Detection of Genomic Polymorphisms Associated with Venous Thrombosis Using the Invader Biplex Assay
2004; Elsevier BV; Volume: 6; Issue: 2 Linguagem: Inglês
10.1016/s1525-1578(10)60502-8
ISSN1943-7811
AutoresMadhumita Patnaik, Jeffrey S. Dlott, Robert N. Fontaine, M.T.R. Subbiah, Martin J. Hessner, Kelly A. Joyner, Marlies Ledford, Eduardo C. Lau, Cynthia Moehlenkamp, Jean Amos, Bailing Zhang, Thomas Williams,
Tópico(s)Hemophilia Treatment and Research
ResumoA multi-site study to assess the accuracy and performance of the biplex Invader assay for genotyping five polymorphisms implicated in venous thrombosis was carried out in seven laboratories. Genotyping results obtained using the Invader biplex assay were compared to those obtained from a reference method, either allele-specific polymerase chain reaction (AS-PCR), restriction fragment length polymorphism (PCR-RFLP) or PCR-mass spectrometry. Results were compared for five loci associated with venous thrombosis: Factor V Leiden, Factor II (prothrombin) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, and plasminogen activator inhibitor (PAI-1) 4G/5G. Of a total of 1448 genotypes tested in this study, there were 22 samples that gave different results between the Invader biplex assay and the PCR-based methods. On further testing, 21 were determined to be correctly genotyped by the Invader Assay and only a single discrepancy was resolved in favor of the PCR-based assays. The compiled results demonstrate that the Invader biplex assay provides results more than 99.9% concordant with standard PCR-based techniques and is a rapid and highly accurate alternative to target amplification-based methods. A multi-site study to assess the accuracy and performance of the biplex Invader assay for genotyping five polymorphisms implicated in venous thrombosis was carried out in seven laboratories. Genotyping results obtained using the Invader biplex assay were compared to those obtained from a reference method, either allele-specific polymerase chain reaction (AS-PCR), restriction fragment length polymorphism (PCR-RFLP) or PCR-mass spectrometry. Results were compared for five loci associated with venous thrombosis: Factor V Leiden, Factor II (prothrombin) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, and plasminogen activator inhibitor (PAI-1) 4G/5G. Of a total of 1448 genotypes tested in this study, there were 22 samples that gave different results between the Invader biplex assay and the PCR-based methods. On further testing, 21 were determined to be correctly genotyped by the Invader Assay and only a single discrepancy was resolved in favor of the PCR-based assays. The compiled results demonstrate that the Invader biplex assay provides results more than 99.9% concordant with standard PCR-based techniques and is a rapid and highly accurate alternative to target amplification-based methods. Venous thrombosis (VT) is a multi-factorial disorder that arises from the combined effects of acquired and inherited risk factors [reviewed in1Lane DA Grant PJ Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease.Blood. 2000; 95: 1517-1532Crossref PubMed Google Scholar]. In this study we compare the accuracy and precision of the Invader-based assays in the analysis of five loci implicated as risk factors for VT. The Factor V Leiden (FVL) mutation, an Arg to Gln change at amino acid 506, leads to resistance to cleavage by activated protein C. It is the most common inherited risk factor for venous thrombosis (VT) and is found in 20 to 40% of cases.2Bertina RM Factor V: Leiden and other coagulation factor mutations affecting thrombotic risk.Clin Chem. 1997; 43: 1678-1683PubMed Google Scholar Another highly characterized variation, the Factor II G20210A mutation in the 3′ untranslated region of the prothrombin gene, is found in fewer cases [18% of thrombosis patients;3Poort SR Rosendaal FR Reitsma PH Bertina RM A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.Blood. 1996; 88: 3698-3703Crossref PubMed Google Scholar], but is viewed as a probable causative factor for VT.1Lane DA Grant PJ Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease.Blood. 2000; 95: 1517-1532Crossref PubMed Google Scholar, 4Kottke-Marchant K Genetic polymorphisms associated with venous and arterial thrombosis: an overview.Arch Pathol Lab Med. 2002; 126: 295-304PubMed Google Scholar A common variation in the methylenetetrahydrofolate reductase (MTHFR) gene, a C to T transition at nucleotide 677 leading to a thermolabile version of the enzyme, has been extensively investigated (reviewed in5Schwahn B Rozen R Polymorphisms in the methylenetetrahydrofolate reductase gene: clinical consequences.Am J PharmacoGenomics. 2001; 1: 189-201Crossref PubMed Scopus (191) Google Scholar). A variation in a putative regulatory domain of MTHFR, A1298C, results in an enzyme with only 60% of the normal level of catalytic activity.6Weisberg I Tran P Christensen B Sibani S Rozen R A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity.Mol Genet Metab. 1998; 64: 169-172Crossref PubMed Scopus (1143) Google Scholar The C677T mutation, alone and in combination with A1298C, is associated with elevated levels of homocysteine (hcy), known to be a risk factor for VT and other cardiovascular diseases.5Schwahn B Rozen R Polymorphisms in the methylenetetrahydrofolate reductase gene: clinical consequences.Am J PharmacoGenomics. 2001; 1: 189-201Crossref PubMed Scopus (191) Google Scholar, 7Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG Boers GJ den Heijer M Kluijtmans LA van den Heuvel LP et al.A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.Nat Genet. 1995; 10: 111-113Crossref PubMed Scopus (5168) Google Scholar, 8Lievers KJ Boers GH Verhoef P den Heijer M Kluijtmans LA van der Put NM Trijbels FJ Blom HJ A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk.J Mol Med. 2001; 79: 522-528Crossref PubMed Scopus (150) Google Scholar Elevated levels of PAI-1 are associated with VT,9Green F Humphries S Genetic determinants of arterial thrombosis.Baillieres Clin Haematol. 1994; 7: 675-692Abstract Full Text PDF PubMed Scopus (32) Google Scholar coronary artery disease,10Anvari A Schuster E Gottsauner-Wolf M Wojta J Huber K PAI-I 4G/5G polymorphism and sudden cardiac death in patients with coronary artery disease.Thromb Res. 2001; 103: 103-107Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar atherothrombotic stroke,11Bang CO Park HK Ahn MY Shin HK Hwang KY Hong SY 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene and insertion/deletion polymorphism of the tissue-type plasminogen activator gene in atherothrombotic stroke.Cerebrovasc Dis. 2001; 11: 294-299Crossref PubMed Scopus (58) Google Scholar and various complications during pregnancy.12Glueck CJ Kupferminc MJ Fontaine RN Wang P Weksler BB Eldor A Genetic hypofibrinolysis in complicated pregnancies.Obstet Gynecol. 2001; 97: 44-48Crossref PubMed Scopus (106) Google Scholar, 13Glueck CJ Phillips H Cameron D Wang P Fontaine RN Moore SK Sieve-Smith L Tracy T The 4G/4G polymorphism of the hypofibrinolytic plasminogen activator inhibitor type 1 gene: an independent risk factor for serious pregnancy complications.Metabolism. 2000; 49: 845-852Abstract Full Text PDF PubMed Scopus (97) Google Scholar A single-base deletion at nucleotide −675 (4G/5G) in the upstream regulatory region of PAI-1 has been associated with increased levels of PAI-1 protein.14Dawson SJ Wiman B Hamsten A Green F Humphries S Henney AM The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in HepG2 cells.J Biol Chem. 1993; 268: 10739-10745Abstract Full Text PDF PubMed Google Scholar Genetic testing has become the standard for determining the presence of these variations.1Lane DA Grant PJ Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease.Blood. 2000; 95: 1517-1532Crossref PubMed Google Scholar The most widely used methods for determining these genotypes use the polymerase chain reaction (PCR), notably restriction fragment length polymorphism (PCR-RFLP)7Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG Boers GJ den Heijer M Kluijtmans LA van den Heuvel LP et al.A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.Nat Genet. 1995; 10: 111-113Crossref PubMed Scopus (5168) Google Scholar, 15Peng F Labelle LA Rainey BJ Tsongalis GJ Single nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene are common in U.S. Caucasian and Hispanic-American populations.Int J Mol Med. 2001; 8: 509-511PubMed Google Scholar and allele-specific-PCR (AS-PCR).16Hessner MJ Luhm RA Pearson SL Endean DJ Friedman KD Montgomery RR Prevalence of prothrombin G20210A, factor V G1691A (Leiden), and methylenetetrahydrofolate reductase (MTHFR) C677T in seven different populations determined by multiplex allele-specific PCR.Thromb Haemost. 1999; 81: 733-738PubMed Google Scholar, 17Falk G Almqvist A Nordenhem A Svensson H Wiman B Allele-specific PCR for detection of a sequence polymorphism in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene.Fibrinolysis. 1995; 9: 170-174Crossref Scopus (75) Google Scholar A novel genotyping method suitable for direct analysis of genomic DNA without target amplification, termed the Invader assay, has been demonstrated to be accurate and effective in detecting FVL.18Ledford M Friedman KD Hessner MJ Moehlenkamp C Williams TM Larson R A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous Invader microtiter plate FRET assay.J Mol Diagn. 2000; 2: 97-104Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Hessner MJ Budish MA Friedman KD Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes.Clin Chem. 2000; 46: 1051-1056PubMed Google Scholar Multi-site studies validated the isothermal microtiter plate-based Invader assay in which wild-type and mutant alleles were assayed individually. In this report, we present results from a multi-site study using an Invader biplex assay in which wild-type and mutant alleles are assayed simultaneously in the same reaction well. Detailed descriptions of the mechanics of the Invader assay have been published elsewhere.20Lyamichev V Mast AL Hall JG Prudent JR Kaiser MW Takova T Kwiatkowski RW Sander TJ de Arruda M Arco DA Neri BP Brow MA Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.Nat Biotechnol. 1999; 17: 292-296Crossref PubMed Scopus (404) Google Scholar, 21Hall JG Eis PS Law SM Reynaldo LP Prudent JR Marshall DJ Allawi HT Mast AL Dahlberg JE Kwiatkowski RW de Arruda M Neri BP Lyamichev VI Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.Proc Natl Acad Sci USA. 2000; 97: 8272-8277Crossref PubMed Scopus (218) Google Scholar, 22Olivier M Chuang L-M Chang M-S Chen Y-T Pei D Ranade K de Witte A Allen J Tran N Curb D Pratt R Neefs H de Arruda Indig M Law S Neri B Wang L Cox DR High-throughput genotyping of single nucleotide polymorphisms using new biplex Invader technology.Nucleic Acids Res. 2002; 30: e53Crossref PubMed Scopus (68) Google Scholar An enhancement to the Invader assay, termed the "biplex" format, has recently been introduced for the direct analysis of genomic DNA. The Invader biplex format differs from previous generations of the assay because it enables simultaneous detection of two DNA sequences in a single well. Most often, two alleles of a mutation or two variants of a particular polymorphism are targeted. The Invader biplex assay uses two different discriminatory primary probes, each with a unique 5′ flap, and two different fluorescence resonance energy transfer (FRET) cassettes, each labeled with a spectrally distinct fluorophore.22Olivier M Chuang L-M Chang M-S Chen Y-T Pei D Ranade K de Witte A Allen J Tran N Curb D Pratt R Neefs H de Arruda Indig M Law S Neri B Wang L Cox DR High-throughput genotyping of single nucleotide polymorphisms using new biplex Invader technology.Nucleic Acids Res. 2002; 30: e53Crossref PubMed Scopus (68) Google Scholar By design, the released 5′ flaps will bind only to their respective FRET cassettes to generate a target-specific signal (Figure 1). This multi-site study designed to evaluate the accuracy and performance of the Invader biplex assay was carried out in seven clinical laboratories. Genotyping results obtained from the Invader biplex assay and AS-PCR or PCR-RFLP were compared for five loci associated with venous thrombosis: Factor V Leiden, prothrombin G20210A, MTHFR C677T and A1298C, and PAI-1 4G/5G. Of a total of 1448 genotypes determined in this study, the Invader biplex assay made only a single incorrect call. Moreover, the Invader assay made correct calls for 21 samples incorrectly genotyped by the PCR-based methods. Thus, the compiled results demonstrate that the Invader biplex assay provides results more than 99.9% concordant with standard PCR-based techniques. Samples used in these studies were chosen randomly. Genomic DNA was archived and stored at 4°C or less for no longer than 24 months. All samples were numbered, unlinked, and tested anonymously. No information was available regarding predisposition to thrombosis, related disorders, or other clinical or demographic factors. All samples were extracted from human whole blood or buffy coat from whole blood collected in EDTA or sodium citrate Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). Genomic DNA was extracted using commercially available kits. Each DNA specimen was quantified using either the Molecular Probes (Eugene, OR) PicoGreen dsDNA quantitation method or the Molecular Probes OliGreen method. Samples extracted by the Generation Capture Plate Kit (Gentra Systems, Minneapolis, MN) were quantified using the Molecular Probes OliGreen Quantitation reagents. Only samples containing final DNA concentrations of ≥10 ng/μl were used in the present study. All sites used PCR-based genotyping reference methods. Each laboratory compared the Invader assay with either the specific PCR-based method currently in use or another widely used method. The primers for each allele as well as the references for the protocols are included in Table 1. Sites one through four determined the FVL and prothrombin genotypes using AS-PCR; sites six and seven determined the PAI-1 genotype by PCR-RFLP; and sites three and five determined the MTHFR C1298T and C677T genotypes by PCR-RFLP. Primers were obtained from Invitrogen (Carlsbad, CA) or Third Wave Technologies, Inc. (Madison, WI).Table 1Protocols for Genotyping Reference MethodsAllele testedAssayPCR primersPositive controls (for AS-PCR) or enzymes (for RFLP)References or reaction conditionsFVLAS-PCR5′-GGGGGACAATTTTCAATATATTTTCTTTCA-GGCAG-3′ (forward)Positive control for the mutant reaction: Prothrombin reverse consensus: 5′-CTC CAA ACT GAT CAA TGA CCT TC-3′.29Hessner MJ Dinauer DM Luhm RA Endres JL Montgomery RR Friedman KD Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers.Br J Haematol. 1999; 106: 237-239Crossref PubMed Scopus (15) Google Scholar30Hessner MJ Pircon RA Johnson ST Luhm RA Prenatal genotyping of the Duffy blood group system by allele-specific polymerase chain reaction.Prenat Diagn. 1999; 19: 41-45Crossref PubMed Scopus (15) Google Scholar31Kirschbaum NE Foster PA The polymerase chain reaction with sequence specific primers for the detection of the factor V mutation associated with activated protein C resistance.Thromb Haemost. 1995; 74: 874-878PubMed Google Scholar5′-GGGGGTTCAAGGACAAAATACCTGTATTCCAC-3′ (WT reverse)Positive control for the WT reaction: human growth hormone (HGH): HGH 1 5′-CCT TCC CAA CCA TTC CCT TA-3′) and HGH2 (5′-CAC GGA TTT CTG TTG TGT TTC-3′5′-GGGGGTTCAAGGACAAAATACCTGTATTCCAT-3′ (mutant reverse)ProthrombinAS-PCR5′-TCT AGA AAC AGT TGC CTG GCA GA-3′ (forward consensus)Positive control for the mutant reaction: Prothrombin reverse consensus: 5′-CTC CAA ACT GAT CAA TGA CCT TC-3′29Hessner MJ Dinauer DM Luhm RA Endres JL Montgomery RR Friedman KD Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers.Br J Haematol. 1999; 106: 237-239Crossref PubMed Scopus (15) Google Scholar30Hessner MJ Pircon RA Johnson ST Luhm RA Prenatal genotyping of the Duffy blood group system by allele-specific polymerase chain reaction.Prenat Diagn. 1999; 19: 41-45Crossref PubMed Scopus (15) Google Scholar31Kirschbaum NE Foster PA The polymerase chain reaction with sequence specific primers for the detection of the factor V mutation associated with activated protein C resistance.Thromb Haemost. 1995; 74: 874-878PubMed Google Scholar5′-CAC TGG GAG CAT TGA GGC AC-3′ (WT reverse)Positive control for the WT reaction: human growth hormone (HGH): HGH1 (5′-CCT TCC CAA CCA TTC CCT TA-3′) and HGH2 (5′-CAC GGA TTT CTG TTG TGT TTC-3′)5′-CAC TGG GAG CAT TGA GGC AT-3′ (mutant reverse)MTHFR C677TPCR-RFLP5′-GCT GAC CTG AAG CAC TTG AAG GAG AAG-3′ (forward)Hinf I7Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG Boers GJ den Heijer M Kluijtmans LA van den Heuvel LP et al.A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.Nat Genet. 1995; 10: 111-113Crossref PubMed Scopus (5168) Google Scholar5′-AGG ACG GTG CGG TGA GAG TG-3′ (reverse)MTHFR A1298CPCR-RFLP5′-CTT TGG GGA GCT GAA GGA CTA CTA C-3′ (forward)Mbo II15Peng F Labelle LA Rainey BJ Tsongalis GJ Single nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene are common in U.S. Caucasian and Hispanic-American populations.Int J Mol Med. 2001; 8: 509-511PubMed Google Scholar32Rady PL Tyring SK Hudnall SD Vargas T Kellner LH Nitowsky H Matalon RK Methylenetetrahydrofolate reductase (MTHFR): the incidence of mutations C677T and A1298C in the Ashkenazi Jewish population.Am J Med Genet. 1999; 86: 380-384Crossref PubMed Scopus (62) Google Scholar5′-CAC TTT GTG ACC ATT CCG GTT TG-3′ (reverse)PAI-1AS-PCR5′-TGC AGC CAG CCA CGT GAT TGT CTA G-3′ (reverse consensus)5′-AAG CTT TTA CCA TGG TAA CCC CTG GT-3′ (positive control forward)17Falk G Almqvist A Nordenhem A Svensson H Wiman B Allele-specific PCR for detection of a sequence polymorphism in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene.Fibrinolysis. 1995; 9: 170-174Crossref Scopus (75) Google Scholar33Segui R Estelles AMY Espana F Villa P Falco C Vaya A Grancha S Ferrando F Aznar J PAI-1 promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects with genetic thrombophilic defects.Br J Haematol. 2000; 111: 122-128Crossref PubMed Scopus (99) Google Scholar5′-GTC TGG ACA CGT GGG GA-3′ (forward; 4G)5′-GTC TGG ACA CGT GGG GG-3′ (forward; 5G)PCR-RFLP5′-GAG AGT CTG GAC ACG TCC GG-3′ (forward) [the two mutagenic bases are underscored]BspEIHomebrew5′-AAC AGC CAC AGG GCA TGC A-3′ (reverse) Open table in a new tab Samples were sequenced on the ABI PRISM 377 DNA Sequencer (Applied Biosystems, Foster City, CA) using primers listed in Table 1 and the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Big Dye Terminator with AmpliTaq FS DNA Polymerase). An existing subset of samples (100 samples) was characterized for PAI-1 4G/5G by PCR-mass spectrometry using the MassARRAY system (Sequenom, San Diego, CA). Genomic DNA was extracted by the Generation Capture Plate kit (Gentra Systems) and amplified by PCR to generate a 98-bp fragment of the PAI-1 gene. The unincorporated deoxynucleotides were digested by shrimp alkaline phosphatase (SAP) enzyme. The amplicon was then mixed with hME extension primer, MassEXTEND enzyme (ie, Thermo Sequenase), and an appropriate termination mixture consisting of dNTPs and ddNTPs, to differentially extend the mutant and wild-type alleles. The final extended product was purified by the addition of SpectroCLEAN (cation-exchange beads), and the purified supernatant was spotted onto a SpectroCHIP. Analysis by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) began when air was removed in high vacuum and a laser pulse caused a spontaneous volatilization and ionization of matrix and associated DNA fragments. These gas-phase ions were accelerated through a voltage potential and their time-of-flight (TOF) was recorded. The TOF results were then converted into mass values, which were calculated, recorded, and interpreted for sample genotype. All Invader assay components were provided by Third Wave Technologies, Inc. Ten μl of genomic DNA samples were dispensed into individual wells of 96-well microtiter plates and denatured at 95°C for 5 minutes. Ten μl of a reaction mix containing the appropriate probes/Invader oligo/MgCl2 mix were added, and reactions were overlaid with 20 μl of molecular biology-grade mineral oil (Sigma Chemicals, St. Louis, MO). Each 20-μl reaction contained 40 ng Cleavase enzyme, 3.5% PEG 8000, 2% glycerol, 0.06% NP 40, 0.06% Tween 20, 12 μg/ml BSA, 0.25 μmol/L each of F (FAM) dye and R (Redmond Red) dye (Epoch Biosciences, Redmond, WA) FRET cassettes, 8 mmol/L MgCl2, 0.5 μmol/L of each allele-specific probe, and 0.05 μmol/L Invader oligo. The sequences of the Invader oligos, wild-type and mutant primary probes, wild-type and mutant FRET probes and corresponding synthetic secondary targets are presented in Table 2. Following the reagent dispensing, plates were incubated at 63°C for 4 hours in a PTC-100 thermal cycler (MJ Research, Waltham, MA). Fluorescence was measured directly at the end of the incubation period using a CytoFluor 4000 fluorescence plate reader (Applied Biosystems, Foster City, CA). The settings used were: 485/20 nm excitation/bandwidth and 530/25 nm emission/bandwidth for F dye detection, and 560/20 nm excitation/bandwidth and 620/40 nm emission/bandwidth for R dye detection. The instrument gain was set for each dye so that the No Target Blank produced between 100 to 200 absolute fluorescence units (AFUs).Table 2Invader Assay Oligonucleotide SequencesFactor V LeidenInvader5′-TCTAATCTGTAAGAGCAGATCCCTGGACAGGCC-3′ (G1691A) FVLInvader (1689)5′-TCTAATCTGTAAGAGCAGATCCCTGGACAGACC-3′WT probe5′-CGCGAGGCCGGAGGAATACAGGTATTTTGTCC-Hexanediol-3′Mut probe5′-AGGCCACGGACGAAGGAATACAGGTATTTTGTC-Hexanediol-3′FRET (WT)5′-R-TCT-Q-AGCCGGTTTTCCGGCTGAGACGGCCTCGCG-Hexanediol-3′FRET (Mut)5′-F-TCT-Q-AGCCGGTTTTCCGGCTGAGACGTCCGTGGCCT-Hexanediol-3′WT target5′-TCAAGGACAAAATACCTGTATTCCTCGCCTGTCCAGGGATCTGCTCTT-ACAGATTAGAAGTGATTT-3′Mut target5′-TCAAGGACAAAATACCTGTATTCCTTGCCTGTCCAGGGATCTGCTCT-TACAGATTAGAAGTGATTT-3′Factor II (G20210A) FIIInvader5′-TATGGTTCCCAATAAAAGTGACTCTCAGCT-3′WT probe5′-ACGGACGCGGAGGAGCCTCAATGCTACCAG-Hexanediol-3′Mut probe5′-AGGCCACGGACGAAGCCTCAATGCTCC-Hexanediol-3′FRET (WT)5′-R-TCT-Q-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-Hexanediol-3′FRET (Mut)5′-F-TCT-Q-AGCCGGTTTTCCGGCTGAGACGTCCGTGGCCT-Hexanediol-3′WT target5′-TAGCACTGGGAGCATTGAGGCTCGCTGAGAGTCACTTTTATTGGGA-ACCATAGTTTTAGAAACACAAAAAT-3′Mut target5′-TAGCACTGGGAGCATTGAGGCTTGCTGAGAGTCACTTTTATTGGGAA-CCATAGTTTTAGAAACACAAAAAT-3′MethylenetetrahydrofolateInvader5′-CAAAGAAAAGCTGCGTGATGATGAAATCGC-3′ reductase (677)Invader (SIP)5′-CAAAGAAAAGCTGCGTGATGATGAAATTGC-3′ MTHFR 677WT probe5′-ACGGACGCGGAGGCTCCCGCAGACAC-Hexanediol-3′Mut probe5′-AGGCCACGGACGACTCCCGCAGACAC-Hexanediol-3′FRET (WT)5′-F-TCT-Q-AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-Hexanediol-3′FRET (Mut)5′-R-TCT-Q-TCGGCCTTTTGGCCGAGAGACGTCCGTGGCCT-Hexanediol-3′WT target5′-GAAGGTGTCTGCGGGAGCCGATTTCATCATCACGCAGCTTTTCTTT-GAGG-3′Mut target5′-GAAGGTGTCTGCGGGAGTCGATTTCATCATCACGCAGCTTTTCTTT-GAGG-3′MethylenetetrahydrofolateInvader5′-CCCGAGAGGTAAAGAACAAAGACTTCAAAGACACTTA-3′ reductase (1298)WT probe5′-AGGCCACGGACGTCTTCACTGGTCAGCT-Hexanediol-3′ MTHFR 1298Mut probe5′-CGCGCCGAGGGCTTCACTGGTCAGC-Hexanediol-3′FRET (WT)5′-F-ACT-Q-AGCCGGTTTTCCGGCTGAGTCGTCCGTGGCCT-Hexanediol-3′FRET (Mut)5′-R-TCT-Q-TCGGCCTTTTGGCCGAGAGACCTCGGCGCG-Hexanediol-3′WT target5′-GGAGCTGACCAGTGAAGAAAGTGTCTTTGAAGTCTTTGTTCTTTACCT-CTCGGGATTT-3′Mut target5′-GGAGCTGACCAGTGAAGCAAGTGTCTTTGAAGTCTTTGTTCTTTACCT-CTCGGGATTT-3′Plasminogen ActivatorInvader5′-GCACAGAGAGAGTCTGGACACGTGGGGT-3′ Inhibitor-1 (PAI-1-675 4G/5G) PAI-1 4G/5GWT probe (5G)5′-AGCTCGTCCGACAGAGTCAGCCGTGTATCA-Hexanediol-3′Mut probe (4G)5′-ACGGACGCGGAGAGTCAGCCGTGTATCA-Hexanediol-3′FRET F5′-F-TCT-Q-AGCCGGTTTTCCGGCTGAGATGTCGGACGAGCT-Hexanediol 3′FRET R5′-R-TCT-Q-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-Hexanediol-3′WT target5′-CCGATGATACACGGCTGACTCCCCCACGTGTCCAGACTCTCTCTGTG-CCCC-3′Mut target5′-CCGATGATACACGGCTGACTCCCCACGTGTCCAGACTCTCTCTGT-GCCCC-3′ Open table in a new tab Each microtiter plate contained synthetic DNA controls provided by Third Wave Technologies, Inc. for each genotype, ie, homozygous wild-type, heterozygous and homozygous mutant, as well as a No Target Blank. Data were analyzed using a Microsoft Excel-based spreadsheet (Microsoft, Redmond, WA). For each allele of a given polymorphism, the net signal/background, or Net Fold Over Zero (FOZ − 1), values were calculated as follows for the signal obtained with each dye: FOZ=Raw counts from sampleRaw counts from No Target Blank The two FOZ values (ie, wildtype and mutant) for each sample were used to calculate the WT:Mut ratio as follows: Ratio=(Net WT FOZ)(Net Mut FOZ)where net FOZ = FOZ − 1 In cases for which the net FOZ was ≤0.04, the value was set to 0.04 to calculate the ratio to avoid division by zero as described elsewhere.23Neville M Seltzer R Aizenstein B Maguire M Hogan K Welsh K Neri B de Arruda M Characterization of cytochrome P450 2D6 alleles using the Invader system.Biotechniques. 2002; 32: S34-S43PubMed Google Scholar In order for a sample run, ie, a microtiter plate containing controls and samples to be deemed valid, the FOZ ratios of the controls had to be within specific ranges. The FOZ ratio criteria were 5 or greater for the normal control, between 0.5 and 2 for the heterozygous control, and 0.2 or lower for the homozygous mutant control (except for MTHFR, where the FOZ ratios had to be between 0.3 and 3 for the heterozygous control). The FOZ values for the controls also had to meet minimum FOZ criteria (1.5 and 0.2 for FII, FVL, MTHFR, or 1.75 and 0.2 for PAI-1). Runs failing these criteria were deemed invalid and were repeated. Samples included in the study had to meet the following two criteria. 1. The ratio of net FOZ had to fall into the WT, Het (heterozygous mutation) or Mut (homozygous mutation) range; and 2. The FOZ value for each signal of the sample had to be greater than or equal to the values shown in Table 3Table 3Data AnalysisRatioMut FOZ*For PAI-1, the FOZ criteria were lowered to 1.75.WT FOZ*For PAI-1, the FOZ criteria were lowered to 1.75.Genotype≥5.0≥2.0≥2.0WT>2.0 to 0.2 to <0.5†For MTHFR, the boundaries were set at 0.3 and 3.0 due to differences in the amounts of signal generated by the Invader assays.——Equivocal (EQ2)≤0.2≥2.0≥0.2Mut* For PAI-1, the FOZ criteria were lowered to 1.75.† For MTHFR, the boundaries were set at 0.3 and 3.0 due to differences in the amounts of signal generated by the Invader assays. Open table in a new tab Results falling into either equivocal range as well as those failing the FOZ criteria were deemed invalid and subject to re-analysis; samples that met the ratio and FOZ criteria on re-testing were included in the study. Genotypes were generated from 1475 samples using the biplex Invader assay and AS-PCR, PCR-RFLP, or PCR-mass spectrometry. Of these samples, 1448, or 98.2%, were suitable for inclusion in the study. Results from a previous study demonstrated that neither the sample type (ie, buffy coat versus whole blood) nor the choice of extraction method affects the accuracy of the Invader assay.18Ledford M Friedman KD Hessner MJ Moehlenkamp C Williams TM Larson R A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous Invader microtiter plate FRET assay.J Mol Diagn. 2000; 2: 97-104Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The results comparing the Invader assay to a reference method from each center are presented in Table 4. Samples were analyzed in separate runs, each comprising four controls and approximately 20 samples. The overall concordance rate for the combined results was greater than 99.9% (1447 of 1448 correct calls). The Invader assay failed to assign a genotype correctly in only one case. The sample was homozygous wildtype but was incorrectly called a heterozygote in the initial Invader assay. The Invader assay yielded the correct result on repetition. DNA sequencing revealed that 21 samples that were initially discordant were correctly genotyped by the Invader assay, but were miscalled by the PCR-based reference method. For the PAI-1 assay, six of the seven samples called incorrectly by PCR were genotyped by the PCR-mass spectrometry assay.Table 4Summary of Invader Genotyping ResultsPolymorphismWild typeHeterozygoteMutantInvader assay totalsPCR-based assay totalsFactor V Leiden300/301 (99.7%)58/58 (100.0%)9/9 (100.0%)367/368 (99.7%)368/368 (100%)Prothrombin338/338 (100%)38/38 (100%)1/1 (100%)377/377 (100%)363/377 (96.3%)MTHFR C677T87/87 (100%)83/83 (100%)25/25 (
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