Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two distinct morphological patterns of cell death have been recognized, termed necrosis and apoptosis. Apoptosis, or programmed cell death, occurs in both physiological and pathological conditions. It arises due to an elevation of cytosolic free calcium concentration resulting in activation of a nuclear endonuclease. Activated endonuclease produces oligonucleosome-length DNA fragments. This DNA cleavage can directly precipitate cell death. Both glucocorticoids and TCDD may induce apoptosis by production of a heat labile factor that mediates calcium influx whereas tributyltin causes the opening of calcium channels. Evidence that perturbation in calcium homeostasis is an important event in cell necrosis is becoming increasingly persuasive, but the events that propagate the lesion are still unclear. Despite evidence for cytoskeletal disruption, activation of degradative enzymes such as proteases and phospholipase A2 and stimulation of other enzymes such as poly (ADP-ribose) polymerase, the exact role that these play in cell killing is not resolved. Indeed, recently the radical dichotomy between apoptosis and necrotic cell death has come into question. It is clear that further work is required to determine the role played by some elements of the apoptotic process in chemically induced cell death.
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PMID:Mechanisms of cell death. 192 63

Resistance to the anticoagulant effects of activated protein C (APC) is now considered the most prevalent cause of inherited thrombophilia. The great majority of patients with activated protein C resistance (APCR) have a missense mutation in the factor V molecule (factor V Leiden, FVR506Q) resulting in defective inactivation of factor Va due to a loss of an APC cleavage site. The diagnosis of APCR has been based upon the inability of APC to prolong the activated partial thromboplastin (aPTT) clotting time in subjects with APCR. However, this assay has a number of deficiencies which limit its general use. We have evaluated a newly described one-stage tissue factor dependent factor V coagulation assay for APCR in 117 patients and controls and compared the results of this assay in a blinded manner to a polymerase chain reaction (PCR) based assay for the molecular defect of factor V Leiden. 43% (50/117) of the patients studied were receiving coumadin or heparin, or had a lupus anticoagulant. The tissue factor dependent factor V assay had 100% specificity and sensitivity for factor V Leiden and successfully predicted a homozygous state in the three documented homozygotes. The PCR-based assay for factor V Leiden resulted in a single false positive assay due to a silent A to C transition at nucleotide 1692 resulting in the loss of the Mnl restriction endonuclease cleavage site. The single-stage tissue factor dependent factor V assay is a highly sensitive and generally applicable assay for APCR.
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PMID:Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance. 894

A new genetic test has been developed for detection of the mutation known as factor V Leiden. The test employs heteroduplex technology and comprises a single PCR reaction followed immediately by PCR product analysis. It therefore represents the minimum practical route from blood/tissue sample to genetic result. A cohort of 100 patients with a history of thrombosis have been screened using both the new heteroduplex test and a previously described PCR-restriction endonuclease test. Results gave 100% correlation: normals 75 (75%), heterozygotes 24 (24%) and homozygotes 1 (1%). The heteroduplex test has been shown to give straightforward diagnosis in three different analytical systems: standard polyacrylamide gel electrophoresis (PAGE), mini-gel PAGE and capillary electrophoresis. The latter system is semiautomated, therefore rapid through-put of large sample numbers is now possible.
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PMID:Genetic diagnosis of factor V Leiden using heteroduplex technology. 903 60

The factor V Leiden mutation, a G-->A transition at position 1691 in exon 10 of the gene that codes for factor V, produces an Arg506Gln substitution and is the most common genetic risk factor for venous thrombosis. We have developed a rapid, sensitive, and specific method to detect the factor V Leiden mutation in genomic DNA from whole blood by PCR amplification and microparticle enzyme immunoassay detection using the Abbott LCx instrument. We compared this automated method with the standard procedure using restriction endonuclease digestion of PCR products followed by gel electrophoresis in blinded experiments. In 130 patients (from Veterans Affairs medical centers) with deep venous thromboses, including 24 heterozygotes with the factor V Leiden mutation, there was complete agreement between the two methods. The assay was also able to distinguish heterozygotes from homozygotes. This method, which carries a low potential for cross-contamination of samples, should be a useful routine test for the factor V Leiden mutation in clinical laboratories with sufficient demand for molecular diagnostic assays using the LCx instrument.
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PMID:Automated detection of the factor V Leiden mutation using the LCx microparticle enzyme immunoassay. 989 36

Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Analysis for both mutations is increasingly being performed on patients exhibiting hypercoagulability. The goal of the current study was to evaluate the performance of primer-engineered multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the simultaneous detection of factor V Leiden and prothrombin G20210A. Primer-engineered multiplex PCR-RFLP methods for the detection of factor V Leiden and prothrombin G20210A from the medical literature were reviewed. A modified method was optimized in which both mutations generate HindIII RFLPs and the prothrombin amplicon contains an invariant HindIII recognition site to assess the completeness of endonuclease digestion. Digested amplification products were analyzed by agarose gel electrophoresis in a single gel lane and visualized by ethidium bromide. Primer-engineered multiplex PCR-RFLP was used to analyze 205 human genomic DNA samples whose factor V Leiden genotypes had been previously determined by MnlI PCR-RFLP. Complete concordance for factor V Leiden genotypes was observed between the two methods in the 205-sample cohort comprising 139 wild-type, 62 heterozygous mutant, and four homozygous mutant individuals. For prothrombin G20210A, primer-engineered multiplex PCR-RFLP identified 196 wild-type and nine heterozygous mutant individuals in the 205-sample cohort. To independently verify prothrombin genotypes, the nine heterozygous mutants and an additional 11 wild-type patient samples (representing 10% of patient samples) were subjected to DNA sequencing. Complete concordance was observed between DNA sequencing and primer-engineered multiplex PCR-RFLP results. In further validation, 123 of the DNA samples consisting of four heterozygous mutant and 119 wild type individuals were genotyped with the Invader Assay for Factor II (prothrombin G20210A). Results showed 100% concordance between the Invader Assay and primer-engineered multiplex PCR-RFLP. A primer-engineered multiplex PCR-RFLP based on single restriction endonuclease digestion has been evaluated and shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations. The method is robust and readily adaptable to the clinical molecular diagnostic laboratory.
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PMID:Analytical evaluation of primer engineered multiplex polymerase chain reaction-restriction fragment length polymorphism for detection of factor V Leiden and prothrombin G20210A. 1122 20

The G20210A mutation variant of prothrombin gene is the second most frequent mutation identified in patients with deep venous thrombosis, after factor V Leiden. The risk for developing deep venous thrombosis is high in patients identified as heterozygous for G20210A mutation. In order to identify this polymorphism in the gene coding prothrombin, the 345bp fragment in the 3'- untranslated region of the prothrombin gene was amplified using amplification by polymerase chain reaction and enzymatic digestion by HindIII (restriction endonuclease enzyme). The products of amplification and enzymatic's digestion were analized using agarose gel electrophoresis. We investigated 20 patients with venous leg ulcers and we found 2 heterozygous (10%) for G20210A mutation. None of the patients in the control group had G20210A mutation. Our study confirms the presence of G20210A mutation in the Romanian population. Our study also shows the link between venous leg ulcers and this polymorphism in the prothrombin gene.
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PMID:G20210A prothrombin gene mutation identified in patients with venous leg ulcers. 1206 73

Genetic abnormalities in hemostatic proteins associated with hypercoagulability are an important hereditary risk factor for venous thrombosis. Several genetic mutations that cause hereditary disorders predisposing to thrombosis have been described, point mutation in the coagulation factor V gene (FV:R506Q), called factor V Leiden, being the most common of them. A new inexpensive and simple polymerase chain reaction-single-strand polymorphism (PCR-SSCP) based method for detection of this genetic abnormality is reported. The study population consisted of 150 subjects whose factor V genotype was previously determined by PCR-RFLP method using the Mnl I restriction endonuclease. A 223-bp fragment containing the G1692-A (Arg 506-Gln) polymorphic site in exon 10 of the factor V gene was amplified, denatured, and run overnight on the commercially available GMA gels for SSCP. PCR-SSCP analysis showed reproducible and uniform band patterns for FV mutant and wild type alleles. Furthermore, PCR-SSCP results were consistent with those obtained with PCR-RFLP analysis (100%). The described PCR-SSCP procedure is reliable, time-saving, and cost-effective. The method may be considered as a potentially powerful new tool in the routine detection of factor V Leiden.
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PMID:Detection of factor V Leiden by PCR-SSCP using GMA precast Elchrom scientific gels. 1450 11

During the last years several genetic markers have appeared which were extensively studied for their clinical consequences and impact. Therefore, we developed 14 new genetic tests using the TaqMan technology. The new test systems detect the alpha1-antitrypsin, ACE, apolipoprotein B-100, apolipoprotein E, factor V Leiden, prothrombin, HFE, MTHFR, COL1A1, VDR and HLA-B27 mutations. These new kits were compared to the established endonuclease restriction digestion and flow cytometry, respectively. The results showed, that the allelic discrimination assays (TaqMan method) were in 100% concordance with the formerly used digestion method. Flow cytometry revealed a lower specificity in contrast to the TaqMan PCR system. Thus, it could be demonstrated that the new TaqMan assays are robust, rapid and automated methods for high throughput applications which avoid time consuming (and therefore expensive) and difficult post-PCR steps.
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PMID:A highly reproducible and economically competitive SNP analysis of several well characterized human mutations. 1520 39

Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Molecular diagnostic testing for factor V Leiden and prothrombin G20210A is widespread, and laboratories use a variety of technical approaches. Here we introduce a multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based on single (Mn/l) restriction endonuclease digestion. The assay was shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations.
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PMID:Multiplex PCR-RFLP assay for detection of factor V Leiden and prothrombin G20210A. 1568 66

The etiology of Perthes' disease is unclear. Recent reports have suggested that inheritable thrombophilic disorders may be one of its pathogenetic causes. The G20210A prothrombin gene, factor V Leiden, and MTHFR C677T mutations have been identified as predisposing genetic factors for thrombosis. Ninety children diagnosed with Perthes' disease were studied. A family history of thrombosis and any other personal thromboembolic events were researched. PCR and endonuclease digestion were used to analyze factor V Leiden, prothrombin G20210A, and MTHFR C677T. Two hundred healthy donors were included as a control group. No patient had a family or personal history of early thrombotic events. Four children with Perthes' disease (4.4%) were heterozygous for G20210A polymorphism compared with controls (odds ratio: 2.07; 95% confidence interval: 0.40-8.46). No association between factor V Leiden and Perthes' disease was observed. Three patients (3.33%) were heterozygous for factor V Leiden (odds ratio: 1.36; 95% confidence interval: 0.32-5.84). The prevalence of different genotypes of C677T MTHFR did not show statistical differences compared with controls. Eleven patients were homozygous for this polymorphism (odds ratio: 1.02; 95% confidence interval: 0.42-2.44). This study does not support the screening of this group of polymorphism in patients with Perthes' disease.
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PMID:Legg-perthes disease and heritable thrombophilia. 1595 94


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