Double Fluorescent-amplification Refractory Mutation Detection (dF-ARMS) of the Factor V Leiden and Prothrombin Mutations

1999 ◽  
Vol 81 (01) ◽  
pp. 76-80 ◽  
Author(s):  
Caroline Maher ◽  
Dolores Crowley ◽  
Carmel Cullen ◽  
Carmel Wall ◽  
David Royston ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Detection System ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Direct Dna Sequencing ◽  
Genescan Analysis ◽  
Assay Design ◽  
Polymerase Chain ◽  
Compound Heterozygotes

SummarySimultaneous fluorescent [F] detection of the factor V Leiden (G1691A) and the prothrombin 3’-untranslated region (G20210A) mutations were performed in a single tube polymerase chain reaction (PCR). Amplification refractory mutation detection system (ARMS) formed the basis of this assay design. Fluorescent-labelled primers incorporated into amplicons during the reaction facilitated detection directly by GeneScan analysis without further manipulation. To test the efficacy of this double [F]-ARMS (dF-ARMS) method, 48 patients with unexplained thrombotic tendencies were investigated for their factor V Leiden and prothrombin genotypes. These results corresponded exactly with data achieved using the more conventional methods of restriction fragment length polymorphism (RFLP)-PCR and direct DNA sequencing. Three out of the 48 patients in this group were found to be compound heterozygotes.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

THROMBOTIC EVENTS IN COMPOUND HETEROZYGOTES FOR A HIGH AFFINITY HEMOGLOBIN VARIANT: Hb MILLEDGEVILLE [α44(CE2)Pro→Leu (α2)] AND FACTOR V LEIDEN

Hemoglobin ◽  
2002 ◽  
Vol 26 (3) ◽  
pp. 285-290 ◽  
Author(s):  
Michel Hanss ◽  
Philippe Lacan ◽  
Martine Aubry ◽  
Anne Lienhard ◽  
Alain Francina
Keyword(s):  
Factor V Leiden ◽  
Factor V ◽  
Hemoglobin Variant ◽  
High Affinity ◽  
Compound Heterozygotes

The Polymerase Chain Reaction with Sequence Specific Primers for the Detection of the Factor V Mutation Associated with Activated Protein C Resistance

1995 ◽  
Vol 74 (03) ◽  
pp. 874-878 ◽  
Author(s):  
Nancy E Kirschbaum ◽  
Paul A Foster
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Chain Reaction ◽  
Specific Primers ◽  
Allele Specific ◽  
Fv Leiden ◽  
Polymerase Chain

SummaryThe prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by MnII digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

scholarly journals Evaluation of Electronic Microarrays for Genotyping Factor V, Factor II, and MTHFR

2003 ◽  
Vol 49 (5) ◽  
pp. 732-739 ◽  
Author(s):  
Maria Erali ◽  
Ben Schmidt ◽  
Elaine Lyon ◽  
Carl Wittwer
Keyword(s):  
Molecular Biology ◽  
Mutation Detection ◽  
Methylenetetrahydrofolate Reductase ◽  
Factor V Leiden ◽  
Factor V ◽  
Wild Type ◽  
Sequence Variations ◽  
Factor Ii ◽  
Additional Sequence ◽  
Detection Of Mutations

Abstract Background: Genetic risk factors associated with venous thrombosis include mutations in the factor V (Leiden), factor II (prothrombin), and methylenetetrahydrofolate reductase (MTHFR) genes. We evaluated a method using electronically addressable microarrays for the detection of mutations in these genes that have been associated with vascular disease. Methods: The NanoChip® Molecular Biology Workstation (Nanogen) uses electronic microarrays for mutation detection. Factor V, factor II, and MTHFR genotypes identified in the NanoChip system on 225 samples were compared with genotypes from LightCycler® assays (Roche). We determined within- and between-cartridge signal and ratio variation and analyzed the effect of additional mutations at or near the detection area used for the NanoChip assays. Results: Genotypes determined for all three mutations on the NanoChip platform were in complete concordance with LightCycler results. Within-cartridge signal variation as measured by the CV of fluorescence signals was <10% for each allele when present. The within-cartridge CV for heterozygous mutant/wild-type ratios was <8.5%, and between-cartridge CV was <18%. A dilution study showed that results could be obtained in this assay with 6 ng of nucleic acid per PCR, the lowest input tested. The presence of additional sequence variations near the expected mutations can produce equivocal or discrepant results. Conclusions: Mutation detection using the NanoChip Molecular Biology Workstation was accurate and reproducible for the three assays evaluated.

Allelic Discrimination of Factor V Leiden Using the GeneAmp® 5700 Sequence Detection System

2002 ◽  
Vol 88 (12) ◽  
pp. 1071-1072 ◽  
Author(s):  
SE Monk ◽  
AW Duckworth ◽  
J Farrugia ◽  
JA Copplestone ◽  
SAJ Rule
Keyword(s):  
Detection System ◽  
Factor V Leiden ◽  
Factor V ◽  
Allelic Discrimination ◽  
Sequence Detection

Acquired Resistance to Activated Protein C (aAPCR) Is Associated with Increased Risk of Deep Vein Thrombosis in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3484-3484
Author(s):  
Francesca Elice ◽  
Louis Fink ◽  
Guido J. Tricot ◽  
Teresa J. Milner ◽  
Bart Barlogie ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Acquired Resistance ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Response To Therapy ◽  
Response To Treatment ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Apc Resistance ◽  
Increased Risk

Abstract Non factor V Leiden APC resistance (aAPCR) has been described in cancer patients and found to be associated with an increased risk of deep venous thrombosis (DVT). We analyzed the incidence and clinical impact of APC resistance in a large group of multiple myeloma patients. A total of 1178 myeloma patients were tested for APC resistance using an aPTT-based assay in the presence of excess of factor V-deficient plasma and the ratio with or without APC was calculated (≤ 2.00 was considered abnormal). PCR amplification of genomic DNA was used to detect Factor V Leiden. Abnormal APC resistance was found in 109 patients (9.3%), 83 of those were tested for factor V Leiden, 31 had the mutation and 52 (63%) did not have it. Analyzing a subgroup of 254 chemotherapy naïve patients, APC ratio was abnormal in 11% of patients and two third of them were not carriers of factor V Leiden mutation. The presence of aAPC resistance was associated with an increased risk for DVT: 27.9% in patients with aAPCR vs.12.3% in the others (P = 0.008); 22.6% in patients with factor V Leiden mutation. In 32 patients with abnormal aAPCR, the test was repeated: 31/32 patients normalized their APC ratio in sequential testing. Correlation between myeloma baseline markers (serum and urine M-component, beta2-microglobulin, CRP, IL-6), response to treatment and APC activity were studied. In this analysis active disease emerged as the most important factor associated with aAPCR, as 19 patients with normalization of the APC ratio had a concomitant clinical response to therapy. We concluded that aAPCR is a transient finding in myeloma patients that showed a significant correlation with development of DVT.

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