Activated protein C resistance, Leiden mutation, anticoagulant proteins and fibrinogen levels in patients with deep venous thrombosis

Resistance to activated protein C (APC-R) is the leading cause of deep vein thrombosis (DVT). Deficiencies of protein C (PC) or its cofactor protein S (PS) are less frequent. Resistance usually results from nucleotide substitution (1691 GA) in the factor V gene (Leiden mutation). We searched for APC-R and the Leiden mutation (FVL) and measured the activities of PC, PS, antithrombin III (AT III) and Fb levels in patients with DVT. The results were analyzed against a history of thrombosis (idiopathic, risk factor related). We enrolled 29 patients aged 50 years or younger, with first symptoms of thrombosis detected before the age of 40 years. The control group consisted of 25 healthy volunteers of similar age. APC-R was established using Accelerimat (bioMerieux) assays. APC-R was diagnosed when the normalized sensitivity coefficient "r" was < 0.9. FVL was detected with the SSP-PCR (sequence specific primers-polymerase chain reaction) method. Abnormal APC-R "r" values were found in seven DVT patients (24%). Heterozygous (G/A genotype) form of the Leiden mutation was confirmed in six of them (all had a history of recurrent thrombosis). FVL carriers demonstrated lower PC levels in comparison with controls and DVT without FVL. Eight patients (27%) had PS activities below the cut-off point (60%). The deficiency in six of them was not associated with other abnormalities. Patients with recurrent thrombosis had markedly higher concentrations of Fb (usually without FVL) and reduced AT III activities. The study has shown that the APC-R test is useful for FVL screening. The Leiden mutation, elevated levels of Fb and reduced activities of PC are the main factors predisposing to DVT and its recurrence. Reduced activity of PS is usually an isolated abnormality tending to predispose to a single DVT episode.     

单碱基延伸法检测乙型肝炎病毒YMDD基序变异

目的 建立一种简便、快速检测乙型肝炎病毒(HBV)P基因区色氨酰-蛋氨酰-天冬氨酰-天冬氨酰(YMDD)基序变异混合株组成的检测方法.方法 聚合酶链反应(PCR)扩增含有YMDD基序的聚合酶(P)基因区片段,将PCR产物捕获至链亲合素包被的微孔板,以探针与PCR产物杂交,该探针3'末端位于待检测变异点上游一位碱基;在4个独立的微孔中分别加入4种荧光素标记的双脱氧核苷酸(Fluorescien-12-ddNTP)及Klenow酶进行单碱基延伸反应;酶标抗荧光素抗体结合Fluorescien-12-ddNTP,加底物显色,测定各孔吸光度,根据公式计算野生株及变异株在混合毒株中所占百分比.结果 以野生株和变异株单克隆质粒不同比例混合的标准品,验证本方法可检测混合株中含量少至10%的毒株,模板的适宜浓度范围为100 fg/ml～1 ng/ml.结论 单碱基延伸法是一种简便、快速、灵敏度和特异性较高的点突变检测方法,且可以检测混合株中各成分所占百分比.     

Factor V Leiden mutation is not a predisposing factor for acute coronary syndromes

BackgroundThe prevalence of Coronary artery disease (CAD) in India has increased considerably over the past few years and could become the number one killer disease if interventions are not done. Factor V Leiden (FVL) mutation and FII G20210A polymorphism are two recently described genetic factors with a propensity towards venous thrombosis. This warrants the investigations for thrombophilia in myocardial infarction patients in India.MethodsThe study cohort consisted of 51 patients aged below 50 years presenting with acute coronary syndromes. In both patient group and normal individuals the major risk factors Protein C deficiency, Protein S deficiency, anticardiolipin antibodies, Fibrinogen and Lipoprotein [a] were studied. Factor V Leiden (FVL) G1691A mutation in both control and patient group was looked by using Polymerase chain reaction (PCR) followed by sequencing of the PCR products.ResultsOur results indicated significantly higher levels of anticardiolipin antibodies and fibrinogen in the patients and absence of FVL (G1691A) mutation in our study cohort. One of the patients (H5) showed insertion of an extra A nucleotide in exon 10 of the Factor V gene resulting in frame shift mutation in this patient.ConclusionThe results of present study showed absence of FVL mutation in our population. However, there is a need to confirm the above findings on patients from different populations from different parts of the country. The insertion of an extra A in exon 10 in the patient needs to be ascertained to confirm that it is one of its kinds or is prevalent in the population.     

Prevalence of factor V Leiden in south Tunisian blood donors

Venous thrombosis (VT) is a common disease with multifactorial pathogenesis. Factor V Leiden mutation (G1691A) (FVL) is the most common risk factor in venous thrombosis. The prevalence of FVL varies according to geography and ethnicity. Hence, in several countries there is a difference in the frequency of this mutation between the southern, central and north. In Tunisia, no data is available about prevalence of FVL mutation by geographical origin. For this reason, we sought the prevalence of FVL mutation in blood donor of south Tunisia population. FVL has been detected by APCR-test and confirmed by PCR-RFLP and sequencing. Two hundred fifty blood donors, different in age and sex were included in this study to determine the prevalence of FVL in blood donors. FVL mutation was found in 13.6% of the studied population. Thirty-one were heterozygous and three persons were homozygous with a rate of 12.4 and 1.2%, respectively. In conclusion, FVL mutation is very common in south Tunisian population.     

Factor V Leiden mutation in venous thrombosis in southeast Turkey

Venous thrombosis (VT) is a common disease, with an annual incidence in the general population of approximately 1 per 1,000. Factor V Leiden mutation (G1691A) (FVL) is the most common risk factor in venous thrombosis. The prevalence of FVL for thrombosis varies greatly in different regions of the world. FVL mutation has been identified both by conventional method and fluorescence resonance energy transfer (FRET) with the LightCycler. Sixty-one patients with VT, different in age and sex, were consecutively entered into this study to assess the prevalence of FVL in VT in southeast Turkey. FVL mutation was found in 24.6% (15/61). Fourteen individuals were heterozygous and 1 homozygous, a rate of 22.9% and 1.6%, respectively. In conclusion, the authors suggest that FVL mutation is common in patients with venous thrombosis in southeast Turkey.     

Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers

The cooperative effects of the GPIa 807TT, MTHFR 677TT and prothrombin 20210GA genotypes with the FV Leiden 1691GA (FVL) genotype were evaluated by comparing these genotype frequencies in 77 asymptomatic and 156 symptomatic heterozygous FVL carriers. The GPIa 807TT and MTHFR 677TT genotypes did not segregate within the symptomatic FVL carrier group and did not contribute to venous thrombotic risk in this patient cohort. There was no difference in the prothrombin 20210GA genotype frequency between asymptomatic FVL carriers and a random Caucasian control group; however, the prothrombin 20210GA genotype was nearly 5 times as prevalent (19/156 v 2/77; P < 0.02) in the symptomatic FVL carriers (odds ratio 5.21; 95% confidence interval 1.20-47.62), demonstrating that this important prothrombotic risk factor acts synergistically with FVL.     

应用免疫-PCR检测弓形虫循环抗原的实验研究

目的　控索弓形虫感染早期诊断方法。方法　用链亲素将生物素化的二抗和生物素化的 DNA偶联起来 ,通过PCR扩增标记 DNA检测固定的抗原 ,建立了弓形虫循环抗原免疫— PCR检测技术 ,用该技术和 EL ISA法平行检测系列稀释的弓形虫的感染鼠阳性血清中的 CAg以及实验感染鼠血清中 CAg的动态变化 ,对比研究二者之间的敏感性。结果　免疫—PCR法检测弓形虫 CAg吴阳性的血清最高稀释度为 1∶ 10 0 0 ,EL ISA法检测弓形虫 CAg呈阳性的血清最高稀释度为 1∶ 5 ,免疫— PCR最早在鼠感染弓形虫后第 3天测到 CAg,EL ISA最早在鼠感染弓形虫后第 5天测到 CAg。结论　免疫— PCR是一种特异性强、敏感性高的弓形虫 CAg检测方法 ,敏感性较 EL ISA法提高了 2 0 0倍 ,免疫— PCR检出阳性结果时间早 ,为弓形虫病早期诊断提供了科学依据     

Hereditary Thrombophilic Factors in Stroke Due to Cerebral Infarct

BackgroundThe stroke is the third most common cause of all deaths. In new studies, the importance of hereditary thrombophilic factors on stroke is emphasized. The aim of this study is to determine the role of hereditary thrombophilic factors including factor V Leiden A1691G (FVL), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T gene mutations in patients with stroke because of cerebral infarct.MethodsTwenty-four patients with stroke and 53 controls with risk factor for stroke were enrolled. Polymerase chain reaction was used to detect these mutations.ResultsHeterozygote FVL mutation in 2 (8.3%) patients and MTHFR mutation in 10 (41.7%) patients were detected. In the control group, there were 2 (3.8%) patients with heterozygote FVL mutation and 15 (28.3%) patients with MTHR mutation. Both FVL and MTHFR gene mutations were detected in 1 patient and 2 controls, respectively. Prothrombin gene mutation was not found in 2 groups. There were not statistically significant differences for all 3 mutations in-between 2 groups (P > 0.05). Odds ratios were 0.431 (0.074–2.504, 95% CI) for FVL mutation and 0.553 (0.221–1.381, 95% CI) for MTHFR mutation, respectively.ConclusionAlthough our study group was small, hereditary thrombophilic factors might not be risk factors for stroke because of cerebral infarct.     

A matrix-assisted laser desorption/ionization time-of-flight based method for screening the 1691G --> A mutation in the factor V gene.

A point mutation, 1691 G --> A in the coagulation factor V gene results in an Arg506 --> Gln amino acid substitution in the factor V molecule. This mutation, defined as factor VLEIDEN, results in activated protein C (APC) resistance and is the most common genetic risk factor for familial thrombophilia. A new mini-sequencing method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed for the screening of the 1691G --> A substitution in factor V. In this method, a fragment of genomic DNA containing the 1691st base is first amplified, followed by mini-sequencing in the presence of dGTP and ddATP, ddCTP, and ddTTP. In this manner, the primer is extended by one base from one allele and two bases from the other allele. The extended products are analyzed using MALDI-TOF mass spectrometry. The base at position 1691 is identified based on the number of nucleotides added. We have used this method to genotype 16 APC-resistant patients previously identified by conventional methods and 11 normal control samples. The genotypes of all samples were correctly identified. This method is accurate, fast, and potentially allows for simultaneous multiplex genotyping of a number of mutation sites associated with thrombophilia and clot formation.     

Clinical evaluation of a functional prothrombin time-based assay for identification of factor V Leiden carriers in a group of Italian patients with venous thrombosis.

The efficiency of a new prothrombin-based activated protein C (APC) resistance test to detect factor V Leiden (FVL) was clinically evaluated in 150 Italian patients with deep venous thrombosis. Patient samples are diluted in factor-V-deficient plasma, an APC-containing reagent, and specific factor V activator; after incubation, clotting is initiated by addition of activated-factor-FV-dependent prothrombin activator. Two prothrombin time determinations were performed under identical assay conditions except that no APC was added to one. A ratio over 4.2 for normal individuals and under 2.0 for FVL patients is expected: between 1.3 and 1.9 for FVL heterozygotes, and between 1.0 and 1.1 for FVL homozygotes. Using a predefined cut-off ratio of 2.0, a specificity and a sensitivity of 1.00 for detection of FVL mutation were found. With a cut-off ratio of 1.1, a specificity of 0.98 and a sensitivity of 1.00 were found for discrimination between FVL heterozygous (n = 60) and homozygous (n = 6). No interferences by heparins, oral contraceptives, oral anticoagulant therapy, protein C, protein S, D-dimer, homocysteine, MTHFR mutations and antiphospholipid autoantibodies were detected. In our experience, this new prothrombin time-based APC resistance assay provides improved discrimination between normal individuals and FVL carriers compared with the classical methods. Moreover, this new assay allows good discrimination between homozygous and heterozygous FVL carriers. In the authors' experience this prothrombin time-based method was not influenced by many factors compared with the classical activated partial thromboplastin time-based method.     

Hereditary thrombophilic risk factors and venous thromboembolism in Istanbul, Turkey: the role in different clinical manifestations of venous thromboembolism.

The aim of this study was to investigate the hereditary thrombophilic risk factors in patients with venous thromboembolism (VTE) and whether these risk factors play a different role in patients with isolated pulmonary embolism (PE) as compared with patients with deep vein thrombosis (DVT) and patients with PE + DVT. The protein C (PC), protein S, antithrombin activities, homocysteine levels, and factor V Leiden (FVL) G1691A and prothrombin G20210A mutations were evaluated in 191 patients with VTE and 191 controls. The prevalence of FVL and PC deficiency were higher in patients (P = .003 and P = .02, respectively). There was no significant difference for the other risk factors. The combination of thrombophilic risk factors was significantly higher in patients with DVT + PE as compared with patients with isolated PE or DVT (P = .04). In conclusion, the most important hereditary risk factors for VTE in this study were the FVL mutation and PC deficiency.     

A novel primer-extension assay for the detection of a G to A mutation in the distal precore region of hepatitis B virus DNA

The roles of genetic heterogeneity of the hepatitis B virus (HBV) precore gene in the pathogenesis of HBV infection are unclear. Various methods have been used to detect nucleotide (nt) 1896 precore mutants. We established a new primer-extension assay to facilitate the detection of these mutants. This assay is based upon the fact that there is no adenine in the distal precore region of wild-type HBV. Polymerase chain reaction (PCR)-amplified template DNA was denatured and annealed to the [γ-³²P]-labelled primer. During primer extension in the presence of DNA polymerase and dCTP, dGTP, dTTP and ddATP, the reaction terminates if there is a nucleotide A. When mixtures of different ratios of wild-type and nt 1896 precore mutants were analysed in the primer-extension assay, correlation between the percentage known amounts and the percentage measured amounts of nt 1896 precore mutants was excellent (r²=0.9669). When the primer-extension assay and direct sequencing were compared in hepatitis B e antigen (HBeAg)-positive and -negative chronic active hepatitis B patients, the primer-extension assay detected a greater number of nt 1896 precore mutants than direct sequencing and thus most HBV infections were found to be mixed infections. In conclusion, the primer-extension assay is a reliable and sensitive method for the detection of nt 1896 precore mutants.     

Genomic subtraction for cloning DNA corresponding to deletion mutations.

We have developed a technique, called genomic subtraction, for isolating the DNA that is absent in deletion mutants. The method removes from wild-type DNA the sequences that are present in both the wild-type and the deletion mutant genomes. The DNA that corresponds to the deleted region remains. Enrichment for the deleted sequences is achieved by allowing a mixture of denatured wild-type and biotinylated mutant DNA to reassociate. After reassociation, the biotinylated sequences are removed by binding to avidin-coated beads. This subtraction process is then repeated several times. In each cycle we hybridize the unbound wild-type DNA from the previous round with fresh biotinylated deletion mutant DNA. The unbound DNA from the final cycle is ligated to adaptors and amplified by using one strand of the adaptor as a primer in the polymerase chain reaction. The amplified sequences can then be used to probe a genomic library. We applied genomic subtraction to a yeast strain that has a 5-kilobase deletion, corresponding to 1/4000th of the genome. In the experiment reported here, three rounds of subtraction were sufficient to accurately identify genomic clones containing sequences that are missing in the deletion mutant. We discuss the limitations and some potential applications of the method.     

乙型肝炎病毒核心蛋白c/el表位处TC标签的基因标记对S和e抗原表达的影响

目的 研究乙型肝炎病毒核心蛋白c/el表位处TC标签的基因标记对S和e抗原表达的影响,探索TC标签标记HBV病毒体的可行性.方法 以含有1.3倍HBV基因组的载体为模板,在HBV C基因中插入编码TC标签的碱基序列,得到重组的突变型HBV载体,其编码的核心蛋白的c/el表位处嵌入了TC标签.将野生型和突变型HBV载体分别瞬时转染HepG2细胞,Westernblot检测野生型和各种TC嵌合型核心蛋白的表达,酶联免疫吸附法分析病毒S和e抗原的表达.多组间比较用单因素方差分析.结果 Western blot显示各种嵌合型核心蛋白在细胞内成功表达,并且同野生型蛋白的表达水平无显著差别,酶联免疫吸附法检测表明各组细胞S和e抗原表达水平没有明显差别.结论 HBV核心蛋白c/el表位处TC标签的基因标记不会明显影响突变HBV载体表达病毒蛋白.     

High prevalence of three prothrombotic polymorphisms among Palestinians: factor V G1691A, factor II G20210A and methylenetetrahydrofolate reductase C677T

Factor V leiden G1691A/R506Q (FVL), prothrombin G20210A (FII) and methylenetetrahydrofolate reductase (MTHFR) C677T are related genetic risk factors for venous thromboembolism. Analysis for those mutations is increasingly being performed on patients exhibiting hypercoagulability. The objective of this study was to determine the prevalence of FVL, FII-G20210A and MTHFR-C677T polymorphisms and their coexistence among apparently healthy Palestinians. After institutional approval, 303 apparently healthy students from An-Najah University representative to North and South regions of West Bank with no previous history of cardiovascular diseases participated in this study. A uniform questionnaire was used to collect relevant information through personal interview with the subjects. The collected information included gender, age, smoking habits, weight and height, diseases such as diabetes, cardiovascular and family history of CVD. The frequencies of allelic distribution of the three prothrombotic polymorphisms factor V G1691A/R506Q), prothrombin G2010A, and MTHFR-C677T were 0.114, 0.050 and 0.071, respectively. The prevalence of the three thrombotic polymorphisms (FVL, FII G20210A and MTHFR-C677T) were 20.1, 9.1 and 13.8?%, respectively. Statistical analysis for factor V leiden showed no significant association between place of residence (P value?=?0.953) and gender (P value?>0.082). The data presented in this study showed the highest prevalence of FVL among healthy Palestinians compared to other populations and this important finding should be followed in terms of clinical significance.     

Genetic risk factors in young adults with 'cryptogenic' ischemic cerebrovascular disease.

Mutations such as factor V Leiden G1691A (FVL), prothrombin G20210A (FIIM), methylenetetrahydrofolate reductase (MTHFR) C677T, cystathionine beta-synthase (CBS) 844ins68 and endothelial cell protein C receptor (EPCR) 4031ins23 are risk factors for thromboembolism. To assess the role of these mutations in young adults with cerebral ischemia of otherwise undetermined etiology, 93 patients younger than 50 years old with thromboembolic strokes or transient ischemic attacks were studied. One hundred and eighty-six healthy age-matched and sex-matched blood donors served as controls. The FVL mutation was detected in 15/93 patients and 13/186 controls. After adjustment for smoking, arterial hypertension, and hyperlipidemia, the association of the FVL mutation with cerebral ischemia [odds ratio (OR), 3.19; 95% confidence interval (CI), 1.38-7.39] remained significant. One of 93 patients and 6/186 controls were carriers of FIIM (OR, 0.33; 95% CI, 0.04-2.75). We detected the MTHFR TT677 genotype in 9/93 patients and 26/186 controls (OR, 0.66; 95% CI, 0.30-1.47), a CBS 844ins68 mutation in 12/93 patients and 19/186 controls (OR, 1.30; 95% CI, 0.60-2.81), and an EPCR 4031ins23 mutation in 1/93 patients and in no control individual (P = 0.33). In conclusion, in younger adults the FVL mutation is a risk factor for cerebrovascular disease. FIIM, the MTHFR TT677 genotype and the CBS 844ins68 mutation did not contribute to the risk in this group of patients. The EPCR 4031ins23 mutation is very rare, its possible role needs further investigation.     

Anticodon-dependent aminoacylation of a noncognate tRNA with isoleucine, valine, and phenylalanine in vivo.

An assay based on the initiation of protein synthesis in Escherichia coli has been used to explore the role of the anticodon in tRNA identity in vivo. Mutations were introduced into the initiator tRNA to change the wild-type anticodon from CAU (methionine) to GAU (isoleucine), GAC (valine), and GAA (phenylalanine), where each derivative differs from the preceding by a single base change in the anticodon (underlined). These changes were sufficient to cause the mutant tRNAs to be aminoacylated by the corresponding aminoacyl-tRNA synthetases based on the amino acid inserted into protein from complementary initiation codons. Construction of additional single base anticodon variants (GUU, GGU, GCC, GUC, GCA, and UAA) showed that all three anticodon bases specify isoleucine and phenylalanine identity and that both the middle and the third anticodon bases are important for valine identity in vivo.