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Abstract(s)
Introdução: A trombose é uma patologia comum e complexa, referente à formação de um coágulo, que dificulta o normal fluxo sanguíneo. Sob condições normais, os mecanismos procoagulantes, anticoagulantes e fibrinolíticos regulam a hemostasia para evitar a formação patológica destes coágulos. No entanto, alterações nos genes de proteínas envolvidas direta ou indiretamente na regulação hemostática podem levar a um aumento anómalo da coagulação e a uma maior predisposição à formação de coágulos obstrutivos. Esta condição é conhecida como trombofília e descreve um estado de hipercoagulabilidade responsável pelo desenvolvimento de eventos trombóticos que comprometem a vida (como o enfarte do miocárdio, acidente vascular cerebral e embolia pulmonar) e que geralmente se desenvolvem na presença de um ou mais fatores de risco herdados (variantes genéticas) e ambientais (adquiridos). A trombose tem, portanto, uma origem multifatorial causada pela interação destes fatores de risco herdados e/ou outros adquiridos (tabagismo, gravidez, uso de contracetivos orais e terapia hormonal), que podem coexistir no mesmo indivíduo e conferir um risco contínuo ao longo da vida. Devido ao seu estado adquirido de hipercoagulabilidade, as grávidas apresentam ainda um risco acrescido à manifestação de tromboses, traduzido pela ocorrência de abortos espontâneos recorrentes. Através da eliminação de fatores de risco adquiridos é possível prevenir (até certo ponto) o desenvolvimento de trombose. É assim importante conhecer o perfil genético da população de forma a reunir dados que permitam a identificação de indivíduos de alto risco e antecipar a ocorrência destes eventos, bem como desenvolver medidas preventivas dirigidas aos mesmos. Os objetivos deste projeto são então estimar a prevalência das variantes trombogénicas FV G1691A (FV Leiden), FII G20210A, AT Cambridge II, PAI-1 4G/5G, MTHFR C677T e MTHFR A1298C na população portuguesa, estabelecer associações entre a presença destas variantes genéticas e outros fatores de risco adquiridos no desenvolvimento de eventos trombóticos e identificar grupos de indivíduos de alto risco em Portugal.
Metodologia: 774 participantes do estudo e_COR, um estudo epidemiológico transversal, previamente desenvolvido no Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) com participantes das 5 regiões de Portugal Continental (Norte, Centro, Lisboa, Alentejo e Algarve), foram aleatoriamente selecionados para o presente estudo de forma a serem representativos da população portuguesa. A todos os participantes foi realizado um exame físico, bem como um questionário com informação clínica e pessoal necessária à realização do estudo. Após recolha das amostras de sangue, os parâmetros bioquímicos foram determinados e o ADN extraído, procedendo-se à genotipagem das variantes FV Leiden, FII G20210A, AT Cambridge II, PAI-1 4G/5G, MTHFR C677T e MTHFR A1298C pela tecnologia TaqMan® OpenArray™. As frequências alélicas e genotípicas das variantes foram determinadas de forma a estimar a sua prevalência na população portuguesa. Realizaram-se correlações entre os genótipos e a informação pessoal, bioquímica e clínica dos participantes, tendo sido também determinadas as frequências de subgrupos em risco acrescido de trombose.
Resultados: Dos indivíduos estudados, apenas 2,2%, 4,1% e 0,6% apresentavam, respetivamente, as variantes genéticas FVL, FII G20210A e AT Cambridge II em heterozigotia, não sendo identificados casos de homozigotia para estas alterações genéticas. Por outro lado, as variantes PAI-1 4G/5G e MTHFR C677T e A1298C revelaram estar amplamente distribuídas pela população: 50,4% e 21,8% dos participantes eram respetivamente heterozigotos e homozigotos para o PAI-1 4G/5G; na mesma ordem, 43.2% e 12,8% eram heterozigotos e homozigotos para a MTHFR C677T; e 39,5% e 9,8% exibiam heterozigotia e homozigotia para a MTHFR A1298C. Apenas 3,6% dos participantes não apresentaram qualquer alelo de risco trombogénico para as variantes em estudo. A maioria dos Portugueses (69% da população) eram portadores de pelo menos 2 variantes genéticas – a combinação mais prevalente foi a variante PAI-1 4G/5G com as variantes MTHFR C677T (24%) ou A1298C (19,8%), englobando ambos os genótipos hetero- e homozigótico. No que diz respeito aos fatores de risco trombogénicos adquiridos, observou-se que: 64,7% dos indivíduos apresentavam excesso de peso, 19,4% hiperhomocisteinemia e 24,3% hábitos tabágicos. Estes fatores revelaram ser mais prevalentes entre o sexo masculino. As mulheres podem apresentar um risco acrescido pelo uso de contracetivos orais (29,5% da população feminina em estudo) ou tratamentos hormonais (12,3%). Foi também analisada a ocorrência de enfarte do miocárdio e acidente vascular cerebral na população, bem como de abortos espontâneos entre as mulheres. Comparando os indivíduos que não desenvolveram nenhum evento trombótico, com o grupo de indivíduos que sofreu de enfarte do miocárdio, verificou-se que estes englobavam, com maior frequência, portadores das variantes FVL (5,3% vs. 2,1%) e FII G20210A (15,8% vs. 3,7%), assim como de homozigotos para a variante PAI-1 4G/5G (31,6% vs. 21,6%). O grupo com diagnóstico de AVC apresentava maior frequência de heterozigotos para o FVL (13,0% vs. 1,9%), de heterozigotos para a MTHFR C677T (65,2% vs. 42,4%), bem como, de homozigotos para o PAI-1 4G/5G (30,4% vs. 21,6%). Por fim, entre as mulheres que sofreram de aborto espontâneo, observou-se uma maior frequência dos genótipos heterozigóticos para o FVL (4,5% vs. 2%) e PAI-1 4G/5G (56,8 % vs. 47%), assim como, para o genótipo homozigótico da variante MTHFR A1298C (15,9% vs. 9,2%, p=0,037). Por outro lado, as mulheres fumadoras que tiveram pelo menos um aborto espontâneo apresentavam maior frequência do genótipo homozigótico da MTHFR C677T (18,2% vs. 3%).
Discussão: A variante genética mais prevalente neste estudo foi a PAI-1 4G/5G, seguida pelas variantes MTHFR C677T e A1298C. A prevalência estimada para as variantes FVL e FII G20210A é claramente menor que as anteriores e a AT Cambridge II revelou-se rara na população Portuguesa. Embora as prevalências estimadas para Portugal se encontrem de acordo com as frequências esperadas para populações Caucasianas, as frequências determinadas para o FVL e FII G20210A diferem entre países Europeus. As variantes FVL, FII G20210A e MTHFR C677T parecem influenciar a ocorrência de trombose arterial. Pela análise realizada neste estudo, 0,3% da população pode estar em risco uma vez que apresenta as três variantes em simultâneo. Apresentar o genótipo PAI-1 4G/4G pode também influenciar estas ocorrências, colocando 21,8% da população portuguesa em maior risco trombótico, particularmente aqueles que apresentam simultaneamente hipercoagulabilidade e hipofibrinólise: 1,9% e 2,8% da população que é também portadora das variantes FVL ou FII G20210A, respetivamente. 43,8% da população Portuguesa apresenta simultaneamente a variante genética PAI-1 4G/5G com uma das variantes da MTHFR, o que sugere um perfil trombofílico caraterizado por fibrinólise anormal, maior procoagulação, possível aumento da inflamação, bem como, disfunção endotelial. As mulheres podem ter ainda acrescido risco pelo uso de contracetivos orais e por serem portadoras de pelo menos um alelo de risco das variantes: PAI-1 4G/5G (presente em cerca de 20% da população feminina), MTHFR C677T (cerca de 15%) e MTHFR A1298C (cerca de 15%). Têm também um risco aumentado 6 a 10% da população feminina portadora de pelo menos um dos alelos de risco para cada uma dessas variantes referidas e sob tratamento hormonal. Verificou-se que as mulheres que tomam contracetivos orais e apresentam as variantes FVL (0,2%) e FII G20210A (0,7%), assim como, as que fazem terapia hormonal e são portadoras do FII G20210A (0,7%) estão em maior risco, especialmente se forem obesas e/ou fumadoras. As grávidas que apresentam a variante MTHFR A1298C correm maior risco de aborto espontâneo, apesar das variantes FVL e PAI-1 4G/5G poderem também influenciar o risco. As variantes FII G20210A e MTHFR C677T (isoladamente) não parecem influenciar a ocorrência de aborto; no entanto, a homozigotia para a MTHFR C677T juntamente com o tabagismo aparentam ter influência nessas incidências. A presença de pelo menos um alelo de risco de qualquer uma das variantes da MTHFR em fumadoras pode também favorecer a ocorrência de aborto.
Conclusão: A população portuguesa apresenta vários fatores de risco genéticos e adquiridos que podem interagir e culminar na incidência de eventos trombóticos. Consciencializar para a trombofilia é crítico para a prevenção, quer seja pela redução de fatores de risco adquiridos (mudanças no estilo de vida) ou pela otimização da terapêutica, que pode melhorar o prognóstico destes indivíduos. Desta forma, os resultados obtidos no presente estudo são importantes para a elaboração de estratégias terapêuticas e diretrizes de saúde para a identificação dos grupos de maior risco trombótico.
Introduction: Thrombosis is a pathology in which a blood clot (thrombus) hampers the normal blood flow to a tissue. Under normal conditions, procoagulant, anticoagulant and fibrinolytic pathways regulate hemostasis to avoid pathological clot formation. However, changes in the genes encoding proteins, directly or indirectly involved in hemostatic regulation, can lead to enhanced abnormal blood coagulation, resulting in an increased disposition to the formation of obstructive clots, known as thrombophilia. This state of hypercoagulability is responsible for the development of life-threatening thromboembolic events (like myocardial infarction, ischemic stroke and pulmonary embolism), which usually develop when one or more of both genetic (inherited) and environmental (acquired) risk factors come into play. Pregnant women are also at major risk to manifest thrombosis by recurrent spontaneous abortion episodes. Thus, thrombosis results from the interplay of inherited DNA changes and/or acquired risk factors (cigarette smoking, pregnancy and oral contraceptive or hormone therapy use) that may coexist in the same individual; however, it is preventable. Hence, it is important to recognize the genetic thrombogenic profile of the population to assemble data for identification of high-risk individuals and to develop local guidelines for prevention. With this in mind, this project aims to estimate the prevalence of the FV G1691A (FV Leiden), FII G20210A, AT Cambridge II, PAI-1 4G/5G and MTHFR C677T and A1298C thrombogenic variants in the Portuguese population, to establish associations between the presence of these genetic variants and the presence of other increasing risk factors in the development of thrombotic-related events and to identify groups of high-risk individuals in Portugal. Methodology: 774 e_COR participants from the North, Center, Lisbon, Alentejo and Algarve regions of Portugal were randomly selected to be representative of the Portuguese population. All participants were physically examined and had their personal and recent clinical data collected, as well as blood samples for biochemical analysis and DNA extraction. Participants were genotyped for the FV Leiden, FII G20210A, AT Cambridge II, PAI-1 4G/5G, MTHFR C677T and MTHFR A1298C variants by the TaqMan® OpenArray™ Technology. Allelic and genotypic frequencies were determined to estimate the prevalence of these thrombogenic variants in the Portuguese population. Correlations were made between the genotype data plus personal, biochemical and clinical information available and frequencies of subgroups of individuals with increased risk of thrombosis were determined. Results: Only 2.2%, 4.1% and 0.6% of the population were carriers of the FVL, the FII G20210A and the AT Cambridge II variants respectively, and no homozygosity was found for these genetic defects. In contrast, the PAI-1 4G/5G and the MTHFR C677T and A1298C variants were largely distributed among the population: 50.4% and 21.8% were, respectively, heterozygous and homozygous for the PAI-1 4G/5G; 43.2% and 12.8% were, in the same order, heterozygous and homozygous for the MTHFR C677T; and 39.5% and 9.8% displayed the heterozygous and homozygous genotype of the MTHFR A1298C variant. Only 3.6% of the participants did not display inherited genetic risk alleles for neither thrombogenic variants, while the majority (69% of the population) were carriers of at least two different genetic variants. The most prevalent coinheritances were the combination of the PAI-1 4G/5G variant with either the MTHFR C677T (24%) or the MTHFR A1298C (19.8%) variants, in heterozygous and/or homozygous genotypes. High frequencies of individuals with acquired thrombogenic risks were also observed: overweight or obesity (64.7%), hyperhomocysteinemia (19.4%) and cigarette smoking habits (24.3%); which were more prevalent among men. Women can be at especially increased risk by using oral contraceptives (29.5% of this study female population) and hormone therapy treatments (12.3%). Some participants had also developed thrombotic-related events. Among those who suffered a myocardial infarction, it was detected an increased heterozygous frequency for the FVL (5.3% vs. 2.1%) and FII G20210A (15.8% vs. 3.7%) and an increased frequency for the PAI-1 4G/5G homozygous genotype (4G/4G) (31.6% vs. 21.6%). The group with stroke diagnosis revealed a higher heterozygous frequency for the FVL (13.0% vs. 1.9%) and MTHFR C677T (65.2% vs. 42.4%) and an increased homozygous frequency for the PAI-1 4G/4G variant (30.4% vs. 21.6%). Finally, among women who had a spontaneous abort, it was observed a higher frequency of FVL (4.5% vs. 2%) and PAI-1 4G/5G (56.8% vs. 47%) heterozygous genotypes and of the MTHFR A1298C homozygous genotype (15.9% vs. 9.2%, p=0.037). Additionally, women smokers who had spontaneous abortion displayed a higher frequency of the MTHFR C677T homozygous genotype (18.2% vs. 3%). Discussion: The PAI-1 4G/5G was the most prevalent variant in mainland Portugal, followed by the MTHFR C677T and A1298C variants. The estimated prevalence of FVL and FII G20210A variants was considerably lower, while the AT Cambridge II was quite rare. Even though the variants’ estimated Portuguese prevalence was within the same range for Caucasian populations, the FVL and FII G20210A frequencies appear to differ among European countries. In this study, the FVL, FII G20210A and MTHFR C677T variants seemed to impact the onset of arterial thrombosis; so, 0.3% of the population may be at increased risk due to coinheritance of these three genetic factors. The PAI-1 4G/4G genotype may also be involved in the arterial thrombosis pathology, putting 21.8% of the Portuguese population at higher thrombotic-risk, particularly those with the synergistic combination of hypercoagulability and hypofibrinolysis: 1.9% and 2.8% of the population who are simultaneously carriers of the FVL or the FII G20210A variants, respectively. However, the most prevalent coinheritances in Portugal were found to be the combination of PAI-1 4G/5G with the MTHFR variants (43.8% of the population), suggesting a thrombophilic genetic profile characterized by impaired fibrinolysis, greater procoagulant activity, possible enhanced inflammation and endothelial dysfunction. Women may also be at particular risk by using oral contraceptives and carrying at least one risk allele of either one of these variants: PAI-1 4G/5G (about 20% of all women), MTHFR C677T (about 15%) and MTHFR A1298C (about 15%). Another female fraction of about 6 to 10%, carriers of PAI-1 4G/5G, MTHFR C677T or MTHFR A1298C variants, is also at risk due to the concurrent use of hormone therapy. Women users of oral contraceptives who were also carriers of the FVL (0.2%) and FII G20210A (0.7%) variants, or those who do hormone therapy and were found to be FII G20210A carriers (0.7%) are expected to be the ones at high-risk, especially if they are obese and/or smokers. Pregnant carriers of the MTHFR A1298C can be at high-risk of spontaneous abortion, whereas the FVL and the PAI-1 4G/5G variants may also have some influence in such incidence. The FII G20210A did not seemed to influence the occurrence of spontaneous abortion, neither the MTHFR C677T, at least by itself. Taking into consideration a possible synergistic risk of smoking habits, the MTHFR C677T homozygous genotype may potentiate the occurrence of spontaneous abortion and the inheritance of at least one risk allele of either one of the MTHFR variants among smokers may indeed enhance the risk of spontaneous abortion. Conclusion: The Portuguese population was shown to present several genetic and acquired thrombotic risk factors, which can interact and culminate in the onset of the disease. Therefore, awareness about thrombophilia can be critical for prevention, either for the elimination of acquired risk factors (lifestyle changes) or to optimize prophylaxis. Our findings might be important to elaborate health guideline strategies and to identify thrombotic high-risk groups that deserve special attention.
Introduction: Thrombosis is a pathology in which a blood clot (thrombus) hampers the normal blood flow to a tissue. Under normal conditions, procoagulant, anticoagulant and fibrinolytic pathways regulate hemostasis to avoid pathological clot formation. However, changes in the genes encoding proteins, directly or indirectly involved in hemostatic regulation, can lead to enhanced abnormal blood coagulation, resulting in an increased disposition to the formation of obstructive clots, known as thrombophilia. This state of hypercoagulability is responsible for the development of life-threatening thromboembolic events (like myocardial infarction, ischemic stroke and pulmonary embolism), which usually develop when one or more of both genetic (inherited) and environmental (acquired) risk factors come into play. Pregnant women are also at major risk to manifest thrombosis by recurrent spontaneous abortion episodes. Thus, thrombosis results from the interplay of inherited DNA changes and/or acquired risk factors (cigarette smoking, pregnancy and oral contraceptive or hormone therapy use) that may coexist in the same individual; however, it is preventable. Hence, it is important to recognize the genetic thrombogenic profile of the population to assemble data for identification of high-risk individuals and to develop local guidelines for prevention. With this in mind, this project aims to estimate the prevalence of the FV G1691A (FV Leiden), FII G20210A, AT Cambridge II, PAI-1 4G/5G and MTHFR C677T and A1298C thrombogenic variants in the Portuguese population, to establish associations between the presence of these genetic variants and the presence of other increasing risk factors in the development of thrombotic-related events and to identify groups of high-risk individuals in Portugal. Methodology: 774 e_COR participants from the North, Center, Lisbon, Alentejo and Algarve regions of Portugal were randomly selected to be representative of the Portuguese population. All participants were physically examined and had their personal and recent clinical data collected, as well as blood samples for biochemical analysis and DNA extraction. Participants were genotyped for the FV Leiden, FII G20210A, AT Cambridge II, PAI-1 4G/5G, MTHFR C677T and MTHFR A1298C variants by the TaqMan® OpenArray™ Technology. Allelic and genotypic frequencies were determined to estimate the prevalence of these thrombogenic variants in the Portuguese population. Correlations were made between the genotype data plus personal, biochemical and clinical information available and frequencies of subgroups of individuals with increased risk of thrombosis were determined. Results: Only 2.2%, 4.1% and 0.6% of the population were carriers of the FVL, the FII G20210A and the AT Cambridge II variants respectively, and no homozygosity was found for these genetic defects. In contrast, the PAI-1 4G/5G and the MTHFR C677T and A1298C variants were largely distributed among the population: 50.4% and 21.8% were, respectively, heterozygous and homozygous for the PAI-1 4G/5G; 43.2% and 12.8% were, in the same order, heterozygous and homozygous for the MTHFR C677T; and 39.5% and 9.8% displayed the heterozygous and homozygous genotype of the MTHFR A1298C variant. Only 3.6% of the participants did not display inherited genetic risk alleles for neither thrombogenic variants, while the majority (69% of the population) were carriers of at least two different genetic variants. The most prevalent coinheritances were the combination of the PAI-1 4G/5G variant with either the MTHFR C677T (24%) or the MTHFR A1298C (19.8%) variants, in heterozygous and/or homozygous genotypes. High frequencies of individuals with acquired thrombogenic risks were also observed: overweight or obesity (64.7%), hyperhomocysteinemia (19.4%) and cigarette smoking habits (24.3%); which were more prevalent among men. Women can be at especially increased risk by using oral contraceptives (29.5% of this study female population) and hormone therapy treatments (12.3%). Some participants had also developed thrombotic-related events. Among those who suffered a myocardial infarction, it was detected an increased heterozygous frequency for the FVL (5.3% vs. 2.1%) and FII G20210A (15.8% vs. 3.7%) and an increased frequency for the PAI-1 4G/5G homozygous genotype (4G/4G) (31.6% vs. 21.6%). The group with stroke diagnosis revealed a higher heterozygous frequency for the FVL (13.0% vs. 1.9%) and MTHFR C677T (65.2% vs. 42.4%) and an increased homozygous frequency for the PAI-1 4G/4G variant (30.4% vs. 21.6%). Finally, among women who had a spontaneous abort, it was observed a higher frequency of FVL (4.5% vs. 2%) and PAI-1 4G/5G (56.8% vs. 47%) heterozygous genotypes and of the MTHFR A1298C homozygous genotype (15.9% vs. 9.2%, p=0.037). Additionally, women smokers who had spontaneous abortion displayed a higher frequency of the MTHFR C677T homozygous genotype (18.2% vs. 3%). Discussion: The PAI-1 4G/5G was the most prevalent variant in mainland Portugal, followed by the MTHFR C677T and A1298C variants. The estimated prevalence of FVL and FII G20210A variants was considerably lower, while the AT Cambridge II was quite rare. Even though the variants’ estimated Portuguese prevalence was within the same range for Caucasian populations, the FVL and FII G20210A frequencies appear to differ among European countries. In this study, the FVL, FII G20210A and MTHFR C677T variants seemed to impact the onset of arterial thrombosis; so, 0.3% of the population may be at increased risk due to coinheritance of these three genetic factors. The PAI-1 4G/4G genotype may also be involved in the arterial thrombosis pathology, putting 21.8% of the Portuguese population at higher thrombotic-risk, particularly those with the synergistic combination of hypercoagulability and hypofibrinolysis: 1.9% and 2.8% of the population who are simultaneously carriers of the FVL or the FII G20210A variants, respectively. However, the most prevalent coinheritances in Portugal were found to be the combination of PAI-1 4G/5G with the MTHFR variants (43.8% of the population), suggesting a thrombophilic genetic profile characterized by impaired fibrinolysis, greater procoagulant activity, possible enhanced inflammation and endothelial dysfunction. Women may also be at particular risk by using oral contraceptives and carrying at least one risk allele of either one of these variants: PAI-1 4G/5G (about 20% of all women), MTHFR C677T (about 15%) and MTHFR A1298C (about 15%). Another female fraction of about 6 to 10%, carriers of PAI-1 4G/5G, MTHFR C677T or MTHFR A1298C variants, is also at risk due to the concurrent use of hormone therapy. Women users of oral contraceptives who were also carriers of the FVL (0.2%) and FII G20210A (0.7%) variants, or those who do hormone therapy and were found to be FII G20210A carriers (0.7%) are expected to be the ones at high-risk, especially if they are obese and/or smokers. Pregnant carriers of the MTHFR A1298C can be at high-risk of spontaneous abortion, whereas the FVL and the PAI-1 4G/5G variants may also have some influence in such incidence. The FII G20210A did not seemed to influence the occurrence of spontaneous abortion, neither the MTHFR C677T, at least by itself. Taking into consideration a possible synergistic risk of smoking habits, the MTHFR C677T homozygous genotype may potentiate the occurrence of spontaneous abortion and the inheritance of at least one risk allele of either one of the MTHFR variants among smokers may indeed enhance the risk of spontaneous abortion. Conclusion: The Portuguese population was shown to present several genetic and acquired thrombotic risk factors, which can interact and culminate in the onset of the disease. Therefore, awareness about thrombophilia can be critical for prevention, either for the elimination of acquired risk factors (lifestyle changes) or to optimize prophylaxis. Our findings might be important to elaborate health guideline strategies and to identify thrombotic high-risk groups that deserve special attention.
Description
Tese de mestrado, Ciências Biofarmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Thrombosis Thrombophilia Hypercoagulability Risk factors Thrombogenic variants Oral contraceptives Cigarette smoking Myocardial infarction Stroke Spontaneous abortion Teses de mestrado - 2020