicas.
Objetivos: Verificar a presença no ADN de mutações no codão 12 do gene KRAS em estudantes tatuados da Egas Moniz School of Health and Science.
Metodologia: Realizou-se um estudo piloto experimental com 16 estudantes tatuados, selecionados com base nos seguintes critérios de inclusão: indivíduos saudáveis, sem doenças dérmicas, com tatuagens pretas e localizadas nos membros superiores, cada tatuagem tinha pelo menos dois centímetros de diâmetro e tinha sido feita há mais de dois anos. O ADN foi recolhido através da raspagem da pele com swab húmida e extraído
utilizando colunas de centrifugação revestidas com sílica (kit NZYtech). O gene KRAS foi amplificado utilizando Nested PCR. A análise da mutação consistiu na digestão do produto amplificado com a enzima de restrição BstNI, seguida de eletroforese em gel de agarose a 4%. A análise dos dados foi realizada com recurso a uma análise descritiva simples.
Resultados: Obteve-se 10 amostras viáveis de ADN com qualidade suficiente para interpretação molecular, correspondentes a 5 participantes. Quatro deles apresentaram um padrão de fragmentação correspondente ao genótipo selvagem (111 pb), indicando a ausência de mutações no codão 12 do gene KRAS. Uma amostra apresentou uma banda de 140 pb, o que é consistente com um genótipo mutante homozigótico. No entanto, os resultados também destacam limitações importantes da metodologia utilizada, particularmente no que diz respeito à qualidade do ADN, que pode ter comprometido a eficiência do processo de amplificação na maioria das amostras.
Conclusão: Os resultados limitados deste estudo reforçam a necessidade de otimizar a metodologia de recolha. Abordagens alternativas poderiam incluir a limpeza prévia da pele com uma solução alcoólica para remover células mortas ou a extração de ADN dos folículos capilares em vez da raspagem da pele. São necessárias mais pesquisas envolvendo uma amostra maior e técnicas laboratoriais mais robustas para validar a viabilidade da genotipagem do gene KRAS em indivíduos tatuados.
Introduction: Mutations in the RAS family of proto-oncogenes, particularly in the KRAS gene, are a key factor in the development of various skin cancers, including melanoma and squamous cell carcinoma. The most common mutation occurs in codon 12, altering the functionality of the GTPase protein, which plays a role in cell signalling. There have also been reports of skin neoplasms in tattooed individuals, raising questions as to whether the ink, the mechanical trauma of the needle or photoaging could be triggering factors. However, despite these observations, there is still a lack of robust scientific evidence directly linking tattoos to specific genetic mutations. Objectives: To verify the presence of mutations in codon 12 of the KRAS gene in the DNA of tattooed students at the Egas Moniz School of Health and Science. Methodology: An experimental pilot study was conducted with 16 tattooed students who met the following inclusion criteria: they were healthy individuals without skin diseases; their tattoos were black; they were located on the upper limbs; and each tattoo was at least two centimetres in diameter and had been completed more than two years ago. DNA was collected by scraping the skin with a wet swab and extracted using silica-coated centrifugation columns (NZYtech kit). The KRAS gene was amplified using nested PCR. Mutation analysis consisted of digesting the amplified product with the BstNI restriction enzyme, followed by electrophoresis on a 4% agarose gel. Data analysis was performed using simple descriptive statistics. Results: Ten viable DNA samples of sufficient quality for molecular interpretation were obtained, corresponding to five participants. Four of these samples exhibited a fragmentation pattern indicative of the wild-type genotype (111 bp), suggesting an absence of mutations in codon 12 of the KRAS gene. One sample exhibited a 140 bp band, consistent with a homozygous mutant genotype. However, the results also highlight important limitations of the methodology used, particularly with regard to DNA quality, which may have compromised the efficiency of the amplification process in most samples. Conclusion: The limited results of this study reinforce the need to optimise the collection methodology. Alternative approaches could include cleaning the skin with an alcoholic solution prior to sampling to remove dead cells, or extracting DNA from hair follicles instead of scraping the skin. Further research involving a larger sample size and more robust laboratory techniques is needed to validate the feasibility of KRAS gene genotyping in tattooed individuals.
Introduction: Mutations in the RAS family of proto-oncogenes, particularly in the KRAS gene, are a key factor in the development of various skin cancers, including melanoma and squamous cell carcinoma. The most common mutation occurs in codon 12, altering the functionality of the GTPase protein, which plays a role in cell signalling. There have also been reports of skin neoplasms in tattooed individuals, raising questions as to whether the ink, the mechanical trauma of the needle or photoaging could be triggering factors. However, despite these observations, there is still a lack of robust scientific evidence directly linking tattoos to specific genetic mutations. Objectives: To verify the presence of mutations in codon 12 of the KRAS gene in the DNA of tattooed students at the Egas Moniz School of Health and Science. Methodology: An experimental pilot study was conducted with 16 tattooed students who met the following inclusion criteria: they were healthy individuals without skin diseases; their tattoos were black; they were located on the upper limbs; and each tattoo was at least two centimetres in diameter and had been completed more than two years ago. DNA was collected by scraping the skin with a wet swab and extracted using silica-coated centrifugation columns (NZYtech kit). The KRAS gene was amplified using nested PCR. Mutation analysis consisted of digesting the amplified product with the BstNI restriction enzyme, followed by electrophoresis on a 4% agarose gel. Data analysis was performed using simple descriptive statistics. Results: Ten viable DNA samples of sufficient quality for molecular interpretation were obtained, corresponding to five participants. Four of these samples exhibited a fragmentation pattern indicative of the wild-type genotype (111 bp), suggesting an absence of mutations in codon 12 of the KRAS gene. One sample exhibited a 140 bp band, consistent with a homozygous mutant genotype. However, the results also highlight important limitations of the methodology used, particularly with regard to DNA quality, which may have compromised the efficiency of the amplification process in most samples. Conclusion: The limited results of this study reinforce the need to optimise the collection methodology. Alternative approaches could include cleaning the skin with an alcoholic solution prior to sampling to remove dead cells, or extracting DNA from hair follicles instead of scraping the skin. Further research involving a larger sample size and more robust laboratory techniques is needed to validate the feasibility of KRAS gene genotyping in tattooed individuals.
Description
Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz
Keywords
Gene KRAS Mutações Tatuagens Variabilidade genética