Associação de polimorfismos genéticos de citocinas pró e anti-inflamatórias e dos seus níveis circulantes com a progressão da doença hepática na hepatite C crónica

Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2020Hepatitis C is a contagious liver disease that is caused by the Hepatitis C Virus (VHC), which can lead to two types of hepatitis: acute, or chronic. This disease is one of the principal causes of liver transplants and liver diseases related deaths. It is known by the scientific community that 60% to 80% of those infected by the VHC will develop a chronic state of Hepatitis C, leading to a 30% risk of developing hepatic cirrhosis or hepatocellular carcinoma. Chronic Hepatitis C (CHC) is a clinical and pathologic syndrome that as several causes and is characterized by different stages of fibrosis and hepatic inflammation. The chronic infection of Hepatitis C is characterized by a slow progression hepatic fibrosis, which begins at the stage of no fibrosis, leading up to the stage of cirrhosis. The hepatic fibrosis is the most important prognostic factor of Chronic HCV infection. The main objective of this work is to determine if the genetic polymorphisms of the studied genes influence the progression of Hepatitis C disease on fibrosis stages. The secondary objectives are to study the influence of the studied polymorphisms in the propensity for the CHC disease; to study the relation of anthropometric, sociodemographic, metabolic, and biochemical parameters with the fibrosis stage; and to relate theses parameters with genetic polymorphisms. This study sample is constituted by 329 individuals with CHC who have been accompanied by Prof Fátima Serejo MD,PhD through the hepathology consultations at the Santa Maria Hospital(CHLNCAML). This sample has the following characteristics: the average age of the individuals is 48.93 years, the average Body Mass Index (BMI) of the individuals is 25.25 Kg/m2, and; the sample is divided in 62.30% males and 37.70% females. The TGF-β -509 C/T and TGF-β 29 T/C polymorphisms were determined by PCR-RFLP; and the TNF-α -308 G/A, TNF-α -238 G/A, IL-6 -174 G/C, IL-10 -1082 T/C, IL-10 -592 G/T and HPSE A/G were determined through the Endpoint genotyping technique. Additionally, it was performed a plasmatic concentration determination of the TNF-α, TGF-β, IL-10 cytokines through an ELISA method. The data statistical analysis was performed through SPSS 25.0, were the statistically significant values were those that had a p<0.05. In this work there were obtained associations between the intermediate phenotypes and genotypes, being them those of TNF-α -308 G/A, TGF-β -509 C/T, TGF-β 29 T/C, IL-6 -174 G/C and HPSE A/G polymorphisms with increased parameters, mainly of the iron metabolism and the hepatic lesion biomarkers. Another associations obtained was those of the genotypes and distant phenotypes, where it was verified that the TGF-β -509 C/T, IL-10 -1082 T/C and IL-10 -592 G/T polymorphisms had a relation with the higher stages of fibrosis. Lastly, in this work were also identified some intermediate markers that could relate with the development and progression of this pathogenesis, being the age, platelets, ALP, AST, ALT, GGT, iron, TIBC,plasma ferritin, plasma haptoglobin and plasma TNF-α. In conclusion, it is of high relevance the existence of epidemiologic studies in human beings that examine the associations between the several genetic polymorphisms and inflammatory biomarkers with the fibrosis stages in individuals with Chronic Hepatitis C. With these studies, it will be possible to predict the individual risks of this disease’s progression, to obtain a more adequate personalized diagnosis and treatment.A hepatite C é uma doença hepática contagiosa que é causada pelo vírus da hepatite C (VHC), vírus este que pode causar dois tipos de hepatite: aguda ou crónica. Esta doença é uma das principais causas de transplante hepático e de morte relacionada com o fígado. Sabe-se que cerca de 60 a 80 % dos indivíduos infetados pelo VHC irão desenvolver um estádio crónico de hepatite C, havendo um risco de 30% de desenvolverem cirrose hepática ou carcinoma hepatocelular. A hepatite C crónica (HCC) é uma síndrome clínica e patológica que tem várias causas e é caracterizada por diferentes estádios de fibrose e inflamação hepatocelular. A infeção crónica pelo vírus da hepatite C é caracterizada por uma fibrose hepática com uma progressão lenta, progressão essa que vai desde do estádio sem fibrose até ao estádio com cirrose. A fibrose hepática é o fator de prognóstico mais importante em indivíduos com a infeção crónica do VHC. O objetivo principal deste estudo é determinar se os polimorfismos genéticos dos genes estudados influenciam a progressão da doença da hepatite C para os estádios de fibrose. Os objetivos secundários foram estudar a influência dos polimorfismos estudados na suscetibilidade para a doença HCC; estudar a relação dos parâmetros antropométricos, sociodemográficos, metabólicos, e bioquímicos com o estádio de fibrose; e, por fim, relacionar estes parâmetros com os polimorfismos genéticos. A amostra foi constituída por 329 indivíduos com HCC seguidos pela Professora Doutora Fátima Serejo nas consultas de Hepatologia no Hospital Santa Maria. Amostra utilizada apresentava uma média de idade de 48,93 anos e de IMC de 25,25 Kg/m2 , e uma distribuição por sexo de 62,30% homens e de 37,70% mulheres. Os polimorfismos TGF-β -509 C/T e TGF-β 29 T/C foram determinados por PCRRFLP e os polimorfismos TNF-α -308 G/A, TNF-α -238 G/A, IL-6 -174 G/C, IL-10 -1082 T/C, IL-10 -592 G/T e HPSE A/G foram determinados pela técnica de Endpoint genotyping. Foi realizado também o doseamento da concentração plasmática das citocinas TNF-α, TGF-β, IL-10 pelo método de ELISA. A análise estatística dos dados foi realizada por SPSS 25.0. Valores estatisticamente significativos para p