Detecção das variações genéticas R702W e L1007finsC no gene NOD2/CARD15 e sua relação com a microbiota intestinal de pacientes com doença de Crohn
Arquivos
Data
2017-10-11
Autores
Alencar Junior, Hagamenon de 
Orientadores
Paiotti, Ana Paula Ribeiro
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Avaliar a incidência das variações R702W e L1007finsC no gene NOD2/CARD15 e sua relação com a diversidade da microbiota intestinal de pacientes com Doença de Crohn. Métodos: Foram estudados 105 indivíduos, sendo 54 com Doença de Crohn e 51 como grupo controle. A análise do polimorfismo do gene NOD2/CARD15 foi utilizada a técnica de PCR-RFLP para avaliar as duas variações genéticas R702W e L1007finsC. A análise da microbiota intestinal foi realizada por PCR em Tempo Real. Foram avaliadas a expressão gênica do filo Bacteroidetes, classe Bacilli (filo Firmicutes), família Bifidobacteriaceae (filo Actinobacteria) e família Enterobacteriaceae (filo Proteobacteria). Os dados foram representados como média ± desvio-padrão, mínimo e máximo e frequência em porcentagem; teste qui-quadrado para as análises dos polimorfismos e teste “t” e Mann-Whitney para as análises de expressão gênica. Na comparação entre a expressão gênica e atividade clínica bem como localização da doença utilizamos ANOVA (Kruskal-Wallis); modelos de regressão lineares (modelo não condicional e modelo de condicionamento parcial) para avaliar a interação entre os genótipos e quantidade de expressão dos grupos bacterianos. Valores de p<0,05 foram considerados estatisticamente significativos. Resultados: Para ambas as variações genéticas, o genótipo selvagem foi o mais comum. No polimorfismo R702W, 13 indivíduos do grupo caso e 4 participantes do grupo controle apresentaram o genótipo heterozigoto e nenhum paciente homozigoto. Para o polimorfismo L1007finsC, a maioria dos participantes apresentaram o genótipo selvagem, 9 participantes do grupo caso e 4 do grupo controle apresentaram o genótipo heterozigoto e 2 indivíduos com Doença de Crohn e 1 individuo do grupo controle apresentaram o genótipo homozigoto. Na análise da expressão gênica dos grupos bacterianos, verificamos um aumento significativo do filo Bacteroidetes nas fezes dos participantes com Doença de Crohn em relação ao grupo controle. Por outro lado, observamos uma redução significativa na expressão da classe Bacilli e da família Bifidobacteriaceae. Em relação a família Enterobacteriaceae, não observamos diferenças. Analisando a interação dos polimorfismos com a expressão gênica dos grupos bacterianos, não observamos resultados significativos. Conclusão: Para ambas as variações genéticas (R702W e L1007finsC), o genótipo selvagem foi o mais frequente tanto nos participantes com Doença de Crohn como no grupo controle. A expressão gênica de bactérias comensais está diminuída nas fezes dos participantes com DC em relação ao grupo controle. Não houve relação entre as variações genéticas estudadas e a expressão da microbiota.
Objective: To evaluate the incidence of R702W and L1007finsC variations in the NOD2 / CARD15 gene and its relation with the intestinal microbiota diversity of patients with Crohn's disease. Methods: We studied stool samples of 105 individuals, 54 with Crohn's Disease and 51 as control group. Polymorphisms in the NOD2/CARD15 gene was performed by PCR-RFLP for two genetic variations: R702W and L1007finsC. The gut microbiota analysis was performed by Real-Time PCR. The Bacteroidetes phylum, class Bacilli (Firmicutes phylum), family Bifidobacteriaceae (Actinobacteri phylum) and family Enterobacteriaceae (Proteobacteria phylum) were evaluated. Data were represented as mean ± standard deviation, minimum and maximum, and frequency as percentage. Chi-square test for was performed for polymorphism analyzes and Mann-Whitney for gene expression. In the comparison between gene expression and clinical activity as well as localization of the disease we used ANOVA (Kruskal-Wallis); and nonconditional model and partial conditioning model to evaluate the interaction between genotypes and expression of bacterial groups. Values of p <0.05 were considered statistically significant. Results: For both genetic variations, wild type genotype was the most common. In the R702W polymorphism, 13 individuals with Crohn´s Disease and 4 participants of control group showed heterozygote genotype. For the polymorphism L1007finsC, the majority of the participants showed wild type genotype, 9 participants with Crohn´s disease and 4 of the control group showed the heterozygous genotype. Only two individuals with Crohn's Disease and 1 individual of the control group showed the homozygous mutant. In the analysis of the gene expression of bacterial groups, we verified a significant increase of Bacteroidetes in fecal samples of participants with Crohn's Disease when compared to the control group. On the other hand, we observed a significant reduction in the expression of Bacilli and Bifidobacteriaceae. Regarding Enterobacteriaceae, no difference was observed. It was not possible to establish an interaction between the polymorphisms and the bacterial gene expression. Conclusion: For both genetic variations (R702W and L1007finsC), wild type genotype was the most frequent in both Crohn's Disease and control group. The gene expression of commensal bacteria was decreased in fecal samples of participants with CD when compared to the control group. There was no relation between the genetic variations studied and the expression of the microbiota.
Objective: To evaluate the incidence of R702W and L1007finsC variations in the NOD2 / CARD15 gene and its relation with the intestinal microbiota diversity of patients with Crohn's disease. Methods: We studied stool samples of 105 individuals, 54 with Crohn's Disease and 51 as control group. Polymorphisms in the NOD2/CARD15 gene was performed by PCR-RFLP for two genetic variations: R702W and L1007finsC. The gut microbiota analysis was performed by Real-Time PCR. The Bacteroidetes phylum, class Bacilli (Firmicutes phylum), family Bifidobacteriaceae (Actinobacteri phylum) and family Enterobacteriaceae (Proteobacteria phylum) were evaluated. Data were represented as mean ± standard deviation, minimum and maximum, and frequency as percentage. Chi-square test for was performed for polymorphism analyzes and Mann-Whitney for gene expression. In the comparison between gene expression and clinical activity as well as localization of the disease we used ANOVA (Kruskal-Wallis); and nonconditional model and partial conditioning model to evaluate the interaction between genotypes and expression of bacterial groups. Values of p <0.05 were considered statistically significant. Results: For both genetic variations, wild type genotype was the most common. In the R702W polymorphism, 13 individuals with Crohn´s Disease and 4 participants of control group showed heterozygote genotype. For the polymorphism L1007finsC, the majority of the participants showed wild type genotype, 9 participants with Crohn´s disease and 4 of the control group showed the heterozygous genotype. Only two individuals with Crohn's Disease and 1 individual of the control group showed the homozygous mutant. In the analysis of the gene expression of bacterial groups, we verified a significant increase of Bacteroidetes in fecal samples of participants with Crohn's Disease when compared to the control group. On the other hand, we observed a significant reduction in the expression of Bacilli and Bifidobacteriaceae. Regarding Enterobacteriaceae, no difference was observed. It was not possible to establish an interaction between the polymorphisms and the bacterial gene expression. Conclusion: For both genetic variations (R702W and L1007finsC), wild type genotype was the most frequent in both Crohn's Disease and control group. The gene expression of commensal bacteria was decreased in fecal samples of participants with CD when compared to the control group. There was no relation between the genetic variations studied and the expression of the microbiota.