Correlation of interaction between genetic polymorphisms with thrombin activity in plasma and pathological stages of esophageal carcinoma / 西安交通大学学报(医学版)

Objective To investigate the correlation of interaction between polymorphisms of prothrombin gene G20210A in 3' untranslated region and tissue factor pathway inhibitor (TFPI) gene C399T in 5' untranslated region with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Methods Based on TNM method,we selected 198 patients with stage Ⅰ esophageal carcinoma,198 with stage Ⅱ,198 with stage Ⅲ,and 198 with stage Ⅳ from the First Affiliated Hospital of Xinxiang Medical College from May 2011 to August 2015 for this study;198 patients with esophageal carcinoma of stage 0 served as the control group.The thrombin activity in plasma were determined by chromogenic substrate assay.The genetic polymorphisms of prothrombin gene G20210A in 3' untranslated region and TFPI gene C399T in 5' untranslated region in peripheral blood leukocytes of the above-mentioned patients were analyzed by PCR-RFLP technique.Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95％ confidence intervals (95％ CI) of polymorphisms prothrombin gene G20210A and TFPI gene C399T polymorphisms and to analyze the interaction of nucleotide polymorphisms with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Results The frequencies of G20210A (GA),G20210A (AA),C399T (CT) and C399T (TT) were 24.24％,26.77％,24.24％ and 25.76％ in stage Ⅰ group;34.34％,37.37％,34.85％ and 36.36％ in stage Ⅱ group;39.90％,42.93％,40.41％ and 41.92％ in stage Ⅲ group;45.45％,46.97％,45.35％ and 46.46 in stage Ⅳ group;and 13.64％,14.14％,13.13％ and 13.64％ in stage 0 group,respectively.Statistical tests showed significant difference in the frequencies among each group (all P＜0.01).The risks of invasion and metastasis of esophageal carcinoma significantly increased in the subjects with G20210A,in those with G20210A(AA) genotype,in those with C399T (CT) genotype and in those with C399T (TT) genotype.Combined analysis of the polymorphisms showed that percentage of G20210A (AA)/C399T (TT) in stage Ⅰ group,stage Ⅱ group,stage Ⅲ group,stage Ⅳ group and stage 0 group was 7.07％,14.14％,18.18％,21.71％ and 1.52％,respectively,and statistical tests showed significant difference in the frequency among each group (all P＜0.01).People who carried G20210A(AA)/C399T(TT) had higher risks of invasion and metastasis of esophageal carcinoma,and statistical analysis suggested a positive interaction between G20210A (AA) and C399T (TT) in increasing the risks of invasion and metastasis of esophageal carcinoma (All γ＞ 1).Likewise,there were also positive interactions in the pathogenesis of invasion and metastasis of esophageal carcinoma between G20210A (GA) and C399T (TT),G20210A (GA) and C399T(CT),G20210A (AA) and C399T (CT) (All γ＞1).The thrombin activities in plasma in stage Ⅰ,Ⅱ,Ⅲ and Ⅳ groups were all significantly higher than those in stage 0 group,and there were significant differences among stage Ⅰ,stage Ⅱ,stage Ⅲ and stage Ⅳ in thrombin activities (all P＜0.01).Patients with mutation genotype had significantly higher thrombin activities than those with wild homozygous in the same TNM stage.Conclusion G20210A and C399T gene mutations are the risk factors in the invasion and metastasis of esophageal carcinoma.Significant interactions between G20210A and C399T mutations increase the risk of invasion and metastasis of esophageal carcinoma,which may be closely related to their increased thrombin activities in plasma.

Mutación g20210a del gen de la protrombina: presentación clínica en dos gestantes / G20210a mutation of the prothrombin gene: clinical presentation in two pregnant women

La trombofilia hereditaria es una enfermedad genética que resulta de dos o más mutaciones en genes involucrados en el sistema hemostático, con variabilidad en la penetrancia del fenotipo trombótico. Entre los genes mutados asociados al incremento del riesgo de trombosis venosa se encuentra la mutación G20210A del gen de la protrombina. Se presentan los casos de dos pacientes jóvenes embarazadas que acudieron a la consulta de Hemostasia del Instituto de Hematología e Inmunología. Una, con historia personal de enfermedad tromboembólica (ETE) asociada al uso de anticonceptivos orales; y la otra, con antecedentes familiares de ETE e historia personal de abortos recurrentes. A ambas pacientes se les realizó estudio de trombofilia en el que se detectó la mutación G20210A del gen de la protrombina en estado heterocigoto. Recibieron seguimiento médico multidisciplinario y tratamiento profiláctico con aspirina a bajas dosis hasta la semana 34 de la gestación; y heparina de bajo peso molecular durante la gestación y seis semanas después del parto. Se lograron dos nacimientos sin complicaciones obstétricas ni fetales. La expresión de gen de la protrombina G20210A es variable, incluso dentro de una misma familia, y puede estar influenciada por factores de riesgo adquiridos, como el uso de anticonceptivos orales, el embarazo y el puerperio(AU)

Hereditary thrombophilia is a genetic disease that results from two or more mutations in genes involved in the hemostatic system, with variable penetrance of the thrombotic phenotype. Among the mutated genes associated with increased risk of venous thrombosis is mutation G20210A prothrombin gene. We present two pregnant young patients who attended the Haemostasis outpatient service at the Institute of Hematology and Immunology. One of them, with personal history of thromboembolic disease (TED) associated with use of oral contraceptives; and the other one, with a family history of TED and personal history of recurrent abortions. In both patients´ thrombophilia studies the G20210A mutation in the prothrombin gene in heterozygous state was detected. The patients received multidisciplinary medical monitoring and prophylactic treatment with low-dose aspirin until week 34 of gestation, and low molecular weight heparin during pregnancy and six weeks after delivery. Two births without obstetric or fetal complications were achieved. The gene expression of prothrombin G20210A is variable, even within the same family and may be influenced by acquired risk factors such as the use of oral contraceptives, pregnancy and the postpartum period(AU)

Comparison of the Real-Time PCR Tests for Factor V G1691A and Prothrombin G20210A with PCR-Restriction Fragment Length Polymorphism and Direct Sequencing Tests / 임상검사와정도관리

BACKGROUND: Factor V (FV) G1691A and prothrombin G20210A mutations are the most common targets of genetic tests for thromboembolism. This study compared the ability of real-time PCR to detect FV G1691A and prothrombin G20210A (BioSewoom, Korea) with that of PCR-restriction fragment length polymorphism (RFLP) and direct sequencing, to evaluate diagnostic equivalency. METHODS: Real-time PCR was compared with PCR-restriction fragment length polymorphism (RFLP) and direct sequencing using patients' samples as well as heterozygous and homozygous World Health Organization (WHO) reference reagent DNA. The limit of detection (LoD) for real-time PCR was determined using WHO reference reagents. RESULTS: All 141 and 156 patient samples were tested for the FV G1691A and prothrombin G20210A mutations, respectively; the results from all three methods (real-time PCR, PCR-RFLP, and direct sequencing) consistently showed that the samples were wild type. Each of the three methods showed the same results in tests using heterozygous and homozygous DNA from the WHO reference reagents. The LoD of wild type and homozygous samples was 65.16 pg/mL for FV G1691A, and 61.3 pg/mL for prothrombin G20210A. The LoD of heterozygous samples was 1,650.0 pg/mL for FV G1691A and 1,640.0 pg/mL for prothrombin G20210A. CONCLUSIONS: The real-time PCR test kits for FV G1691A and prothrombin G20210A showed reliable equivalency with PCR-RFLP and direct sequencing, and could be useful tests to detect gene polymorphisms for thromboembolism.

Predictive value of prothrombin G20210A mutation detection in pulmonary thromboembolism / 吉林大学学报(医学版)

Objective To study the incidence frequency of prothrombin G20210A (FⅡ G20210A)mutation in the patients with pulmonary thromboembolism(PTE)in northeast China,and to clarify the predictive value of FⅡG20210A mutation detection in PTE of the population in northeast China.Methods 60 PTE patients(PTE group) and 80 sex-matched healthy controls(control group)from the same geographic area were selected.All the patients were diagnosed by lung ventilation/perfusion scan and/or multi-slice CT pulmonary angiography(CTPA)as well as medical history.The genome DNA was extracted from the whole blood using alcohol.Polymerase chain reaction (PCR),restriction fragment length polymorphisms(RFLP)analysis with HindⅢ restriction enzyme and sepharose gel electrophoresis were used to identify the F Ⅱ G20210A mutation in PTE group and control group. Results After digested by HindⅢ restriction enzyme,only the fragments of 407 and 99 bp were found in PTE group.The frequency of FⅡ G20210A mutation was 0%,there was no statistical difference compared with contol group(P>0.05).There were no heterozygote and homozygote mutation of FⅡ G202210A gene in PTE group and control group.Conclusion The incidence of FⅡ G20210A mutation in the PTE patients in northeast China is very low,and the detection of FⅡ G20210A mutation may have no predictive value in PTE of the population in northeast China.

Analysis of factor V Leiden and prothrombin mutations in patients with suspected thrombophilia in São Paulo state-Brazil / Análise da mutação no fator V de Leiden e na protrombina em pacientes do estado de São Paulo, Brasil

INTRODUCTION: Prothrombin (factor II) is a thrombin precursor, which induces fibrin formation. A mutation in the prothrombin gene (G20210A) has been described, which is directly associated with high prothrombin levels, hence thrombophilia. G1691A mutation in the factor V Leiden (FVL) gene occurs on exon 10, one of the main cleavage sites for protein C activation, resulting in protein alteration. OBJECTIVE: To identify and estimate the genotype frequency of the three possible genotypes and the frequency of the two existing alleles in the FVL and prothrombin genes in patients with suspected thrombophilia in the state of Sao Paulo. This study may provide more literature and reference data on the incidence of prothrombin genotypes among individuals in Brazil. MATERIAL AND METHODS: Analysis of point mutation by real time polymerase chain reaction (RT-PCR). RESULTS: We obtained a total of 100 individuals, from which 94% had the homozygous G genotype. Only 6% had heterozygous genotype and there was no individual with the homozygous genotype A for FVL gene. As to the prothrombin gene, the frequency was 97% for homozygous G genotype and 3% for the heterozygous genotype. There was no patient with the homozygous A genotype. CONCLUSION: This study demonstrated that the genotype identification of these genes is advisable for patients with suspected thrombophilia in this region.

INTRODUÇÃO: A protrombina (fator II) é a precursora da trombina, que induz a formação de fibrina. Foi descrita uma mutação no gene da protrombina (G20210A), associado diretamente a altos níveis de protrombina no sangue e, consequentemente, a trombofilia. A mutação G1691A no gene do fator V de Leiden (FLV) localiza-se no éxon 10, resultando na alteração da proteína, um dos principais sítios de clivagem para ativação da proteína C. OBJETIVOS: Identificar e estimar a frequência genotípica dos três possíveis genótipos, assim como estimar a frequência dos dois alelos existentes no gene do FLV e na protrombina em pacientes com suspeita de trombofilia no estado de São Paulo. Este estudo poderá fornecer mais dados para a literatura e para consulta da incidência dos genótipos da protrombina em indivíduos no Brasil. MATERIAL E MÉTODOS: Análise de mutação pontual por reação em cadeia da polimerase em tempo real (RT-PCR). RESULTADO: Obtivemos o número de 100 indivíduos e, desse total, 94% possuíam o genótipo para homozigoto G; apenas 6%, genótipo heterozigoto; nenhum indivíduo foi encontrado com genótipo homozigoto A no gene do FLV. No gene da protrombina, a frequência foi de 97% para o genótipo homozigoto G e 3% para o genoma heterozigoto; não foi encontrado nenhum indivíduo com o genoma homozigoto A. CONCLUSÃO: Este estudo mostrou que é recomendável a identificação do genótipo para esses genes em pacientes com suspeita de trombofilia nessa região.

Análise da mutação G20210A no gene da protrombina (fator II) em pacientes com suspeita de trombofilia no sul do Brasil / Analysis of prothrombin G20210A mutation (factor II) in patients with suspected trombophilia in Southern Brazil

INTRODUÇÃO: A protrombina (fator II) é uma proteína sanguínea sintetizada no fígado com a presença de vitamina K. É a precursora da trombina, que induz a formação de fibrina. Foi descrita uma mutação no gene da protrombina G20210A, associada diretamente a altos níveis de protrombina no sangue e, consequentemente, à trombofilia. Essa variante alélica consiste em mutação pontual, também chamada de polimorfismo de nucleotídeo simples (SNP), ocasionando a troca de uma guanina por uma adenina no nucleotídeo 20210, localizado em um sítio de clivagem do precursor do ácido ribonucleico mensageiro (mRNA). Essa troca caracteriza o alelo A e a ausência da mutação do alelo G. OBJETIVO: Quantificar o número de indivíduos homozigotos para alelo G, homozigotos para alelo A e heterozigotos, cujas amostras foram enviadas para o laboratório Genolab Análises Genéticas, abrangendo os estados do Paraná e Santa Catarina, no período de 1º de janeiro de 2009 a 10 de outubro de 2010. MÉTODOS: Análise de mutação pontual por reação em cadeia da polimerase em tempo real (RT-PCR). RESULTADOS: Obtivemos o número de 243 indivíduos e desse total 51,03% eram oriundos do estado do Paraná, enquanto 48,97%, oriundos do estado de Santa Catarina. Do total analisado, 88,89% possuíam o genótipo para homozigoto G, e nenhum indivíduo foi encontrado com mutação para homozigoto A. Apenas 11,11% possuíam genótipo heterozigoto. O estado de Santa Catarina apresentou frequência superior para genótipo heterozigoto em relação ao Paraná. CONCLUSÃO: Este estudo mostrou que é recomendável a identificação do genótipo para esse gene em pacientes com suspeita de trombofilia nos dois estados.

INTRODUCTION: Prothrombin (factor II) is a blood protein synthesized in the liver in the presence of vitamin K. It is a thrombin precursor, which induces fibrin formation. Prothrombin G20210A mutation and high prothrombin levels have been closely associated with thrombophilia. This allelic variant is a single mutation, also denominated single nucleotide polymorphism (SNP), in which guanine is replaced with adenine in the messenger ribonucleic acid (mRNA) cleavage of nucleotide 20210. The replacement is characterized by the presence of allele A and the absence of mutation in allele G. OBJECTIVE: To quantify the number of individuals homozygous for allele G, allele A and heterozygotes. The samples were collected in Paraná and Santa Catarina from January 1st, 2009 to October 10th, 2010 and were sent to Genolab Análises Genéticas. METHODS: Analysis of single mutation by polymerase chain reaction in real time (RT-PCR). RESULTS: From 243 individuals, 51.03% were from Paraná and 48.97% were from Santa Catarina. 88.89% individuals were homozygous for G genotype, none of them were homozygous for A. Only 11.11% were heterozygotes. Santa Catarina presented a higher frequency in heterozygous genotype in comparison with Paraná. CONCLUSION: This study showed that patients with suspected thrombophilia should undergo genotype identification in both states.

Mutação G20210A no gene da protrombina, fator V de Leiden e anticorpos anticardiolipina não influenciam a sobrevida do enxerto renal após o transplante / Prothrombin G20210A gene mutation, factor V Leiden and anticardiolipin antibodies do not influence renal graft survival after transplantation

Introdução: Complicações tromboembólicas são importantes fatores de risco para perda do enxerto e pior evolução após o transplante renal. pacientes com defeito trombofílico apresentam maior risco de complicações tromboembólicas. Foram analisados, entre receptores de transplante renal, a prevalência de defeito trombofílico e o risco atribuído a esta condição para a perda do enxerto e para o desenvolvimento de tromboses intravasculares. Métodos: estudo do tipo coorte incluindo 388 receptores adultos analisados quanto à presença de trombofilia de acordo com a pesquisa de anticorpos anticardiolipidina (aCL) por ELISA e das mutações G1691A no gene do fator V (FV) e G20210A no gene da protrombina (PT) por PCR multiplex. Resultados: Defeito trombofílico foi identificado em 25,8% dos pacientes. As taxas de sobrevida de 2 anos do enxerto foram semelhantes entre os pacientes com e sem defeito trombofílico (94%, p=0,53), bem como a sobrevida dos enxertos livres de tromboses intravasculares (97% versus 97%, p=0,83). pacientes com defeito trombofílico apresentaram prevalência de tromboses intravasculares semelhante à do grupo-controle (3% versus 3,5%, p=0,82). O transplante renal anterior foi associado a maior risco de perda de enxerto (OR 20,8, p<0,001) e de ocorrência de trombose intravasculares (OR 6,8, p=0,008). Conclusões: As prevalências das mutações FVG1691A e PTG20210A na população estudada foram semelhantes às da população geral não transplantada, e a prevalência de anticorpos aCL superou a observada entre os indivíduos sadios. Não houve associação entre os marcadores de trombofilia estudados e a sobrevida em médio prazo do transplante renal.

Introduction: Thromboembolic complications are important risk factors for graft loss and poor outcome after renal transplantation. patients with thrombophilic defects are at increased risk of thromboembolic complications. Were analyzed, among kidney transplant recipients, the prevalence of thrombophilic defects and the risk attributed to this condition for graft loss and the development of intravascular thrombosis. Methods: A cohort study including 388 adult recipients analyzed for the presence of thrombophilia according to anticardiolipidina antibodies (aCL) by ELISA and gene mutations G1691A in factor V (FV) and prothrombin gene G20210A (PT) by multiplex PCR. Results: thrombophilic defect was identified in 25.8% of patients. The survival rates of two years of the graft were similar between patients with and without thrombophilic defect (94%, p = 0.53), and the survival of free grafts of intravascular thrombosis (97% versus 97%, p = 0 , 83). patients with an increased prevalence of thrombophilic defect intravascular thrombosis similar to the control group (3% versus 3.5%, p = 0.82). Previous renal transplantation was associated with increased risk of graft loss (OR 20.8, p <0.001) and intravascular thrombosis (OR 6.8, p = 0.008). Conclusions: The prevalence of mutations and FVG1691A PTG20210A in this study were similar to those of the general population not transplanted, and the prevalence of aCL antibodies exceeded that observed among healthy individuals. There was no association between markers of thrombophilia studied and medium-term survival in renal transplantation.

Low prevalence of Factor V Leiden and the prothrombin G20210A mutation in a healthy population from the central-south region of Chile / Baixa prevalência do Fator V Leiden e da mutação da protrombina G20210A em uma população sã da região centro-sul do Chile

Thrombosis is a result of the interaction between predisposing genetic polymorphisms and acquired risk factors. The two prothrombotic polymorphisms which are most frequently associated with thrombosis are factor V (FV) Leiden and the prothrombin (PT) G20210A mutation. The objective of this work was to investigate the prevalence of both factors in the central-south region of Chile. Determination of the frequency was carried out by means of a genetic analysis of 1200 healthy, non-native individuals. The mutation of FV Leiden was found in 1.25 percent of the population and the PT G20210A mutation in 1.33 percent. None of the individuals were homozygosis or had both polymorphisms. The prevalences of FV Leiden and the PT G20210A mutation are less common in the healthy population.

A trombose pode ser o resultado da interação de polimorfismo genético e fatores de riscos adquiridos. Os polimosfirmos protrombóticos mais frequentes são fator V (FV) Leiden e a mutação da protrombina (PT) G20210A. O objetivo deste trabalho foi investigar a prevalência de ambos os polimorfismos na região centro-sul do Chile. Foram realizadas análises genéticas (PCR RFLP) de 1.200 pessoas saudáveis, não nativas da região. Foram encontrados 1,25 por cento de mutação do Fator V Leiden e 1,33 por cento da mutação da protrombina G20210A. Não foi detectada homozigose em ambos os polimorfismos. A prevalência de FV Leiden e da mutação G20210A é baixa na população estudada.

Factor VLeiden and Prothrombin G20210A Gene Polymorphisms in Patients with Coronary Artery Disease

PURPOSE: The precise molecular mechanisms culminating in coronary artery disease (CAD) are not well understood, despite a wealth of knowledge on predisposing risk factors and pathomechanisms. CAD and myocardial infarction (MI) are complex genetic diseases; neither the environment alone, nor a single gene, cause disease, rather, a mix of environmental and genetic factors lead to atherosclerosis of the coronary arteries. MATERIALS AND METHODS: In the present study, our aim was to investigate the roles of prothrombin G20210A mutation and Factor VLeiden mutation in atherosclerotic coronary artery disease. 287 subjects (106 control subjects, who were angiographically normal, and 181 angiographically documented coronary atherosclerotic patients who exhibited coronary artery narrowing to a degree of > or = 50%) were included in this study. The mutations were assessed with LightCycler Real-Time PCR mutation detection kits (Roche Diagnostics, GmbH, Germany). RESULTS: 6.6% of control subjects, and 6.1% of patients with (50% coronary artery narrowing were determined to have the Factor VLeiden heterozygote mutation. 6.6% of control subjects had the Prothrombin G20210A heterozygote mutation, while 7.7% of patients with (50% coronary artery narrowing had this mutation. The OR for Factor VLeiden was 1.52 (CI: 0.240-9.602) and for Prothrombin G20210A mutation, the OR was 1.415 (CI: 0.287-6.962). CONCLUSION: Although both the heterozygote Factor VLeiden and Prothrombin gene mutations were more frequent in patients with CAD than in control subjects, there was no statistical relationship found to exist between coronary artery disease and the Factor VLeiden and Prothrombin G20210A mutations.

Trombofilias heredadas y pérdida gestacional recurrente / Inherited thrombophilia and recurrent pregnancy loss

Una de las causas de pérdida gestacional recurrente es la trombofilia, que se define como una tendencia a la trombosis o hipercoagulabilidad, con variabilidad en las manifestaciones clínicas dependiente de la región vascular afectada por la ausencia de flujo sanguíneo. Las trombofilias se pueden clasificar como heredadas y adquiridas de acuerdo con la naturaleza de su causa. Entre las trombofilias heredadas están el factor V Leiden, la protrombina G20210A, la metilentetrahidrofolato reductasa C677T, las deficiencias de los anticoagulantes naturales antitrombina III, proteína C y proteína S, las disfibrinogenemias y la homocistinuria. En el grupo de las trombofilias adquiridas se encuentran el síndrome antifosfolípido, la resistencia a la proteína C activada sin alteraciones en el gen del factor V y la hiperhomocisteinemia leve o moderada.Este artículo es una revisión de la literatura de estudios recientes que han buscado la asociación entre las diferentes trombofilias y la pérdida gestacional recurrente. Se incluyen las recomendaciones diagnósticas, profilácticas y terapéuticas para mujeres con trombofilia y pérdida gestacional.