Clin Exp Med (2010) 10:269–272 DOI 10.1007/s10238-010-0096-3
SHORT COMMUNICATION
Comparison of three methods for genotyping of prothrombotic polymorphisms Marika Bianchi • Enzo Emanuele • Annalisa Davin • Stella Gagliardi • Emanuela Cova • Valentina Meli • Rosita Trotti • Cristina Cereda
Received: 26 January 2010 / Accepted: 14 April 2010 / Published online: 29 April 2010 Ó Springer-Verlag 2010
Abstract Several methods have been developed to detect common prothrombotic mutations, including factor V Leiden (G1691), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677C. In this study, we compared the accuracy of three different molecular techniques, i.e.: (1) restriction enzyme digestion (RFLP), (2) real time with hybridization probes and final melting curve (Fluorescence Resonance Energy Transfer, FRET), and (3) real time with hydrolysis probes (TaqManÒ). Sequencing was used as the reference standard. Our data showed that RFLPs analysis for the detection of prothrombotic mutations, albeit easy-to-perform, had a limited reliability for assessing correct genotypes. FRET analysis displayed higher resolution than RFLPs. Additionally, FRET analysis was faster and less tedious than sequencing. Keywords Prothrombotic mutations RFLPs FRET probes Real-time PCR Hydrolysis probes
M. Bianchi (&) A. Davin C. Cereda Laboratory of Neurogenetics, IRCCS Neurological Institute ‘‘C. Mondino’’, Via Mondino 2, 27100 Pavia, Italy e-mail:
[email protected] E. Emanuele Department of Health Sciences, University of Pavia, Via Bassi 21, 27100 Pavia, Italy S. Gagliardi E. Cova C. Cereda Laboratory of Experimental Neurobiology, IRCCS Neurological Institute ‘‘C. Mondino’’, Via Mondino 2, 27100 Pavia, Italy V. Meli R. Trotti Laboratory of Clinical Biochemistry, IRCCS Neurological Institute ‘‘C. Mondino’’, Pavia, Italy
Introduction Multiple complex interactions between genetic and environmental factors occur during the pathological course of thrombosis. Genetic predisposition to thrombosis may be suspected in patients who either develop this condition at a young age, or have recurrent thrombosis, or have thrombosis in an unusual site. In recent years, a number of mutations within genes coding for coagulation proteases have been identified in patients with inherited thrombophilia. The factor V Leiden (G1691A) and prothrombin G20210A (Factor II G20210A) mutations are two such examples that have been found to be strongly associated with spontaneous and recurrent venous thromboembolism [1, 2]. Similarly, a thermolabile variant of methylenetetrahydrofolate reductase (MTHFR C677T), an enzyme involved in the folate-dependent metabolism of homocysteine, has been reported to increase the risk for deep vein thrombosis and pulmonary embolism [3]. In recent years, different molecular approaches, such as restriction fragment length polymorphism (RFLP) and realtime PCR, have been developed to genotype single nucleotide polymorphisms (SNPs) in these genes. Compared with more traditional methods such as RFLP, realtime PCR is a fast, simple, and accurate procedure for SNP genotyping of medium to large collections of samples. Real-time PCR analysis can be performed using various strategies [5]. In the TaqManÒ assay, the reporter dye is released by hydrolysis during PCR amplification and the free reporter’s quantity is proportional to fluorescence [4, 7]. Fluorescence resonance energy transfer (FRET) is a method that distinguishes alleles by melting the products and monitoring the loss of fluorescence using an allelespecific oligonucleotide (ASO) probe, which hybridises to the mutant or wild-type sequences [4, 6, 8–10].
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In this study, we sought to compare the accuracy and speed of three methodologies (RFLP, real-time PCR with FRET probes and real-time PCR with TaqManÒ probes) for the genotyping of three prothrombotic polymorphisms, i.e. factor V Leiden, Factor II G20210A and MTHFR (C677T) variants.
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The MTHFR C677T variant was determined using the AMS52 PCR-RFLP kit (Clonit, Milan, Italy) according to the manufacturer’s protocol. Bands were separated on 3% agarose gels. Wild-type alleles produced a 198-bp fragment, whereas the mutant allele yielded a 175-bp product (Fig. 1). Real-time PCR with FRET probes
Materials and methods Patients A total of 51 subjects (35 women and 16 men, mean age: 33.4 ± 7.1 years) were recruited at the IRCCS Neurological Institute ‘‘C. Mondino’’, Pavia, Italy. Genotyping for prothrombotic polymorphisms was performed due to an enhanced prothrombotic risk associated with the use of oral contraceptive pill, hormone replacement therapy, or because of situations of temporary increased risk (surgery and pregnancy). All patients gave their consent to be included in this study. The study was approved by the local ethical committee and was performed in accordance with the Ethical Standards of the Declaration of Helsinki. DNA extraction Genomic DNA was isolated from 200 ll whole blood using a silica gel spin column method (QIAamp DNA Mini Kit, Qiagen, Milan, Italy) according to the manufacturer’s protocol. The DNA was then resuspended in 50 ll buffer TE, and the quality of DNA was tested by a spectrophotometric measurement (NanoDrop, Celbio, Milan, Italy). After quantification, DNA samples were adjusted to a concentration of 50 ng/ml in 1 9 TE. PCR-RFLP genotyping Factor V Leiden mutation was detected using the Factor V G1691A RFLP mutation detection system (Euroclone, Milan, Italy) following the manufacturer’s instructions. Fragments were separated on a 3% agarose gel. The length of the obtained fragments were the following: 148 bp for homozygous mutant genotype (AA), 123 bp for homozygous wild-type genotype (GG), and double band, one at 148 bp and one at 123 bp for heterozygous genotype (AG). The factor II G20210A mutation was assessed using the Factor II G20210A RFLP mutation detection system (Euroclone, Milan, Italy) according to the manufacturer’s instructions. Fragments were separated on a 3% agarose gel. The length of the fragments was as follows: 199 bp for the mutant homozygous genotype (AA), 177 bp for homozygous wild-type genotype (GG), and double band, one at 199 bp and one at 177 bp for heterozygous genotype (GA).
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FRET genotyping curve analysis was performed on a LightCyclerÒ 480 system (Roche Diagnostic, Mannheim, Germany). Factor V Leiden mutation was detected by realtime PCR using the LightCycler DNA Master Hyprobe (Roche, Mannheim, Germany) in a reaction volume of 20 ll containing 0.20 lM of each primer, 20 pmol/ll of the sensor probe (50 -ggC gAg gAA TAC Agg TAT 30 -FL), 20 pmol/ll of the anchor probe (LC Red 640-50 TgT CCT TgA AgT AAC CTT TCA gAA ATT CTg 30 -PH), and 200 ng of genomic DNA. Primers sequences were as follows: 50 -TCA ggC Agg AAC ACC ACC AT 30 (forward) and 50 -gCC CCA TTA TTT AgC CAg gAg-30 (reverse). After an initial denaturation step and Taq activation at 95°C for 1 min, amplification was performed by using 40 cycles of denaturation (95°C for 1 s), annealing (57°C for 5 s), and extension (72°C for 15 s). After amplification was complete, a final melting curve was recorded by heating to 95°C for 30 s and then cooling to 50°C, followed by a 30-s hold before heating to 85°C (ramp rate 5°C/s) and 40°C for 5 s. Fluorescence was measured continuously during the slow temperature ramp to monitor the dissociation of the hybridization probes. PCR for Factor II G20210A mutation was performed by real-time PCR using the LightCycler DNA Master Hyprobe (Roche, Mannheim, Germany) in a reaction volume of 20 ll containing 2 mM MgCl2 with each primer at a concentration of 0.20 lM, sensor probe at a concentration of 20 pmol/ll (50 -CTC AgC gAg CCT CAA Tg 30 -FL), 20 pmol/ll anchor probe (LC Red 640-50 TCC CAg TgC TAT TCA Tgg gC 30 -PH), and 200 ng of genomic DNA. Primers sequences were as follows: 50 -CCg CTg gTA TCA AAT ggg-30 (forward) and 50 -CCA gTA gTA TTA CTg gCT CTT CCT g-30 (reverse). After an initial denaturation
Fig. 1 Agarose gel depicting typical MTHFR C677T polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) genotype results. The length of the fragments obtained was 175 bp for mutant allele and 198 bp for wild-type allele
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step and Taq activation at 95°C for 30 s, amplification was performed by using 40 cycles of denaturation (95°C for 1 s), annealing (55°C for 5 s), and extension (72°C for 15 s). After amplification was complete, a final melting curve was recorded by heating to 95°C for 40 s and then cooling to 40°C, followed by a 30 s hold before heating to 85°C (ramp rate 5°C/s) and 40°C for 5 s. Fluorescence was measured continuously during the slow temperature ramp to monitor the dissociation of the hybridization probes. The MTHFR C677T variant was determined by using commercial primers and hybridization probes LC set (TIB Molbiol, Genoa, Italy) and the LightCycler FastStart DNA Master HypProbe (Roche, Mannheim, Germany) (Fig. 2). PCR was performed according to the manufacturer’s instructions. Real-time PCR with TaqManÒ probes Taq ManÒ genotyping curve analysis was performed on an iQ5 real-time PCR detection system (BioRad, Segrate, Italy). Genotyping of the factor V Leiden mutation was performed with the DUPLICa Real Time FACTOR V G1691A genotyping kit (EuroClone, Milan, Italy). Genotyping of the factor II (G20210A) mutation was done using the DUPLICa Real Time Factor II G20210A mutation genotyping kit (EuroClone, Milan, Italy) according to the manufacturer’s recommendations. Genotyping of the MTHFR (C677T) variant was performed by using the DUPLICa Real Time MTHFR C677T genotyping kit (EuroClone, Milan, Italy) according to the manufacturer’s protocol. DNA sequencing PCR products were isolated by using the NucleoSpin Extract II PCR purification kit (M-Medical, Milan, Italy). Sequencing was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130 XL DNA sequencer (Applied
Fig. 2 Melting curve data generated by fluorescent hybridization probes specific for the MTHFR C677T mutant allele. The red fluorescent resonance energy transfer (FRET) signal is progressively lost as the mutant probe denatures from the amplicon. The probe
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Biosystems, Foster City, CA, USA). Each fragment was sequenced on both strands. Quality control Wild type and mutated controls were included in each run for quality control purposes. For quality control, genotyping analyses were done blind with respect to the status of the study participants and a random 20% of the samples were repeated. Two investigators independently reviewed all results.
Results No samples failed on the three genotyping analyses. Results of genotyping using the three different techniques are depicted in Table 1. According to chi-square analysis, there was a marginally significant difference in overall genotyping results for factor V Leiden mutation (P = 0.09). In the case of the sequencing and real-time PCR with FRET probes results, genotypes from all samples were in 100% concordance. According to sequencing results, PCR-RFLPs gave incorrect results in 6 cases for factor V Leiden mutation, in 2 cases for factor II (G20210A) mutation, and in 3 cases for the MTHFR C677T variant. Real-time PCR with TaqManÒ probes gave incorrect results in 2 cases for factor V Leiden mutation, whereas all other genotyping results were concordant with the sequencing analysis. For all three polymorphisms, realtime PCR with FRET probes had a 100% concordance with sequence results.
Discussion In this study, we have compared three methods (i.e. RFLPs, Real-time PCR with TaqManÒ probes, Real-time PCR with
denatures from wild-type alleles at 62°C (melting temperature, or Tm), whereas it remains hybridized to mutant alleles until the temperature reaches 52°C (Tm)
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Table 1 Distribution of genotypes for the subset of samples chosen for the genotype methods comparison study Factor V Leiden
Wild type
Prothrombin G20210A
MTHFR C677T
RFLP
FRET–RT
TaqMan–RT
RFLP
FRET–RT
TaqMan–RT
RFLP
FRET–RT
TaqMan–RT
49
43
43
48
50
50
15
12
12
Heterozygous mutation
2
8
6
1
1
1
22
22
22
Homozygous mutation
0
0
2
2
0
0
14
17
17
Study participants = 51
FRET probes) for genotyping of common prothrombotic mutations of major clinical interest. Fluorescence genotyping, using either TaqManÒ or FRET probes, allows simultaneous amplification and analysis of the selected mutations in approximately 60 min without any further manual steps, including either enzyme digestion or electrophoresis. Using these methodologies, we were able to avoid potential problems related to sample tracking and end-products contamination. Unlike conventional restriction enzyme digestion followed by gel electrophoresis, fluorescence genotyping with either hybridization probes or hydrolysis probes yielded identical results for all but two tested samples. Sequencing of the amplification products clearly demonstrated that real-time PCR with FRET probes had a 100% accuracy for correct genotyping. In addition to the highly accurate detection of known mutations, genotyping with hybridization and hydrolysis probes offer important real-world advantages for the molecular diagnostic laboratory. Indeed, fluorescent methods are capable to determine the genotypes of several samples in a single closed tube in approximately 60 min, without the need for hands-on post-PCR analysis. This approach not only reduces the potential for post-PCR amplicon contamination and sample tracking errors, but also lowers labour costs and improves assay turnaround time by eliminating the need for post-PCR digestion and electrophoresis. We conclude that RFLPs analysis, albeit easy-to-perform, has a limited reliability for assessing correct genotypes of known prothrombotic mutations. Real-time analysis displays higher resolution than RFLPs. Importantly, our results suggest that FRET analysis is more accurate compared with real-time PCR with TaqManÒ probes.
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Conflict of interest
None.
References 1. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RG, deRonde H, van der Velden PA, Reitsma PH (1994) Mutation in blood coagulation factor V associated with resistance to activate protein C. Nature 343:64–67 2. Poort SR, Rosendaal FR, Reitsma PH (1996) A common genetic variant in the 3’- untraslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 88:3698–3703 3. Ray JG (1998) Meta-analysis of hyperhomocysteinemia as a risk factor for venous thromboembolic disease. Arch Intern Med 158:2101–2106 4. Gibson NJ (2007) The use of real-time PCR methods in DNA sequence variation analysis. Mol Cell Probes 363:171–176 5. Sevall JS (2001) Rapid allelic discrimination from real-time DNA amplification. Methods 25:452–455 6. Didenko VV (2001) DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques 31(5):1106–1121 7. Holland PM, Abramson RD, Watson R, Gelfand DH (2001) Detection of specific polymerase chain reaction product by utilizing the 50 –30 exonuclease activity of Thermus aquaticus DNA polymerase. Biochemistry 88:7276–7280 8. Lay MJ, Wittwer CT (1997) Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. ClinChem 43(12):2262–2267 9. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50(7):1156–1164 10. Parks SB, Popovich BW, Press RD (2001) Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes. Am J Clin Pathol 115:439–447
