Abbate, Rosanna

Alessandrello Liotta, A.

Battaglini, B.

Fatini, Cinzia

Gensini, Francesca

Sticchi, E.

English

Florence Research

P1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002Biochemical Genetics, Vol. 42, Nos. 3/4, April 2004 ( C° 2004)High-Speed Detection of the G894T Polymorphismin Exon 7 of the eNOS Gene by Real-TimeFluorescence PCR With the Light-CyclerElena Sticchi,1 Cinzia Fatini,1;2;3 Francesca Gensini,1 Beatrice Battaglini,1Agatina Alessandrello Liotta,2 and Rosanna Abbate2Received 15 January 2003—Final 19 June 2003In endothelial cells nitric oxide (NO) is synthesized by endothelial-nitric oxidesynthase (e-NOS), constitutively expressed and encoded by a 26-exon gene, lo-cated on chromosome 7q35-36. The prevalence of the T rare variant of the G894Tpolymorphism in exon 7 of the e-NOS gene (Glu!Asp amino acid substitution)has been reported to be significantly higher in patients with coronary spasm andcoronary artery disease. To date G894T polymorphism detection is performedby PCR-RFLP assay. In order to establish a high-speed genotyping method, wehave taken advantage of the Light Cycler instrument, a thermal cycler that com-bines rapid-cycle DNA amplification with a real-time fluorescence monitoring.This technology is based on hybridization of the adjacent fluorescently labeledprobes with PCR products. This methodology is considered more accurate andless time-consuming than conventional PCR-RFLP assay. To validate this tech-nique we genotyped 270 healthy subjects. The results were consistent with thoseobtained from PCR-RFLP assay.KEY WORDS: Light Cycler; real-time PCR; e-NOS gene; G894T polymorphism.1 Department of Clinical Physiopathology, Unit of Medical Genetics, University of Florence, Florence,Italy.2 Department of Medical and Surgical Critical Care, Section of Clinical Medicine and Cardiology,University of Florence, Thrombosis Center, Azienda Ospedaliera Careggi, Florence, Italy.3 To whom correspondence should be addressed at Dipartimento dell’Area Critica Medico Chirurgica,Sezione Clinica Medica Generale e Cliniche Specialistiche, University of Florence, Centro Trombosi,Azienda Ospedaliera Careggi, Viale G. Morgagni 85, 50134 Firenze, Italy; e-mail: cinziafatini@hotmail.com.1210006-2928/04/0400-0121/0 C° 2004 Plenum Publishing CorporationP1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002122 Sticchi, Fatini, Gensini, Battaglini, Liotta, and AbbateINTRODUCTIONNitric oxide (NO), a potent regulator of the cardiovascular system, is synthesizedfrom L-arginine by a family of three NO synthases (NOS): neuronal (n-NOS,type I), inducible (i-NOS, type II), and endothelial (e-NOS, type III) (Wang andMarsden, 1995). In endothelial cells, NO is synthesized by the e-NOS, consti-tutively expressed, encoded by a 26-exon gene located on chromosome 7q35-36 (Marsden et al., 1993). Endothelium-derived NO controls the vascular tone,inhibits platelet (Radomski et al., 1987) and leucocyte adhesion to vascular en-dothelium (Kubes et al., 1991), inhibits smooth muscle cell migration (Sarkar et al.,1996) and growth (Garg and Hassid, 1989), and limits the oxidation of atherogeniclow-density lipoproteins (Hogg et al., 1993). These mechanisms suggest an impor-tant atheroprotective role, beyond its effect on vessel tone and blood pressure, andthat an alteration in the vascular NO system activity could contribute to the patho-genesis of atherosclerosis. The e-NOS human gene exhibits many polymorphisms,some of which seem related to a significant proportion of the variance of NO plas-matic levels. A point mutation of guanine to thymine at nucleotide 894 in exon 7 ofthe eNOS gene, which results in the replacement of glutamic acid by aspartic acidat codon 298 (Glu298Asp) has been described (Hingorani et al., 1995). It has beendemonstrated that this polymorphism is not silent and may possibly be perceivedas a target for cleavage (Tesauro et al., 2000). Evidence has been presented thatthis polymorphism is associated with coronary spasm (Yoshimura et al., 1998),acute myocardial infarction, and coronary artery disease in Caucasians (Hingoraniet al., 1999).EXPERIMENTAL PROCEDURETo date, G894T polymorphism detection for e-NOS is performed by polymerasechain reaction (PCR), followed by restriction fragment length polymorphism(RFLP) analysis through digestion with the MboI restriction enzyme and resolu-tion by electrophoresis on agarose gel (Hingorani et al., 1999). In order to establisha high-speed and facile genotyping method, we have taken advantage of the LightCycler (Roche Diagnostics), a microvolume fluorometer integrated with a thermalcycler that combines rapid-cycle DNA amplification with real-time fluorescencemonitoring (Wittwer et al., 1997). This technology is based on hybridization ofamplicon-specific oligonucleotide probes, labeled with adjacent fluorophores ca-pable of fluorescence resonance energy transfer (FRET) introduced in a thermalcycler (Light Cycler) that uses glass capillaries and hot air for heating. The probesare designed to hybridize to the same strand between the two unlabeled primers.A detection probe (Sensor probe), which matches the rare allele sequence, is la-beled at its 30-end with fluorescein (FLU). Moreover, a second hybridization probe(Anchor probe) is labeled at the 50-end with a Light Cycler red fluorophore (LCP1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002High-Speed Detection of the G894T Polymorphism 1235’-CACT CCCCACAGCTC TGCAT  3’-GTGAGGGGTGTCGAGACGTA        GCCCCAGATGATCCCCCAGAACTC        CCTTC T GCCCCCCGAGCTGGTCC      CGGGGTCTACTAGGGGGTCTTGAG        GGAAGACGGGGGGCTCGACCAGG                                                                     ACGTGAGCACCAAAGGGATTG -3’                                                                 TGCACTCGTGG TTTC CCTAAC-5’ NOS F (forward primer) NOS R (reverse primer) SENSOR PROBE (3’-FLU) ANCHOR PROBE (5’-LC Red640) Fig. 1. Schematic showing positions of primers and hybridization probes for amplification and geno-typing of eNOS G894T polymorphism site. An amplicon that spans the area of the polymorphism siteis created with the unlabeled primers. Two hybridization probes anneal to the same strand internalto the unlabeled primers. The Sensor probe is labeled at its 30-end with fluorescein (FLU), and theAnchor probe is labeled at its 50-end with a Light Cycler red fluorophore (LC Red 640).Red 640) (Fig. 1). When both probes hybridize in close proximity (in a head-to-tail manner), fluorescence energy transfer between the donor fluorophore (FLU),which is excited by a LED (light emitting dye) light source of the instrument(‚ D 530 nm), and the acceptor fluorophore (LC Red 640) occurs. The acceptorfluorophore emits light of a longer wavelength (‚ D 640 gm), which is detectedin specific channels of the instrument.For e-NOS G894T polymorphism genotyping, genomic DNA was isolatedand purified from EDTA blood using QIAamp spin columns according to the man-ufacturer’s instructions (QIAamp Blood Kit, Qiagen, GmbH, Hilden, Germany).Light Cycler (LC) amplification is performed in a final volume of 20 „L in theLC-glass capillaries. The reaction mixture consisted of 12.4 „L of H2O, 0.8 „Lof MgCl2 (25 mM), 10 pmol of each primer, NOSF (50-CACTCCCCACAGCTCTGCAT-30) and NOSR (50-CAATCCCTTTGGTGCTCACG-30), 4 pmol of An-chor probe, 4 pmol of Sensor probe, and 2 „L of DNA-Master hybridizationprobes (Reaction Buffer). Two microliters of genomic DNA (50–200 ng/„L) wereused for amplification. Fluorescently labeled hybridization probes were synthe-sized by TIB Molbiol (Berlin, Germany). The Anchor probe, 50-LC Red 640-CCTTCTGCCCCCCGAGCTGGTCC-30-P, was 50 labeled with the LC-Red 640fluorophore and phosphorylated (P) at its 30-end to prevent probe elongation by theTaq polymerase. The detection probe, 50-CCCCAGATGATCCCCCAGAACTC-30, was labeled with fluorescein (FLU) at its 30-end.Cycling conditions for e-NOS G894T polymorphism detection include aninitial denaturation at 95–C for 30 s, followed by 45 cycles with a fast denaturationat 95–C with an incubation time of 0 s, annealing at 62–C for 10 s and extensionat 72–C for 12 s with a ramping time of 20–C/s. After amplification, meltingcurves were generated following a fast denaturation of the reaction at 95–C with anP1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002124 Sticchi, Fatini, Gensini, Battaglini, Liotta, and AbbateFig. 2. Melting curves (fi) are converted to melting peaks (fl) by plotting the negative derivative offluorescence with respect to temperature (¡dF/dT). (A) The typical melting curve pattern, obtainedwhen the Sensor probe hybridizes with the specific DNA sequence (894T rare allele), is a single meltingpeak at a temperature of 65.5–C. The picture shows a homozygous genotype for T rare allele (TT). (B)For the common allele (894G) there is a mismatch between the PCR product and the Sensor probe,which leads to strand instability and consequent earlier melting. The result is a single melting peak ata temperature of 60.5–C. The picture shows a homozygous genotype for the common G allele (GG).(C) Heterozygous patients show two melting peaks, one at 60.5–C and one at 65.5–C. The pictureshows a heterozygous genotype (GT).incubation time of 0 s, holding the sample at 52–C for 30 s and then slowly heatingthe sample to 75–C with a ramp rate of 0.1–C/s. Melting curves were convertedto melting peaks by calculating the negative derivative of the fluorescence withrespect to temperature (–dF/dT) against temperature. This plot allows distinctionof the common allele from the rare one.The typical melting curve pattern, obtained when the Sensor probe hybridizeswith the specific DNA sequence (894T rare allele), is a single melting peak atP1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002High-Speed Detection of the G894T Polymorphism 125Fig. 2. (Continued).a temperature of 65.5–C (Fig. 2(A)). For the common allele (894G) there is amismatch between the PCR product and the Sensor probe, which leads to strandinstability and consequent earlier melting. The result is a single melting peak at atemperature of 60.5–C (Fig. 2(B)). Heterozygous patients show two melting peaks(60.5 and 65.5–C) (Fig. 2(C)).To validate this technique, we genotyped 270 healthy subjects recruited fromblood donors: 61 subjects were homozygous for the mutant 894T allele (TT,22%), 172 were heterozygous (GT, 64%), and 145 were homozygous for theG allele (GG, 54%). The 894T allele frequency was 0.33 (Table I). The geno-types were easily and clearly identified by generation of characteristic fluores-cence melting peaks. We have compared this technique with the results obtainedwith a conventional method (PCR-RFLP): in each case, identical genotypes wereobtained.P1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002126 Sticchi, Fatini, Gensini, Battaglini, Liotta, and AbbateFig. 2. (Continued).This methodology gives more accurate results than conventional PCR-RFLP-based assay because it minimizes PCR contamination concerns related to samplehandling and does not require digestion of PCR products with restriction enzymesand/or fragment separation on gels; it also does not require the use of hazardousreagents such as ethidium bromide and permits high-speed genotyping (i.e., 30Table I. Distribution of Genetic Polymorphisms of the eNOS G894T PolymorphismeNOS 894 TT GT GG T GGenotype 61 (22%) 172 (64%) 145 (54%)Allele frequency 0.33 0.67(Healthy Subjects n D 270).P1: GCEBiochemical Genetics [bigi] PP1109-478133-05 March 11, 2004 15:41 Style file version Nov 9th, 2002High-Speed Detection of the G894T Polymorphism 127samples can be analyzed in less than 2 h, instead of 8 h needed for PCR-RFLPassay).In conclusion, this assay has shown its reliability and rapid practicability. Itis especially suitable for molecular diagnostic routine laboratories, where a largenumber of samples must be processed.REFERENCESGarg, U. C., and Hassid, A. (1989). Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosinemonophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells.J. Clin. Invest. 83:1774–1777.Hingorani, A. D., Jia, H., Stevens, P. A., Monteith, P. S., and Brown, M. J. (1995). A common variantin exon 7 of the endothelial constitutive nitric oxide synthase gene. Clin. Sci. 88:21.Hingorani, A. D., Liang, C. F., Fatibene, J., Lyon, A., Monteith, S., Parsons, A., Haydock, S., Hopper,R. V., Stephens, N. G., O’Shaughnnessy, K. M., and Brown, M. J. (1999). A common variantof the endothelial nitric oxide synthase (Glu298 !Asp) is a major risk factor for coronary arterydisease in the UK. Circulation 100:1515–1520.Hogg, N., Kalyanaraman, B., Joseph, J., Struck, A., and Parthasarathy, S. (1993). Inhibition of low-density lipoprotein oxidation by nitric oxide. Potential role in atherogenesis. FEBS Lett. 334:170–174.Kubes, P., Suzuki, M., and Granger, D. N. (1991). Nitric oxide: An endogenous modulator of leukocyteadhesion. Proc. Natl. Acad. Sci. U.S.A. 88:4651–4655.Marsden, P. A., Heng, H. H., Scherer, S. W., Stewart, R. J., Hall, A. V., Shi, X. M., Tsui, L. C.,and Schappert, K. T. (1993). Structure and chromosomal localization of the human constitutiveendothelial nitric oxide synthase gene. J. Biol. Chem. 268:17478–17488.Radomski, M. W., Palmer, R. M., and Moncada, S. (1987). Endogenous nitric oxide inhibits humanplatelet adhesion to vascular endothelium. Lancet 2:1057–1058.Sarkar, R., Meiberg, E. G., Stanley, J. C., Gordon, D., and Webb, R. C. (1996). Nitric oxide reversiblyinhibits the migration of cultured vascular smooth muscle cells. Circ. Res. 78:225–230.Tesauro, M., Thompson, W. C., Rogliani, P., Qi, L., Chaudhary, P. P., and Moss, J. (2000). Intracellularprocessing of endothelial nitric oxide synthase isoforms associated with differences in severityof cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298.Proc. Natl. Acad. Sci. U.S.A. 97:2832–2835.Wang, Y., and Marsden, P. A. (1995). Nitric oxide synthase: Biochemical and molecular regulation.Curr. Opin. Nephrol. Hypertens. 4:12–22.Wittwer, C., Ririe, K., Andrew, R., David, D., Gunndry, R., and Balis, U. (1997). The lightcycler: Amicrovolume multisample fluorimeter with rapid temperature control. Biotechniques 22:176–181.Yoshimura, M., Yasue, H., Nakayama, M., Shimasaki, Y., Sumida, H., Sugiyama, S., Kugiyama, K.,Ogawa, H., Ogawa, Y., Saito, H., Miyamoto, Y., and Nakao, K. (1998). A missense Glu298Aspvariant in the endothelial nitric oxide synthase gene is associated with coronary spasm in theJapanese. Hum. Genet. 103:65–69.

High-speed detection of the G894T polymorphism in the exon 7 of the eNOS gene by real-time fluorescence PCR with the Light-Cycler

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High-speed detection of the G894T polymorphism in the exon 7 of the eNOS gene by real-time fluorescence PCR with the Light-Cycler

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